Dynamics of anterior pituitary immunoreactive gonadotrophs in moulted hens supplemented with protein, symbiotic and probiotics.

The present work delineates redistribution patterns of the hormone-producing cells of the anterior pituitary, after the phase of moulting. Two hundred single comb White Leghorn hens at the end of their first production cycle (Age = 70 week) were purchased from the commercial poultry farm and were induced to moult by high-dietary zinc (3 g/kg feed/day) after 1 week of acclimatization, at the experimental research station, Department of Physiology and Pharmacology, University of Agriculture, Faisalabad. The moulted birds were equally (n = 50) and randomly allocated to their respective groups as G1 (control; CP (Crude protein) 16%, no supplement), G2 (CP18%, no other supplement), G3 (CP16%, symbiotic at does rate of 85 mg/l in drinking water daily) and G4 (CP16%, probiotic at dose rate of 85 mg/l in drinking water daily). Ten birds were slaughtered in each group at 5% and at peak of post-moult production stage to collect their pituitary glands. An earlier post-moult production recovery, sustained and lengthier production span was seen in the G2 as compared to all other groups. The lowest production and an earlier production decline were seen in G1. The cell diameter and area of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) gonadotroph increased (p ≤ 0.01) in G2 and G3 as compared to G1. The FSH gonadotroph nucleus diameter and area did increase (p ≤ 0.01) in G2 and G3, while LH gonadotroph nucleus diameter and area decreased (p ≤ 0.01) in G2 and G3 as compared to G1. The increased FSH and LH gonadotroph diameter in protein and symbiotic supplemented birds is accountable for the increased egg production in these groups.

Effect of vitamins, protein level and probiotics on immune response of moulted male broiler breeders.

This study was planned to investigate the comparative effect of vitamins C (L-ascorbic acid), E (DL-α-tocopherol acetate), probiotics, lower than normal protein level (14%) and combination of these treatments on immune response of male broiler breeders after zinc-induced moulting. One hundred and eighty birds at the age of 65 weeks were induced to moult by mixing zinc oxide (ZnO) in feed at the rate 3000 IU/kg of feed. Upon completion of moulting, birds were divided into six groups (five replicates per group) in a completely randomized design and were fed vitamin C (500 IU/kg), vitamin E (100 IU/kg), lower protein level, probiotics (50 mg/l), and a combination of these components, while one group was kept as control. After completion of moulting phase (5 weeks), the treatment effects were tested as in vitro macrophages engulfment percentage, nitric oxide (NO) production, serum antibody titres against Newcastle disease (ND) and infectious bronchitis (IB). The results showed that in vitro macrophage engulfment percentage in unopsonized conditions was significantly higher in vitamin E-supplemented group. In addition, in opsonized condition, the macrophage engulfment percentage was significantly higher in both vitamin E- and C-supplemented groups. The NO (opsonized and unopsonized) production and antibody titre against ND and IB were significantly higher in vitamin E-supplemented group. It was concluded that vitamin E is a better option for enhanced immune response in broiler breeders after zinc-induced moulting.

Effect of vitamins, probiotics and protein level on semen traits and seminal plasma biochemical parameters of post-moult male broiler breeders.

1. A study was designed to investigate the comparative effect of supplementary vitamin E, vitamin C, probiotics and dietary crude protein concentration on semen traits and seminal plasma biochemical parameters in male broiler breeders after Zn-induced moulting. 2. A total of 180 male broiler breeders were induced to moult at 65 weeks of age by mixing ZnO in diet at the rate 3000 mg/kg of feed. After moulting, the males were divided into six groups that were given feed supplemented with: vitamin C (500 IU/kg), vitamin E (100 IU/kg), protein (140 g CP/kg), probiotics (50 mg/kg) and the combination of these components, while one group was kept as a control. Semen samples were collected weekly and semen volume, spermatozoa concentration, motility and dead spermatozoa percentage were determined. Seminal plasma was separated to determine the concentration of total antioxidant capacity (TAC), homocysteine, paraoxonase (PON1), arylesterase, ceruloplasmin, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities. 3. Semen volume was significantly higher in the vitamin E and C groups compared to the control. Spermatozoa motility was higher in the vitamin E group and dead spermatozoa percentage was significantly lower in the vitamin C group compared with the control group. 4. Seminal plasma TAC was higher in the vitamin E group, homocysteine was lower in the vitamin C and E groups. PON1 was higher in the combination group. Arylesterase increased significantly in the vitamin C and combination groups over time. Seminal plasma AST was significantly lower in the vitamin C and E supplemented groups whereas ALT decreased significantly only in the vitamin E group compared with the control. Higher concentrations of ceruloplasmin were observed in the combination group compared with the other treatments. 5. It was concluded that additional vitamin E and C or their combination was the most potent nutrient treatment for improving the semen traits and seminal plasma biochemical characteristics in male broiler breeders after Zn-induced moulting.

Immunohistochemical localization and morphometry of somatotrophs and lactotrophs in protein, probiotic and symbiotic supplemented molted layers.

Two-hundred Single Comb White Leg-Horn spent hens at the age of 70 weeks were purchased from a commercial layer farm. The birds were shifted to the Poultry Research Station, Department of Physiology and Pharmacology, University of Agriculture, Faisalabad, Pakistan. High dietary zinc (3g/kg feed/day) was used to induce molting in all the birds after one week of acclimatization. Thereafter, birds were divided in groups of 50 birds each, with the following treatments: G1 [control; crude protein (CP)16%, no other supplement], G2 (CP18%, no other supplement), G3 (CP16%, Symbiotic, 85 mg/L drinking water) and G4 (CP16%, Probiotic, 85mg/L in drinking water). Fifteen birds were slaughtered at 5% of peak of production each to collect their pituitary glands. The better egg production was seen in all the supplemented groups as compared to the control. Especially an earlier post molt production recovery and delayed decline was seen in the G2 as compared to all other groups. The immunohistochemistry of the pituitary gland reveals the decrease (P≤0.01) in the cell and nucleus size as well as area of somatotrophs in G2 and G4 as compared to G1. The cell and nucleus size as well as area of lactotrophs decreased (P≤0.01) in G2, G3 and G4 as compared to G1. The better and earlier post molt production in G2 highlights the potential role of protein supplementation in connection with the decreased lactotroph size and area in molted birds.

Effect of protein, probiotic, and symbiotic supplementation on serum biological health markers of molted layers.

Dietary zinc was used to induce molt in 200 White Leghorn birds in caged housing at the age of 70 wk. The birds were equally and randomly allocated to 4 groups each of 50 birds as G1 (control; CP 16%, no supplement), G2 (CP 18%, no other supplement), G3 (CP 16%, symbiotic Perfectin: 85 mg•L(-1) in drinking water daily), and G4 (CP 16%, probiotic Protexin: 85 mg•L(-1) in drinking water daily) after the completion of molt. The sampling was conducted 3 times at 5% production, peak production, and end of production with 15 birds being killed at each sampling from each group to collect the blood for harvesting of serum. Serum health markers including total oxidant status, total antioxidant capacity, homocysteine concentration, and paraoxonase, arylesterase, and ceruloplasmin activity were determined by the prescribed assays. The overall total antioxidant capacity was increased, whereas total oxidant status and homocysteine concentrations were reduced significantly (P ≤ 0.01) in all the supplemented groups compared with the control. The paraoxonase and ceruloplasmin activity were enhanced (P ≤ 0.01) in the supplemented groups compared with the control, and arylesterase activity was increased (P ≤ 0.01) in only G2 compared with the other groups. Although protein supplementation significantly reduced the oxidative stress, supplementation with symbiotic and probiotic also improved the health status by decreasing the oxidative stress in the birds.

Single or combined effects of mannan-oligosaccharides and probiotic supplements on the total oxidants, total antioxidants, enzymatic antioxidants, liver enzymes, and serum trace minerals in cyclic heat-stressed broilers.

This study was intended to explore the oxidative status of broilers under cyclic heat stress (HS) as modulated by supplementation of mannan-oligosaccharides (MOS) and a probiotic mixture (PM). Two hundred fifty 1-d-old chicks were randomly divided into 5 groups. From d 22, birds were either kept in a thermoneutral zone (TN; 26.7°C for 24 h/d) or subjected to HS (35 ± 1.1°C and 75 ± 5% RH for 8 h/d from 1000 to 1800 h) to the conclusion of the study on d 42. Birds were fed either a corn-based diet (TN and HS groups) or the same diet supplemented with 0.5% MOS (HS-MOS group), 0.1% PM (HS-PM group), or their combination as a symbiotic (SYN; HS-SYN group). On d 42, birds were killed by cervical dislocation to collect serum for the determination of total oxidants, total antioxidants, paraoxonase, arylesterase, ceruloplasmin, aspartate aminotransferase, alanine aminotransferase, and trace minerals. Heat stress increased (P < 0.05) total oxidants and total antioxidants and decreased (P < 0.05) paraoxonase and arylesterase, with no change in ceruloplasmin, aspartate aminotransferase, and alanine aminotransferase activities. Dietary supplementation decreased (P < 0.05) total oxidants and total antioxidants, with no effect on the activities of other enzymes. Heat stress did not influence serum copper, zinc, and manganese concentrations of birds when compared with those in the TN group. However, MOS increased (P < 0.05) concentrations of all the trace minerals, whereas SYN increased (P < 0.05) concentrations of only zinc and copper. It was concluded that MOS or PM supplementation, alone or as a SYN, may reduce some of the detrimental effects of HS, whereas MOS alone or as a SYN may improve the absorption of trace minerals.

Somatotrophs and lactotrophs: an immunohistochemical study of Gallus domesticus pituitary gland at different stages of induced moult.

The objective of this study was to determine the distribution of somatotrophs and lactotrophs and conduct a morphometrical analysis of immunoreactive somatotrophs and lactotrophs in the pituitary glands of White Leghorn Hens (Gallus domesticus) during the period of induced moult. We divided the periods of induced moulting into three phases viz. 7, 14 and 21 days. The labeled alkaline-phsphatase method with anti-GH (growth hormone) and anti-PRL (prolactin) as a primary antibody was used to detect somatotrophs and lactotrophs, in the midsagital sections of chicken adenohypophysis. Immunohistochemistry showed that somatotrophs are not only confined to the cephalo-caudal axis but can also be found in the caudal lobe; while lactotrophs were distributed in both lobes of the anterior pituitary gland at all stages of moulting (7, 14 and 21 days). Lactotrophs were of different shapes but somatotrophs were oval to round in morphology. At the given stages of induced moulting, some hypertrophied lactotrophs were also present after 7 days of induced moult in the anterior pituitary gland. However, there were moulting-related changes: from 7 to 21 days of induced moulting the immunoreactive-PRL cell population decreased, while the mean lactotroph size was more than that of somatotrophs. Basic quantitative and morphological information relating to somatotrophs and lactotrophs during the period of induced moult in laying hens is reported here and the changes brought about by induced moulting are restricted to PRL positive cells rather than GH positive cells.

Comparison of the fast-induced and high dietary zinc-induced molting: trace elements dynamic in serum and eggs at different production stages in hens (Gallus domesticus).

A number of 252, 70 week old, single comb white leghorn hens at the end of their first production cycle were divided into two groups: group-I; molted by fasting and group-II; by high dietary zinc to obtain the second and third production cycle. Sampling was carried out at 5%, peak and end of the second production cycle and at 5% and peak of the third production cycle. Serum zinc decreased at the peak of the second production cycle, while increased at the 5% of the third production cycle in zinc-molted group. Serum copper and manganese increased at the 5% and peak of the second production cycle in zinc-molted group. Egg albumin zinc and copper increased at the 5% and peak of the second as well as third production cycles in zinc-molted group. Egg albumin manganese increased at 5% during the second production cycle in fasting molted group. Egg yolk zinc increased at the peak of the second production cycle in zinc-molted group. Egg yolk copper increased at the 5% of second production cycle but decreased during the third production cycle in zinc-molted group. Egg yolk manganese increased at the 5% and at the end of the second production cycle in zinc molted group. Egg yolk manganese and iron decreased during the third production cycle in zinc-molted group. Eggshell zinc decreased at the end of the second production cycle in the zinc-molted group, while eggshell copper increased at the peak of the second as well as third production cycles in fasting molted group.

Changes in luteinizing hormone-containing gonadotrophs after moulting induced by fasting and zinc in laying hens (Gallus domesticus).

The purpose of the present study was to investigate egg production performance, immunohistochemical (IHC) identification, localization and morphological characterization of moulted hen's pars distalis luteinizing hormone (LH) gonadotrophs. This system has been validated for chicken LH by immunodetection of purified hormones on dot blots, western blotting and by specific labelling of cells on sagittal sections of chicken pituitaries. This study showed that egg production and egg weight increased significantly after zinc-induced moult when compared with fasting-induced moult. Luteinizing hormone-gonadotrophs are of numerous shapes and distributed throughout the adenohypophysis, with densely populated regions on the margins of pars distalis. Moulting of laying hens was associated with a significant increase in immunoreactive LH gonadotroph cell count and cell size, which peaked at the subsequent production stages. Zinc-induced moulting also caused a significant increase in LH gonadotroph cell size as compared with fasting-induced moulting. The nuclear size of LH-positive cells was distinct during decreased egg production. Our results validate the use of ovine LHbeta antibodies for the IHC localization of chicken LH gonadotrophs. This technology was further applied to the characterization of the shape and morphological changes of gonadotrophs as a function of the egg production status of the birds.

Estimation of atrazine in the stratum corneum and its toxic effects in skin following topical application to rabbits.

ABSTRACT For risk assessment, a workable non-invasive method for the estimation of atrazine in skin was needed. Moreover, the dermato-toxic effects of different concentrations of atrazine have not been studied so far. Fifteen milligrams of 35% solution of atrazine in ethanol was topically applied to each of six different clipped sites on the back of rabbits (n = 6). Each site was tape-stripped with 10 consecutive adhesive tapes at 0.5, 1, 2, 3, 4, and 6 h of topical dosing to remove the stratum corneum (SC). Atrazine in tapes was extracted with methanol and analyzed with HPLC. The amount of atrazine detected in the SC at 6 h (1955.79 +/- 47.22 mug) is equivalent to 13.03% of the total applied dose. In dermal toxicity studies, 500 mul of 35%, 70% atrazine in ethanol and a commercial preparation of atrazine (Balance(R)) was dosed on clipped backs of rabbits (n = 4) for 4 days under occluded vs non-occluded conditions. Ethanol alone served as control. On the 5th day, rabbits were euthanized and skin was scored for erythema and then examined microscopically. Significant differences (p < 0.05) in erythema scores were observed with 70% atrazine and Balance(R) as compared to the control under occluded conditions. Significant differences in epidermal thickness and cell layers were observed with Balance(R) and 74% atrazine as compared to control in both dosing conditions. There were non-significant differences in erythema, epidermal thickness, or cell layers in occluded vs non-occluded applications of atrazine in ethanol, indicating that atrazine is equally toxic regardless of its application procedures. Moreover, under non-occluded application, very slight erythema was observed but microscopically significant epidermal hyperplasia was noticed. This indicates that even if there are no significant gross skin manifestations of atrazine this can still produce significant damage to the epidermal barrier and hence can result in increase in penetration of self or other toxic substances.

Clonotypic polymerase chain reaction confirms minimal residual disease in CLL nodular PR: results from a sequential treatment CLL protocol.

Patient-tumor-specific oligonucleotides were generated for the detection of minimal residual disease (MRD) in a highly specific and sensitive clonotypic polymerase chain reaction (cPCR). The clone-specific region of highest diversity, CDR-III, was PCR amplified and sequenced. Nested CDR-III clonotypic primers were used in a semi-nested cPCR with a sensitivity of at least 1 in 10(5) cells. Patients with protocol-eligible Rai intermediate or high-risk chronic lymphocytic leukemia (CLL) received induction with fludarabine 25 mg/m(2) per day for 5 days every 4 weeks for 6 cycles, followed by consolidative high-dose cyclophosphamide (1.5, 2.25, or 3g/m(2)). cPCR was performed on peripheral blood and bone marrow mononuclear cells. All 5 patients achieving a clinical partial remission (PR) studied by cPCR were positive. Five patients achieved nodular PR (nPR) (residual nodules or suspicious lymphocytic infiltrates in a bone marrow biopsy as the sole suggestion of residual disease). Five of 5 patients with nPR were cPCR positive. In contrast, flow cytometry for CD5-CD19 dual staining and kappa--lambda clonal excess detected MRD in only 3 of the same 5 nPR patients, all of whom were cPCR positive, and immunohistochemistry detected MRD in only 1 of 4 assessable patients. Three of 7 CR patients evaluable by cPCR had MRD. Only 1 CR patient had MRD by flow cytometry; that patient was also cPCR positive. These data support the conclusions that nodular PR in CLL represents MRD and that clonotypic PCR detects MRD in CLL more frequently than flow cytometry or immunohistochemistry. (Blood. 2001;97:1929-1936)

Selection bias in clinical trials.

All research is subject to bias that may systematically distort results. Though over 50 types of bias in analytical research have been identified and many classification schemes proposed, this review focuses on the special problem of selection bias in clinical trials. We demonstrate the systematic nature of selection bias in clinical research. We describe the common sources of selection bias in clinical trials including the (inappropriate) use of historical controls, stage migration, inclusion/exclusion criteria, the use of multiple subset analyses, and investigator bias. We then move from the general to the specific, using the recent experience of high dose chemotherapy for breast cancer as an illustrative example. Finally, we suggest means to avoid falling into the many selection bias traps that often confront clinical researchers.

Results and long term follow-up for 1581 patients with metastatic breast carcinoma treated with standard dose doxorubicin-containing chemotherapy: a reference.

BACKGROUND: The authors report results and long term follow-up for 1581 patients with metastatic breast carcinoma treated with doxorubicin-containing combination chemotherapy at a single institution; this report is meant to serve as a reliable reference for single-arm studies of newer therapies in this patient population. METHODS: Prospectively collected data from 18 successive doxorubicin-containing protocols for the treatment of metastatic breast carcinoma were evaluated. RESULTS: The response rate was 65.0% (95% confidence interval [CI]: 62.5-67.3%), complete response (CR) rate was 16.6% (95% CI: 14.8-18.6%), and partial response (PR) rate was 48.5% (95% CI: 46.0-50.9%). Median progression free survival (PFS) was 11.5 months (95% CI: 10.9-12.3 months) and median overall survival (OS) was 21.3 months (95% CI: 20.3-22.7 months). Survival correlated with response to therapy; median PFS and OS were 22.4 and 41.8 months, respectively, for the patients who achieved CR (n=263) and 14 and 24.6 months, respectively, for PR patients (n=766). The median OS of patients who had progressive disease during chemotherapy was 3.8 months. The response rate, PFS and OS correlated with number of organs involved and especially with tumor burden. Patients with hormone receptor-positive tumors had a similar response rate to that of patients with hormone receptor negative tumors but had significantly longer PFS (medians of 14.3 and 8.7 months, respectively) and OS (medians of 28.6 and 18.1 months, respectively). CONCLUSIONS: In patients with metastatic breast carcinoma, doxorubicin-containing chemotherapy had a response rate of 65% and a CR rate of 16.6%. PFS and OS were 11.5 months and 21.3 months, respectively, for all responders and 22.4 months and 41.8 months, respectively, for those who had CR.

Impact of selection process on response rate and long-term survival of potential high-dose chemotherapy candidates treated with standard-dose doxorubicin-containing chemotherapy in patients with metastatic breast cancer.

PURPOSE: Most of the data about high-dose chemotherapy (HDCT) for metastatic breast cancer are derived from phase II studies. The interpretation of these data depends on comparisons with data from properly selected historical control patients treated with standard therapy under similar circumstances. We report the long-term results of patients with metastatic breast cancer who were eligible for HDCT but were treated with doxorubicin-containing standard-dose chemotherapy. PATIENTS AND METHODS: Prospectively collected data from 18 successive doxorubicin-containing protocols for the treatment of metastatic breast cancer were evaluated. Using common eligibility criteria for HDCT, we identified patients who would have been candidates for HDCT. We analyzed response rates, progression-free survival (PFS), and overall survival (OS) for all patients, potential HDCT candidates, and noncandidates. RESULTS: A total of 1,581 patients was enrolled onto the 18 studies. Six hundred forty-five were HDCT candidates, and 936 were noncandidates. The complete response rate was 27% for HDCT candidates and 7% for noncandidates; median PFS was 16 and 8 months and median OS was 30 and 17 months, respectively. Survival rates for HDCT candidates and noncandidates, respectively, were 21% and 6% at 5 years and 7% and 2% at 10 years. CONCLUSION: This study suggests that encouraging results of single-arm trials of HDCT could partially be due to selection of patients with better prognoses and further stresses the importance of completing ongoing randomized trials of HDCT to assess the relative efficacy of HDCT in patients with metastatic breast cancer.

Comparative anthelminthic efficacy and safety of Caesalpinia crista seed and piperazine adipate in chickens with artificially induced Ascaridia galli infection.

The antiascarid activity of Caesalpinia crista Linn. seeds, popularly known as Karanjwa, was evaluated in chickens of the Fumi breed, suffering from artificially induced Ascaridia galli infection. Eggs per gram (EPG) counts were determined in the droppings of chickens prior and after treatment with powdered C. crista at doses of 30, 40 and 50 mg/kg of body weight along with its extracts in water and methanol in amounts representing 50 mg/kg of crude powder. The crude drug at the dose rates of 40 and 50 mg/kg and its methanol extract induced a significant (P < 0.001) effect on post-treatment days 10 and 15 while the 30 mg/kg dose was efficacious (P < 0.05) on day 15 only. However, the aqueous extract did not show significant results. These results suggest that a 50 mg/kg dose of C. crista seed powder, its equivalent methanolic extract and piperazine (200 mg/kg) are equieffective in treating the ascarid infection of poultry. The crude C. crista powder appears to be potent and safer than its methanol extract on the basis of the side effects observed.