Bacterial Analysis of Fertilized Eggs of Atlantic Salmon from the Penobscot,Naraguagus, and Machias Rivers,Maine

Serious losses have occurred at the U.S. Fish and Wildlife Service, Craig Brook National Fish Hatchery, East Orland, Maine, among eggs that were taken from Atlantic Salmon Salmo salar, which were held as captive broodfish during their returns to the Penobscot River, Naraguagus River, and Machias River to spawn. Bacterial isolations were attempted from external surfaces and the internal contents of individual eggs. Externally and in all cases, Pseudomonas fluorescens was the predominant bacterium associated with the surface of all eggs. These bacteria were resistant to a surface treatment of 1,667 ppm formalin for 15 min and, therefore, the monoclonal nature of P. fluorescens on egg surfaces was considered to result from its ability to resist the germicidal activity of formalin administered for antifungal treatments. Flavobacterium psychrophilum, the cause of bacterial coldwater disease, was isolated from the interior of 23.6, 18.1, and 29.2% of the dead Atlantic Salmon eggs from Penobscot River egg lots A-98, A-100, and A-101, respectively, and concentrations of this pathogen ranged from 1.0 × 10³ to >5 × 10⁸ CFU per gram of dead egg. Flavobacterium psychrophilum was also isolated from 8.3, 26.7, and 10.0% of the dead eggs from Naraguagus River egg lots N-158, N-161, and N-163, respectively, in which concentrations of this organism ranged from 1.0 × 10³ to 7.5 × 10⁸ CFU per gram of egg. This bacterium was also isolated from within 18.3% and 3.3% of the dead eggs from Machias River egg lots M-128 and M-142, respectively, and its concentrations ranged from 1.0 × 10³ to 1.5 × 10⁸ CFU per gram of egg. The finding of F. psychrophilum from within these eggs is indicative of this pathogen's widespread and persistent prevalence in Atlantic Salmon in New England.

Received December 19, 2014; accepted May 4, 2015     

Use of Penicillin and Streptomycin to Reduce Spread of Bacterial Coldwater Disease II: Efficacy of Using Antibiotics in Diluents and During Water Hardening

Bacterial coldwater disease, caused by Flavobacterium psychrophilum, has lead to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid and can enter the egg during fertilization. The addition of antibiotics to diluents and water-hardening solutions could prevent the spread of the disease. In separate trials, a mixture of 0.197 mg/mL penicillin plus 0.313 mg/mL streptomycin was added to both a 0.5% sodium chloride fertilization diluent and hatchery well water during hardening. Tests showed that the addition of the antibiotics to the diluent and during up to 60 min of water hardening had no effect on the eye-up, hatch and deformity rates of Rainbow Trout Oncorhynchus mykiss eggs compared with the nonantibiotic-treated controls. Also, significant reductions in the prevalence of F. psychrophilum on the surface and inside eggs were observed when compared with controls. These results indicate that the addition of penicillin and streptomycin to diluents and during water hardening can prevent the vertical transmission of bacterial coldwater disease.

Received May 1, 2014; accepted July 10, 2014     

Flavobacterium psychrophilum Infections in Salmonid Broodstock and Hatchery-Propagated Stocks of the Great Lakes Basin

Bacterial coldwater disease (BCWD), caused by Flavobacterium psychrophilum, threatens wild and propagated salmonids worldwide and leads to substantial economic losses. In addition to being horizontally transmitted, F. psychrophilum can be passed from infected parents to their progeny, furthering the negative impacts of this pathogen. In Michigan, both feral and captive salmonid broodstocks are the gamete sources used in fishery propagation efforts. A 5-year study was initiated to follow the prevalence of systemic F. psychrophilum infections in feral broodstocks of four species (steelhead Oncorhynchus mykiss [potadromous Rainbow Trout]; Coho Salmon O. kisutch; Chinook Salmon O. tshawytscha; and Atlantic Salmon Salmo salar) residing in three Great Lakes watersheds. Additionally, captive broodstocks of four species (Rainbow Trout, Brown Trout Salmo trutta, Lake Trout Salvelinus namaycush, and Brook Trout Salvelinus fontinalis) maintained at two facilities were assessed for the presence of F. psychrophilum. The resultant offspring from each broodstock population were sampled for F. psychrophilum infections multiple times throughout hatchery residency. Using selective flavobacterial culture and PCR confirmation, F. psychrophilum was detected in all broodstocks except the captive Lake Trout and Brook Trout. Logistic regression analysis demonstrated that among the infected feral broodstocks, Chinook Salmon from the Lake Michigan watershed had the highest prevalence of systemic F. psychrophilum infection (mean = 63.2%). Among the captive broodstocks, the Gilchrist Creek strain of Brown Trout had the highest infection prevalence (mean = 5%). Collectively, the captive broodstocks were found to have significantly lower infection prevalence than the feral broodstocks. Despite the high prevalence of systemic F. psychrophilum infections in many broodstock populations, the bacterium was rarely detected in their progeny during hatchery rearing. However, heavy losses associated with clinical BCWD outbreaks did occur. Collectively, our results reinforce that BCWD continues to threaten Great Lakes basin salmonids.

Received April 6, 2015; accepted August 25, 2015     

Use of Penicillin and Streptomycin to Reduce Spread of Bacterial Coldwater Disease I: Antibiotics in Sperm Extenders

Bacterial coldwater disease caused by Flavobacterium psychrophilum has led to the loss of significant numbers of hatchery-reared salmonids. The bacteria can be spread from parent to progeny within contaminated sperm and ovarian fluid. Methods for disinfecting ovarian fluid and unfertilized eggs are available, but methods for disinfecting sperm have not been described. In this study we determined whether sperm extenders containing a mixture of penicillin and streptomycin can be used to eliminate F. psychrophilum. In vitro trials demonstrated that when Rainbow Trout Oncorhynchus mykiss sperm is mixed with an extender, a 15-min exposure to 0.197 mg penicillin plus 0.313 mg/mL streptomycin is effective at killing the bacteria and has no effect on sperm motility. Small-scale trials showed that egg fertilization rates were not reduced when sperm held in an extender solution containing the same antibiotic mixture for 15 min was used to fertilize eggs. Production-scale trials, however, showed a roughly 18% decrease in egg fertilization rate when sperm stored in an antibiotic containing extender was used. To determine why a reduction in fertilization capacity was observed, a small-scale experiment testing the fertilization of eggs with larger quantities of sperm was performed and showed that increasing the volume of sperm used did not increase fertilization rates. Our results demonstrate that extenders containing penicillin and streptomycin can be used to disinfect sperm, especially when small quantities of eggs are fertilized, but factors negatively affecting egg fertilization and survival on a production scale still need further investigation.

Received May 1, 2014; accepted August 10, 2014     

Enrichment of conjugated linoleic acid (CLA) in hen eggs and broiler chickens meat by lactic acid bacteria

1. The aim of this work was to compare conjugated linoleic acid (CLA) concentrations in chickens supplemented with 4 American Tissue Culture Collection (ATCC) bacterial strains, Lactobacillus plantarum, Lactobacillus lactis, Lactobacillus casei and Lactobacillus fermentum, and 4 isolates of Lactobacillus reuteri from camel, cattle, sheep and goat rumen extracts.

2. Micro-organisms were grown anaerobically in MRS broth, and 10⁶ CFU/ml of bacteria were administered orally to mixed-sex, 1-d-old broiler chickens weekly for 4 weeks and to 23-week-old layer hens weekly for 6 weeks.

3. The 4 strains were evaluated for their effects on synthesis of CLA in hen eggs and broiler meat cuts.

4. Administration of pure Lactobacillus and isolated L. reuteri strains from camel, cattle, goat and sheep led to significantly increased CLA concentrations of 0.2–1.2 mg/g of fat in eggs and 0.3–1.88 mg/g of fat in broiler chicken flesh homogenates of leg, thigh and breast.

5. These data demonstrate that lactic acid bacteria of animal origin (L. reuteri) significantly enhanced CLA synthesis in both eggs and broiler meat cuts.     

Effect of melamine-contaminated diet on tissue distribution of melamine and cyanuric acid,blood variables,and egg quality in laying hens

1. The aim of the study was to investigate the effect of melamine-contaminated feed (100 mg/kg) on the distribution of melamine and cyanuric acid in the tissues of laying hens. The effect of a 5-week melamine administration in feed on the egg quality and blood variables of layers was also investigated.

2. A total of twenty 36-week-old ISA Brown layers were used in the experiment. The layers were equally divided into an experimental melamine group (n = 10) and a control group without melamine (n = 10). At the end of the experiment, samples of liver, kidney, breast and thigh muscles were collected from all hens and analysed for the presence of melamine and cyanuric acid by gas chromatography and tandem mass spectrometry.

3. Layers receiving the melamine-contaminated diet laid eggs with decreased eggshell strength. The decrease in shell strength was found in weeks 3 and 4 compared to the initial state (week 0) and week 1.

4. Hens receiving the melamine-contaminated diet also exhibited a higher total red blood cell count and lower mean corpuscular haemoglobin compared to the control group. However, melamine at 100 mg/kg feed had no effects on the blood variables of layers.

5. Melamine was detected in all analysed tissues of layers fed on the melamine-contaminated diet, with its mean concentrations decreasing in the following order: kidney (7.43 mg/kg) > breast muscle (3.88 mg/kg) > liver (3.11 mg/kg) > thigh muscle (1.91 mg/kg). The kidney and liver of layers fed on the melamine-contaminated diet also exhibited the presence of cyanuric acid.

6. On the basis of our results, it can be concluded that the biotransformation of melamine into cyanuric acid proceeded mainly in the liver, and cyanuric acid was eliminated in urine.     

Studies on occurrence,characterisation and decontamination of emerging pathogenic Escherichia coli (STEC,ETEC and EIEC) in table eggs

1. Escherichia coli is one of the most common facultative anaerobic species present in the gastrointestinal tract of animals and human beings. Usually they occur as commensals, but some serotypes can cause significant illnesses in humans as well as mammals and birds.

2. The occurrence of E. coli in different categories of table eggs collected from markets was evaluated. Isolates were analysed for the presence of virulence genes, antibiotic susceptibility pattern and efficacy of peracetic acid and chlorine for the purpose of decontaminating table eggs.

3. Significant differences were observed in the occurrence of E. coli between different groups viz. processed (cleaned, washed, sanitised and packed eggs), unprocessed (un-cleaned, un-sanitised and loose eggs) and free range (eggs obtained from backyard poultry) table eggs. Overall, E. coli occurred in table eggs at 28.6% with 22.9, 29.2 and 50.0% occurrence in processed, unprocessed and free-range table eggs, respectively.

4. A total of 24 isolates of E. coli were obtained and screened for virulence genes viz. STH, SLT1/2 and INVE genes. Of the 24 isolates recovered, 10 typeable isolates belonged to O141, O119, O9, O120 and O101 serotypes, while the remaining 14 were untypeable. Antibiograms of the isolates showed multiple antimicrobial resistance (MAR) index in the range of 0.13–0.40.

5. Peracetic acid (PAA) and chlorine (CL) were studied for their sanitisation efficacy; concentrations of 100 mg/kg of PAA and 200 mg/kg of CL completely inactivated E. coli over the egg surface and also resulted in 2.58 and 2.38 log reduction in total viable counts (TVC), respectively.

6. The presence of virulence-associated shiga-like toxin (SLT1/2) and invasion E (INVE) genes and antimicrobial resistance among the emerging serotypes of pathogenic E. coli isolated from table eggs has public health implications. It underscores the need to implement better management practices across the production systems and marketing channels to produce E. coli-free wholesome eggs for consumers.     

Comparative study of the effects of organic selenium on hen performance and productivity of broiler breeders

1. A 10-week experiment was conducted with Ross 308 broiler breeder chickens in cages to evaluate the influence of organic and inorganic sources of selenium (Se) supplementation. A total of 600 birds at 29 weeks of age were divided at random into 4 groups and fed on a maize–soya basal diet supplemented with different forms of Se.

2. The first (control) group was given the basal diet without Se supplementation, whereas the second, third and fourth groups were given, respectively, the basal diet with 0.3 mg/kg of inorganic Se in the form of sodium selenite or 0.3 and 0.5 mg/kg of organic Se in the form of Se enriched yeast (Se-yeast).

3. The experiment was carried out for 10 weeks to compare and evaluate the influence of Se supplementation on breeder performance, egg production, hatchability and the quality of eggs. Samples were collected for analysis at weeks 0, 5 and 10 of the experimental period.

4. At the end of the experiment (39 weeks), there was a reduction in mortality in breeders given diets supplemented with 0.5 mg/kg of Se-yeast. Supplementation of feed with 0.5 mg/kg organic Se increased egg production, percentage of settable eggs and hatchability.

5. Selenium supplementation increased egg weight and specific gravity compared to the control diet. However, no significant variation was found in albumen or yolk protein content at the end of week 10.

6. Selenium accumulation and glutathione peroxidase (GSHPx) activity were lower in the egg albumen and yolk of control compared with Se-supplemented treatments. Se accumulation and GSHPx activity were higher in the group given 0.3 mg/kg organic compared to 0.3 mg/kg of inorganic Se.

7. The results favour the use of Se-yeast at the dose of 0.5 mg/kg in broiler breeder diets for better productivity of eggs, settable eggs, hatchability and higher Se accumulation and antioxidant status in eggs.     

Influence of citrus flavonoids on laying hen performance,inflammatory immune response,egg quality and yolk oxidative stability

1. The objective of this study was to investigate the effects of dietary supplementation with natural flavonoids (naringin and hesperidin) on laying hens’ performance, cellular immunity and egg quality parameters.

2. A total of 72 individually caged laying hens were allocated into 1 of 6 treatment groups: a control (C) group that was fed with a basal diet and groups that were offered the same diet further supplemented with hesperidin at 0.75 g/kg (E1), or 1.5 g/kg (E2), or naringin at 0.75 g/kg (N1), or 1.5 g/kg (N2) or α-tocopheryl acetate at 0.2 mg/kg (VE) for 67 d.

3. Supplementation with naringin or hesperidin did not affect the performance and egg quality (P > 0.05) apart from an improvement in the yolk colour that was more orange in naringin and hesperidin groups in comparison to the controls (P-linear < 0.05). Egg yolk and plasma cholesterol levels were not affected by citrus flavonoids (P-linear > 0.05).

4. Inflammatory immune response, measured by phytohaemagglutinin skin test (PHA), was suppressed in laying hens that were fed with either naringin (P-linear < 0.05) or hesperidin (P < 0.05). Egg yolk oxidative stability was improved from the 4th d after naringin or hesperidin supplementation. This beneficial effect was comparable to that of α-tocopheryl acetate and was observed in eggs that were stored for up to 120 d.

5. In conclusion, naringin and hesperidin may favourably prolong the shelf life of eggs, appear to possess anti-inflammatory properties and could improve the yolk colour without any side effects on the performance or egg quality traits.     

Case Report: Strawberry Disease in Farmed Chilean Rainbow Trout

Strawberry disease is a chronic, nonlethal skin condition that affects Rainbow Trout Oncorhynchus mykiss in the United States and several European countries, where it is also known as red-mark syndrome. We provide the first identification and characterization of three strawberry disease outbreaks occurring at two aquaculture farms in southern Chile. Clinically affected fish weighing an average of 400 g presented multiple bright-red, usually raised, skin lesions on the flank, ventral surface, and dorsal surface. A PCR using Rickettsia-like-organism (RLO)-specific primers was performed on nine affected fish, and all skin samples were positive for the RLO 16S ribosomal RNA sequence. All PCR results for Flavobacterium psychrophilum and other bacterial and viral pathogens were negative. Histopathological examination of the skin lesions revealed extensive dermatitis, with severe lymphocytic infiltration in advanced cases. This report is the first to describe strawberry disease in farmed Chilean Rainbow Trout. Additional studies are needed to evaluate the risk for Rainbow Trout culture; fish challenge experiments should be performed to fulfill Koch’s postulates and to demonstrate that RLO is the cause of this disease.

Received December 27, 2014; accepted October 23, 2015.     

Effects of dietary rosemary and oregano volatile oil mixture on quail performance,egg traits and egg oxidative stability

1. This study was conducted to determine the effects of volatile oil mixture on quail laying performance, egg traits and egg malondialdehyde (MDA) concentration.

2. A total of 260 Pharaoh quails (Coturnix coturnix Pharaoh) aged 6 weeks were equally divided into 5 groups of 65 (4 replicates of 13 quails each). The mixture of diets was as follows: a control treatment with 0 mg volatile oil/kg of diet; (1) 200 mg/kg rosemary volatile oil; (2) 200 mg/kg oregano volatile oil; (3) 40 mg/kg rosemary volatile oil plus 160 mg/kg oregano volatile oil (ratio 20:80) and (4) 160 mg/kg rosemary volatile oil plus 40 mg/kg oregano volatile oil (ratio 80:20). The diets were prepared fresh for each treatment. The experimental period lasted 10 weeks.

3. At the end of the experiment, there were no significant differences amongst the groups in body weight, egg weight, egg mass, egg shape index, Haugh unit, egg shell thickness or egg shell–breaking strength.

4. Diets containing rosemary volatile oil increased the egg production significantly. Feed intake significantly increased in the groups containing volatile oil mixture (groups 4 and 5). The inclusion of rosemary volatile oil at 200 mg/kg improved feed efficiency.

5. Egg albumen and egg yolk index values showed significant increases in the group given diets containing rosemary volatile oil. Egg yolk colour became darker with the addition of rosemary and oregano volatile oil. The treatment group had lower egg yolk MDA concentration than the control group.

6. It is concluded that, alone or in combination, rosemary and oregano volatile oil can be used in quail diets without adverse effects on the measured parameters. Inclusion of rosemary and oregano volatile oil in quail diets enhanced the antioxidant status of eggs.     

Effects of dietary oregano and garlic essential oils on carcass characteristics,meat composition,colour, pH and sensory quality of broiler meat

1. An experiment was conducted to determine the individual and combined effects of two essential oils, oregano and garlic, on carcass characteristics, meat composition, colour, pH and sensory quality of broiler meat.

2. The diets were supplemented with no essential oil (control), oregano essential oil or garlic essential oil at 300 mg/kg and oregano essential oil at 150 mg/kg + garlic essential oil at 150 mg/kg.

3. Dietary oregano and garlic oil supplementation did not affect carcass yields, the relative weight of carcass parts, breast and thigh meat composition, pH or b* value of breast meat. Oregano + garlic oil supplementation significantly decreased the L* value. The a* value of breast meat in birds given a diet supplemented with oregano oil was lower than that in birds given a diet supplemented with garlic oil and oregano oil + garlic oil. The essential oil addition had no positive effect on the oxidative stability. There was no difference between the treatments in breast appearance.

4. The juiciness, flavour, oxidised flavour and acceptability of breast meat samples were affected by treatments.     

Initial Detection and Molecular Characterization of Namaycush Herpesvirus (Salmonid Herpesvirus 5) in Lake Trout

A novel herpesvirus was found by molecular methods in samples of Lake Trout Salvelinus namaycush from Lake Erie, Pennsylvania, and Lake Ontario, Keuka Lake, and Lake Otsego, New York. Based on PCR amplification and partial sequencing of polymerase, terminase, and glycoprotein genes, a number of isolates were identified as a novel virus, which we have named Namaycush herpesvirus (NamHV) salmonid herpesvirus 5 (SalHV5). Phylogenetic analyses of three NamHV genes indicated strong clustering with other members of the genus Salmonivirus, placing these isolates into family Alloherpesviridae. The NamHV isolates were identical in the three partially sequenced genes; however, they varied from other salmonid herpesviruses in nucleotide sequence identity. In all three of the genes sequenced, NamHV shared the highest sequence identity with Atlantic Salmon papillomatosis virus (ASPV; SalHV4) isolated from Atlantic Salmon Salmo salar in northern Europe, including northwestern Russia. These results lead one to believe that NamHV and ASPV have a common ancestor that may have made a relatively recent host jump from Atlantic Salmon to Lake Trout or vice versa. Partial nucleotide sequence comparisons between NamHV and ASPV for the polymerase and glycoprotein genes differ by >5% and >10%, respectively. Additional nucleotide sequence comparisons between NamHV and epizootic epitheliotropic disease virus (EEDV/SalHV3) in the terminase, glycoprotein, and polymerase genes differ by >5%, >20%, and >10%, respectively. Thus, NamHV and EEDV may be occupying discrete ecological niches in Lake Trout. Even though NamHV shared the highest genetic identity with ASPV, each of these viruses has a separate host species, which also implies speciation. Additionally, NamHV has been detected over the last 4 years in four separate water bodies across two states, which suggests that NamHV is a distinct, naturally replicating lineage. This, in combination with a divergence in nucleotide sequence from EEDV, indicates that NamHV is a new species in the genus Salmonivirus.

Received April 20, 2015; accepted October 11, 2015     

Hatchability rate and embryonic growth of broiler chicks following in ovo injection royal jelly

1. The objectives of this study were to compare the hatchability, chick body and internal organs weights and plasma testosterone concentration of hatchlings after in ovo administration of royal jelly (RJ) on d 7 of incubation.

2. Fertile eggs (n = 150) were injected into the air sac or yolk sac with 0.5 ml normal saline solution or normal saline and pure RJ. The eggs were randomly divided into 5 groups of 30 eggs each: NC, control eggs receiving no injection; ASA, air sac–injected eggs given normal saline solution; ARJ, air sac–injected eggs injected with pure RJ; YSA, yolk sac–injected eggs receiving normal saline solution and YRJ, yolk sac–injected eggs given pure RJ.

3. Injection of RJ significantly decreased hatchability (46.7%) compared with injection of saline solution (68.3%). Hatchability was lower in ARJ (33.3 %) and YRJ (60.0%) groups than in the NC group (90.0%). Hatchability in ASA (70.0%) and YSA (66.66%) groups were comparable to the NC group.

4. In ovo injection of RJ into both sacs increased chicks’ absolute and relative body, heart, liver and testes weights compared to the control group whereas plasma testosterone concentration was similar among the different groups.

5. It was concluded that in ovo injection of RJ may be an effective method to increase the body weight of hatched chicks as an absolute value (CWT) and chicks' internal organ weights.     

Assessment of Detoxifying Markers for Florfenicol in Rainbow Trout Liver

Florfenicol (FF) is employed in fish farms to contest or prevent bacterial infections. However, this pharmaceutical may produce reactive oxygen species that may cause biochemical changes in antibiotic-treated fish. The aim of this study was to evaluate the effect of FF on Rainbow Trout Oncorhynchus mykiss treated for 10 d with 7.5 and 15 mg/kg FF followed by a withdrawal period of 5 d. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I and glyoxalase II, total glutathione, lactic dehydrogenase, and alkaline phosphatase were investigated in the livers of treated and untreated fish. A general impairment of antioxidant enzymes and metabolic indicators was measured in FF-treated Rainbow Trout. Onset of oxidative damage may have occurred during the antibiotic treatment as a consequence of the effect of FF toxicity at mainly the highest dose. Nevertheless, the rise in levels of total glutathione and glutathione S-transferase even after the withdrawal period may shield the antibiotic-mediated oxidative processes.

Received December 22, 2015; accepted May 26, 2016 Published online October 28, 2016     

Pasture,multi-enzymes,benzoic acid and essential oils positively influence performance,intestinal organ weight and egg quality in free-range laying hens

1. The objective of this study was to investigate the effect of range type, multi-enzyme applications, and a combination of benzoic acid (BA) and essential oils (EO) on the productive performance, organ weight and egg quality of free-range laying hens.

2. Three hundred laying hens were evaluated for the short-term (6 weeks) and long-term (12 weeks) effects of range type (G = no pasture, P = pasture) and feed additives (T1 = control; T2 = betaglucanase/pectinase/protease; T3 = BA/EO). Body weight, feed intake (FI), feed conversion ratio (FCR), egg production (EP), digestive organ weight, and egg quality (EQ) were evaluated. Data were analysed using SPSS 2.2 in a 2×2×3 factorial arrangement.

3. Hens that ranged on pasture were significantly heavier (2043 g vs. 1996 g; p < 0.001), laid heavier eggs (61.9 g vs. 60.3 g; p < 0.001) and produced darker yolk colour (4.3 vs. 7.0; p < 0.001) compared to hens ranged on gravel. Hens fed T2 were significantly heavier (2050 g) compared to hens fed T1 (2005 g) or T3 (2008 g). Organ weights (gizzard, liver and pancreas) were significantly heavier in hens ranged on pasture (16.8 g/kg BW, 22.3 g/kg BW and 1.89 g/kg BW, respectively) compared to hens ranged on gravel (14.2 g/kg BW, 21.7 g/kg BW and 1.83 g/kg BW, respectively). Over time, body weight (1970–2070 g; p < 0.001) and egg weight (59.5–62.8 g; p < 0.001) increased, FI (123–120 g; p = 0.024) was reduced and FCR (2.36–2.10; p = 0.002) improved

4. In conclusion, hens housed on pasture and fed multi-enzyme supplemented diets had significantly heavier body weight and produced heavier eggs with darker yolk colour. Pasture intake and enzyme supplementation increased digestive organ weight significantly.     

Influence of graded inclusion of white lupin (Lupinus albus) meal on performance,nutrient digestibility and ileal viscosity of laying hens

1. The aim of this study was to investigate the effect of white lupin (Lupinus albus) meal (WLM) addition on the intestinal viscosity, bird performance and nutrient utilisation of laying hens.

2. The experiment was conducted with 360 laying hens aged 21 weeks fed one of 6 treatments, including a corn-soybean meal control diet (CON) and 5 experimental diets containing 60, 120, 180, 240 and 300 g/kg WLM.

3. A linear increase in feed intake (p < 0.001) was observed with higher levels of WLM from 0 to 300 g/kg. Laying rate decreased quadratically (p < 0.05) and egg weight (at 6th, 12th and 18th weeks of the trial) decreased linearly with WLM inclusion from 0 to 300 g/kg. Birds fed 60 g/kg or more of WLM laid lighter eggs (p < 0.05) than CON hens. When 240 g/kg or more WLM was included into the diet, laying rate was affected negatively (p < 0.05).

4. As WLM increased from 0 to 300 g/kg, apparent metabolisable energy and pre-caecal digestibility of dry matter and crude protein decreased quadratically (p < 0.05). When 300 g/kg of WLM was used, there was a tendency (p < 0.1) to decrease pre-caecal starch digestibility. WLM dose exerted a quadratic effect (p < 0.05) on total sialic acid excretion. As WLM increased, the viscosity of ileal digesta linearly increased (p < 0.05).

5. In the 6th and 12th weeks of the experiment (p < 0.05), eggshell thickness decreased linearly when 240 g/kg of WLM was added. At the 6th, 12th and 18th weeks, a linear decrease in eggshell content was observed (p < 0.05) after WLM addition.

6. In conclusion, the graded inclusion of WLM into laying hens’ diets resulted in depressed performance, AME_N and eggshell quality.     

Gill Infection Model for Columnaris Disease in Common Carp and Rainbow Trout

Challenge models generating gill lesions typical for columnaris disease were developed for the fry of both Common Carp Cyprinus carpio and Rainbow Trout Oncorhynchus mykiss by means of an immersion challenge and Flavobacterium columnare field isolates were characterized regarding virulence. Carp inoculated with highly virulent isolates revealed diffuse, whitish discoloration of the gills affecting all arches, while in trout mostly unilateral focal lesions, which were restricted to the first two gill arches, occurred. Light microscopic examination of the gills of carp exposed to highly virulent isolates revealed a diffuse loss of branchial structures and desquamation and necrosis of gill epithelium with fusion of filaments and lamellae. In severe cases, large parts of the filaments were replaced with necrotic debris entangled with massive clusters of F. columnare bacterial cells enwrapped in an eosinophilic matrix. In trout, histopathologic lesions were similar but less extensive and much more focal, and well delineated from apparently healthy tissue. Scanning and transmission electron microscopic observations of the affected gills showed long, slender bacterial cells contained in an extracellular matrix and in close contact with the destructed gill tissue.

This is the first study to reveal gill lesions typical for columnaris disease at macroscopic, light microscopic, and ultrastructural levels in both Common Carp and Rainbow Trout following a challenge with F. columnare. The results provide a basis for research opportunities to examine pathogen–gill interactions.

Received January 10, 2014; accepted July 27, 2014     

Effect of day length on flock behavioural patterns and melatonin rhythms in broilers

1. Ross × Ross 308 male broilers were used to study the impact of 14, 17, 20 and 23 h of light (L) on flock behavioural patterns and 24 h melatonin rhythm synchrony during the photophase.

2. Behaviour in two pens (n = 53) per lighting treatment was digitally recorded for 24 h in trial 1 (27–28 d of age (d 27)) and two (42–43 d (d 42)), and quantified using a scan sampling technique at 10 min intervals. Regression procedures were used to test flock trend analysis between behavioural (resting, standing, walking and feeding) variables and time during the photophase.

3. The presence of a flock melatonin rhythm was determined by radioimmunoassay of blood samples collected at 6 times for birds raised on 23L and 8 times for 14, 17 and 20L birds (n = 6 per time) over a 24 h period (d 21) in experiment 1.

4. Quadratic and linear relationships between time and behaviour during the photophase were frequent in 14L and 17L birds, sporadic in 20L birds and non-existent in 23L birds. Relationships were noted in inactive resting (d 27: 14L and 17L; d 42: 14L and 20L), walking (d 27: 14L and 17L), standing (d 27 and d 42: 14L, 17L and 20L) and feeding (d 27: 14L and 17L). A quadratic time × melatonin relationship existed in 14L, 17L and 20L flocks only.

5. Behaviour during the photophase and 24 h melatonin production indicates that flocks exposed to 23L do not develop synchronised rhythms, increasing the risk of suffering from sleep fragmentation.     

An antimicrobial peptide-A3: effects on growth performance,nutrient retention,intestinal and faecal microflora and intestinal morphology of broilers

1. The present study investigated the effects of dietary supplementation with an antimicrobial peptide-A3 (AMP-A3) on growth performance, nutrient retention, intestinal microflora and intestinal morphology of broilers.

2. A total of 320-d-old chicks (Ross 308, average BW 44.0 ± 3.4 g) were randomly allotted to 4 dietary treatments on the basis of initial body weight (BW). The dietary treatments were negative control (NC; basal diet), positive control (PC; basal diet + 15 mg avilamycin/kg diet) and AMP-A3 (basal diet supplemented with 60 or 90 mg/kg AMP-A3). The NC diet was considered as 0 mg/kg AMP-A3 treatment. Experimental diets were given in two phases: starter phase (d 0–21) and finisher phase (d 22–35).

3. The overall BW gain and retention of dry matter (DM), gross energy (GE; d 19–21) and crude protein (CP; d 19–21 and d 33–35) were greater in birds fed on the PC and 90 mg/kg AMP-A3 diets than in birds fed on the NC diet. Also, an increase in dietary AMP-A3 linearly improved BW gain and retention of DM, GE (d 19–21) and CP (d 19–21 and d 33–35).

4. Birds fed on the PC and 90 mg/kg AMP-A3 diets had fewer excreta coliforms (d 21 and d 35), total anaerobic bacteria (TAB) and Clostridium spp. (d 35) and ileum and caecum coliforms (d 35) than birds fed on the NC diet. In addition, birds fed on the diet supplemented with increasing levels of AMP-A3 had linearly reduced excreta TAB (d 35), Clostridium spp. and coliforms (d 21 and d 35) and ileum and caecum coliforms (d 35).

5. Birds fed on the PC and 90 mg/kg AMP-A3 diets had greater villus height of the duodenum, jejunum and ileum than birds fed on the NC diet. Moreover, birds fed on increasing levels of AMP-A3 diet had increased (linear) villus height of the duodenum, jejunum and ileum.

6. These results indicate that 90 mg/kg AMP-A3 has the potential to improve growth performance, nutrient retention and intestinal morphology and to reduce harmful microorganisms in broilers and can be used as a potential antimicrobial growth promoter.