A Concise Review of Factors Limiting Yolk Sac Nutrient Utilization in Chicken Embryos

Abstract

The yolk sac is generally regarded as a vital and complex organ during embryonic development, the role and relative importance of which vary across different species. In avian species, particularly poultry, this sac plays a crucial role in nutrient absorption, metabolic regulation, and immune defenses. Advances in genetic selection and improvements in management practices have resulted in changes in yolk utilization; therefore, a more detailed understanding of the basic mechanisms of yolk utilization and factors that may limit or improve the embryo’s utilization of yolk sac nutrients is needed. Adequate insights into the role of the yolk sac and the optimization of its functional conditions may be very useful for improving the health, growth, and productivity of poultry, making poultry farming more effective and sustainable. Stressors that may limit yolk sac nutrient utilization, such as temperature, humidity, oxidation, prolonged storage, microorganisms, and chemicals, and, thus, negatively affect the growth and development of avian embryos were briefly reviewed herein.

1. Introduction

The yolk sac (YS) is amongst the first extra-embryonic organs to develop in the embryo during embryogenesis in different species. From an evolutionary perspective, it is a structure that has evolved to meet the nutritional and developmental needs of the embryo through different mechanisms across amniote classes of mammals, reptiles, and birds [1, 2, 3]. The role of YS in the growth and development of the avian embryo is indispensable because it is the source of nutrients for the embryo [4]. The avian embryo derives nourishment from the yolk, albumen, and shell [2]. Yolk contents and YS tissue are classified under the YS category [5, 6]. This sac is not a mere membrane, but it is an organ with multiple functions related to nutrient uptake, metabolism, secretion, and pathogen defenses [2, 7]. YS exhibits the functions of various organs for the developing embryo [8]; it is a multifunctional organ within the embryonic period with equivalent functions to bone marrow in hematopoiesis, the intestines in the digestion and transportation of nutrients, the liver in the production of plasma proteins and metabolism of nutrients, the thyroid in regulating metabolism, and lastly, the immune system in the transfer of antibodies and synthesis of antimicrobial peptides [2, 8].

The dynamic structure of YS in avian species was examined as early as in 1981 [9]. There are three distinct zones. The central area, the pellucida, forms the base of the early embryo and is surrounded by the area vasculosa, the most developed region containing a network of blood vessels [10]. There are no blood vessels in the area vitellina in the outermost region [11]. As development progresses, the area vitellina becomes the area vasculosa [12]. While this transformation continues, epithelial cells grow structures resembling finger-like projections and specialized pits that allow for the highly efficient absorption of YS nutrients. Therefore, the area vasculosa is regarded as the powerhouse for utilizing yolk in the developing chick embryo [6, 10].

The primary role of YS tissue is the absorption of nutrients [13]. YS development starts in the early stages of embryogenesis and stops in the late days of egg incubation. The internalization of YS into the abdominal cavity of the embryo finally starts on approximately embryonic day (E) 19 and is completed about 14 hours before hatching [8]. Any dysfunctions in YS during this final phase will cause nutrient deficiencies, early mortality, and poor chick quality [14]. Different factors may induce changes in YS functions and affect nutrient uptake, gas exchange, waste removal, and increase mortality. Bacterial infection has been shown to damage YS, which results in embryonic mortality [15]. Furthermore, chronic high temperatures during incubation lead to low chick weights and heavy residual YS, indicating changes in the metabolic performance of YS [16]. Therefore, a more detailed understanding of the cellular and molecular mechanisms controlling YS functions is critical for improving embryonic and chick growth and development.

Since avian YS is absorbed by the embryo during hatching, it has received little attention compared to the intraembryonic organs. In this review, we describe that YS conducts the absorption and distribution of yolk nutrients as an essential organ for healthy embryonic development. To elucidate the factors that negatively affect the YS integrity is of great importance. However, we summarize that detailed mechanisms for this unsung extraembryonic organ are little known at present. We encourage our colleagues worldwide to work on the following topics to promote sustainable poultry production.

2. YS primary nutrients

2.1 Lipids

One of the main components of the yolk is lipids, which contribute approximately 90% of the total energy needed for embryonic growth and development [17]. Lipids are mainly available in different lipoprotein forms. Low-density lipoproteins (LDL) account for 68% of all lipids in the yolk [18]. Gene expression profiling of YS from E2 to E4 revealed that many genes involved in lipid metabolism are expressed in YS, among which are the genes for fatty acid binding protein 2, phospholipases, monoacylglycerol acetyltransferase, long-chain acetyl-CoA synthetase, and apolipoproteins [8, 19, 20]. Therefore, YS has the capacity to take up, degrade, repackage, and deliver lipids into the extra-embryonic circulation [20]. Furthermore, the existence of lipase in YS indicates the hydrolyzation of lipoproteins by lipase and their absorption through extra-embryonic endoderm cells [21, 22]. Although bile acids in both the YS membrane and yolk content promote the digestion of lipids, the origin of bile remains unknown; YS may synthesize bile or it may originate from the liver and enter via the yolk stalk [16, 21]. These findings indicate that yolk lipids deposited by the hen and the lipids required by the embryo differ and, thus, YS functions as a lipid interface between the hen and chick embryo [17]. Moreover, the age of hens has been suggested to affect the transfer of lipids from the yolk to the embryo. Towards the end of incubation, the transfer of yolk lipids to the embryo was shown to be lower in younger hens [23].

2.2 Carbohydrates

YS and the liver represent major sites of glucose metabolism during incubation. High rates of gluconeogenesis and glycogenesis are generally observed after E13 to match the increased demand for glucose [24], and a previous study showed that the rate of gluconeogenesis was higher in embryos from smaller than typical-sized eggs [25]. The levels of carbohydrates and mRNA encoding gluconeogenic and glycogenic enzymes in YS tissue have been examined [4]. The decrease in glucose level from E1 to E11 could be a result of its use as a source of energy for embryos during the initial days of incubation. Glucose levels then increased from E11 to E19 in YS as gluconeogenesis proceeds. Key enzymes in the gluconeogenesis pathway, including fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase, were expressed in YS tissue. YS is also the main organ for glycogen storage during incubation [2]. An elevated concentration of glycogen in YS from E11 to E19 was accompanied by increases in the mRNA levels of key enzymes for glycogen synthesis [26]. Shibata et al. [27] showed that glucose-6-phosphatase enzymatic activity and mRNA expression were high and were associated with a low amount of glucose in YS in the late third week of incubation. They suggested that glucose was transported from YS to the vitelline circulation.

Glucose transporters are expressed in YS [4] and may transfer glucose into the vitelline circulation for the developing embryo. Transporters at the apical side of YS are necessary for taking up glucose from the yolk, while those at the basolateral side are required to secrete it into circulation. However, the locations of these transporters have yet to be investigated. During incubation, an increase was observed in the mRNA level of the sodium-glucose transporter SGLT1 in YS, which further prompts histological determination of the transporter’s location [26, 28].

2.3 Proteins

The yolk is a complex system that includes granules (non-soluble protein aggregates) and plasma containing LDL and soluble proteins [29]. Different proteins are viable in the YS membrane; the main egg yolk proteins are apolipoprotein B, apovitellenin-1, serum albumin, vitellogenin-2, and vitellogenin-3 [30]. Rehault-Godbert et al. [31] compared the abundance of proteins in fertilized and unfertilized eggs after a 12-day incubation and noted that only five proteins (vitronectin, α-fetoprotein, thrombin-like protein, apolipoprotein B, and apovitellenin-1) showed a major increase in relative abundance in the yolks of fertilized eggs. The YS endoderm has been suggested as a major site for protein synthesis [19]. Free amino acids are also present in fertile egg yolk and their levels vary with time during incubation [32, 33]. This fluctuation in the amino acid content of egg yolk is associated with the functional requirements of the chicken embryo at different embryonic stages [32]. Furthermore, the mRNA level of aminopeptidase N in YS increased from E11 to E17, but decreased thereafter from E17 to E20 [33]. The capacity to digest yolk peptides was also found to peak at approximately E17 [32]. These findings partially overlap with the expression of the oligopeptide transporter PepT1, the level of which increased from E11 to E15 and decreased from E15 to E20. However, the mRNA level of the cationic amino acid transporter CAT1 decreased from E11 to E13 and then increased from E15 to E17 [34]. Therefore, the different expression profiles of transporters may be due to their localization at either the basolateral or apical side of the YS epithelium or the type of amino acids in yolk [34]. Additionally, Yoshizaki et al. [12] detected the proteolytic enzyme cathepsin D in yolk, which suggests a digestion mechanism outside the YS membrane.

3. Stressors affecting YS nutrients

As described above, the avian YS is a critical component for embryonic development, serving as a site for the concentration of nutrients and driving the growth of embryonic organs. Small changes in the environment or the internal state of the hen could, therefore, markedly impact on how these crucial resources are absorbed by YS and used by the embryo. This might cause serious implications for chick development and survival. Several factors may disrupt this critical process by affecting how efficiently the bird uses the resources in YS. The mechanisms underlying yolk utilization and factors affecting residual yolk weight are essential for the optimization of poultry production. The following section will discuss how various factors limit YS utilization during avian development (Fig.1).

Figure 1: Stressors affecting yolk sac (YS) nutrients and their utilization. Possible typical stressors discussed in the text are shown in boxes. Red boxes indicate stressors affecting YS directly, while blue boxes indicate those affecting via mother hen. Since entire egg yolk (and egg white) derive from the mother hen, stressors to the mother hen potentially affect YS nutrients, hence embryos’ development. The absorption, conversion (metabolism), and secretion process of YS primary nutrients discussed in the section 1 are shown in the insertion. It should be noted that the details of these processes are largely unknown, and that further investigation is required. FABP2: fatty acid binding protein 2, MOGAT: monoacylglycerol acetyltransferase, LACS: long-chain acetyl-CoA synthetase, FBPase: fructose 1,6-bisphosphatase, PEPCK: phosphoenolpyruvate carboxykinase, G6Pase: glucose 6-phosphatase, GLUT: glucose transporter(s), SGLT1: sodium glucose co-transporter 1. (See Section 2 “YS primary nutrients” for details.)

3.1 Environmental stressors

3.1.1 Temperature and heat stress

During egg incubation, heat stress may affect the composition and availability of critical nutrients in YS [35]. High temperatures change the concentrations of proteins, lipids, and carbohydrates in YS, which are necessary for the progression of normal embryonic development and growth [36]. Moreover, heat stress impairs the ability of the embryo to absorb or use these nutrients, which negatively impacts growth and development [35]. The incubation temperature is a critical factor in YS utilization; incubation temperatures of 36.3 and 39.3 °C were compared with the normal incubation temperature of 37.8 °C in terms of YS utilization by comparing the expression of genes responsible for lipid uptake and metabolism [37]. The findings obtained show that hot and cold stress affected gene expression and yolk utilization; at the control temperature, an optimal utilization of residual yolk value of 11.12% was measured on the day of hatching, whereas utilization decreased at cold and hot temperatures with residual yolk values of 18.18 and 29.99%, respectively.

After hatching, heat stress still affects the utilization of YS nutrients. The effects of heat stress during the first five days of a chick’s life on residual YS abstraction and utilization were examined [38]; chicks were exposed to moderately cold conditions of 2–3 °C below the optimum temperature for the chick’s age. The second group was exposed to moderate heat 2–3 °C above the optimum temperature for the chick’s age. Control chicks had the highest body and intestinal masses. The resorption of the YS content was approximately equal in all chick groups, indicating the limited effects of post-hatching temperature on residual YS.

3.1.2 Humidity stress

Although humidity changes were reported to have limited effects on embryo development, particularly on the embryo weight to egg weight ratio [39], they may affect nutrient utilization. However, very few studies have examined the effects of humidity on nutrient utilization and residual YS. Humidity is related to the water content of the egg, which accounts for approximately 75% of the total egg content [40]. When relative humidity is lower than the standard, water loss markedly increases [41], and the yolk-free body mass decreases [42]. A previous study examined the effects of three relative humidity (RH) percentages (43, 53, and 63% RH) during incubation on yolk composition [43]. The findings obtained revealed that the percentage of yolk lipids at E16 was higher at 63% RH than at 53% RH in eggs from 26-wk-old breeders, but was lower in eggs incubated at 43% RH than at 53 and 63% RH from 30-wk-old breeders, suggesting that uptake rate of yolk lipid by the embryo varies depending on RH and hen age.

Moreover, optimal humidity ensures that the pores on the eggshell work correctly by allowing water to vaporize. Too high or too low humidity may affect the concentration of solutes and the osmotic balance in YS [44, 45]. If high humidity decreases the rate of water loss, waste products may accumulate and suffocate the embryo. Low humidity may also result in dehydration, which may affect nutrient diffusion and transport [46, 47, 48].

3.1.3 Gaseous environment stress

The structure of the egg facilitates the processes of diffusion and gaseous exchange, primarily oxygen (O₂) and CO₂, between the developing embryo and external environment [49]. The optimal levels for hatching eggs are a minimum of 16% O₂ and a maximum of 2% CO₂ [50]. Cellular respiration necessitates adequate O₂, which ultimately fuels metabolic processes for the use of nutrients stored in the yolk [48]. A previous study reported that O₂ availability was a critical factor for nutrient metabolism in the egg [21]. Insufficient gas exchange may result in hypoxia for the embryo, which hinders nutrient metabolism and results in developmental issues. Gas exchange and humidity are interrelated factors whereby improper humidity conditions affect chicken embryo gas exchange [51], decreasing the utilization of YS nutrients.

During the early days of incubation, YS is regarded as the embryo’s provisional respiratory organ. By approximately E2, when blood circulation begins, the YS membrane starts gas exchange [52]. However, YS is separated from the inner eggshell membrane by a layer of albumen or egg white. This creates a diffusion distance that increases the difficulty for O₂ to easily reach the embryo [51, 53].

The growing embryo’s demand for O₂ significantly increases from E4 to E6, thus stressing the total capacity of gas transport across YS [52]. During this time, the low partial pressure of O₂ in the embryo’s veins indicates that YS is unable to satisfy the increasing demand for O₂. On E8, the allantoic sac fuses with the chorion to create the chorioallantoic membrane and becomes the most dominant respiratory organ from E14 to E16 [54]. The role of YS is not entirely terminated because it still nourishes the embryo until the chick hatches [52,55].

On the other hand, during the early days of chick development, O₂ is transported by primitive red blood cells (RBCs) generated in YS [56, 57, 58]. RBCs contain embryonic hemoglobins with a higher affinity for O₂ than adult hemoglobins [59]. Their main function is maintaining a suitable O₂ pressure gradient inside the embryo [60]. Failure to maintain O₂ levels, resulting in hypoxia, seriously affects the nutrient utilization of YS during embryonic development. A previous study reported that hypoxic conditions during embryonic development decreased metabolism [61], which had a negative impact on embryonic development. On the other hand, high CO₂ levels (hypercarbia) disrupted the acid-base balance and caused acidosis, which interfered with nutrient transport and utilization, because exposure to 4% CO₂ from E10 to E18 increased pH and bicarbonate levels [62, 63].

3.2 Biological and oxidative stressors

3.2.1 Oxidative stress

Oxidative stress is caused by factors like high temperature during egg storage and egg incubation. If excessive reactive oxygen species (ROS) are produced in tissues in the embryonic phase, antioxidant defenses, such as vitamins A, E, C, and carotenoids, are needed [64], leading to embryonic oxidative stress before hatching [65]. Additionally, high levels of ROS are associated with many diseases [65] through their induction of protein and lipid oxidation [66] and DNA damage [64]. Oxidative stress has also been associated with early embryonic death, malformations, and post-hatch growth retardation in avians and mammals [67,68].

Conversely, maternal dietary supplementation with antioxidants (such as vitamin E, selenium, and canthaxanthin) through YS partly relieved the adverse effects of oxidative stress in chicks. Surai et al. [69] reported that the accumulation of canthaxanthin in egg yolk was proportional to its content in the diet. The transfer of canthaxanthin from egg yolk to the developing embryo was demonstrated, and its concentration in the embryo liver increased on E16 and in 1-d-old chicks. Moreover, metabolites in YS were more enriched in histidine and sulfur on E19 than on E13, suppressing oxidative stress damage to YS through the glutathione system [70].

3.2.2 Microbial stress

Eggs are generally in a non-sterile location during egg formation and after oviposition because specific microorganisms are naturally present in the oviduct. After the egg is laid, environmental microorganisms penetrate the eggshell. Although albumen contains anti-bacterial proteins, such as lysozyme and avidin [71], the volume of albumen decreases during incubation [12]. Pathogenic microorganisms, such as bacteria, viruses, and fungi, may attack YS and inhibit nutrient absorption and utilization. Pathogenic microbes or their toxins may structurally and functionally compromise the integrity of the YS epithelium. This will decrease the effectiveness of the epithelium to transport and absorb nutrients from the yolk for utilization by the developing embryo [15]. Moreover, damage to YS tissues due to bacterial infection correlates with embryonic mortality [15]. Infections by microbes stimulate immune responses that may drain energy and nutrients away from growth and development [72].

One of the nutrients transferred from YS to the embryo is immunoglobin Y (IgY), the maternal antibody [72, 73, 74]. YS plays a critical role in immune responses. Beyond transporting IgY, YS generates CD45+ hematopoietic cells. Macrophages are also produced by YS throughout the embryonic period as early as E2 [75, 76]. Furthermore, YS is a significant physical barrier in innate immunity and expresses antimicrobial peptides to provide additional protection [7, 77].

3.2.3 Parent flock age

As the age of hen increases, the size of yolks becomes larger, shifting their composition towards higher lipid levels, lower protein contents, and a higher yolk to albumen ratio [78]. Furthermore, age has been shown to decrease the efficiency of yolk mobilization and damage the integrity of the YS membrane [16, 79], thereby negatively affecting embryonic growth and development. Older hens produce eggs that are more disproportionate in terms of their yolks, which increase in size, but are nutritionally less optimal [16]. YS functionality also declines, posing potential issues for the developing embryo.

A previous study demonstrated that the weight of the residual YS significantly decreased as the age of hens increased [80]; there was a significant decline in YS weights in eggs from Pekin duck hens aged 42 weeks. Another study examined the effects of the ages of hens (26, 28, and 30 weeks) on YS content [43], and showed that breeder age significantly affected the percentage of YS and different YS fatty acids. The YS weight of the embryo and serum glucose levels in newly hatched chicks were also found to be affected by breeder hen age and may be related to changes in other associated physiological and molecular processes [81].

3.3 Handling and chemical stressors

3.3.1 Egg storage

Egg storage solutions play a crucial role in maintaining the quality and integrity of eggs, with fertilized eggs generally being stored at cool temperatures. If eggs are stored at a cool temperature, the embryos do not grow [55, 82]. The embryo enters a temperature-induced diapause, whereby the embryo maintains reduced cellular activity and suppressed apoptosis. However, the prolonged storage of eggs has an inverse effect and impairs the embryo’s survival. Embryos that survived long-term storage showed a significant delay in hatching by a few hours [83].

The storage period also affects the egg content, with total yolk fat being shown to decrease when eggs were stored at 22 °C for ten days due to lipid peroxidation [84]. A previous study demonstrated that chick weight or yolk-free body mass did not significantly differ between eggs stored for less than seven days and the eggs stored for more than seven days; however, the residual yolk weight was smaller in the eggs stored for more than seven days by 0.9 g [85].

The impact of these egg storage techniques on embryonic development potentially occurs via yolk absorption in YS and subsequent distribution to the embryo. However, to the best of the authors' knowledge, no studies have yet investigated the intra-YS process.

3.3.2 Mechanical stress

Mechanical stress reduces the availability of YS and may be induced during egg transportation and incubation in different manners, such as unsuitable handling, shaking, or turning eggs. These factors may violate the integrity of the membrane of YS and disrupt the transport and utilization of nutrients.

In geese, the egg turning angle during incubation significantly affects yolk utilization and embryonic development. With wider turning angles, yolk utilization was enhanced, and relative yolk mass was reduced by 1.92% on E22. Moreover, the expression of genes related to lipolysis and lipid transportation was significantly higher when eggs were turned at an angle of 70° than at an angle of 50° [86].

Additionally, the mechanical vibration of fertilized eggs was found to affect blood parameters in one-day-old chicks with higher concentrations of glucose and urea being noted with increasing vibrations. However, calcium, cholesterol, and triglyceride levels were reduced, indicating potential metabolic stresses on yolk nutrient utilization during egg incubation [87].

3.3.3 Chemical stress

Eggs may be exposed to various chemicals during the egg laying and hatching processes, including pesticides, antibiotics, veterinary drugs, heavy metals, mycotoxins, disinfectants, and environmental pollutants [88, 89]. Residues of pesticides and herbicides originate from feed and litter contamination and concentrate in egg yolk. Similarly, the residues of antibiotics and other veterinary medications given to laying hens are passed onto eggs. Heavy metals, notably lead, cadmium, and mercury, may also be present due to soil, water, or feed contamination. Mycotoxins are fungal toxins that may be present in feed and bedding and may end up in eggs. Chemicals from cleaning and disinfecting processes in the hatchery environment may leave residues on the eggshell or in the content of eggs.

Therefore, these chemicals (environmental pollutants, synthetic pesticides, and chemicals in therapeutic compounds) may affect YS nutrient transport, causing stunted growth and, in extreme cases, embryonic mortality [90]. The exposure of eggs to most pesticides was previously shown to significantly reduce hatchability [90]. Furthermore, exposure to a combination of insecticides induced DNA damage in chick embryos [89]. Egg disinfectants also significantly affected YS; the effects of five disinfectants on hatching eggs were examined and the findings obtained showed significant YS retention when eggs were contaminated and disinfected, with changes in YS, including YS inflammation, being observed [91].

Despite the importance of chemical agents in hatching eggs, limited research has addressed this topic. A more detailed understanding of how these chemicals impair YS nutrient utilization is attracting increasing interest for the development of proper interventions that optimize hatchability, chick quality, and overall poultry production.

4. Conclusion

The effects of environmental factors on YS functions have yet to be elucidated in detail. Further clarification will contribute to a reduction in resource wastage through the optimization of incubation methods and increases in hatchability rates. YS is absorbed into the chick before hatching and, thus, has received less attention than post-hatching organs; however, it is worthy of both basic and applied research. Future studies need to focus on the cellular and molecular mechanisms underlying YS functions, which involve the expression of key genes and enzymes that help absorb, process, and deliver nutrients and are involved in their regulation. These investigations will lead to the development and improvement of appropriate poultry management for the healthy YS, thereby reducing unhatched egg wastage, increasing poultry productivity and sustainability both in developing countries and advanced nations. In addition, insights from avian YS research may now be related to human health concerning various metabolic and nutritional disorders because YS is an organ common to vertebrates, including humans.

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