The inclusion of CSM in poultry feeds has been studied for decades. It has been found that high levels of CSM in the diet of laying hens can cause a deterioration in egg quality. In our previous research, we demonstrated that the “rubber egg” phenomenon is triggered by high levels of CSM in the diets of laying hens [8]. Gossypol (FG) and cyclopropenoid fatty acids (CPFAs) are the two main toxic substances found in CSM [11]. However, the component within the CSM which is responsible for the phenomenon remains unclear. In the present study, FG and CPFA were added separately to the diets of laying hens to investigate the causes and mechanisms involved in the “rubber egg” phenomenon. A high level of CSM was added to the diet via a different treatment group to act as a positive control. As expected, supplementation of the diet with FG or CPFA caused a reduction in productive performance when compared to the unsupplemented group. The intensity of laying and FCRs of the test groups both decreased but to different extents. Previous studies have shown that hens fed with diets containing FG at inclusion rates between 200 mg/kg and 400 mg/kg can result in a decrease in feed consumption, accompanied by a drop in egg production and egg size[12, 13].
In this study, we further tested the effects of adding 100 mg/kg and 200 mg/kg of FG to the feed but found no significant difference in egg production (data not shown). However, the addition of 400 mg/kg of FG lowered the egg production rate from the fourth week onwards when compared to the control group. It has also been reported that CPFA can reduce egg production only at concentrations unattainable via incorporation of common plant products into laying hen diets[14]. In the present study, we used an artificially high concentration of 300 mg CPFA/kg in the diet to obtain the observed effects on egg production. Moreover, egg production in the FG and CPFA groups were decreased significantly compared with the control group, but the reduction in the size of the eggs was observed only in the FG group.
The effects of FG and CPFA on egg production and discoloration were demonstrated in the present study. Many of the previous studies that investigated egg discoloration in relation to CSM inclusion in the diet used whole ground cottonseeds or CSM produced by screw press processing techniques, which left considerably more oil and FG in the meal [15]. Shenstone and Vickery (1959) [14]noted that egg yolks from hens fed with diets containing either malvalic acid or sterculic acid had surface colors of salmon or orange, a condition attributed to the formation of iron-conalbumin complexes which superimpose themselves on normal yolk pigments and produce pink-white discoloration. In the present study, we observed that a high content of CSM (as a positive control) caused a similar discoloration during cold storage. According to the results of Davis et al. (2002) [16] and Lordelo et al. (2007) [17], egg-yolk discoloration was positively associated with the presence of gossypol in the diet. However, here, we did not observe any apparent egg-yolk discoloration in the treatment group with added FG. This may be due to the lack of responsiveness of the hens to the egg discoloration effects of gossypolm [16]. The CSM and CPFA groups showed an increase in the occurrence and severity of pink albumen and yolk mottling in eggs that were under cold storage. These findings are in agreement with those of Kemmerer et al. (1963) [2]. When we tested the elasticity of the boiled yolks after storage at 4 °C for 2 to 6 weeks, it was found that only eggs from hens fed on diets containing CPFA showed changes (increase). These changes took place concurrently with the appearance of pink discoloration in the albumen.
To further explore the causes of the “rubber egg,” we measured the changes in pH of the yolk and albumen along with the concentrations of Fe and Ca. A modest increase in yolk pH coupled with a small increase in albumen pH was observed in the eggs from the CPFA and CSM groups, which is probably related to the presence of CPFA in the diet. However, the pH, Fe, and Ca concentrations in eggs from the FG and control groups were not affected. The pink albumen discoloration seen in cold storage eggs occurs after a relatively long period of storage because iron from yolk proteins must first diffuse out and combine with the albumen proteins [4]. In addition to iron, we found that other divalent ions, such as calcium, were also exchanged between the yolk and albumen.
Along with the observed changes in cation concentration, a higher yolk water content was also detected in the CPFA and CSM groups when compared to the control. Based on our results, we suggest that the adverse effects of a diet containing CPFA on egg quality can be explained by an increase in permeability of the vitelline membrane. Vitelline is a type of semi-permeable membrane that prevents the passing of large molecules from the yolk into the albumen [18]. Due to the presence of the vitelline membrane, the pH of the yolk changes at a slower rate when compared to albumen. Water in the albumen increases the permeability of the vitelline membrane and facilitates the passage of molecules into the yolk; in turn, the salinity and carbon dioxide present in the yolk permeates into the albumen [19].
We further observed the ultrastructure of the vitelline membrane and yolk granules. It was found that there was a change in the structure of the vitelline membrane in the CPFA and CSM groups, with different-sized holes distributed inside the vitelline membrane. The yolk granules in the CPFA group lost their normal structure and became irregularly shaped with thin slices after more than four weeks of cold storage. The ultrastructure of the granules in the egg yolk depends very much on the physicochemical characteristics of the surrounding medium, such as pH, ionic strength, and concentration of bivalent cations [20]. According to Causeret’s study, the modification of one of these parameters was sufficient to destructure the granules irreversibly. Here, we found that the pH of the yolk in the CPFA and CSM groups increased significantly while the concentration of calcium and iron decreased. These effects were accompanied by a partial destabilization of the microstructure of the yolk granules.