The combined effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and the phytoestrogen genistein on steroid hormone secretion,AhR and ERβ expression and the incidence of apoptosis in granulosa cells of medium porcine follicles

Low doses of endocrine disrupting chemicals (EDCs) used in combination may act in a manner different from that of individual compounds. The objective of the study was to examine in vitro effects of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 100 pM) and genistein (500 nM) on: 1) progesterone (P₄) and estradiol (E₂) secretion (48 h); 2) dynamic changes in aryl hydrocarbon receptor (AhR) mRNA and protein expression (1, 3, 6, 24 and 48 h); 3) dynamic changes in estrogen receptor β (ERβ) mRNA and protein expression (1, 3, 6, 24 and 48 h); and 4) induction of apoptosis in porcine granulosa cells derived from medium follicles (3, 6 and 24 h). TCDD had no effect on P₄ or E₂ production, but potentiated the inhibitory effect of genistein on P₄ production. In contrast to the individual treatments which did not produce any effects, TCDD and genistein administered together decreased ERβ and AhR protein expression in granulosa cells. Moreover, the inhibitory effect of TCDD on AhR mRNA expression was abolished by genistein. The treatments did not induce apoptosis in the cells. In summary, combined effects of low concentrations of TCDD and genistein on follicular function of pigs differed from that of individual compounds. The results presented in the current paper clearly indicate that effects exerted by low doses of EDCs applied in combination must be taken into consideration when studying potential risk effects of EDCs on biological processes.     

Comparison of effects of leptin and ghrelin on porcine ovarian granulosa cells

The aim of our studies was to compare the roles of leptin and ghrelin in the direct control of proliferation, apoptosis, and secretory activity by porcine ovarian cells. In our in vitro experiments, we analyzed the effects of leptin and ghrelin treatments (at 0, 1, 10, or 100 ng/mL medium) on the accumulation of proliferation-related peptides (PCNA, cyclin B1, MAP kinase [MAPK]) and apoptosis-associated peptides (Bax, caspase 3, p53), and on progesterone secretion by cultured porcine granulosa cells, using immunocytochemistry, SDS PAGE-Western immunoblotting, and radioimmunoassay (RIA). Leptin stimulated proliferation (PCNA, cyclin B1, MAPK), apoptosis (Bax, p53), and progesterone secretion. Ghrelin promoted proliferation (PCNA, cyclin B1, MAPK) and progesterone secretion but suppressed apoptosis (Bax, caspase 3, p53). These observations suggest that both leptin and ghrelin directly control proliferation, apoptosis, and secretory activity by porcine ovarian cells. At the level of the ovary, in contrast to the hypothalamo-hypophysial system, leptin and ghrelin may have similar action in promoting granulosa cell proliferation and progesterone secretion, but they may be antagonistic to one another (leptin, stimulator; ghrelin, inhibitor) in controlling apoptosis.     

VEGF modulates the effects of gonadotropins in granulosa cells

Follicle selection is associated with an increase in the expression of vascular endothelial growth factor (VEGF) and its receptors in granulosa cells, however, the roles of VEGF in regulating the function of these or other non-endothelial cells in the ovary have not been explored in detail. The current study used bovine cell cultures to investigate potential roles of VEGF in the regulation of granulosa cell function during follicle development. Granulosa cells were obtained from morphologically healthy follicles 4 to 8 mm or 9 to 14 mm in diameter (corresponding to diameters before and after the establishment of dominance, respectively, during a bovine follicular wave) and exposed to a range of VEGF concentrations (1 to 100 ng/mL) encompassing concentrations found naturally in bovine dominant follicles. A concentration of VEGF of 1 ng/mL induced significant proliferation of granulosa cells from 4- to 8-mm follicles (P = 0.024) and increased the proliferative response of these cells to follicle-stimulating hormone (FSH; P = 0.045); whereas higher doses of VEGF had no effect on proliferation (P = 0.9). Treatment with VEGF induced an overall increase in mean extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation (P = 0.02). In contrast, VEGF, alone or in combination with FSH, had no effect on expression of the steroidogenic enzyme, CYP11A1, by cells from 4- to 8-mm follicles (P = 0.9). Granulosa cells from 9- to 14-mm follicles responded to 1 ng/mL VEGF with an increase in expression of the ovulation-associated gene, PTGS2 (P = 0.003) but higher VEGF doses had no effect (P = 0.9). The PTGS2 response to 1 ng/mL VEGF was similar to that induced by treatment with luteinizing hormone (LH). Interestingly, the stimulatory effects of LH on ERK1/2 phosphorylation (P = 0.003) and PTGS2 expression (P < 0.01) in granulosa cells from 9- to 14-mm follicles were abolished (P = 0.2) by specific chemical inhibition of VEGF receptor 2 (VEGFR2). These results suggest novel and important roles of VEGF and its receptor, VEGFR2, in mediating and/or enhancing the effects of gonadotropins in granulosa cells.     

Plasma ghrelin and oxyntomodulin concentrations in lactating dairy cows receiving abomasal soybean oil,corn starch,and casein infusions

The effects of increased postruminal supply of casein, corn starch, and soybean oil on plasma concentrations of the gastrointestinal hormones ghrelin and oxyntomodulin (OXM) were investigated. Four mid-lactation Holstein cows were used in a 4 × 4 Latin square. Treatments were continuous abomasal infusions (23 h/d) for 7 d of water, soybean oil (500 g/d), corn starch (1100 g/d), or casein (800 g/d). Jugular vein plasma was obtained every 30 min for 7 h on days 1 and 7. Soybean oil and casein infusion decreased preprandial plasma ghrelin concentration by approximately 20% on both d (time-by-treatment P < 0.10); however, dry matter intake (DMI) was depressed only after 7 d of oil infusion. Infusion of soybean oil, corn starch, or casein did not change the plasma OXM concentration (P > 0.20). The present data indicate that plasma ghrelin concentration is depressed immediately before feeding by the postruminal infusion of soybean oil and casein, but it is not affected during the postprandial period. Plasma ghrelin concentration was not altered (P > 0.20), pre- or postfeeding, by increased postruminal supply of corn starch. In addition, plasma OXM concentration did not respond (P > 0.20) to postruminal nutrient infusion. In conclusion, a decrease in DMI when fat is infused could be partially explained by the decrease in prefeeding plasma ghrelin concentration, but a decrease in prefeeding plasma ghrelin concentration is not always associated with a decrease in DMI, as observed for the infusion of casein. Plasma OXM concentration was not affected by postruminal infusion of macronutrients.     

The isoflavone daidzein directly affects porcine ovarian cell functions and modifies the effect of follicle‐stimulating hormone

The key biological active molecule of soya is the isoflavone daidzein, which possesses phytoestrogenic activity. The direct effect of soya and daidzein on ovarian cell functions is not known. This study examined the effect of daidzein on basic porcine ovarian granulosa cell functions and the response to follicle‐stimulating hormone (FSH). We studied the effects of daidzein (0, 1, 10 and 100 μm ), FSH (0, 0.01, 0.1, 1 IU/ml) and combinations of FSH (0, 0.01, 0.1, 1 IU/ml) + daidzein (50 μm ) on proliferation, apoptosis and hormone release from cultured porcine ovarian granulosa cells and ovarian follicles. The expression of a proliferation‐related peptide (PCNA) and an apoptosis‐related peptide (Bax) was analysed using immunocytochemistry. The release of progesterone (P4) and testosterone (T) was detected using EIA. Leptin output was analysed using RIA. Daidzein administration increased granulosa cell proliferation, apoptosis and T and leptin release but inhibited P4 output. Daidzein also increased T release and decreased P4 release from cultured ovarian follicles. Follicle‐stimulating hormone stimulated granulosa cell proliferation, apoptosis and P4, T and leptin release. The addition of daidzein promoted FSH‐stimulated apoptosis (but not proliferation) but suppressed FSH‐stimulated P4, T and leptin release. Our observations of FSH action confirm previous data on the stimulatory effect of FSH on ovarian cell proliferation, apoptosis and steroidogenesis and demonstrate for the first time the involvement of FSH in the upregulation of ovarian leptin release. Our observations of daidzein effects demonstrated for the first time that this soya isoflavone affected basic ovarian cell functions (proliferation, apoptosis and hormones release) and modified the effects of FSH. Daidzein promoted FSH action on ovarian cell proliferation and apoptosis and suppressed, and even inverted, FSH action on hormone release. The direct action of daidzein on basic ovarian cell functions and the ability of these cells to respond to FSH indicate the potential influence of soya‐containing diets on female reproductive processes via direct action on the ovary.     

Effects of the phytoestrogen,genistein, and protein tyrosine kinase inhibitor–dependent mechanisms on steroidogenesis and estrogen receptor expression in porcine granulosa cells of medium follicles

The use of soy-based products in pig diets had raised concerns regarding the reproductive toxicity of genistein, the predominant isoflavone in soybeans. Genistein was reported to exhibit weak estrogenic activity but its mechanism of action is not fully recognized. The aim of the study was to examine the in vitro effects of genistein on (1) progesterone (P₄) and estradiol (E₂) secretion by porcine granulosa cells harvested from medium follicles, (2) the viability of cultured granulosa cells, and (3) the mRNA and protein expression of estrogen receptors α and β (ERα and ERβ) in these cells. In addition, to verify the role of protein tyrosine kinase (PTK)–dependent mechanisms possibly involved in genistein biological action, we tested the effects of lavendustin C, the nonsteroidal PTK inhibitor, on granulosa cell steroidogenesis. We found that genistein inhibited (P < 0.05) basal P₄ secretion by granulosa cells harvested from medium follicles of pigs. In contrast, lavendustin C did not affect basal P₄ secretion by the cells. Moreover, genistein increased (P < 0.05) basal granulosal secretion of E₂. In contrast, lavendustin C did not alter basal E₂ secretion by porcine granulosa cells. In addition, we demonstrated that genistein increased mRNA and protein expression of ERβ (P < 0.05) in the examined cells. The expression of ERα mRNA was not affected by genistein and ERα protein was not detected in the cultured granulosa cells of pigs. In summary, the genistein action on follicular steroidogenesis in pigs involved changes in the granulosal expression of ERβ. However, the genistein action on P₄ and E₂ production by granulosa cells harvested from medium follicles did not seem to be associated with PTK.     

Ghrelin differentially modulates the GH secretory response to GHRH between the fed and fasted states in sheep

The effect of energy balance on the growth hormone (GH) secretory responsiveness to growth hormone-releasing hormone (GHRH) has not been determined in ruminant animals. Therefore, we examined the effects of intravenous injections of 0, 3.3, and 6.6 μg ghrelin/kg body weight (BW), with and without GHRH at 0.25 μg/kg BW, on GH secretory responsiveness in both the fed and fasted sheep. The injections were carried out at 48 h (Fasting state) and 3 h (Satiety state) after feeding. Blood samples were taken every 10 minutes, from 30 minutes before to 120 minutes after the injection. Low (3.3 μg/kg BW) and high (6.6 μg/kg BW) doses of ghrelin stimulated GH secretion significantly (P < .05) greater in the Satiety state than in the Fasting state. Growth hormone-releasing hormone plus both doses of ghrelin stimulated GH secretion significantly (P < .05) greater in the Satiety state than in the Fasting state. Ghrelin and GHRH exerted a synergistic effect in the Satiety state, but not in the Fasting state. Plasma ghrelin levels were maintained significantly (P < .05) greater in the Fasting state than in the Satiety state except the temporal increases after ghrelin administration. Plasma free fatty acid (FFA) concentrations were significantly (P < .01) greater in the Fasting state than in the Satiety state. In conclusion, the present study has demonstrated for the first time that ghrelin differentially modulates GH secretory response to GHRH according to feeding states in ruminant animals.     

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E₂), progesterone (P₄), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E₂-active (EA) and E₂-inactive (EI) follicles were selected according to their E₂-to-P₄ ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E₂ and P₄ concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.     

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: Potential role of estradiol

We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5 h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (± SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 ± 0.09 vs 2.83 ± 0.49 ng/mL (P = 0.004) for LH, 0.43 ± 0.05 vs 0.55 ± 0.03 ng/mL (P = 0.015) for FSH, and 9.34 ± 1.01 vs 11.51 ± 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E₂) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E₂ concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24 h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5 h before the start of infusion), then every 2 h for the following 22 h. Kisspeptin infused for 24 h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12 h = 50%; 6 h = 12.5%). The plasma concentration of E₂ was greater in ewes with an LH surge compared to those without LH surges; mean (± SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 ± 0.16 vs 1.27 ± 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E₂-positive feedback on gonadotropin secretion in acyclic ewes.     

Pharmacokinetics and bioavailability of a long-acting formulation of cephalexin after intramuscular administration to cats

The pharmacokinetic profile and bioavailability of a long-acting formulation of cephalexin after intramuscular administration to cats was investigated. Single intravenous (cephalexin lysine salt) and intramuscular (20% cephalexin monohydrate suspension) were administered to five cats at a dose rate of 10 mg/kg. Serum disposition curves were analyzed by noncompartmental approaches. After intravenous administration, volume of distribution (V_z), total body clearance (Cl_t), elimination constant (λ_z), elimination half-life (t_½λ) and mean residence time (MRT) were: 0.33 ± 0.03 L/kg; 0.14 ± 0.02 L/h kg, 0.42 ± 0.05 h⁻¹, 1.68 ± 0.20 h and 2.11 ± 0.25 h, respectively. Peak serum concentration (C_max), time to peak serum concentration (T_max) and bioavailability after intramuscular administration were 15.67 ± 1.95 μg/mL, 2.00 ± 0.61 h and 83.33 ± 8.74%, respectively.     

Pharmacokinetic and pharmacodynamic interactions of tolfenamic acid and marbofloxacin in goats

Pharmacokinetic and pharmacodynamic properties in goats of the non-steroidal anti-inflammatory drug tolfenamic acid (TA), administered both alone and in combination with the fluoroquinolone marbofloxacin (MB), were established in a tissue cage model of acute inflammation. Both drugs were injected intramuscularly at a dose rate of 2 mg kg⁻¹. After administration of TA alone and TA + MB pharmacokinetic parameters of TA (mean values) were C_max = 1.635 and 1.125 μg ml⁻¹, AUC = 6.451 and 3.967 μg h ml⁻¹, t_1/2K₁₀ = 2.618 and 2.291 h, Vdarea/F = 1.390 and 1.725 L kg⁻¹, and ClB/F = 0.386 and 0.552 L kg⁻¹ h⁻¹, respectively. These differences were not statistically significant. Tolfenamic acid inhibited prostaglandin (PG)E₂ synthesis in vivo in inflammatory exudate by 53-86% for up to 48 h after both TA treatments. Inhibition of synthesis of serum thromboxane (Tx)B₂ ex vivo ranged from 16% to 66% up to 12 h after both TA and TA + MB, with no significant differences between the two treatments.From the pharmacokinetic and eicosanoid inhibition data for TA, pharmacodynamic parameters after dosing with TA alone for serum TxB₂ and exudate PGE₂ expressing efficacy (E_max = 69.4 and 89.7%), potency (IC₅₀ = 0.717 and 0.073 μg ml⁻¹), sensitivity (N = 3.413 and 1.180) and equilibration time (t_1/2K_e0 = 0.702 and 16.52 h), respectively, were determined by PK-PD modeling using an effect compartment model. In this model TA was a preferential inhibitor of COX-2 (COX-1:COX-2 IC₅₀ ratio = 12:1). Tolfenamic acid, both alone and co-administered with MB, did not affect leucocyte numbers in exudate, transudate or blood. Compared to placebo significant attenuation of skin temperature rise over inflamed tissue cages was obtained after administration of TA and TA + MB with no significant differences between the two treatments. Marbofloxacin alone did not significantly affect serum TxB₂ and exudate PGE₂ concentrations or rise in skin temperature over exudate tissue cages. These data provide a basis for the rational use of TA in combination with MB in goat medicine.     

Endometrial and conceptus expression of HoxA10, transforming growth factor β1, leukemia inhibitory factor,and prostaglandin H synthase-2 in early pregnant pigs with gonadotropin-induced estrus

This study was conducted to evaluate the effect of estrus induction with gonadotropins on endometrial and conceptus expression of HoxA10, transforming growth factor (TGF) β1, leukemia inhibitory factor (LIF), and prostaglandin H synthase-2 (PGHS-2) during early pregnancy in pigs. Twenty-four prepubertal gilts received 750 IU of pregnant mare serum gonadotropin (PMSG) and 500 IU of human chorionic gonadotropin (hCG) 72 h later. Gilts in the control group (n = 23) were observed daily for estrus behavior. Endometrial tissue samples, conceptuses, blood serum, and uterine luminal flushings (ULFs) were collected on days 10, 11, 12, and 15 after insemination. There was no effect of estrus induction on estradiol content in ULFs, or on ovulation and fertilization rates in studied gilts. However, the content of progesterone in the blood serum was greater in naturally ovulated gilts in comparison to gonadotropin-treated animals on day 12 of pregnancy (P < 0.05). HoxA10 expression was up-regulated in the endometrium of pregnant gilts, with natural ovulation on days 12 (P < 0.05) and 15 (P < 0.001) in comparison to days 10 and 11. When compared to control gilts, administration of PMSG/hCG resulted in decreased expression of endometrial HoxA10, TGFβ, LIF, and PGHS-2 on day 12 of pregnancy (P < 0.05). Conceptus expression of studied factors was not affected by gonadotropin treatment. Overall, these results suggest improper endometrial preparation for implantation in prepubertal gilts induced to ovulate with PMSG/hCG.     

Determination of a dosage regimen of colistin by pharmacokinetic/pharmacodynamic integration and modeling for treatment of G.I.T. disease in pigs

Colistin is an antimicrobial drug of the polymyxin group and COLIVET SOLUTION is an aqueous solution containing colistin sulphate (2 × 10⁶ IU/mL), formulated for oral administration. The target species is the pig, particularly the suckling and post weaning animal. This investigation was undertaken to provide pharmacokinetic and pharmacodynamic data on which to base the selection of dosage rate and interval of the solution for the treatment of porcine colibacillosis.Colistin absorption from the gastrointestinal tract of young pigs, when administered at dosage rates of 25,000, 50,000 and 1,00,000 IU/kg, was slight or absent. The drug was therefore restricted almost entirely to the required site of action. The colistin concentration-time profile within the jejunum and ileum was established, and this enabled determination of the pharmacokinetic variables, maximum concentration (C_max) and area under curve (AUC) and derivation of the surrogate indices of antibacterial activity, C_max/minimum inhibitory concentration (MIC) and AUC/MIC through integration of in vivo data with the results of in vitro potency studies for four strains of Escherichia coli.In the in vitro bacterial growth inhibition studies colistin acted by a concentration-dependent killing mechanism. Numerical values for the surrogate parameter AUC/MIC producing bactericidal and eradication effects of colistin against four strains of E. coli were established by PK-PD modeling based on the sigmoidal E_max equation. These data were used to predict a daily dosage regimen for colistin.     

Effects of unsaturated fatty acids on progesterone secretion and selected protein kinases in goat granulosa cells

Previous studies in cattle have shown influences of dietary unsaturated fatty acid (UFA) supplementation on ovarian function. However, it is unclear whether these UFA exert direct or indirect effects on ovarian steroid production or their mechanisms of action. We have recently shown that 5′AMP-activated protein kinase (AMPK) regulates progesterone secretion through mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 (MAPK ERK1/2) in rodent granulosa cells. Here, we investigated the effects of 3 UFAs, oleic acid (OA), linoleic acid (LA), and α-linolenic acid (ALA) on progesterone secretion in goat granulosa cells. Finally, we examined the effects of UFAs on MAPK ERK1/2 and AMPK phosphorylation in these granulosa cells. Oleic acid and LA (10 μM each), but not ALA (100 μM), increased progesterone secretion (P < 0.05) in the presence or absence of insulin-like growth factor (IGF)-1 (10^-8 M) or FSH (5 × 10⁻⁸ M). The different AMPK subunits, except for γ3, are present in the goat ovary. Treatment with metformin (10 mM), an activator of AMPK, increased AMPK phosphorylation (P < 0.05) and reduced progesterone secretion by 50% (P < 0.05) in the basal state and in response to IGF-1 or FSH in goat granulosa cells. Oleic acid and LA had no effect on AMPK phosphorylation, whereas they rapidly increased MAPK ERK1/2 phosphorylation (P < 0.05). Finally, U0126, a MAPK ERK1/2 inhibitor, decreased OA- and LA-induced progesterone secretion (P < 0.05), suggesting that these UFAs could stimulate progesterone secretion partly through MAPK ERK1/2 in the absence of IGF-1 and FSH in goat granulosa cells. The involvement of AMPK in this process remains to be demonstrated. Taken together, some fatty acids could improve ovarian steroidogenesis through the MAPK ERK1/2 signaling pathway and, consequently, have beneficial effects on goat fertility.     

Intra-uterine insemination with low numbers of frozen–thawed boar spermatozoa in spontaneous and induced ovulating sows under field conditions

The present study was conducted to evaluate non-return rate (NR), farrowing rate (FR), and number of total pigs born/litter (TB) of weaned sows after intra-uterine insemination (IUI) using low numbers of frozen–thawed (FT) spermatozoa. Semen from 6 boars was cryopreserved individually in a 0.5-ml straw, at a concentration of 1 × 10⁹ spermatozoa/ml. A total of 40 multiparous sows with weaning-to-estrus interval of 3 to 7 days were included. The sows were detected for standing estrus twice daily and randomly assigned to two groups: I) spontaneous ovulation (n = 20) and II) induced ovulation (n = 20) which the sows were given 750 IU human chorionic gonadotrophin (hCG) i.m. immediately at estrus detection. Ovulation was determined every 12 h using transrectal ultrasonography. FT semen containing 1 × 10⁹ motile spermatozoa/dose was used to IUI. In group I, the sows were inseminated at 24 h after the detection of estrus and repeated every 12 h until ovulation. In group II, the sows were inseminated at 36, 42 and/or 48 h after hCG treatment. The results showed that the interval from standing estrus to ovulation (EOI) differed significantly between group I (40.2 h) and group II (35.6 h; P = 0.01). Variation of EOI among sows within each group seemed to be lower in group II (4.5 h SD) than in group I (5.5 h SD; P = 0.5). The number of IUI per sow was 2.9 ± 0.6 times in group I and was 2.4 ± 0.5 times in group II. There were no significant differences (P > 0.05) in the NR (80 vs 85%), FR (60 vs 65%) and the TB (8.0 ± 2.8 vs 9.4 ± 3.7 piglets/litter) between the groups. These results indicated that multiple doses of IUI with a low number of FT boar spermatozoa provided a fairly good NR, and reasonable FR and TB both in spontaneous and induced ovulating sows. The number of inseminations required for attaining acceptable fertility tended to be lower in the weaned sows with induced ovulation.     

巴美肉羊发情期外周血E2和P4浓度变化规律与排卵数关系研究

绵羊在发情周期中各生殖激素浓度变化与卵泡发育、成熟和排卵有着密切的关系。为了研究巴美肉羊血清生殖激素的动态变化及其与排卵数关系,试验采用电化学法,测定了12只成年母羊发情期血清中2种类固醇激素(E₂和P₄)的浓度水平,分析其动态变化规律,并用SAS 9.0的方差分析程序分析激素浓度与排卵数的关系。结果表明,两种激素在排单卵组和排双卵组绵羊间变化规律不同,E₂在排单卵组表现为先下降后升高的变化趋势,在排双卵组表现为持续下降趋势;P₄在排单卵组表现为持续上升的趋势,在排双卵组为先上升后下降的变化趋势。排单卵和排双卵组绵羊在各时间点的E₂和P₄激素浓度差异均不显著(P＞0.05)。     

Effects of ghrelin and its analogues on chicken ovarian granulosa cells

The aim of these in vitro experiments was (1) to examine the effects of ghrelin on the basic functions of ovarian cells (proliferation, apoptosis, secretory activity); (2) to determine the possible involvement of the GHS-R1a receptor and PKA- and MAPK-dependent post-receptor intracellular signalling cascades; (3) to identify the active part of the 28-amino acid molecule responsible for the effects of ghrelin on ovarian cells. We compared the effect of full-length ghrelin 1-28, a synthetic activator of GHS-R1a, GHRP6, and ghrelin molecular fragments 1-18 and 1-5 on cultured chicken ovarian cells. Indices of cell apoptosis (expression of the apoptotic peptide bax and the anti-apoptotic peptide bcl-2), proliferation (expression of proliferation-associated peptide PCNA), and expression of protein kinases (PKA and MAPK) within ovarian granulosa cells were analysed by immunocytochemistry. The secretion of progesterone (P(4)), testosterone (T), estradiol (E(2)) and arginine-vasotocin (AVT) by isolated ovarian follicular fragments was evaluated by RIA/EIA. It was observed that accumulation of bax was increased by ghrelin 1-28, GHRP6 and ghrelin 1-18, but not by ghrelin 1-5. Expression of bcl-2 was suppressed by addition of ghrelin 1-28, GHRP6 and ghrelin 1-5, but promoted by ghrelin 1-18. The occurrence of PCNA was reduced by ghrelin 1-28, GHRP6, ghrelin 1-18 and ghrelin 1-5. An increase in the expression of MAPK/ERK1, 2 was observed after addition of ghrelin 1-28, GHRP6 and ghrelin 1-18, but not ghrelin 1-5. The accumulation of PKA decreased after treatment with ghrelin 1-28 and increased after treatment with GHRP6 and ghrelin 1-18 but not ghrelin 1-5. Secretion of P(4) by ovarian follicular fragments was decreased after addition of ghrelin 1-28 or ghrelin 1-5 but stimulated by GHRP6 and ghrelin 1-18. Testosterone secretion was inhibited by ghrelins 1-28 and 1-18, but not by GHRP6 or ghrelin 1-5. Estradiol secretion was reduced after treatment with ghrelin 1-28 but stimulated by ghrelins 1-18 and 1-5; GHRP6 had no effect. AVT secretion was stimulated by ghrelin 1-28, GHRP6 and ghrelin 1-18, but inhibited by ghrelin 1-5. The comparison of the effects of the four ghrelin analogues on nine parameters of ovarian cells suggest (1) a direct effect of ghrelin on basic ovarian functions-apoptosis, proliferation, steroid and peptide hormone secretion; (2) that the majority of these effects can be mediated through GHS-R1a receptors; (3) an effect of ghrelin on MAPK- and PKA-dependent intracellular mechanisms, which can potentially mediate the action of ghrelin at the post-receptor level; (4) that ghrelin residues 5-18 may be responsible for the major effects of ghrelin on the avian ovary.     

Androgens promote the acquisition of maturation competence in bovine oocytes

Recent studies in mice suggest that androgens are important for normal follicle development. However, there have been few reports concerning the action of androgens in the growth of oocytes from large animals. The purpose of this study was to determine the roles of androgens in bovine oocyte growth in vitro. Oocyte-granulosa cell complexes (OGCs) collected from 0.4−0.7 mm early antral follicles were cultured for 14 days with 17β-estradiol (E₂) and a non-aromatizable androgen, dihydrotestosterone (DHT). We also examined the ability of an androgen receptor (AR) inhibitor, hydroxyflutamide, to antagonize the effect of androgens on the oocytes. During growth culture, the OGC structures collapsed in the medium with DHT alone, while in the presence of E₂, the OGC structures were maintained. In the medium with both androgens and E₂, the mean diameter of oocytes was increased from 95 μm to around 120 μm, larger than those grown with E₂ alone (115 μm). Also in the maturation culture, oocytes grown with androgens (A₄ or DHT) and E₂ showed higher percentages of metaphase II oocytes (63% or 69%, respectively) than those grown with E₂ alone (32%). Moreover, these maturation rates were decreased by hydroxyflutamide in a dose-dependent manner. Immunostaining showed that ARs were expressed in oocytes and granulosa cells in early antral follicles, and the nuclei of granulosa cells showed intense AR expression. In conclusion, although E₂ supports the OGC structure, additional androgens promote oocyte growth and their acquisition of meiotic competence via AR during in vitro growth culture.     

Cytotoxicity of Senecio in macrophages is mediated via its induction of oxidative stress

In Arunachal Pradesh and other sub-Himalayan areas of India, accidental consumption of Senecio plants by yaks is often fatal as the plant contains toxic alkaloids like Seneciophylline. The present investigation was undertaken to demonstrate the pro-oxidant effects of an ethanolic extract of Seneciochrysanthemoides (S-EtOH). S-EtOH impaired viability in macrophages, the IC₅₀ being 13.8 ± 1.11 μg/mL. The effect of S-EtOH (1 μg/mL) on generation of reactive oxygen species (ROS) in macrophages was measured by flow cytometry using 2′,7′-dichlorofluorescein diacetate (H₂DCFDA) where it caused a significant increase in the mean fluorescence channel (MFC) from 8.55 ± 0.03 to 47.32 ± 2.25 (p < 0.001). S-EtOH also effected a 3.8-fold increase in extracellular nitric oxide (NO) generation from 4.90 ± 0.72 μM to 18.79 ± 0.32 μM (p < 0.001), a 2.2-fold increase in intracellular NO production, the MFC increasing from 14.95 ± 0.48 to 33.34 ± 1.66 (p < 0.001), and concomitantly depleted non protein thiols as analyzed by flow cytometry using mercury orange, with a reduction in MFC from 632.5 ± 49.44 to 407.4 ± 12.61 (p < 0.01). Additionally, S-EtOH (14 μg/mL, 24 h) caused apoptosis as evident by increased Annexin V binding and terminal deoxynucleotidyl transferase mediated dUTP DNA nick end labeling. Taken together, the cytotoxicity of S-EtOH can be partly attributed to its capacity to inflict oxidative damage via generation of both reactive oxygen and nitrogen species culminating in apoptosis.