Incubation and embryo development

An improvement in the rate of gain of broilers during the last two decades has made the incubation period a larger percentage of the overall growth period for commercial poultry (Hulet, 2007). Therefore, incubation and the last days toward hatch have gained more relative importance to the successful rearing of meat poultry than ever before. It is expected that anything that supports or limits growth and development during the incubation period will have a marked effect on overall performance and health of poultry. Because of this, many poultry researchers now realize that future gains in poultry production will come from advancements made during the incubation period and embryogenesis.

The last period of incubation is characterized by oral amnion consumption by the embryo, intensive absorption of the yolk, accumulation of glycogen reserves in muscle and liver tissues and their use during pipping and hatching, beginning of pulmonary respiration, abdominal internalization of remaining yolk, shell pipping and emergence (Christensen, et al., 1982, Donaldson and Christensen, 1991, Donaldson, et al., 1991, Christensen, et al., 1999, Christensen, et al., 2001, Christensen, et al., 2003; Moran, 2007).

During this time frame dramatic physiological and metabolic changes occur and any disturbances arising  in these days  (for example delay in feed access, incubation temperature) affect hatchlings' quality and subsequent performance (Christensen et al., 1999;  Collin et al., 2007; Piestun et al., 2009; Willemsen et al., 2010).

One of the major physiological processes occurring during the pre hatch period is the maintenance of glucose homeostasis. The glycogen reserves are withdrawn as embryos go through the hatching process (Christensen et al., 1982; Lu et al., 2007). Insufficient glycogen forces the embryo to mobilize more muscle protein for gluconeogenesis, thereby reducing early growth and development (Vieira and Moran, 1999) until the glycogen reserves start to be replenished when the newly hatched chick has full access to feed (Moran, 2007).

In birds, the pectoral muscle is the predominant source of protein mobilized to supply amino acids for gluconeogenesis if energy reserves are depleted after hatch (Donaldson., 1995; Lu et al., 2007; De Oliveira et al., 2008).  In a low energetic state or during periods of fasting, the pectoral muscle serves as a source for amino acids and energy, resulting in atrophy. Therefore, the liver and muscles are most affected by changes in metabolic pathways existing during last days of incubation period (de Oliveira et al., 2008). In case of late access to first feed post hatch the development and growth of skeletal muscle exhibit irreversible retardation through to marketing age (Halevy et al., 2000, 2003).

Many studies have examined COH metabolism in the liver of the chick embryo and revealed that the liver is responsible for blood glucose homeostasis (Ballard and Oliver, 1963; Freeman, 1969). It is also performs essential processes involved in COH metabolism and glucose supply to the tissues during chicken embryonic development, such as glucose synthesis from non-COH precursors (gluconeogenesis), glycogen synthesis (glycogenesis), and the breakdown of glycogen (glycogenolysis). Based on these data, one of the criteria in assessing embryos' energetic status has been the measurement of liver glycogen levels (Christensen et al., 2001; Uni et al., 2005). Low liver glycogen levels have been associated with prolonged hatching time and with decreased body weight at hatch (Christensen et al., 1999, 2000).

Another significant physiological process takes place in the yolk where, during the last week of incubation, β-oxidation of yolk-derived fatty acids provides the embryo with its main source of fuel (Speake et al., 1998). However, during the last 2 to 3 d of incubation, due to the high energy demand of the hatching process and the relatively low availability of oxygen, fatty acids cannot supply all of the necessary energy (Moran, 2007). The embryo is then driven toward anaerobic catabolism of glucose, which is dependent on the amount of glucose held in the glycogen reserves of the liver, kidneys and muscles, and on the degree of glucose generated by gluconeogenesis from amino acids, glycerol and lactate (Pearce, 1971; Christensen et al., 2001; De Oliveira et al., 2008).

Of great importance is the physiological process of intestine development. Intestinal functions (i.e. digestion and absorption) and intestinal barrier, as the first line of defense against aggressions arising from luminal content, have major roles in poultry performance and production. In broilers, the critical period for the development of an intact mature intestine is the pre-post hatch period (E17 to d 7 post hatch) were a transition from late term embryo to viable chick is occurring.

During incubation bird embryos do not invest much into gut development, but at the end of the incubation period rapid visceral growth and maturation occurs (Uni, et al., 2003, Gilbert, et al., 2007). At this period an intensive development of the intestine is taking place. The small intestine enlarged their absorption surface area by 5 fold during last 6 days of incubation, enterocyte number increases. Goblet cells which produce acidic mucin appears and the tissue develop quickly abilities of digestion and absorption. Changes in incubation conditions (temperature/ oxygen/ ventilation) affect these physiological processes and probably lead to changes in the intestine development and in the quality of hatchlings.

Chicken embryos have capacity to digest and absorb nutrients prior to hatch, as demonstrated by relatively low mRNA levels of sucrase-isomaltase (SI) and l-aminopeptidase and the ATPase and sodium glucose transporter (SGT-1) in the small intestinal mucosa (Tako, et al. 2004; Uni, et al., 2003). The activity of brush border enzymes leucine amino peptidase (LAP) and sucrase-isomaltase (SI) have been reported in turkey embryos at 25E, and glucose transporter (SGLT-1) and alanine transporter (B^o+) activity have been measured as early as 23E (Foye, et al., 2007). This absorption capacity increases close to hatch and continues to increase during the first few days post-hatch (Uni, et al., 1999, Geyra et al., 2001, Uni, et al., 2003, Tako, et al., 2004). Tako, et al. (2004) mentioned that poultry embryo villus height increases by 200-300% from 17 days of incubation until hatch, and small intestinal weight increases faster than body mass ( Sell, et al., 1991, Uni et al., 1999). Rapid intestinal growth is due to great increase in cell numbers and size, due to accelerated enterocyte proliferation and differentiation, and intestinal crypts formation (Uni, et al., 2000, Geyra, et al., 2001). Therefore, intestinal tissue growth, maturation and metabolism become of great importance in the last period on poultry embryonic development.

The sooner the intestine achieves functional capacity the quicker the pouts can utilize dietary nutrients, absorb minerals and vitamins and support the development of critical organs (skeleton, immune system, breast muscle).

A major factor which has great influence on the development of the broiler embryo and on the hatched chick is the deposit levels of the macronutrients and micronutrients in the fertile egg. While the fertile egg has a defined nutrient composition, varying according to age and nutrition of breeding flock, the rate and mechanism of absorption of these nutrients by the embryo is not fully recognized.

During incubation, the chick embryo derives all of its nutrient requirements from the egg resources (yolk, albumen and shall). The albumen represents about 65 to 75% of the egg's total content, and consists of approximately 88% water and 12% protein, both of which are totally consumed by the embryo during incubation (Romannof, 1960; Shenstone, 1968). The yolk consists of approximately 50% water, 15% protein, 33% fat, and less than 1% carbohydrates; however, this composition depends greatly upon egg weight, genetic strain, and hen age (Shenstone, 1968; Vieira and Moran, 1998). During incubation, nutrients pass from the yolk contents to the embryo through the yolk sac membrane and its surrounding vascular system (Noble and Cocchi, 1990). From day 19 of incubation (19E), the yolk sac begins to be internalized into the embryo's body cavity, and at hatch, it constitutes about 15 to 20% of the chick's body weight, providing the hatchling with immediate nourishment until exogenous feed is given in the brooder house (Romanoff, 1960; Noble and Ogunyemi, 1989, Noy and Sklan, 2001).

The yolk is the main source of energy (via fatty acid oxidation) during embryonic development, and the only source of lipids for embryo tissue growth (Speake et al., 1998). The process of yolk fat utilization has been extensively studied. It has been found that most of the yolk fat is taken up by means of nonspecific phagocytosis at the apical surface of the endodermal cells of the YSM (Lambson, 1970; Noble and Cocchi, 1990, Speake et al., 1998). Many factors can affect yolk fat utilization, such as incubation conditions (Burnham et al., 2001), genetic strain (Scheideler et al., 1998), breeder hen diet (Latour et al., 1998) and age (Yadgary et al., 2010).

The uptake of total fat and water from fresh yolk during the incubation period was done 20-30 years ago by Nobel (Noble et al., 1986) and Ar (Ar, 1991).  Recent research in our lab was done with respect to broiler breeder hen age and examined the protein, fat, water and carbohydrates in the yolk from mid incubation (E11) until day of hatch (Yadgary et al 2010). This research was done in light of the increased metabolic rate recorded in today's commercial embryos (Tona et al., 2004; Hamidu et al., 2007). Results demonstrate infiltration of protein and water from the non-yolk egg compartments (albumen and/or amnion) into the yolk. Absorption of yolk fat up to 15E was lower in embryos from younger hens (30 weeks) than in those from mature hens (50 weeks). These differences were explained by the lower initial yolk fat content in the eggs of the younger hens. At the end of incubation period (day of hatch) the residual yolk, in hatchlings from young breeder hens, has less fat content in compare to residual yolk in hatchlings from older hens (Yadgary et al., 2011).

Regardless the age of breeding flock the general picture obtained from the yolk content analysis during period of incubation shows differential uptake of yolk macronutrients during the 21 days of incubation. By E17 almost 50% of the protein was absorbed by the embryo from the yolk. 65% of fat was absorbed in a linear manner from the yolk between E11 to E17. Then, at E17-E20, only a small amount of fat was absorbed while at day of hatch 15% of yolk fat content was absorbed vigorously during just 24 h. Interestingly and unexpectedly, the amount of carbohydrates in the yolk increased significantly during incubation (E15 to E20) reaching a peak at 19E. This raises a question about the role of the yolk and yolk sac membrane in carbohydrates metabolism.

As for the micronutrients, the analysis of the yolk minerals showed that on E19 the levels of Zn, Cu, Mn, and P in the yolk (The major mineral reserve) decreased significantly to approximately 3, 6, 10 and 13% of their levels at day of set (Yair and Uni 2011). This leaves the embryo with low mineral reserves for the last period of incubation and probably leads to a minerals deficiency status of the embryo.

In order to overcome the described physiological limitations and to improve intestinal functionality and nutritional status of hatchlings, a methodology for "feeding the embryo" (in-ovo feeding) was developed. A method of ing nutrient solutions into the embryonic amniotic fluid was created for poultry and patented (Uni and Ferket 2003). The method makes use of the knowledge that neonatal birds naturally consume the amniotic fluids towards hatch (Romanoff 1960). Therefore, addition of a nutrient solution to the embryonic amniotic fluid delivers essential nutrients into the embryo intestine.

Many potential nutrient supplements can be included in the in-ovo feeding solution. Carbohydrates can be used as a source for glucose, which is crucial for the hatching process and hatchling development (Moran, 1985). Na⁺ and Cl^- ions play a major role in the activity of apical and basolateral transporters and in the absorption of glucose and amino acids. β-hydroxy-β-methylbutyrate (HMB), a leucine metabolite which affect muscle satellite cells and increases carcass yield  (Nissen et al 1994; Kornasio et al 2009), is a good candidate for the in-ovo feeding solution, as are minerals and  vitamins which support the development of skeletal, immune and digestive systems in chickens.

Studies have shown that the administration of 1 ml of in-ovo feeding solution including dextrin (as a source of carbohydrates), Na⁺, Cl^-, zinc-methionine and HMB leads to increased total liver glycogen in the pre-hatch period and markedly enhances enteric development (Tako et al 2004; Tako et al 2005; Smirnov et al 2006). Morphological evaluation of enteric sections from embryos and hatchlings revealed a significant acceleration of development 48 h after in-ovo feeding relative to non-injected controls. The in-ovo-fed birds exhibited increased pancreatic capacity for carbohydrate digestion, increased villus dimensions, higher levels of mRNA expression and activity of brush-border digestive enzymes and transporters. It can be concluded that at the time of hatch, the small intestine of in-ovo-fed birds is at a functional stage similar to that in conventionally fed 2-day-old chicks. Several experiments with offspring from young maternal flocks demonstrated that in-ovo feeding increases hatching weights by 5% over controls and elevates relative breast-muscle size (calculated as % of BW) by 6%. These weight advantages were sustained throughout the experiments (d 25). Commercial practice can be adapted to use in-ovo feeding methodology by using existing automated systems for in-ovo vaccination. Adaptation of injection machines to inject 0.6 ml to broiler and turkey embryos at 18E and 24E respectively were done by Embrex-Pfizer and in ovo feeding solutions were formulated and tested.

Recent experiments from various research groups exhibit the benefits of in ovo feeding method on BW and breast meat yield (Kornasio et al., 2011; Zhai et al., 2011; Bottje et al., 2011). In addition research showed that a solution which includes nutrients/ salts/ minerals and vitamins elevates their levels and availability for the embryo (Yair and Uni 2011). This has a great importance in case of sub-optimal levels which may limit the genetic potential of growth of critical organs.  Interestingly, in ovo feeding has an epigenetic effect by inducing expression of genes involved in major metabolic pathways and processes in tissues and organs and by that affect later on performance.

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