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Recently, a P-deficient diet caused rickets in commercial chicks within three days. This study aimed to investigate the duration of onset of rickets in chicks. Data were collected from 3–11 day old chicks raised on 88 commercial farms. Male day-old Arbor Acres Plus broilers (n = 450) were studied in three trials, with three to four treatments each. Each treatment used one of the following crumbled feeds: control feed (calcium (Ca): phosphorus (P)-1.41), slightly high Ca:P feed (SHCa:P, Ca:P-2.69), high Ca:P ratio, P deficient feed (HCa:P, Ca:P-3.08), and HCa:P feed plus 1.5% dicalcium phosphate (HCa:P + DP). Each treatment had three replicates with 15 birds each. Rickets was induced by HCa:P, and cured by HCa:P + DP, confirmed by gross anatomy, gait score, serum P concentration and growth performance. Lameness was not found in control groups, whereas, observed in the HCa:P groups as early as day 2.7 on commercial farms and day 3 in experimental farm. Serum P was reduced in HCa:P (p < 0.01). Bodyweight and feed intake started decreasing at day 3 on commercial farms and in all trials (p < 0.01). The duration of onset of hypophosphatemic rickets in broiler chicks fed HCa:P crumbled feed is approximately three days.

Salmonella Heidelberg (SH) on contaminated poultry causes economic and health risks to producers and consumers. We hypothesized that sodium bisulfate (SBS) would decrease SH biofilm on polyvinyl chloride (PVC) coupons and decrease the horizontal transfer of SH in broilers. Experiment 1: Salmonella Heidelberg biofilm was cultured with PVC coupons, which were treated with SBS at a pH of 3.5 for 10 min, 8 h, and 24 h. Experiment 2: Nine replicate pens per treatment were divided between two rooms. A seeder contact model was used to mimic a natural infection environment. Treatments consisted of tap water or sodium bisulfate in water at a pH of 3.5. Salmonella Heidelberg incidence and enumeration were measured in crops and ceca. Sodium bisulfate significantly reduced biofilm by 2.16 and 1.04 logs when treated for 8 and 24 h, respectively. Crop colonization was significantly decreased in trials 1 and 2 by 0.29 and 0.23 logs, respectively. Crop pH was significantly decreased in trial 2. Ceca colonization was significantly decreased in trial 1 by 0.39 logs. The results from the present study suggest that SBS may be administered to drinking water to decrease SH gut colonization and to reduce biofilm.

Wooden breast (WB) results in significant losses to the broiler industry due to reductions in meat quality. While the etiology of WB is unknown, it is believed to be associated with localized hypoxia and decreased lactate levels in skeletal muscles, indicating the presence of altered lactate metabolism in WB. We hypothesized that the expression levels of the major signaling molecules that control lactate metabolism, including lactate dehydrogenases (LDHA and LDHB) and monocarboxylate transporters (MCT1 and MCT4), were altered in WB. Therefore, the objectives of this study were to evaluate whether there were changes in mRNA and protein levels of LDHA, LDHB, MCT1, and MCT4 in WB compared to normal breast (NB) muscles. Biochemical analysis for LDH enzyme activity in NB and WB muscles was studied. MicroRNA375 (miR-375) expression, known to be inversely associated with LDHB protein expression in human cells, was also investigated. The level of LDHA mRNA was 1.7-fold lower in WB tissues than in NB tissues (P < 0.0001). However, the LDHA protein levels were similar in WB and NB tissues. In contrast, the levels of LDHB mRNA and protein were 8.4-fold higher (P < 0.002) and 13.6-fold higher (P < 0.02) in WB than in NB tissues, respectively. The level of miR-375 was not different between WB and NB muscles. The specific LDH isoenzyme activity that converted lactate to pyruvate was 1.8-fold lower in WB compared to NB tissues (P < 0.01). The level of MCT1 mRNA was 2.3-fold higher in WB than those in NB muscles (P < 0.02). However, this upregulation was not observed with MCT1 protein expression levels. The expression levels of MCT4 mRNA and protein were elevated 2.8-fold (P < 0.02) and 3.5-fold (P < 0.004) in WB compared to NB tissues, respectively. Our current findings suggest the potential roles of LDHB and MCT4 on lactate metabolism and provide a unique molecular elucidation for altered lactate homeostasis in WB muscles of broilers.

Monitoring antimicrobial resistance of foodborne pathogens in poultry is critical for food safety. We aimed to compare antimicrobial resistance phenotypes in Salmonella isolated from poultry samples as influenced by isolation and antimicrobial susceptibility testing methods. Salmonella isolates were cultured from a convenience sample of commercial broiler ceca with and without selective broth enrichment, and resistance phenotypes were determined for 14 antimicrobials using the Sensititre® platform and a qualitative broth breakpoint assay. The broth breakpoint method reported higher resistance to chloramphenicol, sulfisoxazole, and the combination of trimethoprim and sulfamethoxazole, and lower resistance to streptomycin as compared to the Sensititre® assay in trial one. Selective enrichment of samples containing Salmonella in Rappaport-Vassiliadis broth reported lowered detectable resistance to amoxicillin/clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftriaxone, nalidixic acid, and meropenem, and increased resistance to streptomycin and tetracycline than direct-plating samples in trial one. Using matched isolates in trial two, the Sensititre® assay reported higher resistance to chloramphenicol and gentamicin, and lower resistance to nalidixic acid as compared to the broth breakpoint method. These results suggest methodology is a critical consideration in the detection and surveillance of antimicrobial resistance phenotypes in Salmonella isolates from poultry samples and could affect the accuracy of population or industry surveillance insights and intervention strategies.

Salmonellosis is a zoonotic infection caused by Salmonella enterica serotypes contracted from contaminated products. We hypothesized that competitive exclusion between Salmonella serotypes in neonatal broilers would reduce colonization and affect the host immune response. Day of hatch broilers were randomly allocated to one of six treatment groups: (1) control, which received saline, (2) Salmonella Kentucky (SK) only on day 1 (D1), (3) Salmonella Typhimurium (ST) or Salmonella Enteritidis (SE) only on D1, (4) SK on D1 then ST or SE on day 2 (D2), (5) ST or SE on D1 then SK on D2, and (6) SK and ST or SE concurrently on D1. Salmonella gut colonization and incidence were measured from cecal contents. Livers and spleens were combined and macerated to determine systemic translocation. Relative mRNA levels of interleukin-1β (IL-1β), IL-6, IL-10, IL-18, and gamma interferon (IFN-γ) were measured in cecal tonsils and liver to investigate local and systemic immune responses. When a serotype was administered first, it was able to significantly reduce colonization of the following serotype. Significant changes were found in mRNA expression of cytokines. These results suggest competitive exclusion by Salmonella enterica serotypes affect local and systemic immune responses.

Rational: Salmonella Enteritidis (S. Enteritidis) is a globally significant zoonotic foodborne pathogen which has led to large numbers of deaths in humans and caused economic losses in animal husbandry. S. Enteritidis invades host cells and survives within the cells, causing resistance to antibiotic treatment. Effective methods of elimination and eradication of intracellular S. Enteritidis are still very limited. Here we evaluated whether a new intracellular antibacterial strategy using iron oxide nanozymes (IONzymes) exerted highly antibacterial efficacy via its intrinsic peroxidase-like activity in vitro and in vivo. Methods: The antibacterial activities of IONzymes against planktonic S. Enteritidis, intracellular S. Enteritidis in Leghorn Male Hepatoma-derived cells (LMH), and liver from specific pathogen free (SPF) chicks were investigated by spread-plate colony count method and cell viability assay. Changes in levels of microtubule-associated protein light chain 3 (LC3), a widely used marker for autophagosomes, were analyzed by immunoblotting, immunofluorescence, and electron microscopy. Reactive oxygen species (ROS) production was also assessed in vitro. High-throughput RNA sequencing was used to investigate the effects of IONzymes on liver transcriptome of S. Enteritidis-infected chicks. Results: We demonstrated that IONzymes had high biocompatibility with cultured LMH cells and chickens, which significantly inhibited intracellular S. Enteritidis survival in vitro and in vivo. In addition, co-localization of IONzymes with S. Enteritidis were observed in autophagic vacuoles of LMH cells and liver of chickens infected by S. Enteritidis, indicating that IONzymes mediated antibacterial reaction of S. Enteritidis with autophagic pathway. We found ROS level was significantly increased in infected LMH cells treated with IONzymes, which might enhance the autophagic elimination of intracellular S. Enteritidis. Moreover, orally administered IONzymes decreased S. Enteritidis organ invasion of the liver and prevented pathological lesions in a chicken-infection model. Non-target transcriptomic profiling also discovered IONzymes could change hepatic oxidation-reduction and autophagy related gene expressions in the S. Enteritidis infected chickens. Conclusion: These data suggest that IONzymes can increase ROS levels to promote the antibacterial effects of acid autophagic vacuoles, and thus suppress the establishment and survival of invading intracellular S. Enteritidis. As a result, IONzymes may be a novel alternative to current antibiotics for the control of intractable S. Enteritidis infections.

SUMMARY A rapid and effective means to clean and disinfect premises is needed by the poultry industry. We hypothesized that commercially available foaming disinfectants and cleaners applied via a compressed air foam system (CAFS) may be used to significantly reduce aerobic bacteria in a commercial caged layer complex. Six field trials were conducted to evaluate current industry cleaning and disinfection protocols and the proposed CAFS application. A commercially available chlorinated alkaline foaming cleaner (CHL/ALK) was applied in trials 1 and 2 by CAFS to one half of the house, and the other half of the house was not treated. The entire house was then washed with a high-pressure water rinse (HPWR). A commercially available peroxyacetic acid (PAA) in trials 3 and 4 or a 14% glutaraldehyde (HI GLUT)/2.5% quaternary ammonia (QAC) blended disinfectant in trials 5 and 6 was applied by CAFS to one half of a washed house. The remainder of the house was then treated with a 7% glutaraldehyde (LO GLUT)/26% QAC, which was spray-applied to cages by the integrator. Environmental swabs of drinker cups and cage floors were collected pre- and post-treatment to determine if aerobic bacteria were reduced. The HPWR and the CHL/ALK treatments did not consistently reduce bacteria on treated surfaces. Significant differences were observed with each of the CAFS applications of the PAA product, resulting in bacterial reductions of 1.83 to 2.27 log10 cfu/sample. Although inconsistent, differences (P

SUMMARY Layer cage cleaning and disinfection are important for disease prevention; however, published research is limited. The objective of this study was to determine the efficacy of commonly used products on soiled layer cages. Trial one was conducted at a university poultry research farm, and trial two was completed in a commercial pullet house. All products were mixed according to manufacturer recommendations. Treatments consisted of a negative control, a low-pressure water rinse (LPWR, garden hose), a high-pressure water rinse (HPWR, power washer), a soap, a chlorinated cleaner, a quaternary ammonium, a glutaraldehyde, a peroxyacetic acid, a phenolic, a potassium peroxymonosulfate, a hydrogen peroxide, and a quaternary/glutaraldehyde blend product. Swabs of cage floors were collected post treatment to determine if bacterial loads were reduced (P

Limited research has been performed to evaluate the effects of high-frequency electrical stunning (ES) methods on the lipid oxidative stability of the meat goose livers. This study was conducted to evaluate the effects of high-frequency-ES current intensities on lipid oxidative stability and antioxidant capacity in the liver of Yangzhou goose (Anser cygnoides domesticus). Forty 92-day-old male Yangzhou geese were randomly divided into five treatments (n = 8). Geese were not stunned (control) or exposed to ES for 10 s with alternating current (AC) at 500 Hz in a water bath. Current intensities were set at 30 V/20 mA (E30V), 60 V/40 mA (E60V), 90 V/70 mA (E90V), or 120 V/100 mA (E120V), respectively. The malondialdehyde level at day 0 was the highest in 120 V (p &lt, 0.05). Antioxidant enzymes&rsquo, activity on day 2 was the highest in E60V. The 1, 1-diphenyl-2-picrylhydrazyl free radical (DPPH&middot, ) elimination ability was lower in the E120V than that in the E60V at two days and four days postmortem (p &lt, 0.05). A combination of 60 V/40 mA/ 500 Hz/ 10 s per bird could be applied in the ES of Yangzhou geese to improve the lipid oxidative stability and antioxidant capacity in the livers.

Simple Summary Male layer chicks do not have economic value and are humanely killed after hatching. The layer industry is seeking alternative methods to humanely kill recently hatched male chicks. This study evaluated the use of gases or negative air pressure as a means of humane and viable alternatives to maceration. The treatments included carbon dioxide, nitrogen, reduced air pressure, and a negative control. The study showed that chicks exposed to treatments, gases or negative air pressure, did not differ significantly in terms of the physiological stress response. The use of carbon dioxide resulted in a faster onset of unconsciousness and ultimately death as compared to nitrogen or negative air pressure treatments. Abstract Hatched male layer chicks are currently euthanized by maceration in the United States. Public concerns on the use of maceration have led to the search for alternative methods. We hypothesized that gas inhalation and low atmospheric pressure stunning (LAPS) are viable and humane alternatives to instantaneous mechanical destruction. The objective of this study was to evaluate the physiological and behavioral responses of recently hatched male layer chicks when subjected to carbon dioxide, nitrogen inhalation, or LAPS. The study consisted of seven treatments: breathing air (NEG), 25% carbon dioxide (CO2), 50% CO2, 75% CO2, 90% CO2, 100% nitrogen (N2), or LAPS. Ten day-of-hatch, male layer chicks were randomly assigned to each treatment, and each treatment was replicated on ten different days. A custom-made vacuum system was used to reduce air pressure inside the chamber from 100.12 kPa to 15.3 kPa for the LAPS treatment. Serum corticosterone and serotonin levels were measured using commercially available competitive enzyme linked immunosorbent assay (ELISA). Latencies to loss of posture and motionlessness were determined from video recordings. The 25% and 50% CO2 treatments were discontinued after the first replication, as the majority of the chicks recovered. The chicks in the negative (NEG) group had significantly higher levels of corticosterone than the other four euthanasia treatments. On the other hand, the serotonin levels of chicks in the NEG group was significantly lower when compared to the other four euthanasia treatments. The latencies to loss of posture and motionlessness of chicks exposed to 75% and 90% CO2 were significantly shorter than those in the LAPS and N2 inhalation treatments. These data suggest that the stress responses of chicks to the CO2, N2, and LAPS treatments do not differ among each other. However, the CO2 inhalation method was faster in inducing loss of posture and motionlessness in chicks than the LAPS and N2 inhalation treatments.

Simple Summary Compressed air, detergent, and water make up compressed air foam. Our laboratory has previously reported that compressed air foam may be an effective method for mass depopulation of caged layer hens. Gases, such as carbon dioxide and nitrogen, have also been used for poultry euthanasia and depopulation. The objective of this study was to produce compressed air foam infused with carbon dioxide or nitrogen to compare its efficacy against foam with air and gas inhalation methods (carbon dioxide or nitrogen) for depopulation of caged laying hens. The study showed that a carbon dioxide-air mixture or 100% nitrogen can replace air to make compressed air foam. However, the foam with carbon dioxide had poor foam quality compared to the foam with air or nitrogen. The physiological stress response of hens subjected to foam treatments with and without gas infusion did not differ significantly. Hens exposed to foam with nitrogen died earlier as compared to methods such as foam with air and carbon dioxide. The authors conclude that infusion of nitrogen into compressed air foam results in better foam quality and shortened time to death as compared to the addition of carbon dioxide. Abstract Depopulation of infected poultry flocks is a key strategy to control and contain reportable diseases. Water-based foam, carbon dioxide inhalation, and ventilation shutdown are depopulation methods available to the poultry industry. Unfortunately, these methods have limited usage in caged layer hen operations. Personnel safety and welfare of birds are equally important factors to consider during emergency depopulation procedures. We have previously reported that compressed air foam (CAF) is an alternative method for depopulation of caged layer hens. We hypothesized that infusion of gases, such as carbon dioxide (CO2) and nitrogen (N2), into the CAF would reduce physiological stress and shorten time to cessation of movement. The study had six treatments, namely a negative control, CO2 inhalation, N2 inhalation, CAF with air (CAF Air), CAF with 50% CO2 (CAF CO2), and CAF with 100% N2 (CAF N2). Four spent hens were randomly assigned to one of these treatments on each of the eight replication days. A total of 192 spent hens were used in this study. Serum corticosterone and serotonin levels were measured and compared between treatments. Time to cessation of movement of spent hens was determined using accelerometers. The addition of CO2 in CAF significantly reduced the foam quality while the addition of N2 did not. The corticosterone and serotonin levels of spent hens subjected to foam (CAF, CAF CO2, CAF N2) and gas inhalation (CO2, N2) treatments did not differ significantly. The time to cessation of movement of spent hens in the CAF N2 treatment was significantly shorter than CAF and CAF CO2 treatments but longer than the gas inhalation treatments. These data suggest that the addition of N2 is advantageous in terms of shortening time to death and improved foam quality as compared to the CAF CO2 treatment.

Simple Summary Reportable diseases, such as avian influenza, spread rapidly among poultry, resulting in the death of a large number of birds. Once such a disease has been diagnosed at a farm, infected and susceptible birds are rapidly killed to prevent the spread of the disease. The methods to eliminate infected caged laying hens are limited. An experiment was conducted to study the effectiveness of foam made from compressed air, water, and soap to kill laying hens in cages. The study found that stress levels of the hens killed using compressed air foam in cages to be similar to the hens killed by carbon dioxide or the negative control. Hens exposed to carbon dioxide died earlier as compared to the foam methods. The authors conclude that application of compressed air foam in cages is an alternative to methods such as gas inhalation and ventilation shutdown to rapidly and humanely kill laying hens during epidemics. Abstract During the 2014–2015 US highly pathogenic avian influenza (HPAI) outbreak, 50.4 million commercial layers and turkeys were affected, resulting in economic losses of $3.3 billion. Rapid depopulation of infected poultry is vital to contain and eradicate reportable diseases like HPAI. The hypothesis of the experiment was that a compressed air foam (CAF) system may be used as an alternative to carbon dioxide (CO2) inhalation for depopulating caged layer hens. The objective of this study was to evaluate corticosterone (CORT) and time to cessation of movement (COM) of hens subjected to CAF, CO2 inhalation, and negative control (NEG) treatments. In Experiment 1, two independent trials were conducted using young and spent hens. Experiment 1 consisted of five treatments: NEG, CO2 added to a chamber, a CO2 pre-charged chamber, CAF in cages, and CAF in a chamber. In Experiment 2, only spent hens were randomly assigned to three treatments: CAF in cages, CO2 added to a chamber, and aspirated foam. Serum CORT levels of young hens were not significantly different among the CAF in cages, CAF in a chamber, NEG control, and CO2 inhalation treatments. However, spent hens subjected to the CAF in a chamber had significantly higher CORT levels than birds in the rest of the treatments. Times to COM of spent hens subjected to CAF in cages and aspirated foam were significantly greater than of birds exposed to the CO2 in a chamber treatment. These data suggest that applying CAF in cages is a viable alternative for layer hen depopulation during a reportable disease outbreak.

In a companion study, the effects of dietary antibiotic alternative and coccidial vaccination on the growth performance of male broilers have been reported. In this paper, the effects of dietary probiotics and coccidial vaccination on diversity and composition of cecal microbiota were investigated using a 3 (diets) × 2 (vaccinated or non-vaccinated) factorial setting of treatments. Three diets, including a corn and soybean-meal control diet, an antibiotic diet (a control diet supplemented with bacitracin and salinomycin), and a probiotic diet (a control diet supplemented with Bacillus subtilis) were provided to broiler chicken from day 0 to 42. To simulate an Eimeria challenge in the field, all chicks were gavaged with a 20× dose of commercial coccidial vaccine containing live Eimeria oocysts on day 14. Cecal contents were collected on day 42. High-throughput sequencing of the 16S rRNA gene was used to determine microbial diversity and composition. Coccidial vaccination to broilers reduced bacterial diversity (Shannon index) of the cecal microbiota. There was a significant interaction between the dietary additive and coccidial vaccination on the observed bacterial species number. Diets supplemented with B. subtilis increased bacterial species of non-vaccinated broilers but decreased bacterial species of vaccinated broilers. In contrast, diets supplemented with antibiotics reduced bacterial species of broilers from both groups. Interactions between dietary additive and coccidial vaccination were also observed on microbial composition. Vaccinated broilers fed the B. subtilis diet exhibited the lowest Firmicutes percentage and highest Bacteroidetes percentage within the microbial community. In addition, vaccinated broilers fed the B. subtilis diet exhibited the highest Rikenella microfusus percentage. From this study, the coccidial vaccination on the day of hatch reduced the microbial diversity of broilers at a later age. The inclusion of B. subtilis-probiotics in the feed of vaccinated broilers may reduce microbial diversity in cecal content by increasing the proportion of a predominant bacterial species, R. microfusus, in the microbial community.

Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC α1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC α1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns ofrPer2andrBmal1expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.

While peripheral tissues and serum-shocked fibroblasts express rhythmic oscillations in clock gene expression, only the suprachiasmatic nucleus (SCN) is capable of endogenous, self-sustained rhythmicity and of functioning as a pacemaker by imposing rhythmic properties upon other cells. To differentially examine the molecular elements necessary for the distinctive rhythm-generating and pacemaking properties of the SCN, the effects of antisense inhibition of Clock expression on the rhythms in 2-deoxyglucose uptake and Per gene expression were compared in immortalized SCN cells and a fibroblast cell line. Similar to changes in molecular and physiological rhythmicity observed in the SCN of Clock mutant mice, the rhythmic pattern of Per2 expression was disrupted and the period of metabolic rhythmicity was increased in SCN2.2 cells subjected to antisense inhibition of Clock. NIH/3T3 fibroblasts cocultured with antisense-treated SCN2.2 cells showed metabolic rhythms with comparable increases in period and decreases in rhythm amplitude. Per2 expression in these cocultured fibroblasts exhibited a similar reduction in peak levels, but was marked by non-24 h or irregular peak-to-peak intervals. In serum-shocked NIH/3T3 fibroblasts, oscillations in Per2, Bmal1, and Cry1 expression persisted with some change in rhythm amplitude during antisense inhibition of CLOCK, demonstrating that feedback interactions between Clock and other core components of the clock mechanism may be regulated differently in SCN2.2 cells and fibroblasts. The present results suggest that CLOCK is differentially involved in the generation of endogenous molecular and metabolic rhythmicity within SCN2.2 cells and in the regulation of their specific outputs that control rhythmic processes in NIH/3T3 cells.

Three selective estrogen receptor modulator (SERM) drugs which included 4-OH-tamoxifen (Tam), EM-800 (EM) and GW 5638 (GW) were investigated to determine their ability to inhibit estradiol-responsive gene expression in sheep endometrium. The uteri of ovariectomized ewes (10 ewes per SERM group) were infused with 10−7 M SERMs for 24 h prior to hysterectomy. Five ewes from each group received 50 μg 17β-estradiol (E2) and the remaining five ewes received vehicle 18 h prior to hysterectomy. Northern blot analyses and in situ hybridization demonstrated that E2 treatment increased estrogen receptor (ER), progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and cyclophilin (CYC) mRNA levels in most endometrial cells examined. Tam and GW exhibited characteristics similar to E2 by increasing ER gene expression, but they antagonized the E2-induced increases in PR and CYC mRNA levels. EM acted as an E2-agonist of GAPDH gene expression, but antagonized the E2 up-regulation of ER, PR and CYC gene expression in most endometrial cells. Immunohistochemistry determined that EM decreased ER protein levels in the glandular epithelium, and the SERMs investigated antagonized increases in PR protein levels in endometrium. In conclusion, GW and EM exhibit fewer agonist effects than Tam on endometrial gene expression. EM demonstrated the greatest antagonism of E2-enhanced levels of ER, PR and CYC, likely due to the inhibition of ER gene expression at both mRNA and protein levels.

The purpose of this study was to identify an endometrial cell line that maintained the E2 up-regulation of estrogen receptor (ER) mRNA by enhanced message stability and to assess its dependence on ER protein. Estradiol (E2) effects on gene expression were measured in three cell lines: one immortalized from sheep endometrial stroma (ST) and two from human endometrial adenocarcinomas (Ishikawa and ECC-1). E2 up-regulated ER mRNA levels in ST and Ishikawa cells, but down-regulated ER mRNA levels in ECC-1 cells. E2 up-regulated progesterone receptor (PR), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and transforming growth factor-alpha (TGF-alpha) in both Ishikawa and ECC-1 cells. The selective estrogen receptor modulator ICI 182,780 antagonized the E2-induced up-regulation of ER and/or PR mRNA levels in all three cells, while another, GW 5638, antagonized the up-regulation of PR mRNA in Ishikawa and ECC-1 cells. In mechanistic studies, E2 had no effect on ER mRNA stability in ST cells and it destabilized ER mRNA in ECC-1 cells. Thus, Ishikawa cells appear to be the most physiologically relevant cell line in which to study the up-regulation of ER mRNA levels by enhanced mRNA stability. Its antagonism by ICI 182,780 reveals that ER protein is involved in this E2 response.

Neonatal alcohol exposure produces long-term changes in the suprachiasmatic nucleus (SCN) that are presumably responsible for disturbances in the light–dark regulation of circadian behavior in adult rats, including the pattern of photoentrainment, rate of re-entrainment to shifted light–dark cycles, and phase-shifting responses to light. Because SCN neurons containing vasoactive intestinal polypeptide (VIP) receive direct photic input via the retinohypothalamic tract and thus play an important role in the circadian regulation of the SCN clock mechanism by light, the present study examined the long-term effects of neonatal alcohol exposure on VIP neuronal populations within the SCN of adult rats. Male Sprague-Dawley rat pups were exposed to alcohol (EtOH; 3.0, 4.5, or 6.0 g/kg/day) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4–9 using artificial-rearing methods. At 2–3 months of age, animals from the suckle control (SC), GC, and EtOH groups were exposed to constant darkness (DD) and SCN tissue was harvested for subsequent analysis of either VIP mRNA expression by quantitative polymerase chain reaction (PCR) and in situ hybridization or of VIP-immunoreactive (ir) neurons using stereological methods. Neonatal alcohol exposure had no impact on VIP mRNA expression but dramatically altered immunostaining of neurons containing this peptide within the SCN of adult rats. The relative abundance of VIP mRNA and anatomical distribution of neurons expressing this transcript were similar among all control- and EtOH-treated groups. However, the total number and density of VIP-ir neurons within the SCN were significantly decreased by about 35% in rats exposed to alcohol at a dose of 6.0 g/kg/day relative to that observed in both control groups. These results demonstrate that VIP neuronal populations in the SCN are vulnerable to EtOH-induced insult during brain development. The observed alterations in SCN neurons containing VIP may have an impact upon clock responses to light input and thus contribute to the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior.

In humans, prenatal alcohol exposure produces long-term changes in the neural regulation of behavior including learning difficulties, attention deficits, sleep–wake disturbances, bipolar affective disorder and depression (Roebuck et al., 1999). Animal studies have yielded further details on the neurobehavioral consequences of alcohol (EtOH) exposure during critical stages of brain development. In rats, exposure to alcohol during the neonatal period, which corresponds to the third trimester equivalent in human brain development (Dobbing and Sands, 1979), induces chronic deficits on learning /memory and motor performance tasks and these behavioral manifestations are associated with alcohol-induced damage to the hippocampus and cerebellum (Goodlett et al., 1987, 1988; Kelly et al., 1988; Thomas et al., 1996, 1998). Recent applications of this animal model have revealed that circadian or 24-hour behavioral rhythmicity is also vulnerable to alcohol-induced insult during CNS development. Specifically, neonatal alcohol exposure alters fundamental properties of the activity rhythm in adult rats including its free-running period, phase-shifting responses to light and entrainment to light–dark cycles (Allen et al., 2005a,b; Farnell et al., 2004). At present, the molecular or cellular basis for these alcohol-induced disturbances in the neural regulation of rat circadian behavior is unknown. Because modifications in the molecular “gears” of the circadian clockworks produce tangible changes in the circadian regulation of behavior (Reppert and Weaver, 2002), it is possible that neonatal alcohol exposure may permanently modulate circadian properties of the rat activity rhythm by disrupting the clock mechanism perhaps through altered expression or temporal configuration of its molecular components. In mammals, the internal biological clock mediating the generation of circadian rhythms and their entrainment or synchronization to light–dark cycles is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The “gears” of the molecular clockworks in the SCN consist of interlocked transcription–translation feedback loops in which the gene products of core components rhythmically regulate their own transcription or the expression of other clock genes. Clock, Period1 (Per1), Per2, Cryptochrome1 (Cry1), Cry2, and Bmal1 (Mop3) have been identified as core molecular elements of the circadian clock mechanism in the SCN based on studies demonstrating that targeted disruption or knockout of these genes in mice alters or abolishes circadian behavior (Reppert and Weaver, 2002). For example, Per1-, Per2- and Cry1-deficient mice show activity rhythms in which the free-running period is shorter than that observed in wild type animals (Bae et al., 2001; van der Horst et al., 1999; Vitaterna et al., 1999; Zheng et al., 2001). Although the SCN are essential for the generation of mammalian circadian rhythms in many biochemical, physiological, and behavioral processes, the expression and rhythmic regulation of these core clock genes are not spatially confined to the SCN. Instead, rhythmic fluctuations in the same clock genes comprising the molecular core of the SCN clockworks occur widely in other brain regions and many peripheral tissues, including the liver, heart, kidney, endocrine organs, and skeletal muscles (Shearman et al., 1997; Yamazaki et al., 2000; Zylka et al., 1998). These clock gene oscillations in other tissues are currently thought to provide for their “local” organization in time through a hierarchical system in which the SCN functions as a pacemaker linking periodic environmental cues with the internal circadian timekeeping mechanism in peripheral oscillators throughout the body. Consequently, the effects of neonatal alcohol exposure on the regulation of adult circadian behavior may be coupled with alcohol-induced changes in molecular components of the clockworks not only in the SCN pacemaker but also in peripheral oscillators. To determine whether the molecular mechanism for circadian timekeeping is vulnerable to alcohol-induced insult during the brain growth spurt, we examined the long-term impact of neonatal alcohol treatment on the temporal regulation of core clock genes in the SCN pacemaker and other brain or peripheral tissue oscillators. In this study, rats were exposed to alcohol during the neonatal period and the clock genes Per1, Per2, Cry1, Bmal1, and Rev-erbα were assessed for evidence of alcohol-induced changes in their circadian expression within the SCN, cerebellum, and liver. Because alterations in specific genetic components of the circadian clockworks have been linked to abnormal regulation of human sleep–wake cycles (Toh et al., 2001), which is a known neurobehavioral correlate of prenatal alcohol exposure in humans (Sher, 2004), information from this study may have important implications in understanding how alcohol-induced injury during rapid brain growth produces pathologies in the circadian regulation of sleep and other body processes.

In rats, neonatal alcohol (EtOH) exposure coinciding with the period of rapid brain growth produces structural damage in some brain regions that often persists into adulthood and thus may have long-term consequences in the neural regulation of behavior. Because recent findings indicate that the circadian clock located in the rat suprachiasmatic nucleus is vulnerable to alcohol-induced insults during development, the present study examined the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior in adult rats. Rat pups were exposed to alcohol (3.0, 4.5, or 6.0g·kg −1 ·day −1 ) or isocaloric milk formula on postnatal days 4–9 using artificial-rearing methods. At 2 months of age, animals were housed individually and circadian wheel-running behavior was continuously analyzed to determine the effects of neonatal alcohol treatment on the rate of reentrainment to a 6-h advance in the 12-h light:12-h dark photoperiod and the phase-shifting properties of free-running rhythms in response to discrete light pulses on a background of constant darkness. For all doses, neonatal alcohol exposure had a significant effect in reducing the time for reentrainment such that EtOH-treated rats required four to five fewer days than control animals for stable realignment of the activity rhythm to the shifted light–dark cycle. Coupled with the accelerated rate of reentrainment, the amplitude of light-evoked phase delays at circadian time 14 and advances at circadian time 22 in the 4.5 and 6.0g·kg −1 ·day −1 EtOH groups was almost twofold greater than that observed in control animals. The present observations indicate that the mechanisms by which photic signals regulate circadian behavior are permanently altered following alcohol exposure during the period of rapid brain development. These long-term alterations in the photic regulation of circadian rhythms may account, at least partially, for some neurobehavioral consequences of prenatal alcohol exposure in humans such as depression.

IN HUMANS, THE central nervous system is susceptible to structural damage from alcohol (EtOH) exposure during critical stages of brain development (Clarren et al., 1978; Jones and Smith, 1973; Mattson et al., 1996; Sowell et al., 1996). Detailed animal studies have revealed that developmental EtOH exposure similarly disrupts the structural organization of different brain regions. In rats, EtOH exposure during the brain growth spurt (early postnatal period), which corresponds to the equivalent period of human brain development during the third trimester of gestation (Dobbing and Sands, 1979), has been shown to retard brain growth; produce microencephaly; cause neuronal loss in the hippocampus, cerebellum, and olfactory bulb; and alter neuronal circuitry (Bonthius et al., 1992; Bonthius and West, 1990; Chen et al., 1998; Goodlett et al., 1998; Goodlett and Johnson, 1999; Kelly et al., 1988; Maier et al., 1996; West et al., 1981). Furthermore, hippocampal and cerebellar cell loss in rats exposed to EtOH during the early postnatal period have been observed in association with behavioral deficits in learning/memory and motor performance tasks (Goodlett et al., 1987, 1988; Kelly et al., 1988; Thomas et al., 1996, 1998). However, further analysis is necessary to fully determine the scope of the developmental EtOH-induced damage to the brain and whether the resulting neuroanatomical changes have long-term neurobehavioral consequences. The circadian clock and its regulation of circadian behavior have recently emerged as a significant area of interest in the analysis of specific neurobehavioral disturbances associated with developmental EtOH exposure. The internal biological clock responsible for the generation of mammalian circadian rhythms is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The endogenous timekeeping function of the SCN is complemented by its role in mediating the entrainment or synchronization of mammalian circadian rhythms to light/dark cycles. Entraining light/dark signals are transduced by the retina and conveyed to the SCN via the retinohypothalamic tract (RHT), a monosynaptic projection from a subpopulation of retinal ganglion cells that terminates bilaterally within the ventrolateral subfield of each nucleus (Moore, 1983; Pickard, 1982). In rodents, complete ablation of the SCN abolishes circadian rhythmicity in various physiologic and behavioral processes (Moore, 1983; Turek, 1985), whereas destruction of the RHT eliminates entrainment of the activity rhythm to light/dark cycles without affecting other visual functions (Johnson et al., 1988). Initial evidence for EtOH-induced disturbances of circadian clock function has emanated from rodent studies indicating that prenatal EtOH exposure alters the light/dark regulation of circadian rhythms (Sei et al., 2003). Consistent with these effects of developmental EtOH on the SCN clock, our preliminary findings demonstrate that postnatal EtOH treatment disrupts endogenous neurochemical rhythmicity in the SCN and alters the free-running period and light/dark entrainment of circadian rhythms (Earnest et al., 1997; Marchette et al., 2003). On the basis of the effects of developmental EtOH exposure on circadian entrainment to light/dark cycles, this study was conducted to determine whether early postnatal EtOH exposure permanently alters other aspects of the photic regulation of circadian behavior. Photoentrainment of circadian rhythms requires daily adjustment of the SCN circadian clock by an amount equal to the difference between its circadian period and the length (usually 24 hr) of the entraining light/dark cycle. Light is thought to mediate these adjustments by inducing time-dependent phase shifts of the SCN clock mechanism. Circadian rhythms freerunning in constant darkness (DD) are phase-delayed or reset to a later time in response to a brief light exposure during the early subjective night (i.e., coinciding with a previous dark phase or the animal’s active period) but are phase-advanced or displaced to an earlier time when light pulses are administered during the late subjective night. Because phase-shifting responses to light are directly correlated with circadian period (Daan and Pittendrigh, 1976, Pittendrigh and Daan, 1976), it is anticipated that light-induced phase shifts of the activity rhythm in adult rats will be altered in association with the long-term changes in free-running period caused by early postnatal EtOH exposure. Consequently, the circadian rhythm of wheel-running behavior was assessed in adult rats for evidence of developmental EtOH-related alterations in phase-shifting responses to light pulses administered at either 2 or 10 hr after the onset of activity [i.e., during the early subjective night at circadian time (CT) 14 or during the late subjective night at CT 22, respectively], because light exposure at these times induces maximal phase delays or advances of the rat activity rhythm. Because partial damage to the SCN alters circadian clock properties (e.g., period) that influence phase-shifting responses to light (Davis and Gorski, 1984; Pickard and Turek, 1982, 1985), this study also examined whether long-term changes in the photic regulation of circadian behavior are accompanied by developmental EtOH-induced insults on SCN integrity. Specifically, neuronal number and density in the SCN were analyzed for evidence of cell loss in adult rats exposed to EtOH during the early neonatal period.

We examined in vivo effects of selective estrogen receptor modulators (SERMs) 4-OH-tamoxifen (Tam), GW 5638 (GW) and EM-800 (EM) on myometrial gene expression. The uteri of ovariectomized ewes were infused with 10(-7)M of one SERM via indwelling catheters for 24h preceding hysterectomy. Half of the ewes in each SERM group received an intramuscular injection of 50 microg 17beta-estradiol (E2) 18 h prior to hysterectomy. Northern blot analysis and in situ hybridization demonstrated that E2 increased estrogen receptor (ER), progesterone receptor (PR) and cyclophilin (CYC) gene expression in the cells of both inner layer of myometrium (IM) and outer layer of myometrium (OM) as well as glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene expression in OM. Tam also increased ER mRNA levels in OM. EM appeared to increase ER gene expression, but antagonized E2's up-regulation of PR and CYC gene expression in both IM and OM. Tam and GW also antagonized E2 up-regulation of PR gene expression in OM but not IM. No SERM affected GAPDH gene expression with or without E2. Immunohistochemistry indicated that E2 increased nuclear ER and PR protein levels in both IM and OM. EM was unique in up-regulating ER protein levels, opposite to its effects in endometrial cells. All SERMs tested antagonized this increase in PR immunostaining preferentially in OM compared to the IM layer. These results illustrate gene and cell layer-specific effects of SERMs in sheep myometrium.

Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.

Estrogens up-regulate expression of the estrogen receptor alpha (ER) gene in most mammalian tissues studied. Using the ovariectomized ewe as a model, we determined that estradiol (E(2)) acted post-transcriptionally to increase endometrial ER mRNA concentrations by enhancing the stability of the message. The purpose of this study was to determine whether a similar E(2) effect occurs in Ishikawa cells, a well-differentiated human endometrial adenocarcinoma cell line. The presence and function of ER protein in Ishikawa cells was demonstrated by transactivation of a transfected plasmid (ERE(2)tkCAT) in response to 10(-)(9) M E(2), resulting in a 550% increase in reporter gene RNA. Ishikawa cells also responded to E(2) by up-regulating their ER mRNA concentration an average of 100% between 7 and 24 h of treatment. The effect of E(2) on ER mRNA stability was measured after blocking transcription with actinomycin D to find that the half-life increased from 6 to 10 h in control and E(2)-treated cells respectively. These results are consistent with cell-free studies which showed significant enhancement of the half-life of radiolabeled ER 3' untranslated region (3'UTR) RNA in extracts from E(2)-treated cells versus those from control cells. Thus, Ishikawa cells provide a relevant model system for the study of E(2)-regulated endometrial gene expression.

Estrogens upregulate estrogen receptor (ER) and progesterone receptor (PR) gene expression in endometrium immediately before ovulation to prepare, it for nurturing embryos. Most in vitro model systems have lost the ability to upregulate expression of the ER gene in response to estradiol (E2) or the ability to express the ER gene at all. Here, we used explant cultures from control and E2-treated ewes and assessed expression of four genes (ER, PR, glyceraldehyde 3-phosphate dehydrogenase [GAPDH], and cyclophilin [CYC] genes) that are upregulated by E2 in vivo on Northern blots. In cultures from control and E2-treated ewes, ER and PR messenger ribonucleic acid (mRNA) levels dropped significantly during 24 h of culture in the absence of E2. Glyceraldehyde 3-phosphate dehydrogenase mRNA levels increased 300% in explants from control ewes to match the higher levels in the endometrium of the E2-treated ewe (in vivo and in explant culture). The only effect of E2 in the explant cultures was to prevent the decrease in PR mRNA. The new selective ER modulator, EM-800 (EM), decreased ER and PR mRNA levels in explants from control ewes but upregulated GAPDH and CYC mRNA levels. The EM treatment in vitro mimicked that of E2 by increasing the half-life of ER mRNA in endometrial explants. These data illustrate distinct, gene-specific effects of the explant culture process, E2, and EM on the expression of endometrial genes.