Georgia’s College of Agricultural and Environmental Sciences are using molecular genetics to determine how pastured and free-range chickens influence the ecosystem on farms where crops and livestock are raised together. 

Supported by a $749,000 grant from the U.S. Department of Agriculture’s National Institute of Food and Agriculture, entomology doctoral student Sofia Varriano and her thesis advisor, agroecologist and systems biology Professor Bill Snyder, are testing the promise of integrated crop-livestock agriculture to increase the sustainability of smaller farms. 

“We’re interested in this group of farmers that have livestock and crop production within the same farms for their operation. Nowadays we call that integrated crop-livestock agriculture, but 100 years ago what did we call that? We called it farming,” Snyder said. “Every farm, pretty much everywhere in the world, had that model. We’re interested in whether there are aspects of that farming approach that can be useful in a modern setting.” 

Using DNA analysis to demystify diets 

Called “Chicken Ecology and Ecosystem Services,” or ChickEES, the three-year project will detail what the birds on each farm are consuming by analyzing the DNA remains in chicken feces. With this information, producers can determine how their chickens are helping to control pest insects and weeds. 

While research on poultry nutrition in controlled production facilities is plentiful, Snyder said there is little available information on what chickens on smaller farms with integrated poultry production practices are eating in a natural setting. 

“That got us interested in what these chickens eat. Chickens are what we call generalist predators, which means they’re hungry, and they don’t care what they eat, whether it’s a plant or a spider or an aphid or a pest caterpillar — they eat everything. Likewise, they eat things like earthworms, which are beneficial to the soil,” he explained. “Do they mostly eat pests? What weeds do they eat? Are there particular pest species or particular weed species that they prefer? These are the questions we’re interested in.” 

Natural pest control and soil amendment benefits 

With this knowledge, producers can modify their weed and pest control practices to work in tandem with pastured or free-range chickens. 

“The farmers we’re working with have these diversified farms, and a lot of them intuitively feel that it probably has some pest control advantage. This will answer that,” Snyder added. “The other thing the animals do is poop, so that’s manure, that’s fertilizer. The animals and the crops can kind of complement one another in that way.” 

Varriano, who came to CAES to study wild birds, pivoted to poultry research when the pandemic closed labs in 2020. 

This project focuses on chickens with outdoor access, whether they are pastured, free-range or otherwise have the ability to roam outside. DNA samples collected from the chickens are sent off for analysis to identify arthropods and plants through sequencing. By comparing the sequences to a species database, researchers aim to determine what the chickens are consuming, Varriano explained.  

“The end goal with these data is to give growers this information so it can benefit whatever farming they’re doing. One system helps support the other. Using chickens to control pests and enrich the soil to benefit your crops would be one integrative strategy,” Varriano said.  

“We want to help growers take full advantage of the chickens that they have on their farms.” 

Tracking pathogens in free-range and pastured systems 

In addition to DNA sampling, the project will uncover likely sources of chicken exposure to Salmonella and Campylobacter bacteria, which can make people sick, using whole-genome pathogen sequencing and landscape modeling. Exploring technology to help small farmers improve and diversify their production practices is one step toward improving the sustainability of smaller-scale producers. 

“The farmers we’re working with are often selling directly to restaurants, farmers markets and the public through community-supported agriculture. To bring people to their farms or serve the needs of restaurants or fill their stand at the farmer’s market, they need to have all different types of products,” Snyder explained. “The diversity of products helps to make their farms more sustainable.” 

A pilot study Varriano performed in Georgia revealed that chickens can consume hundreds of different types of prey on farms. 

The team has performed similar work in the Western U.S. between Southern California and Canada to create a broad footprint in different climates to determine what is typical for the Southeast versus other regions. 

“If you have a chicken that’s stationary versus a chicken that is free-roaming, we’re trying to predict what their differences in diet will be. In a certain setting, are they going to really focus on one certain pest or weed?” Snyder said.  

Gathering data from smaller flocks through citizen science 

Later in the study, Varriano and Snyder will solicit citizen-scientist input to the study by asking farmers throughout the region to self-submit information about their operations as well as fecal samples from their chickens. 

Providing kits similar to those used for genetic genealogy, the researchers will solicit samples from small or backyard flocks. 

“We’ll extract the DNA and do the sequencing, and then we’ll be able to tell them exactly what chickens on their farms —  these are the five top weeds they’re eating, these are the five top pests and insects that they’re eating,” Snyder said. “That helps the farmers because they usually don’t know what additional benefit they are really getting from these systems; we’ll be able to tell them that.” 

The team plans to create a service center with testing technology to quickly analyze the DNA samples and the many contributors to each sample, whether plant or insect. 

“We can get back, from one farm, more than 200 weeds and insects and other arthropods that the chickens are eating, so we are trying to automate that process,” he said, adding that preliminary studies in Georgia have shown that chickens are eating Palmer amaranth, one of the most damaging weeds for producers. 

 “It does really seem like there are some certain things they really are picking out. When they’re really confined, chickens do just clear out everything. But when they’re foraging freely, it does seem like chickens have things they really like, such as pest caterpillars,” Snyder said. 

Examining how diversification contributes to sustainable farming 

 The researchers also will monitor how soil quality changes when pastured chickens are moved from area to area on farms by performing full soil-quality tests to look at the weed seed bank and extracting DNA from the soil to look at microbial biodiversity.  

“Chickens, presumably, when they’re coming through and pooping, they’re dropping a lot of nitrogen, and that’s probably changing the soil microbiome. And is that beneficial? Is it building up?” Snyder said. “There’s some decent evidence that when you have greater biodiversity and microbes, it makes it harder for pathogens to persist. So are they actually building up the health of the soil in a really general way, not just with nitrogen, but also microbial biodiversity.” 

A collaborator in the UGA College of Veterinary Medicine, Nikki Shariat, an associate professor in the Department of Population Health, Poultry Diagnostic and Research Center, will test samples for the presence of Salmonella and Campylobacter. By performing whole-genome sequencing on the bacteria, they can determine where the foodborne illness-causing bacteria originated in the farm environment. 

“Where on the farm might they be picking these things up? We want to see how often they are present to get some idea of what the incidence would be,” Snyder added. “We want to give the growers a full picture — here are the pests the chickens are eating, here are the beneficials they’re eating, here’s your food safety risk.” 

 Enhancing local food systems and farm profitability 

 The findings have the potential to benefit growing local food systems in populated areas. 

“Economically, if you have 10 acres that you’re farming instead of 1,000 acres, you have to know how to generate enough income. That comes down to diversification,” Snyder said, adding that “chickens in these systems are providing eggs or meat that you can sell, they’re providing fertilizer and they’re providing pest and weed control. 

“When you’re trying to keep costs low and reduce off-farm inputs as much as you can, if more of that can be produced on farm, it’s better for the producer. We are helping those things work together,” he said.  

“The better these producers can function, the more opportunity they have for people in the public to enjoy what they’re doing and for them to make a profit.” 

Source: University of Georgia

At hatch, all chicks were weighed from each treatment group (data not shown). Nine (9) chicks from each treatment were humanely sacrificed by cervical dislocation and the fresh weight of the yolk sac, heart, liver and lungs was taken. Yolk-free chick weight was then estimated and used to calculate the relative organ weights using the equation:

Chorioallantoic Membrane Vessel Measurements

On ED 16, 1-2 mL of 10% formalin was injected through the airspace into three (3) eggs from each treatment group for retention of erythrocytes into vessels of the CAM. After 24 h, the egg was cut into 2 longitudinal sections along the equatorial region using a Dremel drill with a round blade. The embryo, yolk and albumen were carefully removed from the eggshell with little disturbance to the adhered CAM on the eggshell. A beam of 100W halogen light in an all-closed box-like device with a conical opening with a diameter 3 cm on 1 side was passed through the eggshell from the outer part. Digital images were taken with a Canon (EOS Rebel T5i) camera with a focused lens (EF-S 18-55 mm) at 2 different spots in each half section of the longitudinally divided eggshell. A spot represents the circular region covered by the beam of light that passes through the conical-sided opening of the box. From each circular spot image, 2 regions of interest (ROI), each 400 × 400 pixels were cropped for analysis. The ROI is the subset of the spotted image that was manually identified based on clear vessel branches. This procedure followed slight modification from Verhoelst et al. (2011) and Fernandes et al. (2017).

Eight cropped ROI images were obtained from each egg. A total of 24 images/treatment group were processed and analysed as described by Fernandes et al. (2017), using Image J software (version 1.54j, NIH, USA) for the vascular fraction (VF) (%), and fractal dimension (FD) of the vascular network of the CAM (Figure 1). The FD is the measure of the degree of branching of vascular network while the VF is determined as a measure of vessel density, which is in percentage (%) (Verhoelst et al., 2011; Fernandes et al., 2017).

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Figure 1. Image processing procedure using Image J software. Abbreviation: ROI, region of interest: The region of interest (ROI) is the subset area of the main initial image cropped to 400 × 400 pixels for analysis. The region was manually chosen based on clearly defined major and minor vessel branching.

RESULTS AND DISCUSSION

Relative Organ Weights

Table 1 summarizes the impact of breeder age (A_b) and oxygen concentration (O₂) level on embryo and chick organ weight taken at ED 11 afterexposure and d 1 respectively. The relative weight of organs of the embryo was with regards to embryo weight while those of the chicks were with regards to yolk-free chick weight (data not shown for embryo weight and yolk-free chick weight). No significant (P > 0.05) changes were noted in relative heart weight at ED 11, but the relative liver weight was affected by O₂ levels, with the 21% O₂ group having significantly (P = 0.003) lower weight than low O₂ levels. Interactively, chicks from the 50 wk breeders incubated at 21% O₂ level exhibit superior relative heart weight (P < 0.001) compared to 15% and 17% low O₂ levels of the same breeder age. Additionally, the 33 wk breeders had relatively higher heart weights (P = 0.013) than the 50 wk breeders. The main effect of O₂ levels on relative heart weight reveals significantly higher weight (P < 0.001) at 21% O₂ compared to 15% and 17% low O₂ levels. The interactive effect of A_b and O₂ levels significantly influenced relative liver weight, with 33 wk breeder embryos at 21% O₂ and 50 wk breeder eggs at 15% O₂ having heavier weights (P < 0.001) than the other interactive groups. The main effect of O₂ levels showed a significantly decreased relative liver weight (P = 0.002) for 17% O₂ compared to other precedent O₂ levels. No significant (P > 0.05) main or interactive effect was observed for relative lung weight across treatment groups.

Table 1. Effect of breeder age and reduced oxygen concentration level from ED 7- 9 on embryo and chick relative internal organ weights at ED11 and hatch.

Parameters	Rel. embryo heart weight (ED11) (%)	Rel. embryo liver weight (ED11) (%)	Rel. DOC heart weight (%)	Rel. DOC liver weight (%)	Rel DOC lung weight (%)
Breeder age (Ab)
33 wk	5.77	9.86	0.95a	2.74	0.65
50 wk	7.01	15.85	0.91b	2.83	0.60
SEM1	0.540	1.240	0.017	0.041	0.048
Oxygen level (O2)
15%	6.45	14.16a	0.91b	2.77a	0.63
17%	6.86	14.04a	0.88b	2.64b	0.69
21%	5.87	10.37b	1.00a	2.95a	0.56
SEM1	0.660	1.510	0.021	0.059	0.051
Interaction (Ab * O2)
33 wk * 15%	5.32	9.17	0.96ab	2.41b	0.71
33 wk * 17%	6.23	10.08	0.95ab	2.73b	0.75
33 wk * 21%	5.76	10.33	0.95ab	3.09a	0.49
50 wk * 15%	7.57	19.14	0.86b	3.14a	0.54
50 wk * 17%	7.49	18.00	0.81bc	2.54b	0.62
50 wk * 21%	5.98	10.40	1.06a	2.80b	0.64
SEM1	0.930	2.140	0.030	0.083	0.083
P-value2
Ab	0.118	0.160	0.106	0.242	0.465
O2	0.573	0.003	<0.001	0.003	0.342
Ab * O2	0.560	0.076	<0.001	<0.001	0.123

Abbreviations: Rel., relative; DOC, day-old chick; ED, embryonic day.

a-d

Means within the same column with different superscripts are significant at P < 0.05.

1

SEM, pooled standard error of means.

2

P: probability value.

The effect of breeder age (A_b) and oxygen (O₂) levels on hypoxia-induced embryogenesis and internal organ development was investigated. The current research is consistent with previous studies that under hypoxic conditions of 17% and 15% O₂ levels, relative embryo weight is decreased (result not shown). Literature presents conflicting results on the effects of hypoxia on embryo heart weight. A hypoxic environment may increase relative heart weight (Ben-Gigi et al., 2021; Lindgren and Altimiras, 2011), cause no change (Altimiras and Phu, 2000; Druyan et al., 2012) or decrease (Ruijtenbeek et al., 2000; Itani et al., 2016). Druyan et al. (2012) showed that 17% of O₂ hypoxia did not affect the relative heart weight of embryos, but 15% of O₂ did. In the study by Haron et al. (2021), neither the heart nor the liver weights were affected by low O₂ levels. The interaction between A_b and O₂ levels indicates a change in the function of the liver as an oxygen elicitor on ED 11. The metabolic pathways in older breeder flocks may be different from those in younger breeder flocks, explaining the differences in organ weights (Nangsuay et al., 2021).

Vascularization in CAM

In Figure 2, the fractal dimension (FD) and vascular fraction (VF) of blood vessels in the CAM were compared for eggs incubated under normoxic conditions of 21% and hypoxic conditions of 15% and 17% O₂ levels. The results for the FD (the degree of branching of the vessels, Fig 2a) showed there was an interaction (P = 0.001) between A_b and O₂ levels and likewise for the individual main effect of A_b and the different O₂ levels of incubation (P < 0.001). Eggs from 50 wk breeders had higher FD under 15% and 17% O₂ levels compared to the 33 wk. As showed in Fig 2b, no significant interaction (P = 0.156) was found for the VF (degree of vessel density, %) in CAM, however, an increase (P < 0.001) was observed for exposure to 15 and 17% O₂ levels (hypoxic) as compared to the 21% (normoxic) condition, and also for 50 wk compared to 33 wk breeder ages.

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Figure 2. Effect of breeder age and reduced oxygen concentration levels from ED 7-9 on CAM vascularization parameters. ^a-e Letters on each error bar means significance at P < 0.05.

The CAM was seen to cover a large area with increased vessel branching for embryos exposed to 15 and 17% O₂ levels (hypoxia) than normoxic incubations at both breeder ages. This agrees with Druyan et al. (2012) who observed a significant increase in the vessel density of the CAM of embryos that were incubated in environments with lower O₂ concentrations from 5 to 12 d. However, the higher effect in 50 wk breeders indicated that older breeders were more receptive to hypoxic stimulation, probably due to large surface area and thickness of eggshell. This is an adaptive response that is geared towards phenotypic plasticity in embryos. According to Druyan et al. (2012), embryos that develop under hypoxic conditions are expected to have improved gas diffusion and blood transport abilities, which can be attributed to increased CAM vascularization, and, therefore, a greater supply of oxygen to the embryo. A stimulating effect of hypercapnia and systemic acidosis on angiogenesis, as described by Everaert et al. (2008) lowers the pH of egg albumen. As a result, the embryo is forced to adapt by altering their cardiac output and redistributing oxygenated blood to vital organs, including the brain, heart, and adrenal glands for growth (Mulder et al., 1998). These findings suggest that the age of the breeder hens and the oxygen levels during incubation can significantly impact the development of the embryonic vasculature (Lin et al., 2008; Yalçın et al., 2012; Almeida et al., 2016). Older breeders may produce eggs with a greater capacity for vascular branching under hypoxic conditions, potentially enhancing the embryo’s ability to adapt to low oxygen environments (Fasenko et al., 1999). Further research is needed to clarify the underlying mechanisms and the implications for embryonic and posthatch development.

Haematological Profile

Table 2 outlines the effect of A_b and O₂ levels on the haematological profile of hatchlings. The interactive effects indicate that chicks hatched from 33 wk * 15% to 50 wk * 17% O₂ level exhibited significantly higher (P = 0.001) white blood cell (WBC) counts than their precedent counterparts. Lymphocyte (LYMP) count from 33 wk * 21% to 50 wk * 15% O₂ level was also statistically (P = 0.005) higher in percentages compared to chicks hatched from 33 wk * 15% to 33 wk * 17% O₂ level. Granulocyte (GRAN) counts were statistically (P < 0.001) higher in eggs from 33 wk * 15% and 33 wk * 17% O₂ levels and 50 wk * 21% O₂ had markedly higher levels compared to other interaction groups. Haemoglobin (HGB) concentration interactively significantly (P = 0.020) increased in this order; 33 wk * 21% ≤ 33 wk * 17% < 33 wk * 15% < 50 wk * 21% < 50 wk * 17% < 50 wk * 15% O₂ level. Likewise, Hematocrit (HCT) levels also took a significant (P = 0.031) increasing order as follows; 33 wk * 21% ≤ 33 wk * 17% = 33 wk * 15% ≤ 50 wk * 21% = 50 wk * 17% = 50 wk * 15% O₂ level. Mean corpuscular volume (MCV) was significantly greater (P = 0.040) in the 50 wk * 15% and 50 wk * 21% O₂ levels compared to the 33 wk * 15%, 17%, and 21% O₂ levels. Mean cell haemoglobin concentration (MCHC) was significantly higher (P = 0.006) in the 33 wk * 15% and 50 wk * 17% O₂ levels compared to all the other interaction groups. A significantly higher main effect of O₂ level is observed for WBC count for 15% O₂ level being higher compared to other counterparts which were not different among each other. LYMP was also influenced (P = 0.019) by the O₂ level. Neither A_b nor O₂ levels, interactively or individually (P > 0.05) affected the blood proportions of mean cell haemoglobin (MCH) and platelets (PLT).

Table 2. Effect of breeder age and reduced oxygen concentration level from ED 7- 9 on the haematological indices of chicks at hatch.

Parameters	WBC (10^9/L)	LYMP (%)	GRAN (%)	RBC (10^12/L)	HGB (g/L)	HCT (%)	MCV (fL)	MCH (pg)	MCHC (g/L)	PLT (10^9/L)
Breeder age (Ab)
33 wk	65.11	0.90b	0.02a	2.18b	135.33b	0.28b	127.66b	61.99	485.78	2.72
50 wk	69.54	0.91a	0.01b	2.62a	163.89a	0.34a	131.62a	62.85	477.50	1.94
SEM1	1.550	0.002	0.001	0.051	3.460	0.007	0.730	0.570	3.560	0.290
Oxygen level (O2)
15%	72.04a	0.91ab	0.02	2.46	155.50	0.32	129.47	63.22	488.67	2.42
17%	67.95ab	0.90b	0.02	2.41	149.50	0.31	128.75	62.09	482.25	2.17
21%	61.98b	0.91a	0.02	2.32	143.83	0.30	130.70	61.95	474.00	2.42
SEM1	1.900	0.003	0.001	0.062	4.240	0.009	0.900	0.700	4.360	0.360
Interaction (Ab * O2)
33 wk * 15%	76.00a	0.89b	0.02a	2.27	143.67c	0.28b	125.57b	63.50	505.33a	3.33
33 wk * 17%	60.90b	0.90b	0.02a	2.16	133.00d	0.28b	128.10ab	61.43	480.00bc	2.33
33 wk * 21%	58.43b	0.92a	0.01b	2.12	129.33de	0.27bc	129.30ab	61.03	472.00c	2.50
50 wk * 15%	68.07ab	0.92a	0.01b	2.65	167.33a	0.35a	133.37a	62.93	472.00c	1.50
50 wk * 17%	75.00a	0.91ab	0.01b	2.65	166.00a	0.34a	129.40ab	62.75	484.50b	2.00
50 wk * 21%	65.54ab	0.91ab	0.02a	2.51	158.33b	0.33ab	132.10a	62.87	476.00bc	2.33
SEM1	2.690	0.004	0.002	0.088	5.990	0.012	1.270	0.990	6.160	0.510
P-value2
Ab	0.053	0.024	0.013	<0.001	<0.001	<0.001	0.001	0.295	0.110	0.069
O2	0.003	0.019	0.219	0.272	0.168	0.425	0.314	0.385	0.074	0.850
Ab * O2	0.001	0.005	<0.001	0.806	0.020	0.031	0.040	0.452	0.006	0.209

Abbreviations: WBC, white blood cell; LYMP, lymphocyte; GRAN, granulocyte; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet.

a-e

Means within the same column with different superscripts are significant at P < 0.05.

1

SEM, pooled standard error of means.

2

P: probability value.

Chick blood profile measurements showed an interaction between breeder age and O₂ levels on WBC, HGB, HCT, MCV and MCHC. The current finding agrees on HGB and HCT with several studies that embryos exposed to hypoxia during early or late development had elevated HGB concentrations and HCT (Dzialowski et al., 2002; Ruijtenbeek et al., 2000; Chan and Burggren, 2005; Haron et al., 2021, 2022). The higher interaction effect observed for 50 wk breeders compared to 33 wk breeders suggests an increase in oxygen-carrying capacity for older breeders than younger ones. Other studies have also reported that neither RBC count nor PCV or HGB values were affected by high O₂ and CO₂ (Maxwell et al., 1987; Tong et al., 2015; Okur et al., 2022). In avian embryos, the RBC transport oxygen and CO₂ through direct diffusion (Mueller et al., 2022). Tazawa et al. (2012) attributed the increase in HCT (ED 11-ED 19) to increased MCV and not likely due to O₂ transport. This could be true for older breeders but not for younger breeders. An increase in blood O₂-carrying capacity can be achieved by various means: polycythemia (Dusseau and Hutchins, 1988), modification of HGB (Liu et al., 2009), increased vascularization and angiogenesis (Dusseau and Hutchins, 1988; Zhang et al., 2017) or combinations of any of the above factors. Hypoxia during incubation is a known developmental stressor for an embryo (Haron et al., 2021). The combined effect on LYMP and GRAN counts serves as evidence of the impact of A_b and reduced O₂ levels on the immunity and developmental stress response of chicks during hatching. On the contrary, Beker et al. (1995) reported no effect of low O₂ on leukocytes.

Biochemical Profile

The effects of A_b and O₂ levels on serum biochemical indices are presented in Table 3. The results indicated that neither A_b nor O₂ levels, individually or interactively, had a significant (P > 0.05) effect on total protein and albumen concentrations (P > 0.05). Triglyceride concentrations showed a statistical increase (P = 0.046) in the 50 wk breeders incubated at 15%, 17%, and 21% O₂ levels compared to those at 33 wk, irrespective of O₂ levels. The interactive effect on high-density lipoprotein was significantly higher (P = 0.036) in the 33 wk * 21%, 50 wk * 15, 21% O₂ level compared to the other groups. However, low density lipoprotein was markedly superior (P = 0.011) in the order; 33 wk * 15% > 50 wk * 21% ≥ 33 wk * 17% = 33 wk * 21%, 50 wk * 17% < 50 wk * 15% O₂ level compared to the other interaction groups. No interactive (P > 0.05) effect on total cholesterol concentration was found, nevertheless, the main effects of O₂ levels influenced (P = 0.002) its concentrations with 15%, and 21% O₂ levels being different from 17% O₂ levels but not each other. The breeder age effect showed a significantly lower (P = 0.038) concentration of total cholesterol observed in the 50 compared to the 33 wk breeder group.

Table 3. Effect of breeder age and reduced oxygen concentration level from ED 7- 9 on the serum biochemical indices of chicks at hatch.

Parameters	TP (g/l)	ALB (g/l)	TG (g/l)	HDL (g/l)	TC (g/l)	LDL (g/l)
Breeder age (Ab)
33 wk	28.79	9.77	0.81b	1.26	3.97a	2.53
50 wk	28.03	9.73	1.26a	1.04	3.60b	2.31
SEM1	1.360	0.580	0.061	0.100	0.120	0.120
Oxygen level (O2)
15%	28.33	9.70	1.03	1.10ab	3.91a	2.58
17%	26.05	9.48	1.12	0.94b	3.33b	2.17
21%	30.85	10.07	0.96	1.41a	4.12a	2.52
SEM1	1.670	0.712	0.074	0.130	0.150	0.150
Interaction (Ab * O2)
33 wk * 15%	27.20	9.10	0.73b	1.01b	4.26	3.07a
33 wk * 17%	27.47	10.33	0.83b	0.99b	3.33	2.18bc
33 wk * 21%	31.70	9.87	0.88b	1.78a	4.32	2.36bc
50 wk * 15%	29.47	10.30	1.33a	1.20ab	3.55	2.08c
50 wk * 17%	24.63	8.63	1.41a	0.89b	3.34	2.16bc
50 wk * 21%	30.00	10.27	1.04ab	1.03ab	3.91	2.67b
SEM1	2.360	1.010	0.110	0.180	0.210	0.210
P-value2
Ab	0.697	0.968	<0.001	0.143	0.038	0.193
O2	0.143	0.843	0.319	0.041	0.002	0.130
Ab * O2	0.532	0.344	0.046	0.036	0.248	0.011

Abbreviations: TP, total protein; ALB; albumen; TG, triglycerides; HDL, high-density lipoprotein; TC, total cholesterol; LDL, low-density lipoprotein.

a-b

Means within the same column with different superscripts are significant at P < 0.05.

1

SEM, pooled standard error of means.

2

P: probability value.

During incubation, lipid metabolism plays an important role in the growth of the embryo. Our findings agree with Okasha et al., 2021 that there is a decreased TC but disagree with the high TG reported in our results for chicks hatched from 50 wk breeder flocks. The higher level of TG observed in the 50 wk breeder flock can be attributed to yolk size and the level of nutrients in the yolks as a result of the age of the breeder flocks. High absorption of this yolk sac is evident in 50 wk breeders exposed to hypoxic conditions. The lower TC and higher TG levels in the 50 wk breeder compared to the 33 wk group suggest age-related differences in cholesterol metabolism, and in our case, slightly impacted by hypoxic condition. In recent times, Jiang et al. (2023) reported a significant increase in TG and lipoprotein lipase in the liver under hypoxic stress for 6 h in golden pompano. Environmental conditions of hypoxia and normoxia substantially influence plasma TC, TG and HDL (Olanrewaju et al., 2010; Okasha et al., 2021) . According to Nangsuay et al. (2013), albumin from an old breeder flock contains relatively fewer proteins than albumin from a young breeder flock. This result is not clear in our finding and the main reason could be due to differences in breeds and the age of breeders used in both researches. Unlike our study which was in layers, Nangsuay et al. (2013)’s study was on broilers.

Hormonal Profile

Table 4 shows the effect of A_b and O₂ levels on the hormonal profile of hatchling chicks. T₃ concentration was significantly higher (P = 0.001) in the 33 wk * 15%, 50 wk * 15%, 50 wk * 21% O₂ levels compared to the other O₂ levels. A similar trend was observed for T₄ concentration, with a statistically different level (P = 0.021) noted in the 33 wk breeders compared to the 50 wk breeders incubated at 17% O₂ level. Furthermore, a significantly higher (P = 0.026) ratio of T₃ and T₄ was observed in the 50 wk breeders incubated at 17% O₂ level compared to its 15% O₂ level and the 33 wk * 21% O₂ level.

Table 4. Effect of breeder age and reduced oxygen concentration from ED 7- 9 on the thyroid hormone indices of chicks at hatch.

Parameters	T3 (ng/ml)	T4 (ng/ml)	T3/T4
Breeder age (Ab)
33 wk	1.16	8.52	0.14
50 wk	1.24	8.49	0.15
SEM1	0.072	0.470	0.009
Oxygen level (O2)
15%	1.37	9.97a	0.14ab
17%	1.13	7.12b	0.17a
21%	1.10	8.43ab	0.13b
SEM1	0.088	0.570	0.011
Interaction (Ab * O2)
33 wk * 15%	1.63a	10.95a	0.15ab
33 wk * 17%	0.99b	7.49b	0.14ab
33 wk * 21%	0.86b	7.11b	0.12b
50 wk * 15%	1.10ab	8.99ab	0.13b
50 wk * 17%	1.27ab	6.74b	0.20a
50 wk * 21%	1.34ab	9.75ab	0.14ab
SEM1	0.130	0.810	0.015
P-value2
Ab	0.460	0.967	0.170
O2	0.079	0.005	0.039
Ab * O2	0.001	0.021	0.026

Abbreviations: T₃, triiodothyronine; T₄, thyroxine

a-b

Means within the same column with different superscripts are significant at P < 0.05.

1

SEM, pooled standard error of means.

2

P: probability value.

Thyroid hormones are vital for the maturation of essential organ structures, behavioural development (McNabb and Darras, 2015), pipping and hatching (Tullett, 2009) because they ensure a process of transition from allantoic to pulmonary respiration and also play a part in the length of the incubation process (Dewil et al., 1996). The current finding for T₃, T₄ and T₃/T₄ levels of chicks reveals a combined influence of A_b and O₂ levels on T₄ level and breeder eggs hatched from 15% O₂ than other interactive counterparts. The T₄ level was elevated at 15% O₂ compared with 17% but not different from the 21% O₂ level during incubation. Şahan et al (2011) reported a higher T₃, T₄ and T₃/T₄ ratio for high-altitude (hypoxic) hatched chicks than low-altitude (normoxic) chicks. In partial agreement with Hassanzadeh et al. (2004), no effect of both high and low altitudes on T₃, but breeder age and O₂ levels interacted to obtain higher levels for hypoxic for both 33 and 50 wk breeder eggs. Bahadoran et al. (2010) found higher plasma T₃ concentrations with a lower T₃/T₄ ratio for high-attitude hatched chicks. Hypoxia due to high altitude during early embryogenesis may change the endocrine functions of the embryo, enhance growth, or shorten the hatching process of chickens (Bahadoran et al., 2010). The interactive significant impact of high T₄ over T₃ and T₃/T₄ ratio for stimulated hypoxic conditions could indicate that embryos from different breeder ages may have different metabolic mechanisms or may require different levels of energy necessary for pipping and hatching.

Histological Analysis

The histological formation of neo-vascularization in the heart, lungs and liver is presented in Table 5 and Figure 3, Figure 4 to 5. The current study shows that with the exception of the liver tissue of 33 wk * 17% and 21% O₂ level and lungs at 17% O₂ level having normal tissue development, all the other tissues of both breeder ages had a high (vessels seat moderate congestion, “+++”) to a very high (ectatic vessels with high congestion, “++++”). Very high congestion was seen in the tissue of the lungs and liver of 50 wk embryos exposed to 15% and 17% O₂ levels. The same is also observed for the heart and lungs of 33 wk breeder age. High and moderate congestion was seen in the heart tissue under 17% O₂ level for 33 wk breeders while under 15% and 17% O₂ level for 50 wk breeders and in the 15% and 21% O₂ of the lungs.

Table 5. Neo-vascularization in internal organ tissue of chicks of 33 and 50 wk breeder age exposed to reduced oxygen concentration levels from ED 7- 9.

Groups		Neo-vascularization score
Breeder age (Ab)	Oxygen level (O2)	Heart	Lung	Liver
33 wk	15%	++++	++++	+++
	17%	+++	++	++
	21%	++++	++++	++
50 wk	15%	+++	++++	++++
	17%	+++	++++	++++
	21%	++++	++++	+++

Breeder age and Oxygen concentration (A_b * O₂): 33 wk * 15% O₂ (33 weeks breeder eggs incubated under 15% O₂); 33 wk * 17% O₂ (33 weeks breeder eggs incubated under 17% O₂), 33 wk * 21% O₂ (33 weeks breeder eggs incubated under 21% O₂), 50 wk * 15% O₂ (50weeks breeder eggs incubated under 15% O₂), 50 wk * 17% O₂ (50weeks breeder eggs incubated under 17% O₂), 50 wk * 21% O₂ (50 weeks breeder eggs incubated under 21% O₂).

Neo-vascularization grading; ++++ = Very high (ectatic vessels with high congestion); +++ = high (vessels seat of moderate congestion); ++ = Normal (normal tissues)

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Figure 3. Histology of the heart of chicks showing neo-vascularization of embryos exposed to reduced oxygen concentration level from ED 7- 9. ^abcdef: Image of breeder age * oxygen concentration level (A_b * O₂); a: 33 wk * 15% O₂ (33 weeks breeder eggs incubated under 15% O₂); b: 33 wk * 17% O₂ (33 weeks breeder eggs incubated under 17% O₂), c: 33 wk * 21% O₂ (33 weeks breeder eggs incubated under 21% O₂), d: 50 wk * 15% O₂ (50 weeks breeder eggs incubated under 15% O₂), e: 50 wk * 17% O₂ (50 weeks breeder eggs incubated under 17% O₂), f: 50 wk * 21% O₂ (50 weeks breeder eggs incubated under 21% O₂). Neo-vascularization grading: ++++ = Very high (ectatic vessels with high congestion); +++ = high (vessels seat of moderate congestion); ++ = Normal (normal tissues).

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Figure 4. Histology of the lungs of chicks showing neo-vascularization of embryos exposed to reduced oxygen concentration level from ED 7- 9 ^abcdef: Image of breeder age * oxygen concentration level (A_b * O₂); a: 33 wk * 15% O₂ (33 weeks breeder eggs incubated under 15% O₂); b: 33 wk * 17% O₂ (33 weeks breeder eggs incubated under 17% O₂), c: 33 wk * 21% O₂ (33 weeks breeder eggs incubated under 21% O₂), d: 50 wk * 15% O₂ (50 weeks breeder eggs incubated under 15% O₂), e: 50 wk * 17% O₂ (50 weeks breeder eggs incubated under 17% O₂), f: 50 wk * 21% O₂ (50 weeks breeder eggs incubated under 21% O₂).

Neo-vascularization grading: ++++ = Very high (ectatic vessels with high congestion); +++ = high (vessels seat of moderate congestion); ++ = Normal (normal tissues).

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Figure 5. Histology of the liver of chicks showing neo-vascularization of embryos exposed to reduced oxygen concentration level from ED 7- 9. ^abcdef: Image of breeder age * oxygen concentration level (A_b * O₂); a: 33 wk * 15% O₂ (33 weeks breeder eggs incubated under 15% O₂); b: 33 wk * 17% O₂ (33 weeks breeder eggs incubated under 17% O₂), c: 33 wk * 21% O₂ (33 weeks breeder eggs incubated under 21% O₂), d: 50 wk * 15% O₂ (50 weeks breeder eggs incubated under 15% O₂), e: 50 wk * 17% O₂ (50 weeks breeder eggs incubated under 17% O₂), f: 50 wk * 21% O₂ (50 weeks breeder eggs incubated under 21% O₂).

Neo-vascularization grading: ++++ = Very high (ectatic vessels with high congestion); +++ = high (vessels seat of moderate congestion); ++ = Normal (normal tissues).

Neovascularization is the formation of new blood vessels irrespective of the type or size of the organ. The neo-vascular differentiation in the tissue shows that the liver and lungs of older flocks may have a distinct response to hypoxia under 15% and 17% O₂ levels compared to the heart which might require a higher hypoxic level of 15% O₂ level to respond, regardless of the observed tissue congestion under 21% O₂ level. The varying levels of congestion in the heart, lungs and liver tissues of embryos suggest a highly intricate relationship between breeder age and O₂ levels in response to hepatic neovascularization. Adair et al. (1987) reported that sustained hypoxia in the embryo causes vasodilatation and decreases systemic vascular resistance. Literature is limited to explaining the above findings. Nonetheless, in some close research, Hao et al. (2014) noted a notable increase in miR-15a expression in embryonic lung tissue under low oxygen (hypoxic) conditions, emphasizing tissue-specific responses to hypoxia. This could confirm why the heart, lungs and liver have varying congestion under 15% and 17% O₂ levels. Hypoxia affects cardiac and vascular disease in chick embryos by adaptive transcriptional changes in the lungs and hearts of high-altitude animals (Salinas et al., 2009; Ge et al., 2021). By observing elevated plasma lactic acid levels in high-altitude embryos, Hassanzadeh et al. (2004) attributed the change in tissue to anaerobic metabolism of embryo organs caused by hypoxic conditions. Hypoxia plays a role in the vascularization of the heart via its vasodilatory effects, once the coronary circulation is functional (Tomanek et al., 2003). This response is in line with the chick’s body’s attempt to increase oxygen supply or provide rapid support for angiogenesis under conditions of tissue damage especially to tissues of the heart and lungs, when reduced oxygen availability is present (Hsia et al., 2013).

Low oxygen levels may have been compensated for by both breeder ages, resulting in a more controlled and normal neovascular response in the heart and lung tissues of the chicks (Pearce, 2006; Miller and Zachary, 2017). This observation could be true for younger breeders compared to older birds as observed in the current finding of 33 wk breeders at 17% O₂ level. The process occurring within tissues may be an adaptive response to oxygen deprivation or low O₂ levels and that triggers hypoxia-induced neovascularization (Michiels, 2004). Abdollahi et al. (2011) also discussed how micro-environmental conditions such as hypoxia regulate stem cell differentiation, and this could be pertinent to the neovascular response observed in heart and lung tissues. Hypoxia has been reported to influence the development (morphological and physiological) of chick embryos and their effects may depend on the timing of their application during incubation (Altimiras and Phu, 2000; Chan and Burggren, 2005; Onagbesan et al., 2007) since various organs develop and mature at different stages of embryo development.