Commensal or pathogen – a challenge to fulfil Koch’s Postulates

1. Infectious diseases have a large impact on poultry health and economics. Elucidating the pathogenesis of a certain disease is crucial to implement control strategies.

2. Multiplication of a pathogen and its characterisation in vitro are basic requirements to perform experimental studies. However, passaging of the pathogen in vitro can influence the pathogenicity, a process targeted for live vaccine development, but limits the reproduction of clinical signs.

3. Numerous factors can influence the outcome of experimental infections with some importance on the pathogen, application route and host as exemplarily outlined for Histomonas meleagridis, Gallibacterium anatis and fowl aviadenoviruses (FAdVs).

4. In future, more comprehensive and detailed settings are needed to obtain as much information as possible from animal experiments. Processing of samples with modern diagnostic tools provides the option to closely monitor the host–pathogen interaction.     

Maturation and ovulation of Japanese quail oocytes under in vitro conditions

1. An in vitro system for ovulation and maturation of Japanese quail oocytes is described.

2. Ovarian follicles removed from the ovary at 2, 4 or 6 h before the estimated time of ovulation may ovulate under in vitro conditions.

3. The presence of progesterone in the medium had a stimulatory effect on the process of maturation, as has been shown for Xenopus oocytes.     

Effects of phytoestrogens on testosterone secretion by Leydig cells from Biłgoraj ganders (Anser anser)

1. The aim of this study was to examine ganders at different stages of the reproductive season and the effect of: (1) diets with high phytoestrogen content and (2) in vitro phytoestrogen treatment on testosterone secretion by isolated Leydig cells.

2. Thirty-six male Bi?goraj geese were fed control diets with low phytoestrogen content (containing grass meal) and diets with high phytoestrogens (containing alfalfa meal and soy).

3. Testes were obtained from both groups of ganders at three different times of the breeding season: peak of reproductive activity (March), second half of reproductive activity (May) and beginning of photorefractoriness (July).

4. Isolated Leydig cells were incubated with LH as well as genistein, daidzein, equol and coumestrol and the concentration of testosterone in the medium was determined by radioimmunoassay.

5. The mean weight of testes from ganders in May and July decreased relative to their weights in March, but no significant differences among experimental groups were noted.

6. No differences were observed in basal and LH-stimulated testosterone secretion by Leydig cells of ganders fed the control diets and the diets with higher phytoestrogen content. In July, LH did not stimulate testosterone secretion in either group. In vitro treatment with genistein, daidzein and equol (5 and 50?µM) inhibited basal and LH-stimulated testosterone production by Leydig cells from both groups. Coumestrol (5 and 50?µM) inhibited basal testosterone secretion only in March in the control group.

7. Dietary exposure to phytoestrogens had a slight effect on in vitro testicular secretion in ganders. In vitro treatment with phytoestrogen inhibited testosterone production by Leydig cells. Genistein showed the strongest effect and coumestrol had the weakest influence on testicular secretion.     

An in vitro culture method for chick embryos obtained from the anterior portion of the magnum of oviduct

1. Chick embryos, obtained from the anterior portion of the magnum of the oviduct 60 to 80 min after the preceding egg had been laid, were cultured in vitro in small and large recipient eggshells until hatching.

2. Of 82 embryos cultured, 46.3% had survived to day 4 of incubation, and 19.5% survived to hatching.

3. The method for culturing embryos used in this experiment could facilitate research on the in vitro manipulation of early chick embryos.     

In vitro initiation of the acrosome reaction in the emu (Dromaius novaehollandiae)

1. An assessment of the efficiency of the acrosome reaction (AR) provides an important predictor of the fertilizing potential of semen and for diagnosis of the causes of infertility. A standardized protocol was therefore developed for initiation of the acrosome reaction in emu spermatozoa in vitro, and the role of CaCl₂ or perivitelline membrane (PVM) proteins in determining the outcome of the reaction was investigated.

2. The acrosome reaction (assessed by FITC-PNA) was successfully induced in live spermatozoa by incubation for 2 min in NaCl-TES medium supplemented with 5 mM CaCl₂. The maximum response was 32% live acrosome-reacted spermatozoa (LAR) achieved after 10 min incubation.

3. Compared to the outcome with 5 mM CaCl₂ or PVM protein alone, the response was significantly better with a combination of PVM protein and CaCl₂.

4. A significant variation in the percentage of LAR spermatozoa among individual males was observed. No treatment affected the percentage of dead acrosome-reacted spermatozoa.

5. The results emphasize the important role played by both PVM proteins and Ca²⁺ in the in vitro initiation of the acrosome reaction.     

Antimicrobial substances and chick growth promotion: Comparative studies on selected compounds in vitro and in vivo

1. Antibacterial activity of selected compounds in vitro and their ability to promote growth when added to the diet of chicks were not correlated.

2. The difference in growth‐promoting activity between streptomycin, which was active, and kanamycin which was not, was not related to their effects on the flora adhering to the wall of the crop, jejunum or ileum, to their relative toxicity to the chick nor to differences in their stability in the diet.

3. When low concentrations ofkanamycin or streptomycin were given, the antibiotic was concentrated in the caecal contents. In contrast, penicillin was undetectable in the caeca of birds given high concentrations of benzyl penicillin.

4. In chicks given benzyl penicillin, the numbers of Streptococcus faecium increased at the expense of Strep, faecalis. This probably resulted from changes in the crop flora.

5. Birds given high dietary concentrations of benzyl penicillin showed improved weight gains, in spite of increased numbers of Escherichia coli in the small intestine.     

In vitro and in vivo protective effects of three mycotoxin adsorbents against ochratoxin A in broiler chickens

1. The objective of this study was to investigate in vitro and in vivo (in broiler chickens) ochratoxin A (OTA) adsorption efficiency of three different adsorbents: inorganic (modified zeolite); organic (esterified glucomannans) and mixed (inorganic and organic components plus enzymes).

2. The aim of the study was to investigate which of these adsorbents provided the best protection against the presence of residues of OTA in the pectoral muscle and liver of broilers given an OTA-contaminated diet. In addition, it was important to test and compare the results of adsorbent efficiency using two different in vitro methods.

3. The results from classical in vitro investigations carried out in the artificial intestinal fluid, showed that the inorganic adsorbent (Mz), exhibited the highest adsorption, having adsorbed 80.86 ± 1.85% of OTA, whereas average in vitro adsorption abilities of organic (30.52 ± 3.50%) and mixed (32.00 ± 2.60%) adsorbents were significantly lower.

4. In the investigation of absorption in everted sacs of broiler duodenal segments (Everted Duodenal Sacs Procedure), higher OTA adsorption in gut was exhibited by organic adsorbent, 74.26 ± 4.48%. Furthermore, the mean adsorption efficiency of mixed and inorganic adsorbent was 65.26 ± 4.76% and 45.75 ± 7.14%, respectively.

5. In the in vivo investigation, broilers were fed for 21 d on diets containing 2 mg/kg of OTA and supplemented with inorganic (Mz), organic (Ms) or mixed adsorbent (Mf) at the recommended concentration of 2 g/kg of feed. All three adsorbents significantly decreased OTA residue concentrations in the pectoral muscle and livers, but the order of effectiveness was mixed > organic > inorganic. The most efficient was the mixed adsorbent which decreased residue concentration by 72.50% in pectoral muscle and 94.47% in livers.

6. The Everted Duodenal Sac in vitro method provided results similar to those obtained in the in vivo study. However, further studies are required to investigate the efficiencies of adsorbents against various mycotoxins using this method.     

Inhibitory effects of epidermal growth factor on progesterone production of ovarian granulosa cells in Tsaiya duck (Anas platyrhynchos var. domestica)

1.?The purpose of this study was to investigate the effects of epidermal growth factor (EGF) on progesterone (P₄) secretion from ovarian granulosa cells in Tsaiya ducks (Anas platyrhynchos var. domestica).

2.?We obtained the largest (F1) follicle from Tsaiya duck, the granulosa layer was separated and the cells were isolated according to their proximity to the germinal disc.

3.?The granulosa cells were cultured in vitro, the culture media and the cells were used to determine P₄ and steroidogenic enzyme concentrations, respectively.

4.?P₄ concentrations were decreased in cultured granulosa cells taken proximal to the germinal disc (GD) compared to those distal to the germinal disc (NGD).

5.?EGF inhibited both basal and ovine luteinising hormone (oLH)-induced P₄ concentrations. It also inhibited the P₄ secretion via protein kinase A (PKA) pathway when cultured with GD and NGD granulose cells (mixed together) in vitro.

6.?Western blot results showed decreased concentrations of cytochrome P450 side-chain cleavage (P450scc) enzyme, 3β-hydroxysteroid dehydrogenase (3β-HSD) enzyme and steroid acute regulatory (StAR) protein expression, when the cells were co-treated with EGF and oLH.

7.?The inhibitory effect of oLH-induced P₄ production was attenuated by EGF by the addition of MAP-erk kinase (MEK) inhibitor (PD98059) suggests EGF may inhibit P₄ production by affecting via the mitogen-activated protein kinase (MAPK) pathway.     

Influence of dietary lactose on the gut flora of chicks

1. The gut microflora of chicks fed on a purified diet containing 300 g lactose plus 300 g starch/kg was compared with that of control birds receiving a diet containing 600 g starch/kg.

2. In 14‐d‐old conventional chicks, lactose in the diet decreased the incidence of lactobacilli and clostridia in the caecal contents, although when present in lactose‐fed chicks the counts of lactobacilli exceeded those of control chicks.

3. High counts of Proteus sp. were present in the caeca of control birds but they were completely suppressed in conventional birds fed on the lactose diet. In vitro tests showed that this inhibition was partially due to Escher‐ichia coli and Streptococcus faecalis.

4. The growth of Lactobacillus acidophilus was inhibited by lactose when gnotobiotic chicks were monoassociated but not when polyassociated. The protective effect was shown in vitro to be due to L. salivarius.

5. The pH was markedly lowered in the caecum of conventional and polyassociated chicks receiving dietary lactose. Of the strains used in gnotobiotic experiments E. coli, S. faecalis and L. salivarius produced the lowest pH values in the caeca.     

A 31P topical magnetic resonance study of embryonic development in hens' eggs

1. A ³¹P topical magnetic resonance study of whole chicken's eggs in vivo is described.

2. Resonances from adenosine triphosphate, phosphocreatine, inorganic phosphate and phosphorous storage protein were observed. These changed in relative intensity as the embryo developed.

3. No ill effects due to exposure to radiofrequency radiation or magnetic fields were observed.

4. It is concluded that topical magnetic resonance can make a useful contribution to the study of embryonic development in avian eggs.     

In vitro versus in situ evaluation of the effect of phytase supplementation on calcium and phosphorus solubility in soya bean and rapeseed meal broiler diets

1. In vitro assays provide a rapid and economical tool to evaluate dietary effects, but have limitations. In this study, the effect of phytase supplementation on solubility, and presumed availability, of calcium (Ca) and phosphorus (P) in soya bean meal (SBM) and rapeseed meal (RSM) based diets were evaluated both in situ and by a two-step in vitro digestion assay that simulated the gastric and small intestine (SI) phases of digestion.

2. Comparison of the in vitro findings to in situ findings was used to evaluate the in vitro assay. Ross 308 broilers (n = 192) were fed on one of 6 SBM or RSM diets supplemented with 0, 500 or 5000 FTU/kg phytase from 0 to 28 d post hatch. The 6 diets and raw SBM and RSM were exposed to a two-step in vitro assay. Ca and P solubility and pH in the gizzard and jejunal digesta and in the gastric and SI phase of in vitro digestion were measured.

3. Both in vitro and in situ analyses detected that Ca solubility was lowest when diets were supplemented with 500 FTU/kg phytase, compared to the control diets and diets supplemented with 5000 FTU/kg phytase. Phosphorus solubility increased with increasing phytase level. Both methods also identified that mineral solubility plateaus in the gastric phase.

4. Overall relationship of the two methods was strong for both determination of gastric phase Ca and P solubility (r = 0.96 and 0.92, respectively) and also SI phase Ca and P solubility (r = 0.71 and 0.82, respectively). However, mineral solubility and pH were higher when measured in vitro than in situ, and the in situ assay identified an interaction among the effects of phase, protein source and phytase inclusion level on Ca solubility that the in vitro assay did not detect.

5. This two-step in vitro assay successfully predicted phytase efficacy, but to determine detailed response effects in the animal, in situ data is still required.     

Potential of a commercially available water acidification product for reducing Campylobacter in broilers prior to slaughter

1. This study investigated the potential of a commercially available acidified water treatment (PWT) for reducing the number of Campylobacter in vitro and other bacteria in the gut of live broilers.

2. In vitro tests indicated that PWT was highly effective for reducing Campylobacter jejuni and Campylobacter coli at the recommended concentration in water, reducing populations by greater than 7 log₁₀ CFU/ml after 24 h exposure. The decrease in the number of Salmonella serovar Enteritidis and Escherichia coli was not significant.

3. Addition of PWT to the broiler drinking water for the first 7 d, 2 d before and 2 d after each feed change and at feed withdrawal prior to slaughter or only after feed withdrawal had no effect on the number of Campylobacter in caecal samples on farm before thinning and depopulation compared to untreated controls.

4. Although PWT was effective for reducing Campylobacter in water, the results suggest that it does not reduce the number of Campylobacter in the caeca of broilers prior to slaughter under the conditions used in the study.     

The activities of amylase and a trypsin‐like protease in the gut contents of germ‐free and conventional chickens

1. Proteolytic and amylase activities were assayed in the gut contents of germ‐free and conventional chickens aged 3 and 14 d.

2. The tendency was for the proteolytic activity to be increased and the amylase activity to be decreased in conventional chicks. These differences were statistically significant in the caecum.

3. All of the additional proteolytic activity detectable in the caecum of conventional chicks was inhibited by soyabean trypsin inhibitor. However, the increased activity was not detectable in the small intestine and was, therefore, not due to pancreatic trypsin.

4. Attempts to demonstrate in vitro bacterial production of a trypsin‐like enzyme were unsuccessful.     

The effect of dietary bacitracin on the incidence of streptococcus faecalis subspecies liquefaciens and related streptococci in the intestines of young chicks

1. A study has been made of changes occurring in the numbers and types of faecal streptococci in the intestines of chicks from hatching to 23 d of age, when fed on a diet containing 50 mg bacitracin/kg.

2. It was found that Streptococcus faecalis sub‐species liquefaciens predominated in chicks prior to feeding and in control birds the organism remained as a significant part of the flora throughout the experiment.

3. Strep. faecalis s.s. liquefaciens was not found after the first week in birds given dietary bacitracin.

4. Although outnumbered initially in the caeca by Strep, faecalis s.s. liquefaciens, other streptococci were present at 10⁶ to 10⁷/g contents from 14 d onwards in both control and bacitracin‐treated birds; these included Strep. faecium biotypes and two other unidentified but related groups.

5. Tests made in vitro showed that Strep. faecalis s.s. liquefaciens was more sensitive to bacitracin than Strep. faecium.

6. The possible effects of Strep. faecalis s.s. liquefaciens on the developing bird are discussed.     

Gene expression profile of IGF1 and MSTN mRNA and their correlation with carcass traits in different breeds of geese at 70 d of age

1. The expression of insulin-like growth factor-1 (IGF1) and myostatin (MSTN) mRNA in breast and leg muscle was quantified in 70-d-old Taihu and Wanxi geese by using a Multiplex Competitive Fluorescent–PCR method and the correlations between mRNA levels and carcass traits were analysed.

2. IGF1 mRNA expression in breast muscle in Taihu geese was significantly higher than that in Wanxi geese and the MSTN mRNA level in leg muscle in Taihu geese was significantly higher than that in Wanxi geese.

3. There was no significant difference in breast muscle MSTN or leg muscle IGF1 mRNA expression between the two breeds.

4. Within the same breed, the IGF1 mRNA expression in leg muscle of male geese was significantly higher than that in female geese, and MSTN mRNA expression in leg muscle was significantly higher than that in breast muscle.

5. There was no difference in the IGF1 mRNA expression between tissues.

6. There was a positive correlation between IGF1 mRNA and MSTN mRNA and a negative correlation between IGF1 mRNA expression of breast muscle and leg muscle ratio.

7. In Wanxi geese, MSTN mRNA expression in leg muscle was negatively associated with body weight and leg muscle weight.     

The occurrence of giant fibres in different muscles of two chicken genotypes

1. The occurrence of Giant Fibres (GF) in three muscles (Pectoralis major (PM), Iliotibialis lateralis and Semimembranosus) with different types of energy metabolism was studied in slow- and fast-growing chicken strains.

2. A total of 20 one-day-old Leghorn chicks (slow-growing) and 20 broiler (Ross 508) chicks (fast-growing) were reared to 100 and 45 d, respectively.

3. A small percentage of GF was seen in pre rigor muscle samples even at 3 min post mortem in both genotypes and in all muscle types studied.

4. From 3 min to 24 h post mortem GF increased both in Leghorn and broiler chickens but to a different extent according to muscle type and genotype.

5. The highest GF 24 h post mortem value was found in the PM muscles belonging to the fast-growing broiler line.

6. It was concluded that every type of muscle can develop GF, but this phenomenon is more evident in the PM especially in animals selected for increased growth rate.     

Effect of dietary protein and urea on in vitro caecal ammonia production from urea and uric acid in cockerels

1. Single Comb White Leghorn adult cockerels were fed on 50 g/kg protein diet, 200 g/kg protein diet or 50 g/kg protein diet plus urea and in vitro ammoniagenesis from urea and uric acid in the caeca was determined.

2. Four‐fold protein intake caused about 4.6‐fold increase in caecal ammonia production from urea (P < 0.05), and tended to increase it from uric acid as compared with 50 g/kg protein‐fed birds.

3. Dietary urea significantly increased caecal ammonia production from urea and uric acid by about 2 and 3 times as much as those of control birds, respectively (P < 0.05).

4. It is concluded that increased protein intake and the feeding of urea are able to induce ammoniagenesis from urea and uric acid in the caeca of fowls.     

Behaviour of egg shell under compression in relation to deformation measurements1

1. The egg does not completely obey Hook's law when compressed between flat surfaces.

2. Deformation was not completely reversible due to crumbling at the points of contact with the force.

3. There are three components of non‐destructive deformation: (a) local deformation at the points of contact, (b) local surface crumbling and (c) deformation of the over‐all shell structure.

4. The latter two are uncontrolled, contributing to errors in the prediction of fracture force and depending on the non‐destructive force applied.

5. These effects should be considered in test selection and interpretation.

6. The optimum non‐destructive force was found to be 1 kg.     

Paired box 7 (Pax7) gene: molecular characterisation,polymorphism and its association with growth performance in goose (Anser cygnoides)

1. Paired box (Pax7) gene is a member of the paired box family and plays a critical role in animal growth and muscle development. However, the molecular characterisation of the goose Pax7 gene is unknown.

2. The open-reading frame of goose Pax7 is composed of 1509 bp, which encodes a protein of 503 amino acids and shares high homology with Pax7 of other birds.

3. Ten single-nucleotide polymorphisms were identified in the genomic DNA sequence, 8 located in the intron region and two located in the exon region.

4. Association analysis showed the C122T locus was significantly associated with the body weight of Zhedong-White geese in week 4, 6, 8, 10 and 12.

5. It was concluded that the goose Pax7 gene may be an important candidate gene for goose growth traits and marker-assisted selection.     

Effects of short intermittent lighting on food consumption and performance of dwarf broiler breeders and progeny

1. Two hundred and sixty four dwarf broiler breeder hens were subjected to ad libitum or restricted feeding and to four lighting patterns: 15L (bright light):9D (dark), 15L:9d (dim light), 2L:10d: 1L:11d, (0.5L:3.5d) × 6.

2. Breeder hens fed ad libitum and subjected to either conventional or intermittent lighting ingested respectively, 25 g and 14 to 17 g more than hens restricted to 115 g/d.

3. Body weight was greater in hens fed ad libitum irrespective of the lighting pattern and of the amount of food intake.

4. Persistency of egg production was impaired by intermittent lighting.

5. Ad libitum feeding reduced egg fertility. The decrease was larger in breeder hens on intermittent lighting.

6. Hatchability was increased in hens submitted to the symmetrical lighting pattern (0.5L:3.5d).

7. Egg and chick weights were higher in hens fed ad libitum.

8. Shell index and shell breaking strength increased in restricted hens on the intermittent lighting pattern (0.5L:3.5d) × 6.

9. The best performance was obtained in restricted hens on the conventional lighting pattern.