Interaction between ascites susceptibility and CO during the second half of incubation of two broiler lines. Effect on embryonic development and hatching process

1.?Because CO₂ during the second half of incubation is known to influence air cell and blood gases, and embryo development, it is postulated that post-hatch development and ascites sensitivity could also be influenced.

2.?An ascites susceptible (A) and an ascites resistant (E) broiler line were incubated under standard incubation or high CO₂ conditions (up to 4%) from embryonic day (ED) 10 onwards. The embryonic development and the hatching process of these two lines were compared when incubated under standard or high CO₂ conditions from over the second half of incubation.

3.?The A line, selected for high post-hatch growth rate, exhibited a higher relative embryo weight from ED10 until ED16, which was supported by a higher air cell pCO₂, lower air cell pO₂, higher corticosterone and thyroid hormones and earlier hatching time.

4.?Incubation under high CO₂ increased air cell pCO₂, retarded yolk consumption, and decreased glycogen concentration in the liver at hatch. Hatchability decreased in both lines when incubated under high CO₂, due to an increased late mortality of embryos that died before IP.

5.?These results suggest that the development and metabolism of CO₂-incubated embryos differ from control incubated embryos.     

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.     

The hatching sequence of Ostrich (Struthio camelus) embryos with notes on development as observed by candling

1. The hatching sequence of the ostrich (Struthio camelus) was determined by observing embryos during candling, those in the process of hatching and in dead‐in‐shell eggs.

2. Candling showed that there was a progressive increase in dark shadowing within the egg but fine details were not easily seen. Nevertheless candling is considered to be an important method of assessing development of ostrich eggs during incubation.

3. The ostrich embryo adopts a hatching position and follows a hatching sequence different from that of the fowl. During internal pipping, the air space is pulled towards the beak in the former but the beak moves to the air space in the latter. The right foot has an important role in breaking the shell during hatching.

4. The hatching process is considered to reflect an adaptation to the egg having a hard, brittle shell.

5. Malpositions in the ostrich may have been misinterpreted in the past because of the unusual hatching position. The commonest malposition described here was head‐in‐the‐small‐end.     

Storage of hatching eggs: The effect on total incubation period

1. A time‐lapse photographic technique has been developed which enables routine recording of the hatching times of up to 192 chicken embryos.

2. Storage of eggs for 14 d caused a delay of 134 h in the mean total incubation period.

3. Female embryos hatched about 3 h earlier than males in the non‐stored eggs but there was no difference between the sexes in eggs stored for 14 d.

4. Rate of weight loss during incubation was similar in both non‐stored and stored eggs: there was no relationship between weight loss at 19 d incubation and total incubation period.

5. Hatchability of fertile eggs was 19.5% lower in eggs stored for 14 d and proportionately more embryos died between 4 d and 18 d incubation than in the non‐stored eggs.     

Effects of interaction of incubator CO2 levels and mixing hatching eggs of different embryo growth trajectory on embryo physiological and hatching parameters

1. Two experiments were designed to investigate the effects of mixing eggs of different genotype and incubating with different CO₂ concentrations on embryonic parameters. Half of the eggs were incubated in a CO₂ controlled incubator (VCO₂) during the first 10 d of incubation and the other half was incubated at standard incubation ventilation rate (SV).

2. From 10 to 18 d of incubation, thick albumen and embryos were weighed. Blood samples were collected at d 18 of incubation, at internal pipping and at hatch for determination of triiodothyronine (T₃), thyroxine (T₄) and corticosterone concentration. During the last 2 d of incubation, hatching events of individual eggs were monitored every 2 h.

3. From 4 to 5 d of incubation in the SV group of Experiment 1, pH values of Ross strain chickens were lower than those of Isa Brown. From d 12 of incubation onwards, Ross embryos grew faster than those of Isa Brown. At 14 and 16 d of incubation, Ross eggs in the CO₂ controlled incubator had lower albumen weights compared to all other treatments.

4. T₃ concentrations in Ross embryos were higher than those of Isa Brown embryos. Chicken weight at hatch was in the following order: Isa Brown SV < Isa Brown VCO₂ = Ross SV < Ross VCO₂.

5. In Experiment 2, incubation time of VCO₂ eggs until internal pipping, external pipping and hatch was significantly shorter than that of SV eggs.

6. It was concluded that mixing of hatching eggs of differential embryo developmental trajectory affects their hatching process.     

Explanted embryo culture: In vitro and in ovo techniques for domestic fowl

1. Embryos of the domestic fowl (72 h old) have been explanted into shell‐less cultures or ‘surrogate’ eggshells, in order to investigate the possibility of rearing these embryos to hatching.

2. Rocking embryo cultures during the first half of incubation enhanced embryo growth.

3. Embryos explanted into ‘surrogate’ eggshells of either other individuals or other species have been successfully ‘hatched’.

4. A normal chorioallantois is formed in these surrogate eggshells. This enables a functional albumen sac to form and eggshell resorption to be achieved.

5. Embryos grown in ‘surrogate’ eggshells are slightly smaller than controls but otherwise normal.

6. The technique provides opportunities for genetic engineering experiments.     

Performance of lines selected for fast‐and slow‐hatching times and crosses among them 1

1. Males of a control line and two lines selected for fast‐ and slow‐hatching were mated to females of the same three pure lines and three crosses between them. Hatchability, egg weight and hatching time were measured.

2. No significant differences were found between genetic groups in hatchability.

3. Groups containing the fast‐hatching line genotypes were significantly smaller in egg weight than those not containing this line.

4. Significant differences in hatching time existed between male lines and between female lines within pure and cross‐line parent types while no differences were observed between the female parent types and no interactions of male by female lines occurred.

5. It was concluded that inbreeding in the pure lines (10%) did not affect any of the variables measured.

6. The results on hatching time support the conclusion that little genetic variance other than additive variance is involved for this trait.     

In ovo oral vaccination with cmpylobacter jejuni establishes early development of intestinal immunity in chickens

1. Chick embryos were orally immunised at day 16 of incubation by injection of heat‐killed Campylobacter jejuni organisms into the amniotic fluid. The response to vaccination was observed at 5 d after hatching or, in some birds which received a postnatal oral booster vaccination, at 7 d after hatching, and the response was observed at 14 d of age.

2. The titres of antibody in serum, bile and intestinal scrapings, the distribution of immunoglobulin‐containing cells in the spleen, duodenum and ileum and the expression on peripheral blood leukocytes (PBL) of the T cell surface markers CD3, CD4 and CD8 were determined.

3. Whereas low titres of anti‐flagellin antibody were detected in serum, bile and intestinal scrapings of unimmunised birds, high titres were observed in immunised birds.

4. An increase in antibody of all isotypes was detectable in serum but the elevation in IgA antibody in intestinal scrapings and bile was particularly striking. This response was reflected in a dramatic increase in immunoglobulin‐containing cells, detected by fluorescent histology, particularly diose associated with IgA and IgM isotypes in the spleen and intestine of immunised birds.

5. Secondary oral boosting after hatching resulted in a depression in serum anti‐flagellin antibody in immunised birds compared to pre‐boosting titres (although still significandy higher than in non‐immunised controls) but an increase in IgA antibody in intestinal scrapings and bile. The number of immunoglobulin‐containing cells was also increased after boosting.

6. Neither immunisation regimen caused a significant change in the numbers of circulating CD3, CD4 or CD8 T cells.

7. These results indicate that in ovo oral immunisation with C. jejuni antigens stimulates the precocious development of immunity in chicks.     

Effects of ethylnitrosourea on the hatchability and survival of chicks

1. Two experiments were carried out to study the effects of the mutagen ethylnitrosourea (ENU) at concentrations of 1.4 to 3 500 μg in 0.3 ml of saline (experiment 1) and 585 to 1 170 μg in 0.1 ml of saline (experiment 2) on hatching eggs of two breeds of fowls. Hatchability (experiment 1) and survival up to 7 d of age without food (experiment 2) were assessed. In experiment 1 the hens were fed on different diets prior to providing the hatching eggs.

2. The mutagen showed a non‐linear effect upon the hatchability, higher doses being less effective than small doses.

3. In the majority of cases, female embryos were more sensitive to the effect of ENU then male.

4. The mortality after hatching was also affected by ENU; the chicks from treated eggs surviving without food better than controls.     

Protective effect of ubiquinone (coenzyme Q9) on ascites in broiler chickens

1. The effect of ubiquinone (C0Q9) on ascites in broiler chickens was investigated.

2. The commercial broilers were divided into 2 groups of 100 birds each; CoQ9‐treated group and non‐treated group.

3. The chickens were grown in a positive‐pressured house with double high efficiency particulate air filtered intakes and exhaust, and thus were strictly isolated from infectious agents.

4. The chickens (15 to 21 d old) were exposed to cold stress in order to induce ascites.

5. The number of birds with ascites in the C0Q3‐treated group was significantly lower than in the non‐treated group.

6. Survival and production rates were better in the CoQ^‐treated group than in the non‐treated group.     

Traits influencing the hatching performance of Japanese quail eggs

1. Japanese quail eggs from moderately heavier sires showed superior fertility; while fertile eggs from moderately heavier dams hatched slightly better than the eggs from lighter dams.

2. Higher rates of fertility and hatchability of Japanese quail eggs were observed from parents of 10 to 19 weeks of age, with peak fertility and hatchability at 14 and 12 weeks of age, respectively.

3. Sex ratios of 1:2 to 1:5 gave comparable fertility and hatchability results.

4. The hatching performance of quail eggs from cage and deep litter reared breeders was comparable.

5. Fertility and hatchability were directly proportional to the egg weight.

6. Quail egg shell colour, tints and blotches were found to influence hatching performance.

7. Storing quail eggs at 16 ± 2°C and 75 ± 5% relative humidity for more than 4 d reduced hatchability.

8. Hatchability of eggs stored at room temperature was improved if they were sealed in polyethylene bags.

9. Provision of light during the first 14 d of incubation resulted in a photo‐acceleration of about 3.2 h.     

Similar developmental fluctuations of hepato-renal xanthine oxidoreductase gene expression and xanthine oxidase activity in layer and broiler chicken embryos

1. Xanthine oxidase (XO) has many physiological functions associated with the synthesis of both antioxidant (uric acid: UA) and numerous oxidants (e.g. H₂O₂), which makes it an important regulator of the cellular redox potential involving organogenesis. The ontogenetic study of hepatic and renal XO makes a better understanding of the putative role of this enzyme in the development of these tissues.

2. Developmental changes of gene expression of xanthine oxidoreductase (XOR), XO activity and UA content of liver and kidney tissues in both broiler and layer chicken embryos were examined during incubation d 14–21.

3. In both strains, hepatic XOR gene expression peaked on d 21 while renal XOR gene expression did not change.

4. The XO activity was higher in kidney than liver in both strains. Hepatic XO activity of both strains peaked on d 18 and thereafter was decreased on d 21. Renal XO activity peaked on d 18 and from then on did not show any significant changes until d 21 in both strains.

5. The UA content was higher in kidney vs. liver in both strains. The hepatic and renal UA values of the both strains increased significantly from d 14 to d 21.

6. The present results showed dissimilar behaviour of XOR gene expression, XO activity and UA content of liver and kidney tissues in both broiler and layer chicken embryos.     

Optimum incubation conditions of the domestic guinea fowl egg

1. During artificial incubation of 8000 guinea fowl eggs, the effects of temperature, relative humidity, rate of inflowing air and age of the laying flocks were determined.

2. Total duration of incubation was divided into setter (0 to 24 days) and hatcher (25 to 28 days) periods. Eggs transferred at the end of the setter period, (ET, % of fertile eggs) and hatching rate (HR, % of transferred eggs) were calculated, as well as the total hatching rate expressed as percentage of fertile eggs.

3. In the range 36 to 39°C, temperature affected significantly (P< 0.001) ET and HR, with optima at 37.2 and 37.0°C ± 0.1°C respectively.

4. During incubating period, relative humidity in the range 40 to 64% (water vapour partial pressure, Ph₂o ⁼ 17 to 34 Torr at 36 to 39°C), significantly (P< 0.001) affected the total diffusive water loss and hatchling mass, both expressed as percentage of fresh egg mass. ET was significantly affected by water loss, the highest ET being for water loss of 13.3 ± 0.5%. Optimal relative humidity was calculated to be 48 to 52% (Ph₂o = 23 to 25 Torr at 37.2°G).

5. The rate of inflowing air significantly (P< 0.001) affected HR, with an optimum at 3.1 Lstpd/(h . egg).

6. The age of the laying flock significantly (P< 0.001) increased water loss; this was explained by a parallel increase of the mass specific shell conductance to water vapour.

7. Finally optimum incubation conditions were deduced, giving total hatching rates of 78 to 81% of fertile eggs, improving by 5 to 8% the best results obtained routinely in commercial practice.     

Effect of egg composition on hatchability and on growth and slaughter characteristics of meat-type chicks

1.?Using the so-called TOBEC (Total Body Electrical Conductivity) method, which allows the determination of egg composition in vivo, correlations between egg composition, hatchability and hatched chicks’ development were studied.

2.?A total of 1500 hen eggs (Ross-308) were measured by TOBEC, and eggs with extremely high (10%, n = 150), extremely low (10%, n = 150) and average (10%, n = 150) electrical conductivity values were chosen for further investigation.

3.?During the incubation period, it was observed that eggs with high electrical conductivity had a significantly higher mortality than eggs with low electrical conductivity.

4.?It was observed that both the increase in electrical conductivity at the same egg weight, and the increase in egg weight at the same electrical conductivity resulted in an increase in the hatching weight.

5.?It was found that the dry matter, protein and fat content of the chicks hatched from eggs with low electrical conductivity was higher at hatching than that of the chicks hatched from eggs with high electrical conductivity.

6.?At 42 d of age the liveweight of cocks and pullets hatched from eggs with low electrical conductivity was 3·2 and 8·2% higher than the liveweight of cocks and pullets hatched from eggs with high electrical conductivity.

7.?Because of the higher liveweight at slaughter, there was a significant superiority of the chicks hatched from eggs with low electrical conductivity in the case of the examined carcase traits at slaughter.

8.?Similar tendencies were found also in the ratios of carcase variables to liveweight, but the between group differences were not statistically significant in this case.

9.?Based on the results it was concluded that TOBEC seems to be a useful method for separating eggs with different composition.

10.?This could be a good starting point for further in vivo investigations in order to clarify the effect of egg composition on hatchability and further development.     

Manipulations of germ‐cell populations in the gonad of the fowl

1. Embryos of the domestic fowl have been partially sterilised by injecting the drug busulphan into 24‐h incubated eggs.

2. Some of these embryos were injected with primordial germ cells (PGCs) after 55 h of incubation to attempt to repopulate the gonads.

3. Primordial germ cells transfected with a defective retrovirus containing the reporter gene lac Z were shown to settle in these sterilised gonads.

4. Quantitative histology of 6‐d embryos showed that busulphan produced 75% sterilisation but that PGCs could repopulate these gonads.

5. The technique of producing such germ line chimaeras is of value in studying cell kinetics, gonad differentiation and the production of transgenics.     

Haematology and blood composition at two ambient temperatures in genetically fat and lean adult broiler breeder females fed ad libitum or restricted throughout life

1. Genetically fat and lean adult broiler breeder females were fed ad libitum or restricted throughout life. At one year of age, comparisons of blood viscosity and haematology and changes in blood chemistry after exposure to thermal stress were conducted.

2. Whole blood viscosity was more than twice as high, and plasma triglyc‐eride and haemoglobin concentrations were over 30% higher in fat line than in lean line females fed ad libitum or in restricted birds of both genotypes.

3. Mean cell haemoglobin concentration was higher and mean cell volume lower in fat compared with lean line females.

4. Food restriction was associated with lower plasma triglyceride concentrations, lower numbers of heterophils and monocytes and a lower heterophil‐lymphocyte ratio.

5. Fat line birds had a higher blood pCO₂ and lower pH than lean line birds.

6. Thermal stress was associated in ad libitum‐fed birds with a rise in blood pH and a decrease in pCO₂, and in restricted birds with a decline in blood pH and an increase in pCO₂.

7. Blood creatine kinase activity increased in all groups under thermal stress.     

Effect of dietary flax oil and hypobaric hypoxia on right ventricular hypertrophy and ascites in broiler chickens

1. The effect of dietary flax oil on growth rate, blood haemoglobin content, mortality and incidence of pulmonary hypertension and ascites in broilers at ambient pressure and at reduced atmospheric pressure was examined.

2. Birds were housed either in hypobaric chambers simulating 1000, 1500 or 2200 m altitude or in pens at ambient atmospheric pressure and fed on diets containing 100 g/kg added fat as either an animal/vegetable (A/V) blend or flax oil.

3. Birds raised under hypobaric conditions had a decreased growth rate and increased mortality, blood haemoglobin content, and incidence of pulmonary hypertension and ascites compared to the groups at normal atmospheric pressure.

4. Broilers fed on the diet containing flax oil snowed no difference in growth rate or blood haemoglobin content compared to birds fed on the A/V fat diet raised at the same altitude.

5. Inclusion of flax oil in the diet decreased mortality and the incidence of ascites at 2200 m and pulmonary hypertension at 1500 m.

6. Flax oil may be an effective method of reducing ascites and pulmonary hypertension in broilers without affecting performance.     

Increased sensitivity to triiodothyronine (T3) of broiler lines with a high susceptibility for ascites

1. In the studies reported here, broiler lines divergently selected for susceptibility to ascites under low temperature conditions were tested for their sensitivity to 3,3’,5‐triiodothyronine (T3) with respect to growth rate, rate of mortality, plasma concentrations of T3, right ventricular hypertrophy (RVH) and incidence of ascites.

2. Mean body weight of the ascites‐susceptible line (BC‐line) was higher than that of the ascites‐resistant line (A‐line). Adding 0.5 mg T₃/kg of the diet depressed growth rate to the same extent in both lines. The effect of T3 on growth was more pronounced for males than for females.

3. T3‐supplementation increased the relative weight of the heart and the incidence of RVH to the same extent in both lines. More of the T3‐treated BC‐line chickens had fluid accumulation in the abdominal cavity than the T3‐treated A‐line chickens.

4. Dietary T3‐treatment depressed the plasma concentration of growth hormone (GH) profoundly and insulin‐like growth factor (IGF‐I) slightly but to the same extent in both lines. The coefficient of variation of GH concentrations indicate that T3 treatment mainly decreased GH‐pulsatility in young growing broilers.

5. Higher doses of dietary T3 (1 and 2 mg/kg) increased mortality in a dose‐dependent manner. With 2 mg T₃/kg, mortality in the BC‐line was almost double that in the A‐line.

6. These studies indicate that the development of ascites could be linked with thyroid function. Moreover, dietary T3 supplementation could be used to help identify ascites‐inducing factors or genetic lines with differential sensitivity for ascites.     

Ostrich (Struthio camelus) embryonic development from 7 to 42 days of incubation

1. Hatching success of ostrich eggs is poor (50–60% of fertile eggs). The current study was designed to identify the timing of key stages in the development of the ostrich embryo.

2. Growth of both embryo and wing length during 42 d of incubation was comparable and approximately linear, with a more or less weekly doubling in size up to 35 d of incubation.

3. The embryo eye size increased more rapidly than beak length and reached a maximum of ~16.2 mm by 28 d of incubation, whereas beak length increased continuously until hatching at 42 d.

4. Linear regression equations were derived from morphometric measurements of embryos between 7 and 42 d.

5. Information stemming from these results can be used to estimate the age of dead-in-shell embryos in an attempt to identify timing of incubation problems that potentially result in low hatchability of fertile eggs.     

Potential role of serum troponin t in cardiomyocyte injury in the broiler ascites syndrome

1. Cardiac troponin T is a recently developed serological marker used as a sensitive diagnostic tool for early myocardial damage in ‘at risk’ human patients.

2. Serum troponin T values were measured in young 30‐d‐old broilers with ascites and in healthy age‐matched flockmates.

3. The data showed a significant increase (P< 0.001) in troponin T values in ascitic broilers compared with control birds. A similar increase in the arterial pressure index in ascitic birds indicated a good positive correlation at this age.

4. The results suggest that this new assay cross‐reacts with chicken cardiac troponin T antigen and that it may be an important indicator of myocardial cell damage for use in future genetic selection programmes in the combat against ascites.