In a recent study, researchers shed light on the effect of high incubation temperature from embryonic day 12 to day of hatch on the expression and abundance of tight junction proteins, junctional adhesion molecules and heat shock proteins in the yolk sac tissue and small intestine of embryonic broilers.
During the transition from incubation to hatch, chicks shift from obtaining nutrients from the yolk sac to the intestine. The yolk sac tissue and small intestine serve as biological barriers between the yolk or gut contents and blood circulation. These barriers must maintain their structural integrity for optimal nutrient uptake, as well as protection from pathogens.
Studies show that environmental factors such as suboptimal incubation temperature play an important role in embryonic chick growth, with some studies indicating that an elevated incubation temperature (>37.8°C) negatively affects the development of the embryo, as demonstrated by lower yolk-free body mass and higher residual yolk weight at hatch. However, the researchers of the current study published in Poultry Science, argue that there is limited information available on the effect of incubation temperature on the development of the yolk sac tissue and embryonic small intestines in broilers.
The yolk sac tissue plays an important role in protecting the embryo from pathogens by acting as a physiological barrier and by expressing the avian β−defensin 10 peptide that acts against pathogenic bacteria, for example. Because the yolk sac tissue is derived from the small intestine, the structural and functional properties of the yolk sac tissue are similar to the embryonic small intestine, as both have tight junction proteins.
The researchers of the current study state that previous studies have examined the effect of heat stress on tight junction mRNA expression in the small intestine of post-hatch chickens, but not in the yolk sac tissue or embryonic intestine. Thus, in this study, the researchers examined the effect of high incubation temperature (39.5°C) on the mRNA abundance of the tight junction proteins (zona occludens 1 (ZO1), occludin (OCLN), claudin 1 (CLDN1)), junctional adhesion molecules A and 2 (JAMA, and JAM2) and the heat shock proteins (HSP) 70 and 90 in the yolk sac tissue and small intestine of embryonic broiler chickens.
Broiler eggs were incubated at 37.5°C. On embryonic day 12 (E12), half of the eggs were switched to 39.5°C. Yolk sac tissue samples were collected from E7 to day of hatch (DOH), while small intestine samples were collected from E17 to DOH. Measurements to assess the effects of incubation temperature were taken at E13, E15, E17, E19, and day-of-hatch (DOH).
Incubation at 39.5°C from E12 to DOH affected mRNA abundance in the yolk sac tissue of tight junction proteins and heat shock proteins compared to 37.5°C. ZO1 mRNA increased from E13 to DOH and was greater at 39.5°C than at 37.5°C. Both OCLN and CLDN1 mRNA increased from E13 to DOH with increasing temperature. At 37.5°C, JAMA mRNA increased from E13 to E15, from E15 to E19 and from E19 to DOH and, by contrast, at 39.5°C JAMA mRNA increased from E13 to E15, did not change from E15 to E19, but then increased from E19 to DOH. JAM2 mRNA increased from E13 to E19 and was greater at 39.5°C than at 37.5°C. For the heat shock proteins, both HSP70 and HSP90 increased from E13 to DOH with rising incubation temperature.
The high incubation temperature from E12 to DOH altered the mRNA abundance of TJ and HSP proteins in the small intestine. ZO1 mRNA decreased from E17 to DOH and was greater at 39.5°C than at 37.5 °C. OCLN mRNA was not affected by high incubation temperature. However, CLDN1 mRNA decreased from E17 to E19, while JAMA mRNA increased from E17 to DOH. JAM2 mRNA decreased from E17 to DOH but was greater at 39.5°C than at 37.5°C. For the heat shock proteins, HSP70 mRNA was greater at 39.5°C than at 37.5°C, while HSP90 mRNA showed an increase from E19 to DOH, with increasing incubation temperature.
The researchers expected the outcome of high incubation temperature on tight junction proteins and junctional adhesion molecules, as previous studies had shown that high incubation temperatures alter the temporal patterns of mRNA abundance for genes related to lipid uptake and metabolism, oligopeptide uptake, gluconeogenesis and thyroid hormone regulation in yolk sac tissue.
“In our study, incubation at 39.5°C increased ZO1 and JAM2 mRNA in both the yolk sac tissue and small intestine. It is important to know that ZO1 localises near the cytoplasmic membrane and serves as an important link between the actin cytoskeleton and the tight junction, which includes the transmembrane JAM proteins,” the researchers said.
In line with the current study, they stated that previous research work found lower yolk-free body mass and higher residual yolk weight at hatch due to higher incubation temperatures and concluded that the observation was related to an imbalance between metabolic rate and oxygen availability.
In the current study, the hatchability of chicks was dramatically lower at 39.5°C compared to 37.5°C. Incubation at 39.5°C from E12 to DOH reduced hatchability to 42%, compared to 93% at 37.5°C. On day 21 of incubation, chicks hatched from eggs incubated at 39.5°C were delayed compared to chicks hatched from eggs incubated at 37.5°C. As reported in other studies, high incubation temperature was associated with increased embryonic mortality and reduced hatchability and yolk utilisation.
To illustrate differences in heat stress between the embryonic chick and the post-hatch chick, the researchers pointed to previous studies that showed heat stress from day 22 to day 42 by exposure to 33°C for 10 hours per day decreases the jejunal protein expression level of OCLN and ZO1.
“By contrast, in our study there were increases of ZO1 and JAM2 mRNA in the small intestine at 39.5°C. Thus, for embryonic chicks, the increase in TJ mRNA may enhance the structural integrity of the epithelial barrier of the small intestine, whereas in the post-hatch chick the decrease in TJ mRNA may lead to a weakening of the epithelial barrier, perhaps leading to leaky gut syndrome,” they remarked.
In conclusion, the yolk sac tissue expresses zona occludens 1, occludin, claudin 1 and junctional adhesion molecule-A mRNA, which are important for maintaining the tight junction between epithelial cells that line the yolk sac tissue, similar to that of the small intestine. High incubation temperature (39.5°C) from E12 to DOH significantly affected the expression of zona occludens 1 and junctional adhesion molecule-1 mRNA in both the yolk sac tissue and the embryonic small intestine.
It was concluded: “This suggests that the tight junction in the vasculature of the yolk sac tissue and embryonic small intestine of broilers is affected by high incubation temperature.”