Long-term estradiol-17β administration decreases the number of neurons in the caudal mesenteric ganglion innervating the ovary in sexually mature gilts

The effect of estradiol-17β (E(2)) on the number and distribution of neurons in the caudal mesenteric ganglion (CaMG) supplying the ovary of adult pigs was investigated. Also, the numbers of ovarian dopamine-β-hydroxylase (DβH-), neuropeptide Y (NPY-), somatostatin (SOM-), galanin (GAL-) and estrogen receptor (ER)-immunoreactive perikarya as well as the density of the intraganglionic nerve fibers containing DβH and/or NPY, SOM, GAL were determined. E(2) was administered i.m. from day 4 of the first studied estrous cycle to the expected day 20 of the second studied cycle. Injections of E(2) (1) increased the E(2) level in the peripheral blood approximately 4-5 fold, (2) decreased the number of small-sized Fast Blue-positive postganglionic neurons in the CaMG, (3) decreased the number of small perikarya in the ventral, dorsal and central regions of the CaMG, (4) decreased the number of large perikarya in the dorsal and central regions, (5) decreased the number of small and large perikarya in the CaMG that were DβH(+)/NPY(+), (6) decreased the number of small DβH(+) but NPY(-) perikarya, (7) decreased the number of small perikarya coded DβH(+)/SOM(+) and DβH(+)/SOM(-), (8) decreased the number of small DβH(+)/GAL(-) perikarya, (9) decreased the number of small and large perikarya expressing ER subtypes α and β and (10) decreased the total number of nerve fibers in the CaMG containing DβH and/or NPY and DβH and/or GAL. These results show that long-term E(2) treatment of adult gilts downregulates the populations of both noradrenergic and ERs expressing ovarian neurons in the CaMG. Our findings suggest also that elevated E(2) levels that occur during pathological states may regulate gonadal function(s) by affecting ovary supplying neurons.     

Progesterone and estradiol-17β as a potential method for pregnancy diagnosis in the collared peccary (Pecari tajacu)

In this study, the period of pregnancy of nine collared peccary females has been monitored through the analysis of serum progesterone and estradiol-17β profiles. Serum concentrations of progesterone increased by Day 4 after conception, reaching concentrations of 33.4±5.6ng/mL on Day 10. Between Days 10 and 130 progesterone values were maintained between 20 and 60ng/mL. In the collared peccary, embryonic estradiol synthesis is first observed in the systemic circulation by Day 15 of pregnancy. Between Days 0 and 50 of pregnancy, average estradiol-17β concentrations were between 0 and 30pg/mL. From Day 75 of pregnancy onwards, estradiol concentrations were constantly increasing, reaching maximum concentrations (131.4±40.8pg/mL) on the day of parturition. The combined study of serum progesterone and estradiol-17β concentrations as a potential method for early pregnancy diagnosis presented the best overall accuracy (73%) when the threshold was established at 20ng/mL serum progesterone and 20pg/mL serum estradiol. Nevertheless, the accuracy for diagnosing pregnancy of females at mid and late pregnancy was 78% and 95%, respectively. The analysis of the sexual hormones during pregnancy could be a useful tool as a potential pregnancy diagnosis and an efficient predictor of the day of parturition in the captive collared peccary.     

Transport of L‐valine by the chicken caecum

1. We have studied L‐valine transport by the caecal segments of 6‐ to 8‐week‐old chickens. Isolated enterocytes from the proximal caecum incubated with 0.1 mM L‐valine can accumulate the substrate against a concentration gradient. After 50 min incubation, the intracellular L‐valine concentration reached 0.53 mM, a value higher than that observed in enterocytes from the jejunum (0.34 mM; P< 0.01).

2. Enterocytes from the medial and distal caecal regions are unable to transport L‐valine uphill (cell concentration: 0.1 mM).

3. Amino acid accumulation by proximal caecal cells was Na⁺‐dependent and was inhibited by ouabain and 2,4‐dinitrophenol. L‐methionine inhibits L‐valine uptake and a 2.5 mM concentration abolishes the capacity of enterocytes to accumulate the substrate.

4. The high accumulation ratios shown by the proximal caecum for L‐valine suggest a role for this intestinal segment in the absorption of neutral amino acids present in the caecal chamber.     

Plasma fluctuation in estradiol-17β and bone resorption markers around parturition in dairy cows

Blood samples were obtained sequentially from 10 dairy cows around the time of parturition to assess plasma fluctuations in estradiol-17β (E₂) levels in association with those of several bone resorption markers. Plasma E₂ concentration increased sharply a few days prepartum and decreased quickly after parturition. In terms of bone resorption markers, the plasma level of tartrate-resistant acid phosphatase isoform 5b (TRAP5b) rose significantly, commencing 1 week prepartum, and was maintained at this level to a few days postpartum. The plasma concentration of carboxyterminal collagen cross-links of type-I collagen (CTx) increased significantly after parturition. These observations suggest that osteoclast-mediated bone resorption was activated after parturition when plasma E₂ concentrations decreased.     

Utilization of Dietary β-Carotene and Retinol Acetate by Goslings and Young Geese

One-day-old and 3-month-old geese were fed different concentrations of g -carotene (5, 10, 50 or 150 mg kg -1 feed) or retinol acetate (10 000, 20 000 or 40 000 IU kg -1 feed) for about 3 weeks to assess their ability to convert g -carotene to vitamin A. Blood serum concentrations of g -carotene, retinol, f -tocopherol, triacylglycerides and cholesterol, as well as liver concentrations of g -carotene, retinol, triacylglycerides and cholesterol, were included as response factors. g Carotene was not detectable in serum and liver. Serum and liver retinol concentrations were positively correlated with dietary concentrations of g -carotene and retinol acetate ( R 2 = 0.84 and 0.95, respectively). In goslings 1 mg of g -carotene corresponded to 63 IU retinol and in young geese to 1216 IU, equivalent to 3.8% and 72.9%, respectively, of the theoretical value of 1667 IU retinol. Cholesterol concentrations in blood serum and liver were not affected ( P > 0.05) by the dietary levels of g -carotene and retinol acetate. The concentration of f -tocopherol in blood serum decreased with increasing dietary levels of g -carotene and retinol acetate. In the goslings the concentration of triacylglycerides in serum and liver was significantly ( P < 0.05) influenced by the intake of g -carotene, but not by the intake of retinol acetate. In the young geese the serum concentration of triacylglycerides decreased ( P < 0.05) with increasing dietary levels of g -carotene, but was not affected by the dietary concentrations of retinol acetate. The concentration of triacylglycerides in liver tissue of young geese was not affected by the dietary concentration of either g -carotene or retinol acetate ( P > 0.05).     

The effect of tannic acid and polyethylene glycol on the absorption capacity of chicken intestine for d-xylose and β-carotene

In an experiment, the possible influence of tannic acid (TA) and polyethylene glycol (PEG) on the absorption capacity of intestine for d-xylose and β-carotene in broiler chicken was investigated. Four groups of nine 28-day-old broiler cockerels received d-xylose (500 mg) and β-carotene (52 μg) solutions (Group 1 to 4) with TA (1 g, Group 2 to 4) and PEG (500 mg Group 3 and 1 g Group 4), orally. One blood sample prior to, and four others after the administration of test materials, were collected from wing vein on 40 min basis, for 160 min and the concentration of plasma d-xylose was determined. The concentration of β-carotene was also measured in plasma of blood samples taken prior to and 160 min post-administration of the test materials. Plasma d-xylose concentration of all groups showed quadratic correlations with time (p < 0.001, r(2) = 0.84, 0.60, 0.70 and 0.74 for Group 1 to 4, respectively). Administration of TA reduced the plasma concentration of d-xylose in Group 2. However, feeding PEG after TA raised the concentration of d-xylose in Group 4 to the level that there was no difference in that variable between this group and Group 1. Although the plasma concentration of β-carotene was increased in 160 min post-ingestion of the test material, no difference was found in that variable among the experimental groups. In conclusion, TA and its interaction with PEG have impacts on the absorption capacity of intestine for d-xylose and highly likely other simple sugars, but TA or PEG have no influence on the absorption of β-carotene and most probably other fat soluble vitamins.     

Relationship between the peripheral concentrations of estradiol-17β (E2) and preovulatory characteristics of cumulus-oocyte complexes (COCs) during superovulation treatment in Japanese Black cows

The relationship between the peripheral concentrations of estradiol-17β (E(2)) and the preovulatory characteristics of cumulus oocyte complexes (COCs) during superovulation treatment was investigated in Japanese Black cows. A superovulation regimen with FSH treatment in a descending manner was commenced on day 7 (n=3) or day 10 (n=2) of the estrous cycle (day 0=estrus). Peripheral blood was collected to measure E(2) concentrations twice a day throughout the treatment. Ovariectomies were performed at 100 h after the initial FSH treatment in five cows. Every follicle more than 8 mm in diameter was isolated from the ovaries, and cumulus-oocyte complexes (COCs) were gently aspirated. The COCs were then separated into three groups based on the characteristics of the cumulus (compact, expanded and denuded) and subgrouped based on the stage of the nucleus in the oocytes (GV, GVBD). Plasma E(2) concentrations tended to increase gradually and reached the peak level at around 84 h (E(2)-84: n=3) or 96 h (E(2)-96: n=2) after the initial FSH treatment. The ratio of COCs with expanded cumulus was significantly higher in E(2)-84 than in E(2)-96 (P<0.01). However, there was no difference in the ratio of oocytes showing GVBD between E(2)-84 and E(2)-96 (P=0.73), and the characteristics of the cumulus did not affect the stage of the nucleus in the oocytes in either groups (compact, expanded and nude; P=0.61, 0.81 and 1.00). It was possible that the time until the peak plasma E(2) concentrations after the FSH treatment could become an indicator for the maturation of follicles and oocytes in preovulatory follicles during superovulation treatment in Japanese Black cows.     

Comparison of plasma concentrations of estradiol-17β and progesterone, and conception in dairy cows with cystic ovarian diseases between Ovsynch and Ovsynch plus CIDR timed AI protocols

The objectives of this study were 1) to determine the effects of adding a CIDR to the Ovsynch protocol on plasma concentrations of estradiol-17β and progesterone and conception in dairy cows with cystic ovarian diseases and 2) to examine associations among the estradiol-17β and progesterone concentrations and conception. Cows were diagnosed as having cystic ovarian diseases if they were found to have a cystic follicle (diameter ≥25 mm) without a corpus luteum by two palpations per rectum with an interval for 7 to 14 days. They were treated with either the Ovsynch (GnRH on Day 0, PGF(2α) on Day 7 and GnRH on Day 9, with AI on Day 10; n=15) or Ovsynch+CIDR protocol (Ovsynch protocol plus a CIDR from Day 0 to Day 7; n=23). Plasma estradiol-17β concentrations were determined on Days 0, 7 and 9, and plasma progesterone concentrations were determined on Days 0, 7, 9 and 17. The plasma estradiol-17β and progesterone concentrations at all of the days examined and conception rates did not differ significantly between the two timed AI protocols. The progesterone concentrations on Day 17 and conception rates were lower (P<0.05) for cows with low concentrations of estradiol-17β (<2 pg/ml) on Day 9 than for cows with high concentrations of estradiol-17β (≥2 pg/ml). The present study suggests that, in dairy cows with cystic ovarian diseases, addition of a CIDR to the Ovsynch protocol had no remarkable effects on plasma estradiol-17β and progesterone concentrations during and after the treatments or on conception after timed AI. This study indicates that the low plasma estradiol-17β concentration at the second administration of GnRH in the protocols can be a predictor for impaired luteal formation and lower likelihood of pregnancy in dairy cows with cystic ovarian diseases.     

Ileal amino acid digestibility assay for the growing meat chicken—effect of the imposition of a fasting period and the nature of the test diet

1. The objective of this study was to investigate the effects of cold‐pelleting, the length of the fasting period before feeding of the test diet and the nature of die test diet on apparent ileal nitrogen (N) digestibility in the broiler chicken.

2. Four‐week‐old broiler chickens were given a pelleted or non‐pelleted maize/soyabean meal (basal) diet. The birds were starved for 24 h, given a single test meal (25 g) by intubation and killed 4 h after the start of feeding by administration of a barbiturate, to allow sampling of ileal digesta (terminal 15 cm). Cold‐pelleting did not affect apparent ileal N digestibility.

3. Four‐week‐old broiler chickens were fasted for 12 or 24 h and then received a test meal (1 h free access) of either a pelleted soyabean meal or a pelleted meat‐and‐bone meal diet or were continuously fed on one of the two diets. The imposition of a fast did not affect apparent ileal N digestibility. However, a 24 h fasting procedure was preferred, as the between animal variation for apparent ileal N digestibility was lower than for the 12 h fast or for continuous feeding.

4. Four‐week‐old broiler chickens were given either semi‐synthetic starch‐based diets containing maize, wheat bran, meat‐and‐bone meal or fish meal as the sole sources of protein or each of these diets in combination with the basal diet (50:50 on a dry matter basis). With the exception of the maize diet, the apparent ileal N digestibility values calculated by correcting for the digestibility of the basal dietary component were significantly lower than when digestibility was determined directly using a diet in which the respective proteins were the sole protein source. This implies that interactions between the dietary ingredients influence estimates of apparent ileal N digestibility.     

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-��

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-γ were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-γ (ChIFN-γ) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-γ did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.     

Transcriptional profiling of antimicrobial peptides avian β-defensins in the chicken ovary during sexual maturation and in response to Salmonella enteritidis infection

Avian β-defensins (AvβDs) are antimicrobial peptides that play significant roles in the innate immune system in chickens. The aim of this study was to identify the types of AvβDs expressed in the chicken ovary, to investigate the effects of sexual maturation in the ovarian mRNA abundance and to determine the changes in their expression levels as a result to Salmonella enteritidis (SE) infection. RNA was extracted from the ovary of healthy prepubertal, sexually mature and aged birds, as well as from sexually mature and aged SE infected birds. Real-time PCR analysis revealed that 11 AvβDs genes were expressed in the chicken ovary. A significant up regulation of AvβD1, 3, 4, 5, 7 and 11 was observed in the ovary of sexually mature and aged birds. Furthermore, a significant up-regulation of AvβD4, 5, 7, 11 and 12 was observed in the ovary of SE infected sexually mature birds. These results suggest that the mRNA expression of at least six AvβDs increase with age in the ovary of laying hens, and that at least five AvβDs show an induction in their expression in response to SE infection, indicating an AvβD-mediated immune response mechanism in the chicken ovary.     

Effect of aglepristone (RU534) administration during follicular phase on progesterone,estradiol-17β and LH serum concentrations in bitches

Aglepristone was administered in bitches during the follicular phase to evaluate its effects on progesterone, estradiol-17β and LH serum concentrations. Ten German Shepherds were divided into two groups (treated n = 5; control n = 5). Treated bitches received 10 mg/kg BW of aglepristone subcutaneously during the early follicular phase, 24 hr after and then 7 days later. The control group was injected, at the same time periods, with saline solution (0.3 ml/kg BW). For the steroid evaluations, blood was collected daily from the onset of proestrus until the first day of cytological dioestrus. For LH base-line serum determination, blood was also collected every 20 min for 2 hr at the onset of proestrus. For LH surge identification, blood was collected daily (every 6 hr) starting from the day of the first administration of aglepristone or saline solution until the first day of dioestrus. All animals ovulated but the treated group presented longer ovulation-dioestrus intervals than the control group (5.2 ± 2.2 days p < .05). Serum concentrations of the evaluated hormones were similar between experimental animals except for serum LH. Indeed, no LH peaks were detected in the treated group while LH surges were clearly observed in the control group (9 ± 1 days after the beginning of proestrus. In particular, the area under the curve for LH was significantly lower in treated than control animals (12 ± 4 ng/ml x Day; p = .01). In conclusion, administrations of aglepristone during the follicular phase of the bitch does not affect the steroid hormone patterns but does prevent the occurrence of a LH surge. This work raises significant questions and opens perspectives concerning the mechanisms of ovulation in bitches.     

Age associated changes in sodium,potassium and moisture contents of Turkey skeletal muscles and a note on the effects of testosterone and 17‐β‐oestradiol

1. Sodium concentration declined and potassium concentration increased sharply in turkey skeletal muscles during the first 3 weeks of life.

2. Testosterone and 17‐β‐oestradiol implantation of 2‐d‐old male turkeys resulted in increased moisture and Na concentrations in leg muscle.

3. Testosterone but not 17‐β‐oestradiol implantation also increased the moisture content of breast muscle.     

Downregulation of the expression of inhibin α subunit and betaglycan in porcine cystic follicles

Inhibins, as members of the transforming growth factor beta (TGF-β) superfamily, downregulate the synthesis and secretion of follicle-stimulating hormone (FSH) in an endocrine manner. The role of inhibin/betaglycan in the ovary regulation recently gained attention. To date, no data exist on the function of inhibin α subunit and betaglycan in cystic follicles. In this study, the expressions of inhibin α subunit and betaglycan in cystic follicles were investigated using immunohistochemistry, real-time PCR and Western blot analysis. Both inhibin α subunit and betaglycan immunoreactivities were mainly localized in the granulosa cells of follicles. Expression of inhibin α subunit and betaglycan was inferior in cystic follicles compared with that in normal large follicles. However, the result of enzyme-linked immunosorbent assay showed no significant difference in the decreasing in concentration of inhibin α subunit in cystic follicular fluid compared with the control (P>0.05). In this study, we explored the effects of FSH on betaglycan expression in granulosa cells in vitro. As expected, a significant increase in the expressions of betaglycan mRNA and protein in granulosa cells was observed in response to exogenous FSH (30 ng/ml) (P<0.05) compared with the control. Consequently, this study provides evidence that the expressions of inhibin α subunit and betaglycan are inferior in cystic follicles, and this may be caused by the decrease in FSH in the presence of a cystic follicle.     

Development of an Heal amino acid digestibility assay for the growing chicken—effects of time after feeding and site of sampling

1. The study aimed to establish the optimum time after ingestion and optimum sampling site for the development of an ileal amino acid digestibility assay for broiler chickens.

2. To establish the optimal sampling time, 4‐week‐old broiler chickens were given one of 6 protein sources (meat‐and‐bone, soyabean, cottonseed, fish, maize and wheat meals) as the sole source of protein in a test diet. The diets contained chromic oxide as an indigestible marker. The birds were starved for 24 h, fed and subsequently killed for sampling of ileal digesta (terminal 15 cm) at 2, 3, 4, 5 and 6 h after the start of feeding.

3. For the soyabean, fish, wheat and maize meal diets, sampling time had no significant effect on apparent ileal nitrogen digestibility, whereas for the meat‐and‐bone and cottonseed meal diets there was a significant quadratic effect of sampling time. The amount of digesta collected was maximised and the mean apparent ileal nitrogen digestibility had the lowest variation around the 4 h sampling time.

4. To establish the optimum sampling site, 4‐week‐old chickens were given either a meat‐and‐bone, a soyabean or a wheat bran meal‐based diet. The birds were killed 4 h after the start of feeding, and digesta were sampled from 0.10, 0.15, 0.20 or 0.25 cm of terminal ileum.

5. There was no significant effect of sampling site on the apparent ileal digestibility of dietary nitrogen. The terminal 15 cm of ileum was considered a preferred site for sampling ileal digesta from broiler chickens.     

Effect of genistein on the gene and protein expressions of CXCL–12 and EGR–1 in the rat ovary

The effect of genistein (GEN) on the gene expression level of stromal cell-derived factor-1/CXCL-12 and early growth response gene-1 was studied in ovarian tissue of young and initially ageing (early stages in the ageing process) female rats. Forty, young female Sprague Dawley (SD) rats of 2–3 months old (200 ±20 g) and forty, initially ageing female SD rats of 10–12 months (490 ± 20 g) old were selected. According to the weight, rats were divided into control group, low-dose group (L), medium-dose group (M) and a high-dose group (H) and were given 15, 30 and 60 mg/kg GEN respectively. The positive control (Oestrogen) group was given 0.5 mg/kg diethylstilbestrol. The treatment lasted for 30 days. The mRNA expression of C-X-C motif chemokine ligand 12 (CXCL-12) and early growth response factor-1 (EGR-1) was measured by real-time PCR, and protein expression of EGR-1 was detected by Western blot. When compared to the negative control group (NC), the ovary/body weight ratio in the young rats decreased in the GEN group, but the difference was not significant. Similarly, compared with NC, the ovary/body weight ratio in the initially ageing rats also decreased with the increase in GEN concentration, but the decrease was significant in M and H groups (p < .01). The administration of GEN enhanced both the gene and protein expression levels of CXCL-12 and EGR-1 in the ovary. Pearson's correlation analysis showed a synergistic effect between CXCL-12 and EGR-1. Thus, we conclude that the effect of GEN on CXCL-12 and EGR-1 in the initially ageing group was obvious than that in the younger group.