Non-Invasive PPG-Based System for Continuous Heart Rate Monitoring of Incubated Avian Embryo.

The chicken embryo is a widely used experimental animal model in many studies, including in the field of developmental biology, of the physiological responses and adaptation to altered environments, and for cancer and neurobiology research. The embryonic heart rate is an important physiological variable used as an index reflecting the embryo's natural activity and is considered one of the most difficult parameters to measure. An acceptable measurement technique of embryonic heart rate should provide a reliable cardiac signal quality while maintaining adequate gas exchange through the eggshell during the incubation and embryonic developmental period. In this paper, we present a detailed design and methodology for a non-invasive photoplethysmography (PPG)-based prototype (Egg-PPG) for real-time and continuous monitoring of embryonic heart rate during incubation. An automatic embryonic cardiac wave detection algorithm, based on normalised spectral entropy, is described. The developed algorithm successfully estimated the embryonic heart rate with 98.7% accuracy. We believe that the system presented in this paper is a promising solution for non-invasive, real-time monitoring of the embryonic cardiac signal. The proposed system can be used in both experimental studies (e.g., developmental embryology and cardiovascular research) and in industrial incubation applications.

Neither altered incubation temperature during fetal development nor preferred rearing temperature improves leg bone characteristics of broilers.

The present study evaluated whether broiler femoral and tibiotarsal characteristics (as assessed at slaughter age) could be improved if birds were reared under their preferred temperature and whether continuous high or low incubation temperature during the fetal period improves bone characteristics of broilers reared under heat stress or thermal preference. Broiler breeder eggs were incubated from day 13 until hatching under cold (36 °C), control (37.5 °C), or hot (39 °C) temperatures. Under these conditions, the eggshell temperatures were 37.4 ± 0.1°C, 37.8 ± 0.15°C, and 38.8 ± 0.3°C, respectively. Then, broiler chicks were reared under control, preferred (determined previously in thermal preference test), or high temperatures. At day 42 of age, the broilers were weighed and euthanized, and femora and tibiotarsi collected to measure weight, length, diaphysis perimeter, breaking strength, maximum flexion, rigidity, ash, phosphorus, and calcium. Rearing under the preferred temperature did not affect broiler body weight or femoral and tibiotarsal characteristics (P > 0.05). In contrast, high rearing temperature, decreased the body weight, mineral contents of both bones, femoral breaking strength, and tibiotarsal rigidity (P < 0.05). Regarding incubation temperature effects, egg exposure to cold and hot temperatures during the fetal period minimized or avoided a few effects of high rearing temperature, such as those on femoral and tibiotarsal morphological characteristics, mineral composition, and mechanical properties at slaughter age (P < 0.05), but not all. In conclusion, rearing under the preferred broiler temperature did not improve the bone characteristics, and the negative effects of high rearing temperature on bone development were minimized but not completely prevented by high or low temperature incubation during the fetal period.

Smartphone-Based Device for Non-Invasive Heart-Rate Measurement of Chicken Embryos.

Heart rate (HR) is an important parameter in the study of the developmental physiology of chicken embryos and a crucial indicator of dead or live embryo grading in artificial incubation processes. A non-invasive HR measurement technique is required for long-term and routine HR assessment with minimal influence on embryo development. Accordingly, in this study, a non-invasive HR measurement technique of chicken embryos using a smartphone is demonstrated. The detection method of the proposed device is based on the photoplethysmography principle in which a smartphone camera is used for video recording, and the chicken embryonic HR is obtained from the recorded video images using a custom Android application. We used a smartphone to measure the embryonic HR of 60 native chicken eggs and found that it can measure the chicken embryonic HR from day 4 to day 20. The proposed smartphone HR device will be beneficial for scientific research and industrial applications. With internet connectivity, users can utilize their smartphone to measure the HR, display, share, and store the results.

Ankle muscle tenotomy does not alter ankle flexor muscle recruitment bias during locomotor-related repetitive limb movement in late-stage chick embryos.

In ovo, late-stage chick embryos repetitively step spontaneously, a locomotor-related behavior also identified as repetitive limb movement (RLM). During RLMs, there is a flexor bias in recruitment and drive of leg muscle activity. The flexor biased activity occurs as embryos assume an extremely flexed posture in a spatially restrictive environment 2-3 days before hatching. We hypothesized that muscle afferent feedback under normal mechanical constraint is a significant input to the flexor bias observed during RLMs on embryonic day (E) 20. To test this hypothesis, muscle afference was altered either by performing a tenotomy of ankle muscles or removing the shell wall restricting leg movement at E20. Results indicated that neither ankle muscle tenotomy nor unilateral release of limb constraint by shell removal altered parameters indicative of flexor bias. We conclude that ankle muscle afference is not essential to ankle flexor bias characteristic of RLMs under normal postural conditions at E20.

The method of chicken whole embryo culture using the eggshell windowing, surrogate eggshell and ex ovo culture system.

1. The unique accessibility of the avian embryo have made them an ideal model for the study of development and genome editing. Chicken whole embryo culture has provided important insights into toxicity tests, gene manipulation, clarifying gene functions, cell transplantation and cell tracking. 2. A simple technique for chicken manipulation is eggshell windowing, without or with seal, the latter having demonstrated some improvement in hatching rates. 3. Likewise, a surrogate eggshell system provides an accessible model for manipulation during chicken and quail development, with a higher hatchability compared to the simple windowing method. 4. The development of the chicken ex ovo culture systems in a synthetic environment as an efficient technique for imaging and microsurgery applications has enabled the study of important events of live chicken embryos at a specific time point. 5. This short review illustrates recent applications of well-designed whole embryo culture systems as a robust model for research into numerous biological mechanism, drug discovery, gene manipulating and production of functional proteins.

Effect of adjuvants on in ovo vaccination against Newcastle disease on hatchability, performance and antibody titres in commercial pullets.

This study was conducted to investigate the efficacy of in ovo administration of aluminium hydroxide (AH) and/or mannan oligosaccharide (MOS) adjuvants along with lentogenic VG/GA strain-Avinew to alleviate the embryonic pathogenicity of Newcastle disease virus. Six hundred and thirty fertilized Bovans eggs were divided into nine groups of 70 each incubated in a commercial hatchery and administered with eight types of in ovo injections in a factorial design of 2 × 2 × 2 including with/without AH, MOS and Newcastle disease vaccine (NDV), and one uninjected group on day 18 of incubation. Hatchability was higher in the eggs received MOS and/or AH adjuvants plus NDV compared those injected with NDV alone which confirmed the attenuation of NDV. However, the average daily feed intake and feed conversion ratio of pullets hatched from NDV-injected eggs were significantly reduced, but did not affect growth performance during 0-42 days of age. The performance of pullets hatched from eggs injected with AH, MOS or their mixture with NDV was not significantly different during all growth periods. Pullets from MOS + vaccine injected eggs had significantly higher antibody titres against NDV compared to those hatched from either injected with saline or uninjected on d 28 (p < .05). In addition, AH plus vaccine and MOS significantly improved total anti-SRBC and IgG respectively. Histological observation revealed that injection of MOS adjuvant into eggs led to increase crypt depth, whereas AH injection caused a reduction in villus surface area of jejunum in chicks on d 14 post-hatch. It is concluded that in ovo MOS injection as compared to AH may be more effective to attenuate the embryonic pathogenicity of in ovo NDV injection.

Eye and heart morphogenesis are dependent on melatonin signaling in chick embryos.

Calmodulin is vital for chick embryos morphogenesis in the incubation time 48-66 h when the rudimentary C-shaped heart attains an S-shaped pattern and the optic vesicles develop into optic cups. Melatonin is in the extraembryonic yolk sac of the avian egg; melatonin binds calmodulin. The aim of this study was to investigate the function of melatonin in the formation of the chick embryo optic cups and S-shaped heart, by pharmacological methods and immunoassays. Mel1a melatonin receptor immunofluorescence was distributed in the optic cups and rudimentary hearts. We separated embryonated chicken eggs at 48 h of incubation into basal, control and drug-treated groups, with treatment applied in the egg air sac. At 66 h of incubation, embryos were excised from the eggs and analyzed. Embryos from the basal, control (distilled water), melatonin and 6-chloromelatonin (melatonin receptor agonist) groups had regular optic cups and an S-shaped heart, while those from the calmidazolium (calmodulin inhibitor) group did not. Embryos from the luzindole (melatonin receptor antagonist) and prazosin (Mel1c melatonin receptor antagonist) groups did not have regular optic cups. Embryos from the 4-P-PDOT (Mel1b melatonin receptor antagonist) group did not have an S-shaped heart. Previous application of the melatonin, 6-chloromelatonin or forskolin (adenylate cyclase enhancer) prevented the abnormal appearance of chick embryos from the calmidazolium, luzindole, prazosin and 4-P-PDOT groups. However, 6-chloromelatonin and forskolin only partially prevented the development of defective eye cups in embryos from the calmidazolium group. The results suggested that melatonin modulates chick embryo morphogenesis via calmodulin and membrane receptors.

Investigating right ovary degeneration in chick embryos by transcriptome sequencing.

In asymmetric chick gonads, the left and right female gonads undergo distinct programs during development, generating a functional ovary on the left side only. Despite some progress being made in recent years, the mechanisms of molecular regulation remain incompletely understood, and little genomic information is available regarding the degeneration of the right ovary in the chick embryo testis. In this study, we performed transcriptome sequencing to investigate differentially expressed genes in the left and right ovaries and gene functions at two critical time points; embryonic days 6 (E6) and 10 (E10). Using high-throughput RNA-sequencing technologies, 539 and 1046 genes were identified as being significantly differentially expressed between 6R-VS-6L and 10R-VS-10L. Gene ontology analysis of the differentially expressed genes revealed enrichment in functional pathways. Among these, candidate genes associated with degeneration of the right ovary in the chick embryo were identified. Identification of a pathway involved in ovarian degeneration provides an important resource for the further study of its molecular mechanisms and functions.

Differences in flexor and extensor activity during locomotor-related leg movements in chick embryos.

Prior to hatching, chick embryos spontaneously produce repetitive limb movements (RLMs), a developmental precursor to walking. During RLMs, flexor and extensor muscles are alternately active as during stance and swing phases of gait. However, previous studies of RLMs observed that flexor muscles were rhythmically active for many cycles, whereas extensors often failed to be recruited. Thus, we asked if flexor muscles are preferentially recruited during RLMs in chick embryos 1 day before hatching and onset of walking. Using a within-subject design, we compared EMG burst parameters for flexor and extensor muscles acting at the hip or ankle. Findings indicated that flexor burst count exceeded extensor count. Also, flexor muscles were consistently recruited at the lowest levels of neural drive. We conclude that there is a bias favoring flexor muscle recruitment and drive during spontaneously produced RLMs. Potential neural mechanisms and developmental implications of our findings are discussed.

Apical constriction initiates new bud formation during monopodial branching of the embryonic chicken lung.

Branching morphogenesis sculpts the airway epithelium of the lung into a tree-like structure to conduct air and promote gas exchange after birth. In the avian lung, a series of buds emerges from the dorsal surface of the primary bronchus via monopodial branching to form the conducting airways; anatomically, these buds are similar to those formed by domain branching in the mammalian lung. Here, we show that monopodial branching is initiated by apical constriction of the airway epithelium, and not by differential cell proliferation, using computational modeling and quantitative imaging of embryonic chicken lung explants. Both filamentous actin and phosphorylated myosin light chain were enriched at the apical surface of the airway epithelium during monopodial branching. Consistently, inhibiting actomyosin contractility prevented apical constriction and blocked branch initiation. Although cell proliferation was enhanced along the dorsal and ventral aspects of the primary bronchus, especially before branch formation, inhibiting proliferation had no effect on the initiation of branches. To test whether the physical forces from apical constriction alone are sufficient to drive the formation of new buds, we constructed a nonlinear, three-dimensional finite element model of the airway epithelium and used it to simulate apical constriction and proliferation in the primary bronchus. Our results suggest that, consistent with the experimental results, apical constriction is sufficient to drive the early stages of monopodial branching whereas cell proliferation is dispensable. We propose that initial folding of the airway epithelium is driven primarily by apical constriction during monopodial branching of the avian lung.

Temperature during the last week of incubation. I. Effects on hatching pattern and broiler chicken embryonic organ development.

We investigated the effects of an eggshell temperature (EST) of 35.6, 36.7, 37.8, and 38.9°C applied from d of incubation (E) 15, E17, and E19 on hatching pattern and embryonic organ development. A total of 2,850 first-grade eggs of a 43-week-old Ross 308 broiler breeder flock were incubated at an EST of 37.8°C until E15. From E15, E17, or E19 onward, eggs were incubated at an EST of 35.6, 36.7, 37.8, or 38.9°C. Moment of internal pipping (IP), external pipping (EP), and hatch was determined, and organ development was measured at E15, E17, E19, IP, EP, and hatch. A lower EST extended incubation duration compared to a higher EST. The lower incubation duration was mainly caused by the extended time until IP, whereas time between IP and hatch hardly varied between treatments. Relative heart weight was affected by EST already from 2 d after the start of EST treatment on E15, and effects became more pronounced at longer exposure time to various EST treatments. At hatch, the largest difference in relative heart weight was found between an EST of 35.6 and 38.9°C started at E15 (Δ=64.4%). From E17 onward, EST affected yolk-free body mass (YFBM) and relative stomach weight, where a lower EST resulted in a lower YFBM and relative stomach weight before IP and a higher YFBM and relative stomach weight after IP. From E19 onward, a lower EST resulted in a higher relative liver and spleen weight regardless of start time of treatment. Yolk weight and relative intestine weight were not affected by EST before and at E19, but a higher EST resulted in a higher yolk weight and lower relative intestine weight from IP onward. Based on the higher YFBM and higher relative organ growth found at hatch, we concluded that an EST lower than 37.8°C from E15 onward appears to be beneficial for optimal embryo development.

Sharpening of the anterior neural border in the chick by rostral endoderm signalling.

The anterior border of the neural plate, presumed to contain the prospective peripheral portion (roof) of the prospective telencephalon, emerges within a vaguely defined proneural ectodermal region. Fate maps carried out at HH4 in the chick reveal that this region still produces indistinctly neural, placodal and non-neural derivatives; it does not express neural markers. We examined how the definitive anterior border domain of the rostral forebrain becomes established and comes to display a neural molecular profile, whereas local non-neural derivatives become separated. The process, interpreted as a border sharpening mechanism via intercalatory cell movements, was studied using fate mapping, time-lapse microscopy and in situ hybridization. Separation of neural and non-neural domains proceeds along stages HH4-HH4+, is well advanced at HH5, and is accompanied by a novel dorsoventral intercalation, oriented orthogonal to the border, that distributes transitional cells into molecularly distinct neural and non-neural fields. Meanwhile, neuroectodermal Sox2 expression spreads peripherally from the neighbourhood of the node, reaching the nascent anterior border domain at HH5. We also show that concurrent signals from the endodermal layer are necessary to position and sharpen the neural border, and suggest that FGF8 might be a component of this signalling.

Biochemical and molecular investigation of thermal manipulation protocols during broiler embryogenesis and subsequent thermal challenge.

BACKGROUND: The aim of the current study was to evaluate the effect of different thermal manipulation (TM) protocols during embryogenesis on thermotolerance acquisition parameters during subsequent thermal challenge (TC) at posthatch day 28. A total of 1500 fertile chicken eggs were divided randomly into five treatments (300 eggs each): control was maintained at 37.8 °C and 56 % relative humidity (RH) whereas, TM1, TM2, TM3 and TM4 were subjected to 38.5, 39, 39.5 and 40 °C for 18 h and 65 % RH daily during embryonic days ED 12-18. Hatched chicks from each treatment group allocated randomly into two sub-treatment groups (thermo-neutral, naïve (TN) and thermal challenge (TC). At day 28 of age, chicks subjected to TC by adjusting room temperature to 42 °C for 6 h while naïve chicks kept under regular conditions (22 ± 1 °C and 50-60 % RH). Chick's response to TC evaluated by determination of plasma T3, T4, corticosterone, total proteins, albumin, selected enzymes and some electrolytes at the beginning (0 h) and after 1, 3 and 5 h of TC in TM and TN chicks. Furthermore, pectoral and thigh muscles mRNA expression of Atrogin-1, CK, avUCP, DIO3, DIO2 were evaluated in TC and TN sub-treatment groups. RESULTS: TM induced a significant reduction in free T3 and elevation in total proteins and albumin in plasma with significant down-regulation of Atrogin-1 and DIO2 and significant up-regulation of DIO3 mRNA expression in muscle of TM chicks compare to control. During TC at day 28, decrease in the concentrations of plasma free T3, total proteins and albumin with increase in T4 have been detected in control and TM chicks. TC induced up-regulation of Atrogin-1 and DIO3 with down-regulation of DIO2 gene expression in muscles of all TC chicks. CONCLUSION: The present study indicated that, TM improved thermotolerance acquisition by decreasing basal metabolic rate and muscle injury during thermal stress. Basal metabolic rate decreased via reduction of plasma T3 concentration with up and down regulation of expression of DIO3 and DIO2, respectively in muscles. Muscle injury protected by stimulation of protein biosynthesis and down-regulation of Atrogin-1 expression.

Pyridoxine treatment alters embryonic motility in chicks: Implications for the role of proprioception.

Somatosensory feedback is important for the modulation of normal locomotion in adult animals, but we do not have a good understanding of when somatosensory information is first used to modulate motility during embryogenesis or how somatosensation is first used to regulate motor output. We used pyridoxine administration (vitamin B6 ), which is known to mostly kill proprioceptive neurons in adult mammals and embryonic chicks, to explore the role of proprioceptive feedback during early embryonic motility in the chick. Injection of pyridoxine on embryonic day 7 (E7) and E8 reduced the amplitude of leg movements recorded on E9 and the number of large, healthy neurons in the ventral-lateral portion of the DRGs. We conclude that proprioception is initially used during embryogenesis to modulate the strength of motor output, but that it is not incorporated into other aspects of pattern generation until later in development as poly-synaptic pathways develop.

Acute and chronic eggshell temperature manipulations during hatching term influence hatchability, broiler performance, and ascites incidence.

The aim of the current study was to determine how a control temperature and acute and chronic high eggshell temperatures during the last three days of incubation, can affect hatchability, chick quality, and organ development on day of hatch as well as broiler performance and ascites incidence in later life. The eggshell temperature manipulations were applied during hatching term (days 19 to 21) as follows: control EST (37.3 to 38.0°C), acute high eggshell temperature manipulations (38.4- to 39.0°C for three hours daily) and chronic high eggshell temperature manipulations (38.4 to 39.0°C). The lowest hatchability and the highest cull chick rate were in the chronic high eggshell temperature manipulations group. Lower chick quality parameters correlated with lower chick weights and heavier residual yolk sac weights that were in the chronic high eggshell temperature manipulations group depending on hatch time. The live weights on the 1(st) day of the growing period were higher in the control and acute high eggshell temperature manipulations groups than the chronic high eggshell temperature manipulations group. At 6 wk of age, live weights of broilers were the highest in the control than in the acute and chronic high eggshell temperature manipulations groups. The total mortality was 2.5, 9.2, and 13.3%, the mortality due to ascites was 2.1, 8.3, and 12.9% in the control, acute ,and chronic high eggshell temperature manipulations groups, respectively. The right ventricular/total ventricular ratios for the control, acute and chronic high eggshell temperature manipulations groups were 0.22, 0.28, and 0.30%, respectively. In conclusion, short-term and long-term higher temperatures during the hatching term affect embryo development, incubation results, broiler performance, and ascites incidence. Although the acute high eggshell temperature manipulations did not affect the chick quality parameters at hatch, it negatively affected incubation results and broiler performance, especially mortality due to ascites.

Effects of egg storage on hatchability, chick quality, performance and immunocompetence parameters of broiler chickens.

Pre-incubation egg storage is a necessity for the poultry industry. This study evaluated the effects of pre-incubation storage length of broiler eggs on hatchability, 1-day-old chick quality, subsequent performance, and immunocompetence. To this end, a total of 360 hatching eggs were stored for 4, 12, or 16 d prior to incubation. Hatchability and chick quality were assessed at hatch, and growth performance and immunocompetence parameters were assessed during a 35 d rearing period. Hatchability of set and fertile eggs, and embryonic mortality, were not affected by egg storage. On the contrary, 1-day-old chick BW and length were linearly negatively correlated with egg storage length (P-linear<0.05). Nevertheless, BW corrected for egg weight prior to setting was unaffected, and corrected chick length was positively affected by storage length. One-day-old chick Tona score, navel quality, and post-hatch growth performance (BW at 7 and 35 d, cumulative feed intake, and feed conversion ratio at 35 d) were unaffected by egg storage (P, P-linear>0.05). Lymphoid organ weights at 2 and 35 d, the titre of maternal anti-NDV antibodies, most of the thymocyte subpopulations defined by CD3, CD4, and CD8 cell surface expression in the thymus of 2-d-old chicks, cellular responses to the PHA skin test, humoral responses to primary SRBC, and NDV immunizations were also not influenced by length of storage (P, P-linear>0.05). On the contrary, the length of egg storage was found to negatively influence the abundance of CD3+CD4-CD8- thymocytes that represent the majority of γδ-T cells in the thymus of 2-day-old chicks, as well as the humoral response to booster NDV immunization of the birds. In brief, pre-incubation storage of broiler hatching eggs for up to 16 d did not affect most developmental and growth parameters investigated, except for BW and length at hatch. Egg storage was found to suppress some aspects of the immunocompetence of the birds, particularly aspects of acquired immunity.

Effects of the in ovo injection of 25-hydroxycholecalciferol on the yolk and serum characteristics of male and female broiler embryos.

Serum concentrations of 25-hydroxycholecalciferol [25(OH)D3] in broiler embryos at 19 d of incubation:doi:have been shown to increase 3 times by the in ovo injection of 0.60 µg 25(OH)D3 on 18 doi. In this trial, effects of the injection of 25(OH)D3 at 18 doi on the yolks and sera of male and female Ross×Ross 708 broiler embryos were assessed. On 18 doi, embryonated eggs that were set in a single stage incubator were assigned to 6 replicate trays within each of 2 injection treatment groups. Treatments included those injected with 100 µL commercial diluent (control) and those injected with 0.60 µg 25(OH)D3 in 100 µL commercial diluent. On 19 doi, embryos and their yolk sacs were extracted for determination of sex, yolk weight, BW, serum CA and phosphorous concentrations, and yolk CA, phosphorous, moisture, dry matter, and lipid concentrations. The weight of female embryos with their attached yolk sacs as a percentage of set egg weight was greater than that of males (P=0.03). There were treatment×sex interactions for the weight of embryos with their attached yolk sacs relative to 19 doi egg weight (P=0.05) and for yolk CA concentration (P=0.004). In eggs that received 25(OH)D3, the weight of female embryos with their attached yolk sacs relative to 19 doi egg weight was higher than that of males (P=0.005), and percentage yolk CA was higher in control eggs containing female embryos in comparison to those containing males (P=0.007). An injection of 0.60 µg 25(OH)D3 at 18 doi eliminated yolk CA differences associated with embryo sex, suggesting that 25(OH)D3 may influence sex-related differences in the rate of yolk CA absorption by broiler embryos and may be related to subsequent sex-related differences in posthatch bone strength.

Effects of growth patterns and dietary protein levels during rearing of broiler breeders on fertility, hatchability, embryonic mortality, and offspring performance.

The objective of this study was to determine the effects of different growth patterns and dietary crude protein levels during rearing in broiler breeder females on fertility, hatchability, embryonic mortality, and offspring performance. A 2×3 factorial arrangement of treatments was used, with 2 growth patterns to reach a target body weight at 20 wk of age of 2,200 g (standard=standard growth pattern) or 2,400 g (high=high growth pattern), and 3 dietary protein levels (high=crude protein, high), (medium=crude protein, medium), and low=crude protein, low). Fresh egg composition and organ development in hatchlings were determined. Offspring of the different groups were reared until an age of 34 d and feed intake, body weight gain, mortality, and carcass composition were determined. In 29-wk-old high growth pattern breeders compared to standard growth pattern breeders, fertility and hatchability of set eggs were increased; embryonic mortality between d 1 and 9 was decreased whereas hatchability of fertile eggs was not affected. Breeders fed the medium crude protein diet showed a decreased hatchability of fertile eggs caused by an increased embryonic mortality between d 18 and 21 compared to breeders fed the high crude protein and low crude protein diets. Offspring of 29-wk-old high growth pattern breeders tended (P=0.059) to have a higher body weight at d 34 than offspring of standard growth pattern breeders, which was achieved by a tendency to a higher body weight gain (P=0.057). Offspring of breeders fed the medium and low crude protein diet showed a higher feed intake between d 18 and 27 and during the total growth period, as compared to offspring of high crude protein breeders. Male broilers of low crude protein breeders had higher breast meat yield than male broilers of high crude protein breeders, while breast meat yield of female broilers was not affected by dietary protein levels. This experiment showed that a higher growth pattern during the rearing period increased fertility, decreased embryonic mortality, and improved offspring performance in young breeders, whereas decreased dietary protein level had no or less pronounced effects on these traits.

Higher levels of CO2 during late incubation alter the hatch time of chicken embryos.

1. It has been reported that the increasing CO2 tension triggers the embryo to pip the air cell and emerge from the egg. However, the mechanism by which higher CO2 concentrations during the last few days of incubation affect chick physiology and the hatching process is unclear. This study investigated the effect of CO2 concentrations up to 1% during pipping, on the onset and length of the hatch window (HW) and chick quality. 2. Four batches of Ross 308 broiler eggs (600 eggs per batch) were incubated in two small-scale custom-built incubators (Petersime NV). During the final 3 d of incubation, control eggs were exposed to a lower CO2 concentration (0.3%), while the test eggs experienced a higher CO2 concentration programme (peak of 1%). 3. There were no significant differences in blood values, organ weight and body weight. There was also no difference in hatchability between control and test groups. However, a small increase in the chick weight and the percentage of first class chicks was found in the test groups. Furthermore, plasma corticosterone profiles during hatching were altered in embryos exposed to higher CO2; however, they dropped to normal levels at d 21 of incubation. Importantly, the hatching process was delayed and synchronised in the test group, resulting in a narrowed HW which was 2.7 h shorter and 5.3 h later than the control group. 4. These results showed that exposing chicks to 1% CO2 concentration during pipping did not have negative impacts on physiological status of newly hatched chicks. In addition, it may have a significant impact on the physiological mechanisms controlling hatching and have benefits for the health and welfare of chickens by reducing the waiting time after hatching.

Effect of species-specific sound stimulation on the development and hatching of broiler chicks.

1. Previous research has reported that chicken embryos develop a functionary auditory system during incubation and that prenatal sound may play an important role in embryo development and alter the hatch time. In this study the effects of prenatal auditory stimulation on hatch process, hatch performance, the development of embryo and blood parameters were investigated. 2. Four batches of Ross 308 broiler breeder eggs were incubated either in control or in sound-stimulated groups. The sound-stimulated embryos were exposed to a discontinuous sound of species-specific calls by means of a speaker at 72 dB for 16 h a day: maternal calls from d 10 to d 19 of incubation time and embryo/chick calls from d 19 until hatching. The species-specific sound was excluded from the control group. 3. The onset of hatch was delayed in the sound-stimulated group compared to the controls. This was also supported by comparison of the exact hatching time of individual focal chicks within the two groups. However, the sound-stimulated embryos had a lower hatchability than the control group, mainly due to significantly increased numbers of late deaths. 4. The embryos exhibited a similar growth pattern between the sound-stimulated group and the control group. Although sound exposure decreased body weight at d 16, no consistent effect of sound on body weight at incubation stage was observed. Species-specific sound stimulation also had no impact on chick quality, blood values and plasma corticosterone concentrations during hatch.