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The paradoxical effects of progesterone on the eggshell quality of laying hens
Journal Pre-proofs The paradoxical effects of progesterone on the eggshell quality of laying hens Jiacai Zhang, Zhiyun Wang, Xu Wang, Lvhui Sun, Shahid Ali Rajput, Desheng Qi PII: DOI: Reference:
S1047-8477(19)30256-4 https://doi.org/10.1016/j.jsb.2019.107430 YJSBI 107430
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Journal of Structural Biology
Received Date: Revised Date: Accepted Date:
29 September 2019 21 November 2019 22 November 2019
Please cite this article as: Zhang, J., Wang, Z., Wang, X., Sun, L., Rajput, S.A., Qi, D., The paradoxical effects of progesterone on the eggshell quality of laying hens, Journal of Structural Biology (2019), doi: https://doi.org/ 10.1016/j.jsb.2019.107430
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Title Page The paradoxical effects of progesterone on the eggshell quality of laying hens Jiacai Zhang, Zhiyun Wang, Xu Wang, Lvhui Sun, Shahid Ali Rajput, Desheng Qi* Department of Animal Nutrition and Feed Science, Huazhong Agricultural University, Wuhan 430070, Hubei, China. [email protected] (Jiacai Zhang); [email protected] (Zhiyun Wang); [email protected] (Xu Wang); [email protected] (Lvhui Sun); [email protected] (Shahid Ali Rajput); *Corresponding author: [email protected]; Tel.: +86-27-8728-1793
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Abstract: This study demonstrates the effects of progesterone on eggshell quality and ultrastructure by injecting progesterone into laying hens 2 and 5 h post-oviposition, respectively. Progesterone injected 2 h post-oviposition (P4-2 h) improved eggshell quality with a significant decrease (P < 0.01) in the thickness of the mammillary layer and a significant increase (P < 0.01) in the thickness of the effective layer in the eggshell ultrastructure compared to the control. Progesterone injected 5 h postoviposition (P4-5 h) damaged the eggshell quality by significantly reducing (P < 0.01) the effective layer thickness. Progesterone injected delayed obviously (P < 0.01) the following oviposition. Moreover, the concentrations of Thr, Cys, Leu, Lys, and His in the eggshell membranes were significantly higher (P < 0.05) in the P4-2 h treated hens whereas Val and Lys were significantly lower (P < 0.05) in P4-5 h treated hens compared to the control. Therefore, progesterone shows paradoxical effects on eggshell quality depending on the injection time-points post-oviposition, which could explain the contradictions in previous related reports. P4 injected affected the content of amino acids in eggshell membranes, especially lysine which contributed to eggshell quality. In addition, P4 injected 2 h after oviposition improved eggshell quality by promoting the premature fusion of mammillary knobs. This work contributed to a novel insight to understanding the mechanism of improving eggshell quality. Keywords: eggshell quality, eggshell ultrastructure, progesterone, amino acid, laying hens. 1. Introduction One of the common problems in the poultry industry is the decrease in eggshell
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quality, including eggshell thickness and strength, especially in the late stage of laying hens (Bar et al., 1999). The quantity of broken eggs increased and accounted for 7.5 % in aged laying hens (David et al., 1988), which is attributed to the reduced quality of the eggshell and leads to great economic losses. The eggshell is calcified in the uterus of laying hens and is mainly composed of 95 % calcium carbonate and 3.5 % organic matrix proteins (Marie et al., 2014). The eggshell protects the embryo from the harmful influence of outside environmental factors and regulates gas and water exchange (Narushina and Romanova, 2002.). Many studies have reported that eggshell quality is determined by the eggshell ultrastructure (Ahmed et al., 2005; Zhang et al., 2017). The eggshell consists of a organic membrane and a mineral layer. In the mineral layer, different regions can be differentiated: mammillary, palisade and vertical crystal layer, which is related to the eggshell strength (Carnarius et al., 1996). Progesterone (P4) is one of the most important gonadal hormones that maintain the reproduction of laying hens. Blood P4 in laying hens regularly fluctuates from ovulation to oviposition and achieves its highest level 4–6 h before ovulation (Haynes et al., 1973). A previous study reported that when 1 mg/kg body weight (BW) of P4 was injected 4 h after oviposition, the eggshell weight was increased (Nys et al., 1987), which implies that the P4 promoted calcium deposition in the eggshell. However, this result is inconsistent with other research which has shown that 1 mg/kg BW of P4 injected 2–3 h after oviposition inhibits calcium ion transport and reduces the abundance of calbindin mRNA in the uterus (Bar et al., 1996). In the above studies, there is a contradiction between the effects of P4 on eggshell weight and
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uterine calcium ion transport. Interestingly, a high incidence of hens producing hardshelled eggs was observed following P4 injection in another study (Liu & Bacon, 2005). In these studies, the time-points of P4 were different and the dose of P4 was 1 mg/kg BW which maybe was very high. In addition, in our previous study, the plasma P4 level was higher in the laying hens laid hard-shelled eggs than that laid weakshelled eggs during initiation period of calcification (Zhang er al., 2019). It suggested that P4 maybe regulate eggshell calcification before calcification occurs. According to the previous studies discussed above, it is unclear how P4 affects eggshell quality and the mechanism by which P4 affects eggshell quality remains to be determined. Thus, we hypothesized that injecting P4 affects eggshell quality and ultrastructure, and that the effects are different depending on the injection time-points after oviposition. Therefore, in present study, the same dose of P4 (0.15 mg/kg BW) was injected into laying hens 2 and 5 h after oviposition. The objective was to investigate the changes in eggshell ultrastructure caused by P4 injected at different time-points postoviposition. This study contributes to our understanding of the process of eggshell calcification in the uterus and the molecular mechanism by which P4 regulates eggshell quality. 2. Material and methods 2.1. Ethical statement and birds The experimental animal procedure was approved by the Scientific Ethics Committee of Huazhong Agricultural University on 10 July 2018. The ethical approval code is HZAUCH-2018-007. A total of 305 45-week-old Hy-Line Brown
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laying hens were caged individually and fed a layer mash ad libitum as recommended by the NRC (1994). The laying hens were subjected to 16 h light/day, namely the lights were turned on from 05:00 am to 08:00 am and from 17:00 pm to 21:00 pm, respectively. The oviposition time of each laying hen was observed and recorded from 05:00 am to 17:00 pm for 14 consecutive days, which contributed to the selection of hens with a stable oviposition time (with a fluctuation of less than 30 minutes). During this period, the eggshell strength of eggs from each hen was measured every day, which contributed to the selection of hens that laid eggs with an eggshell strength of 25.0–30.0 N. There were 100 laying hens that meet the above two conditions and these were used in the study. 2.2. Progesterone treatment P4 (P0130, Sigma-Aldrich, Saint Louis, Missouri, USA) was dissolved in peanut oil and then injected into the tibial muscle of laying hens. The 100 laying hens that met the selection criteria were randomly divided into 5 groups each with 20 birds, namely control, oil-2 h, oil-5 h, P4-2 h, and P4-5 h groups. The control group received no injection. The oil-2 h and oil-5 h groups received an injection of 0.2 mL peanut oil without P4 2 and 5 h after oviposition, respectively. The P4-2 h and P4-5 h groups received an injection of 0.2 mL peanut oil containing P4 2 and 5 h after oviposition, respectively. Series of different concentrations of progesterone dissolved in peanut oil were used to ensure the same injected volume (0.2 mL) and that the dose of P4 injected was 0.15 mg/kg BW. 2.3. Sample collection
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After the treatments, 10 hens per group were slaughtered during the growth period of eggshell calcification (14-16 h post-oviposition) according to the method described in previous studies (Marie et al., 2015; Rodríguez-Navarro et al., 2015) and the eggs were expelled from the uterus at the growth period of calcification (Expelled eggs). The eggs laid normally at completion by the other 10 hens per group were collected (Normal eggs) and the oviposition time of each hen was recorded. 2.4. The analysis of eggshell quality and ultrastructure Eggshell strength was determined using the Eggshell Force Gauge (EFG-0503, Robotmation Co., Ltd., Tokyo, Japan). Eggshell thickness was measured using an electronic digital micrometer (Shanghai Shenhan Measuring Tools Co., Ltd., Shanghai, China). The eggshell ultrastructures were scanned using a scanning electron microscope (JSM-6390LV, JEOL Ltd., Tokyo, Japan). All the collected eggs were broken manually after being washed with distilled water to remove dirt on the outer surface. Then the content of the interior was discarded and the inside of the shell was cleaned with distilled water to remove residual egg white. The eggshell membranes were removed according to the method described by Kaplan and Siegesmund (1973). Three fragments of eggshell were used to determine the eggshell thickness at three points: the blunt end, equator, and sharp end, and the eggshell thickness was determined by the mean of the three points (Chen et al., 2019). The equator fragments of eggshell without membranes were coated with gold powder and the transverse and inner surfaces were imaged (Gongruttananun, 2011). 2.5. Amino acid analysis of eggshell membranes
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The eggshell membranes of the normal eggs were submitted to an amino acid analysis using an amino acid analyzer (L-8900, Hitachi Ltd., Tokyo, Japan). The eggshell membrane was rinsed with distilled water and dried at room temperature ( 25.0-30.0 ℃ ) for 3 days. The dried eggshell membrane was ground into powder using a pestle and mortar. A sample of 10 mg powdered membrane was acid hydrolyzed according to the method described by Blake et al. (1985). A volume of 1 mL of the hydrolysate was dried with nitrogen, and 0.5 mL of 0.02 M HCl was added to dissolve the residue. The dissolved solution was filtered through Syringe Millex Filters (0.22 μm, Tianjin Alega Ltd., Tianjin, China). The filtrate was used for the amino acid analysis. The amino acid mixture standard solution (013-08391, Wako Ltd., Tokyo, Japan) was used for quantification. 2.6. Statistical analysis All values were analyzed by a one-way ANOVA followed by a Duncan’s test and presented as the mean ± standard deviation (SD). The statistical analysis was conducted using IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY, USA). P values < 0.05, were considered statistically significant. 3. Results and Discussion 3.1. Eggshell quality The expelled eggs and normal eggs are shown in Figure 1. The eggshell appearance of the expelled eggs in the control, oil-2 h, and oil-5 h groups is white. This is because the cuticle layer of the eggshell has not been formed and pigment has not been deposited on the surface of the eggshell. However, the eggshells of the
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expelled eggs are slightly brown in the P4-2 h group and dark brown in P4-5 h group. It may be that progesterone promoted the secretion of pigments in uterus, which caused the partial pigments were deposited into the eggshell in advance.In contrast, the normal eggs have darker eggshells in P4-2 h group and lighter eggshells in P4-5 h group than the control, oil-2 h, and oil-5 h groups. The eggshell strength, eggshell thickness, and the interval between ovipositions are presented in Figure 2. The expelled eggs were too weak to be able to detect their eggshell strength. Nonsignificant differences were observed for all parameters between the control, oil-2 h, and oil-5 h groups, which indicated that the injection of peanut oil without P4 did not affect eggshell calcification. The eggshell thickness of the expelled eggs (Figure 2A) was not affected by P4 injection. However, the eggshell strength and thickness of the normal eggs (Figure 2B, 2C) were significantly higher (P < 0.01) (86.1 % and 39.4 %, respectively) in P4-2 h group than the oil-2 h and significantly lower (P < 0.01) (53.0 % and 32.0 %, respectively) in P4-5 h group than the oil-5 h. This indicates that the effects of the injected P4 on the eggshell quality depended on the injection time. This result could explain the contradiction between previous studies in which the injection times after oviposition were different (Bar et al., 1996; Liu et al., 2005; Nys, 1987). In addition, the eggshell thickness of the expelled eggs was similar to that of the normal eggs in the P4-5 h group, and the cuticle layer of the expelled eggs (expelled eggs E in Figure 1) had begun to be formed, which indicates that P4 injected 5 h after oviposition inhibited and terminated eggshell calcification prematurely. The interval (Figure 2D) between ovipositions was increased significantly (P <
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0.01) both in the P4-2 h and P4-5 h groups compared to oil-2 h, and oil-5 h groups, respectively. This is consistent with the previous study by Nys (1987) that demonstrated that the length of the delay increased with the dose of P4. However, in present study, the length of the delay is related to injection time-points of P4. The delay might be attributed to P4 stimulating prostaglandin in the uterus (Lundholm, 1992). Moreover, the egg weight was not affected by P4 injection as shown in Figure 2E. Although eggshell calcification was affected, both egg white and egg yolk have been formed prior to P4 injection. The eggshell only accounts for a minor portion of the egg weight, resulting in no significant difference in egg weight between groups. 3.2. Eggshell ultrastructure Scanning electron microscopy photographs of the transverse surface of eggshell from expelled eggs and normal eggs are shown in Figures 3 and 4, respectively. The previous study reported that mammillary layer was formed in the initiation period and palisade layer was formed in the growth period of eggshell calcification (Hincke et al., 2011). As shown in Figure 3, the mammillary layer has formed and palisade layer was being formed. The thicknesses of the palisade layers are similar, indicating that eggshell calcification in each group is in the growth period. In addition, The outer surfaces of eggs removed in the initiation period of calcification were shown in Supplementary 1. The mammillary knobs were being formed, which indicated the eggs were in the initiation period of calcification. The mammillary layer thickness, mammillary knob width, and effective layer thickness were quantified and the data are provided in Figure 5. The mammillary
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layer thickness and mammillary knob width of the expelled eggs (Figure 5A) in the P4-2 h group were obviously decreased (P < 0.05) by P4 injected compared to expelled eggs in the oil-2 h group and were unchanged in the P4-5 h group compared to oil-5 h group. However, the effective layer thickness of the expelled eggs was significantly lower (P < 0.05) in the P4-5 h group than oil-5 h group. A similar trend among the normal eggs (Figure 5B) was observed, with a lower mammillary layer thickness and mammillary knob width in P4-2 h group compared to the oil-2 h group (P < 0.01 and P < 0.05, respectively) and no change in the P4-5 h group compared to oil-5 h group. Moreover, the effective layer thickness of the normal eggs increased (P < 0.01) in the P4-2 h group compared to the oil-2 h group and decreased significantly (P < 0.01) in the P4-5 h group compared to the oil-5 h group. The mammillary knobs of expelled eggs and normal eggs are shown in Supplementary 2 and 3, respectively. The density of mammillary knobs was not affected by P4 injection. In general, the density of mammillary knobs should be increased because of the reduction in mammillary knob width in P4-2 h group. However, it really was not reduced as shown in the supplementary figures. Mammillary knob is an inverted cone. Its upper part is wider than the lower part. The mammillary knob width was measured near the fusion area of the inverted cone according to a previous study (Dun er al., 2011). It indicated that if the mammillary knobs were fused early, the mammillary knob width near the fusion area is more narrow. Therefore, the mammillary knob width was decreased while the density of mammillary knobs was not affected in P4-2h group in the present study.
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It is consistent with the authors’ hypothesis that P4 would show paradoxical effects on eggshell quality according to the injection time-points after oviposition. Eggshell strength is positively correlated with eggshell thickness (Carnarius et al., 1996). A previous study also reported that weak eggshells with normal thickness as well as hard eggshells had an abnormal ultrastructure in which the mammillae were formed poorly (Bennett et al., 1988). Therefore, in addition to thickness, eggshell strength is also closely related to eggshell ultrastructure (Bain, 2005). The general structural organization of different avian eggshells is similar, although specific differences have been observed in the ultrastructure of the mammillary layer, which suggests that the mammillary layer plays a key role in determining eggshell strength (Panheleux et al., 1999). This has been demonstrated by many studies. Toledo et al. (1982) reported that mammillary knobs of weak eggshell were smaller and denser than hard eggshell. However, inconsistencies in the literature came to light when it was reported that the eggshell quality of aged hens was reduced with a decrease in the density of mammillary knobs compared to young hens (Sohn & Park, 2018) and that eggshell from post-molt laying hens had smaller mammillary knobs accompanied by a higher density of mammillae than pre-moult laying hens (Ahmed et al., 2005; Gongruttananun, 2018). However, in present study, no significant change caused by the P4 injection was observed in the density of mammillary knobs. The reduction in the thickness of the mammillary layer in the P4-2 h group might result from mammillary knobs fusing earlier than in the oil-2 h group, followed by formation of the palisade layer, which increases the thickness of the effective layer. This scenario
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is supported by previous research which showed that early fusion of the forming mammillary knobs is key to eggshell strength by reducing the penetration of intermammillary clefts (Parsons, 1982). Although the slight decrease in the mammillary layer thickness of the P4-5 h group might have been due to the injection time-point of P4, the mammillary layer is formed in the initiation period of eggshell calcification (Nys et al., 2010). The mammillae begin to be deposited on the outer shell membrane in the tubular shell gland 5 h after ovulation (Stemberger et al., 1977). When P4 injected is absorbed and acts, the deposition of the mammillary layer may be nearing the end in P4-5 h group. Thus, the efficacy of P4 injected 5 h after oviposition is minor. P4 injected prolonged the time the eggs remained in the uterus. The metabolism of P4 might induce opposite changes in the P4-2 h and P4-5 h groups. P4 is rapidly metabolized (Wells, 1971) and its direct effect is to inhibit calcium ion transport in the uterus (Bar et al., 1996). Therefore, P4 injected 2 h after oviposition would have been completely metabolized and the serum P4 returned to a normal physiological level when the eggshell calcification was initiated. Uterine calcium ion transport was not inhibited and the calcification period was prolonged, which led to the increase in eggshell thickness. In contrast, when P4 was injected 5 h after oviposition, uterine calcium ion transport was inhibited, which led to the eggshell calcification that has occurred has been suppressed. Thus, eggshell calcification could not proceed normally, which resulted in the decrease in eggshell thickness. The changes in the eggshell ultrastructure indicate that the fusion of the mammillary knobs is the key process in eggshell calcification that determines the
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eggshell strength. It guides us to study the mechanism by which P4 regulates the eggshell quality. Moreover, the finding of paradoxical effects of P4 injection on eggshell quality related to the timing of injection after oviposition indicates that it is difficult to improve eggshell quality by exogenous P4. In practice, farmers are unable to provide exogenous P4 at specific time-points after oviposition. However, the regulation of endogenous P4 at specific time-points is a new avenue to be explored to improve eggshell quality. 3.3. Amino acids in eggshell membrane The concentrations of amino acids in eggshell membranes are shown in Figure 6. Sixteen amino acids were quantified. Of these, the concentration of Thr, Cys, Leu, Lys, and His were significantly higher (P < 0.05) in the P4-2 h group than in the oil-2 h group. In addition, Val and Lys were lower (P < 0.05) in the P4-5 h group than in the oil-5 h group. The eggshell membrane is formed in the white isthmus of the oviduct (Hincke et al., 2012). Nucleation sites of mammillae are located in the outer shell membrane and their density determines the density of the mammillary knobs (Dunn et al., 2011). The components of the eggshell membrane affect the eggshell quality by altering the ultrastructure of the mammillary layer (Xiao et al., 2014). It has been reported that the isolated shell membrane can regulate calcium carbonate crystal deposition in vitro (Wu et al., 1995; Fernandez, et al., 2004). In addition, eggshell quality decreases with age and the eggshell membrane weight of old hens is lower than that of young hens (Britton, 1977; Kemps et al., 2006). These studies demonstrated that the eggshell
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membrane is a determinant of eggshell quality (Blake et al., 1985). However, research has indicated that the amino acid composition of the eggshell membranes of old and young hens show relatively small differences (Britton & Hale, 1977). Furthermore, there were no significant differences in the concentration of amino acids analyzed among hard and soft-shelled eggs (Klingensmith et al., 1988). However, the effects of P4 on the composition and concentration of amino acids in the eggshell membrane have not been reported. Therefore, this study investigated whether the composition and concentration of amino acids in the eggshell membranes were affected by P4 injection. The results showed that several amino acids were affected by P4 injection. Of the affected amino acids, Lys might play a key role. Lys forms desmosine and isodesmosine under the catalysis of lysyl oxidase which cross-links the amino acids, thus increasing the compactness of the eggshell membrane (Starcher and King, 1980). Desmosine and isodesmosine have been shown to play essential roles in normal eggshell formation and affect the size, shape, and eggshell texture of eggs (Arias et al., 1997; Chowdhurya, 1990). When formation of the cross-links was inhibited, large interstitial spaces and late fusion of the mammillary knobs occurred during eggshell calcification (Chowdhury & Davis, 1995). Thus, the high concentration of Lys contributes to eggshell strength. However, the mechanism by which P4 exerts an effect on Lys in the eggshell membrane requires further study. 4. Conclusion P4 injection produced two different effects on eggshell quality depending on the injection time after oviposition. P4 injected 2 h after oviposition improved eggshell
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quality by promoting the premature fusion of mammillary knobs and prolonging the calcification period of the eggshell in the uterus. This resulted in a significant reduction in the mammillary layer thickness and increase in the effective layer thickness, respectively. In contrast, P4 injected 5 h after oviposition reduced eggshell quality by decreasing the effective layer thickness and its mechanism was not clear while we speculated that uterine calcium ion transport was inhibited. In addition, injection of P4 affected the concentration of several amino acids in eggshell membranes, especially lysine. The paradoxical effects might be related to the rapid metabolism of P4. Acknowledgments This work was financially supported by the National Natural Science Foundation of China [program number 31572438]. In addition to providing funds, the foundation does not play any role in present study. We are also grateful to the Experimental and Teaching Center of college of animal nutrition and feed science for providing the instrument for determining eggshell strength. Authors' contributions Jiacai Zhang designed the study, carried out the experiments and analyses, wrote the paper. Zhiyun Wang, Xu Wang and Shahid Ali Rajput were involved in the feeding of laying hens and sample collection. Lvhui Sun contributed to detect mechanical properties of eggshells. Desheng Qi provided the strategy and idea and approve the version to be submitted. Appendix 15
Supplementary 1: Scanning electron microscopy images of outer surface of the eggs removed from uterus in initiation period. Supplementary 2: Scanning electron microscopy images of mammillary knobs of expelled eggs. MK: mammillary knobs. The figure shows the density of mammillary knobs. Supplementary 3: Scanning electron microscopy images of mammillary knobs of normal eggs. MK: mammillary knobs. The figure shows the density of mammillary knobs. Ethics approval The experiment was approved by the Scientific Ethics Committee of Huazhong Agricultural University. The ethical approval code is HZAUCH-2018-007. Conflict of interest The authors declare no competing financial interest. References Ahmed, A. M. H., Rodriguez-Navarro, A. B., Vidal, M. L., Gautron, J., García-Ruiz, J. M., & Nys, Y., 2005. Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens. Br. Poult. Sci. 46, 268-279. Arias, J. L., Cataldo, M., Fernandez, M. S., Kessi, E., 1997. Effect of betaaminoproprionitrile on eggshell formation. Br. Poult. Sci. 38, 349-354. Bain, M. M., 2005. Recent advances in the assessment of eggshell quality and their future application. Worlds Poult. Sci. J. 61, 268-277.
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Marie, P., Labas, V., Brionne, A., Harichaux, G., Hennequet-Antier, C., Nys, Y., Gautron, J., 2015. Quantitative proteomics and bioinformatic analysis provide new insight into protein function during avian eggshell biomineralization. J. Proteomics 126, 140-154. Marie, P., Labas, V., Brionne, A., Harichaux, G., Hennequet-Antier, C., Nys, Y., Gautron, J., 2014. Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization. Data Brief 1, 65-69. Narushina, V.G., Romanova, M.N., 2002. Egg physical characteristics and hatchability. Worlds Poult. Sci. J. 58, 297-303. NRC (National Research Council). 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Sci., Washington, DC Nys, Y., Hincke, M. T., Hernandez-Hernandez, A., Rodriguez-Navarro, A. B., Gomez-Morales, J., Jonchere, V., Garcia-Ruiz, J. M. Gautron, J., 2010. Eggshell ultrastructure, properties and the process of mineralization: Involvement of organic matrix in the eggshell fabric. Productions Anim. 23, 143-154. Nys, Y., 1987. Progesterone and testosterone elicit increases in the duration of shell formation in domestic hens. Br. Poult. Sci. 28, 57-68. Panheleux, M., Bain, M., Fernandez, M. S., Morales, I., Gautron, J., Arias, J. L., Solomon, S. E. Hincke, M., Nys, Y., 1999. Organic matrix composition and ultrastructure of eggshell: a comparative study. Br. Poult. Sci. 40, 240-252. Parsons, A. H., 1982. Structure of the eggshell. Poult. Sci. 61, 2013-2021. Rodr í guez-Navarro, A. B., Marie, P., Nys, Y., Hincke, M. T., Gautron, J., 2015.
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Amorphous calcium carbonate controls avian eggshell mineralization:A new paradigm for understanding rapid eggshell calcification. J. Struct. Biol. 190, 291303. Sohn, S.,Park, J., 2018. Ultrastructure and elementary composition of the eggshell according to hen age. J. Anim. Sci. 96, 518-519. Starcher, B. C., King, G. S., 1980. The presence of desmosine and isodesmosine in eggshell membrane protein. Connect Tissue Res. 8, 53-55. Stemberger, B. H., Mueller, W. J., Roland, M. L., 1977. Microscopic study of the initial stages of egg shell calcification. Poultry Science 56, 537-543. Toledo, B. V. , Parsons, A. H., Combs, G. F., 1982. Role of ultrastructure in determining eggshell strength. Poult. Sci. 61, 569-572. Wells, J. W., 1971. Metabolism of progesterone in the laying hen (Gallus domesticus). Comp. Biochem. Physiol. A Comp. Physiol. 40, 61-70. Wu, T.M., Rodriguez, J. P., Fink, D. J., Carrino, D. A., Blackwell, J.,Capalan, A. I., Heuer A. H., 1995. Crystallization studies on avian eggshell membranes: implications for the molecular factors controlling eggshell formation. Matrix Biol. 14, 507-513. Xiao, J. F., Zhang, Y. N., Wu, S. G., Zhang, H. J., Yue, H. Y., Qi, G. H., 2014. Manganese supplementation enhances the synthesis of glycosaminoglycan in eggshell membrane: A strategy to improve eggshell quality in laying hens. Poult. Sci. 93, 380-388. Zhang, J., Wang, Y., Zhang, C., Xiong, M., Rajput, S. A., Liu, Y., Qi, D., 2019. The
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differences of gonadal hormones and uterine transcriptome during shell calcification of hens laying hard or weakshelled eggs. BMC Genomics. 20, 707. Zhang, Y. N., Zhang, H. J., Wu, S. G., Wang, J., Qi, G. H., 2017. Dietary manganese supplementation modulated mechanical and ultrastructural changes during eggshell formation in laying hens. Poult. Sci. 96, 2699-2707. Figure Legends Figure 1：The eggshell appearance of expelled and normal eggs. A: control; B: oil-2 h; C: oil-5 h; D: P4-2 h; E: P4-5 h. Figure 2. The effects of progesterone on eggshell quality and the interval between ovipositions. All data were expressed as mean ± SD (n=8). Different symbols among groups indicate significant difference. **(P < 0.01) significant differences compared to the oil-2 h group and ##(P < 0.01) significant differences compared to the oil-5 h group. A: Expelled eggs; B, C, D, E: Normal eggs. Figure 3: Scanning electron microscopy images showing the transverse view of the eggshell ultrastructure from Expelled eggs. MK: mammillary knob; EL: effective layer; ML: mammillary layer. Figure 4: Scanning electron microscopy images showing the transverse view of the eggshell ultrastructure from normal eggs. MK: mammillary knob; EL: effective layer; ML: mammillary layer. Figure 5. Parameters of the mechanical properties of eggshell ultrastructure from expelled eggs and normal eggs. Results are reported as the mean ± SD (n=8). Different symbols among groups indicate significant difference. *,**Means in P4-
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2 h group differ significantly compared to oil-2 h group (*P < 0.05; **P < 0.01) and #,##Means in P4-2 h group differ significantly compared to oil-2 h group (#P < 0.05; ##P < 0.01). A: Expelled eggs; B: Normal eggs. MK: mammillary knob; EL: effective layer; ML: mammillary layer. Figure 6. The concentrations of amino acids in eggshell membranes from normal eggs. Results are reported as the mean ± SD (n=8). Different symbols among groups indicate significant difference. *Means in P4-2h group differ significantly compared to oil-2h group (*P < 0.05) and #Means in P4-5h group differ significantly compared to oil-5h group (#P < 0.05).
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Progesterone shown the paradoxical effects on eggshell quality.
The paradoxical effects depended on injected time of progesterone after oviposition.
Progesterone injected 2 h post-oviposition promoted the fusion of mammillary knobs.
Progesterone injected 2 h post-oviposition improved eggshell quality.
Progesterone injected 5 h post-oviposition reduced eggshell quality.
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S1047-8477(19)30256-4 https://doi.org/10.1016/j.jsb.2019.107430 YJSBI 107430
To appear in:
Journal of Structural Biology
Received Date: Revised Date: Accepted Date:
29 September 2019 21 November 2019 22 November 2019
Please cite this article as: Zhang, J., Wang, Z., Wang, X., Sun, L., Rajput, S.A., Qi, D., The paradoxical effects of progesterone on the eggshell quality of laying hens, Journal of Structural Biology (2019), doi: https://doi.org/ 10.1016/j.jsb.2019.107430
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Title Page The paradoxical effects of progesterone on the eggshell quality of laying hens Jiacai Zhang, Zhiyun Wang, Xu Wang, Lvhui Sun, Shahid Ali Rajput, Desheng Qi* Department of Animal Nutrition and Feed Science, Huazhong Agricultural University, Wuhan 430070, Hubei, China. [email protected] (Jiacai Zhang); [email protected] (Zhiyun Wang); [email protected] (Xu Wang); [email protected] (Lvhui Sun); [email protected] (Shahid Ali Rajput); *Corresponding author: [email protected]; Tel.: +86-27-8728-1793
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Abstract: This study demonstrates the effects of progesterone on eggshell quality and ultrastructure by injecting progesterone into laying hens 2 and 5 h post-oviposition, respectively. Progesterone injected 2 h post-oviposition (P4-2 h) improved eggshell quality with a significant decrease (P < 0.01) in the thickness of the mammillary layer and a significant increase (P < 0.01) in the thickness of the effective layer in the eggshell ultrastructure compared to the control. Progesterone injected 5 h postoviposition (P4-5 h) damaged the eggshell quality by significantly reducing (P < 0.01) the effective layer thickness. Progesterone injected delayed obviously (P < 0.01) the following oviposition. Moreover, the concentrations of Thr, Cys, Leu, Lys, and His in the eggshell membranes were significantly higher (P < 0.05) in the P4-2 h treated hens whereas Val and Lys were significantly lower (P < 0.05) in P4-5 h treated hens compared to the control. Therefore, progesterone shows paradoxical effects on eggshell quality depending on the injection time-points post-oviposition, which could explain the contradictions in previous related reports. P4 injected affected the content of amino acids in eggshell membranes, especially lysine which contributed to eggshell quality. In addition, P4 injected 2 h after oviposition improved eggshell quality by promoting the premature fusion of mammillary knobs. This work contributed to a novel insight to understanding the mechanism of improving eggshell quality. Keywords: eggshell quality, eggshell ultrastructure, progesterone, amino acid, laying hens. 1. Introduction One of the common problems in the poultry industry is the decrease in eggshell
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quality, including eggshell thickness and strength, especially in the late stage of laying hens (Bar et al., 1999). The quantity of broken eggs increased and accounted for 7.5 % in aged laying hens (David et al., 1988), which is attributed to the reduced quality of the eggshell and leads to great economic losses. The eggshell is calcified in the uterus of laying hens and is mainly composed of 95 % calcium carbonate and 3.5 % organic matrix proteins (Marie et al., 2014). The eggshell protects the embryo from the harmful influence of outside environmental factors and regulates gas and water exchange (Narushina and Romanova, 2002.). Many studies have reported that eggshell quality is determined by the eggshell ultrastructure (Ahmed et al., 2005; Zhang et al., 2017). The eggshell consists of a organic membrane and a mineral layer. In the mineral layer, different regions can be differentiated: mammillary, palisade and vertical crystal layer, which is related to the eggshell strength (Carnarius et al., 1996). Progesterone (P4) is one of the most important gonadal hormones that maintain the reproduction of laying hens. Blood P4 in laying hens regularly fluctuates from ovulation to oviposition and achieves its highest level 4–6 h before ovulation (Haynes et al., 1973). A previous study reported that when 1 mg/kg body weight (BW) of P4 was injected 4 h after oviposition, the eggshell weight was increased (Nys et al., 1987), which implies that the P4 promoted calcium deposition in the eggshell. However, this result is inconsistent with other research which has shown that 1 mg/kg BW of P4 injected 2–3 h after oviposition inhibits calcium ion transport and reduces the abundance of calbindin mRNA in the uterus (Bar et al., 1996). In the above studies, there is a contradiction between the effects of P4 on eggshell weight and
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uterine calcium ion transport. Interestingly, a high incidence of hens producing hardshelled eggs was observed following P4 injection in another study (Liu & Bacon, 2005). In these studies, the time-points of P4 were different and the dose of P4 was 1 mg/kg BW which maybe was very high. In addition, in our previous study, the plasma P4 level was higher in the laying hens laid hard-shelled eggs than that laid weakshelled eggs during initiation period of calcification (Zhang er al., 2019). It suggested that P4 maybe regulate eggshell calcification before calcification occurs. According to the previous studies discussed above, it is unclear how P4 affects eggshell quality and the mechanism by which P4 affects eggshell quality remains to be determined. Thus, we hypothesized that injecting P4 affects eggshell quality and ultrastructure, and that the effects are different depending on the injection time-points after oviposition. Therefore, in present study, the same dose of P4 (0.15 mg/kg BW) was injected into laying hens 2 and 5 h after oviposition. The objective was to investigate the changes in eggshell ultrastructure caused by P4 injected at different time-points postoviposition. This study contributes to our understanding of the process of eggshell calcification in the uterus and the molecular mechanism by which P4 regulates eggshell quality. 2. Material and methods 2.1. Ethical statement and birds The experimental animal procedure was approved by the Scientific Ethics Committee of Huazhong Agricultural University on 10 July 2018. The ethical approval code is HZAUCH-2018-007. A total of 305 45-week-old Hy-Line Brown
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laying hens were caged individually and fed a layer mash ad libitum as recommended by the NRC (1994). The laying hens were subjected to 16 h light/day, namely the lights were turned on from 05:00 am to 08:00 am and from 17:00 pm to 21:00 pm, respectively. The oviposition time of each laying hen was observed and recorded from 05:00 am to 17:00 pm for 14 consecutive days, which contributed to the selection of hens with a stable oviposition time (with a fluctuation of less than 30 minutes). During this period, the eggshell strength of eggs from each hen was measured every day, which contributed to the selection of hens that laid eggs with an eggshell strength of 25.0–30.0 N. There were 100 laying hens that meet the above two conditions and these were used in the study. 2.2. Progesterone treatment P4 (P0130, Sigma-Aldrich, Saint Louis, Missouri, USA) was dissolved in peanut oil and then injected into the tibial muscle of laying hens. The 100 laying hens that met the selection criteria were randomly divided into 5 groups each with 20 birds, namely control, oil-2 h, oil-5 h, P4-2 h, and P4-5 h groups. The control group received no injection. The oil-2 h and oil-5 h groups received an injection of 0.2 mL peanut oil without P4 2 and 5 h after oviposition, respectively. The P4-2 h and P4-5 h groups received an injection of 0.2 mL peanut oil containing P4 2 and 5 h after oviposition, respectively. Series of different concentrations of progesterone dissolved in peanut oil were used to ensure the same injected volume (0.2 mL) and that the dose of P4 injected was 0.15 mg/kg BW. 2.3. Sample collection
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After the treatments, 10 hens per group were slaughtered during the growth period of eggshell calcification (14-16 h post-oviposition) according to the method described in previous studies (Marie et al., 2015; Rodríguez-Navarro et al., 2015) and the eggs were expelled from the uterus at the growth period of calcification (Expelled eggs). The eggs laid normally at completion by the other 10 hens per group were collected (Normal eggs) and the oviposition time of each hen was recorded. 2.4. The analysis of eggshell quality and ultrastructure Eggshell strength was determined using the Eggshell Force Gauge (EFG-0503, Robotmation Co., Ltd., Tokyo, Japan). Eggshell thickness was measured using an electronic digital micrometer (Shanghai Shenhan Measuring Tools Co., Ltd., Shanghai, China). The eggshell ultrastructures were scanned using a scanning electron microscope (JSM-6390LV, JEOL Ltd., Tokyo, Japan). All the collected eggs were broken manually after being washed with distilled water to remove dirt on the outer surface. Then the content of the interior was discarded and the inside of the shell was cleaned with distilled water to remove residual egg white. The eggshell membranes were removed according to the method described by Kaplan and Siegesmund (1973). Three fragments of eggshell were used to determine the eggshell thickness at three points: the blunt end, equator, and sharp end, and the eggshell thickness was determined by the mean of the three points (Chen et al., 2019). The equator fragments of eggshell without membranes were coated with gold powder and the transverse and inner surfaces were imaged (Gongruttananun, 2011). 2.5. Amino acid analysis of eggshell membranes
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The eggshell membranes of the normal eggs were submitted to an amino acid analysis using an amino acid analyzer (L-8900, Hitachi Ltd., Tokyo, Japan). The eggshell membrane was rinsed with distilled water and dried at room temperature ( 25.0-30.0 ℃ ) for 3 days. The dried eggshell membrane was ground into powder using a pestle and mortar. A sample of 10 mg powdered membrane was acid hydrolyzed according to the method described by Blake et al. (1985). A volume of 1 mL of the hydrolysate was dried with nitrogen, and 0.5 mL of 0.02 M HCl was added to dissolve the residue. The dissolved solution was filtered through Syringe Millex Filters (0.22 μm, Tianjin Alega Ltd., Tianjin, China). The filtrate was used for the amino acid analysis. The amino acid mixture standard solution (013-08391, Wako Ltd., Tokyo, Japan) was used for quantification. 2.6. Statistical analysis All values were analyzed by a one-way ANOVA followed by a Duncan’s test and presented as the mean ± standard deviation (SD). The statistical analysis was conducted using IBM SPSS Statistics 20 (IBM Corporation, Armonk, NY, USA). P values < 0.05, were considered statistically significant. 3. Results and Discussion 3.1. Eggshell quality The expelled eggs and normal eggs are shown in Figure 1. The eggshell appearance of the expelled eggs in the control, oil-2 h, and oil-5 h groups is white. This is because the cuticle layer of the eggshell has not been formed and pigment has not been deposited on the surface of the eggshell. However, the eggshells of the
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expelled eggs are slightly brown in the P4-2 h group and dark brown in P4-5 h group. It may be that progesterone promoted the secretion of pigments in uterus, which caused the partial pigments were deposited into the eggshell in advance.In contrast, the normal eggs have darker eggshells in P4-2 h group and lighter eggshells in P4-5 h group than the control, oil-2 h, and oil-5 h groups. The eggshell strength, eggshell thickness, and the interval between ovipositions are presented in Figure 2. The expelled eggs were too weak to be able to detect their eggshell strength. Nonsignificant differences were observed for all parameters between the control, oil-2 h, and oil-5 h groups, which indicated that the injection of peanut oil without P4 did not affect eggshell calcification. The eggshell thickness of the expelled eggs (Figure 2A) was not affected by P4 injection. However, the eggshell strength and thickness of the normal eggs (Figure 2B, 2C) were significantly higher (P < 0.01) (86.1 % and 39.4 %, respectively) in P4-2 h group than the oil-2 h and significantly lower (P < 0.01) (53.0 % and 32.0 %, respectively) in P4-5 h group than the oil-5 h. This indicates that the effects of the injected P4 on the eggshell quality depended on the injection time. This result could explain the contradiction between previous studies in which the injection times after oviposition were different (Bar et al., 1996; Liu et al., 2005; Nys, 1987). In addition, the eggshell thickness of the expelled eggs was similar to that of the normal eggs in the P4-5 h group, and the cuticle layer of the expelled eggs (expelled eggs E in Figure 1) had begun to be formed, which indicates that P4 injected 5 h after oviposition inhibited and terminated eggshell calcification prematurely. The interval (Figure 2D) between ovipositions was increased significantly (P <
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0.01) both in the P4-2 h and P4-5 h groups compared to oil-2 h, and oil-5 h groups, respectively. This is consistent with the previous study by Nys (1987) that demonstrated that the length of the delay increased with the dose of P4. However, in present study, the length of the delay is related to injection time-points of P4. The delay might be attributed to P4 stimulating prostaglandin in the uterus (Lundholm, 1992). Moreover, the egg weight was not affected by P4 injection as shown in Figure 2E. Although eggshell calcification was affected, both egg white and egg yolk have been formed prior to P4 injection. The eggshell only accounts for a minor portion of the egg weight, resulting in no significant difference in egg weight between groups. 3.2. Eggshell ultrastructure Scanning electron microscopy photographs of the transverse surface of eggshell from expelled eggs and normal eggs are shown in Figures 3 and 4, respectively. The previous study reported that mammillary layer was formed in the initiation period and palisade layer was formed in the growth period of eggshell calcification (Hincke et al., 2011). As shown in Figure 3, the mammillary layer has formed and palisade layer was being formed. The thicknesses of the palisade layers are similar, indicating that eggshell calcification in each group is in the growth period. In addition, The outer surfaces of eggs removed in the initiation period of calcification were shown in Supplementary 1. The mammillary knobs were being formed, which indicated the eggs were in the initiation period of calcification. The mammillary layer thickness, mammillary knob width, and effective layer thickness were quantified and the data are provided in Figure 5. The mammillary
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layer thickness and mammillary knob width of the expelled eggs (Figure 5A) in the P4-2 h group were obviously decreased (P < 0.05) by P4 injected compared to expelled eggs in the oil-2 h group and were unchanged in the P4-5 h group compared to oil-5 h group. However, the effective layer thickness of the expelled eggs was significantly lower (P < 0.05) in the P4-5 h group than oil-5 h group. A similar trend among the normal eggs (Figure 5B) was observed, with a lower mammillary layer thickness and mammillary knob width in P4-2 h group compared to the oil-2 h group (P < 0.01 and P < 0.05, respectively) and no change in the P4-5 h group compared to oil-5 h group. Moreover, the effective layer thickness of the normal eggs increased (P < 0.01) in the P4-2 h group compared to the oil-2 h group and decreased significantly (P < 0.01) in the P4-5 h group compared to the oil-5 h group. The mammillary knobs of expelled eggs and normal eggs are shown in Supplementary 2 and 3, respectively. The density of mammillary knobs was not affected by P4 injection. In general, the density of mammillary knobs should be increased because of the reduction in mammillary knob width in P4-2 h group. However, it really was not reduced as shown in the supplementary figures. Mammillary knob is an inverted cone. Its upper part is wider than the lower part. The mammillary knob width was measured near the fusion area of the inverted cone according to a previous study (Dun er al., 2011). It indicated that if the mammillary knobs were fused early, the mammillary knob width near the fusion area is more narrow. Therefore, the mammillary knob width was decreased while the density of mammillary knobs was not affected in P4-2h group in the present study.
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It is consistent with the authors’ hypothesis that P4 would show paradoxical effects on eggshell quality according to the injection time-points after oviposition. Eggshell strength is positively correlated with eggshell thickness (Carnarius et al., 1996). A previous study also reported that weak eggshells with normal thickness as well as hard eggshells had an abnormal ultrastructure in which the mammillae were formed poorly (Bennett et al., 1988). Therefore, in addition to thickness, eggshell strength is also closely related to eggshell ultrastructure (Bain, 2005). The general structural organization of different avian eggshells is similar, although specific differences have been observed in the ultrastructure of the mammillary layer, which suggests that the mammillary layer plays a key role in determining eggshell strength (Panheleux et al., 1999). This has been demonstrated by many studies. Toledo et al. (1982) reported that mammillary knobs of weak eggshell were smaller and denser than hard eggshell. However, inconsistencies in the literature came to light when it was reported that the eggshell quality of aged hens was reduced with a decrease in the density of mammillary knobs compared to young hens (Sohn & Park, 2018) and that eggshell from post-molt laying hens had smaller mammillary knobs accompanied by a higher density of mammillae than pre-moult laying hens (Ahmed et al., 2005; Gongruttananun, 2018). However, in present study, no significant change caused by the P4 injection was observed in the density of mammillary knobs. The reduction in the thickness of the mammillary layer in the P4-2 h group might result from mammillary knobs fusing earlier than in the oil-2 h group, followed by formation of the palisade layer, which increases the thickness of the effective layer. This scenario
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is supported by previous research which showed that early fusion of the forming mammillary knobs is key to eggshell strength by reducing the penetration of intermammillary clefts (Parsons, 1982). Although the slight decrease in the mammillary layer thickness of the P4-5 h group might have been due to the injection time-point of P4, the mammillary layer is formed in the initiation period of eggshell calcification (Nys et al., 2010). The mammillae begin to be deposited on the outer shell membrane in the tubular shell gland 5 h after ovulation (Stemberger et al., 1977). When P4 injected is absorbed and acts, the deposition of the mammillary layer may be nearing the end in P4-5 h group. Thus, the efficacy of P4 injected 5 h after oviposition is minor. P4 injected prolonged the time the eggs remained in the uterus. The metabolism of P4 might induce opposite changes in the P4-2 h and P4-5 h groups. P4 is rapidly metabolized (Wells, 1971) and its direct effect is to inhibit calcium ion transport in the uterus (Bar et al., 1996). Therefore, P4 injected 2 h after oviposition would have been completely metabolized and the serum P4 returned to a normal physiological level when the eggshell calcification was initiated. Uterine calcium ion transport was not inhibited and the calcification period was prolonged, which led to the increase in eggshell thickness. In contrast, when P4 was injected 5 h after oviposition, uterine calcium ion transport was inhibited, which led to the eggshell calcification that has occurred has been suppressed. Thus, eggshell calcification could not proceed normally, which resulted in the decrease in eggshell thickness. The changes in the eggshell ultrastructure indicate that the fusion of the mammillary knobs is the key process in eggshell calcification that determines the
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eggshell strength. It guides us to study the mechanism by which P4 regulates the eggshell quality. Moreover, the finding of paradoxical effects of P4 injection on eggshell quality related to the timing of injection after oviposition indicates that it is difficult to improve eggshell quality by exogenous P4. In practice, farmers are unable to provide exogenous P4 at specific time-points after oviposition. However, the regulation of endogenous P4 at specific time-points is a new avenue to be explored to improve eggshell quality. 3.3. Amino acids in eggshell membrane The concentrations of amino acids in eggshell membranes are shown in Figure 6. Sixteen amino acids were quantified. Of these, the concentration of Thr, Cys, Leu, Lys, and His were significantly higher (P < 0.05) in the P4-2 h group than in the oil-2 h group. In addition, Val and Lys were lower (P < 0.05) in the P4-5 h group than in the oil-5 h group. The eggshell membrane is formed in the white isthmus of the oviduct (Hincke et al., 2012). Nucleation sites of mammillae are located in the outer shell membrane and their density determines the density of the mammillary knobs (Dunn et al., 2011). The components of the eggshell membrane affect the eggshell quality by altering the ultrastructure of the mammillary layer (Xiao et al., 2014). It has been reported that the isolated shell membrane can regulate calcium carbonate crystal deposition in vitro (Wu et al., 1995; Fernandez, et al., 2004). In addition, eggshell quality decreases with age and the eggshell membrane weight of old hens is lower than that of young hens (Britton, 1977; Kemps et al., 2006). These studies demonstrated that the eggshell
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membrane is a determinant of eggshell quality (Blake et al., 1985). However, research has indicated that the amino acid composition of the eggshell membranes of old and young hens show relatively small differences (Britton & Hale, 1977). Furthermore, there were no significant differences in the concentration of amino acids analyzed among hard and soft-shelled eggs (Klingensmith et al., 1988). However, the effects of P4 on the composition and concentration of amino acids in the eggshell membrane have not been reported. Therefore, this study investigated whether the composition and concentration of amino acids in the eggshell membranes were affected by P4 injection. The results showed that several amino acids were affected by P4 injection. Of the affected amino acids, Lys might play a key role. Lys forms desmosine and isodesmosine under the catalysis of lysyl oxidase which cross-links the amino acids, thus increasing the compactness of the eggshell membrane (Starcher and King, 1980). Desmosine and isodesmosine have been shown to play essential roles in normal eggshell formation and affect the size, shape, and eggshell texture of eggs (Arias et al., 1997; Chowdhurya, 1990). When formation of the cross-links was inhibited, large interstitial spaces and late fusion of the mammillary knobs occurred during eggshell calcification (Chowdhury & Davis, 1995). Thus, the high concentration of Lys contributes to eggshell strength. However, the mechanism by which P4 exerts an effect on Lys in the eggshell membrane requires further study. 4. Conclusion P4 injection produced two different effects on eggshell quality depending on the injection time after oviposition. P4 injected 2 h after oviposition improved eggshell
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quality by promoting the premature fusion of mammillary knobs and prolonging the calcification period of the eggshell in the uterus. This resulted in a significant reduction in the mammillary layer thickness and increase in the effective layer thickness, respectively. In contrast, P4 injected 5 h after oviposition reduced eggshell quality by decreasing the effective layer thickness and its mechanism was not clear while we speculated that uterine calcium ion transport was inhibited. In addition, injection of P4 affected the concentration of several amino acids in eggshell membranes, especially lysine. The paradoxical effects might be related to the rapid metabolism of P4. Acknowledgments This work was financially supported by the National Natural Science Foundation of China [program number 31572438]. In addition to providing funds, the foundation does not play any role in present study. We are also grateful to the Experimental and Teaching Center of college of animal nutrition and feed science for providing the instrument for determining eggshell strength. Authors' contributions Jiacai Zhang designed the study, carried out the experiments and analyses, wrote the paper. Zhiyun Wang, Xu Wang and Shahid Ali Rajput were involved in the feeding of laying hens and sample collection. Lvhui Sun contributed to detect mechanical properties of eggshells. Desheng Qi provided the strategy and idea and approve the version to be submitted. Appendix 15
Supplementary 1: Scanning electron microscopy images of outer surface of the eggs removed from uterus in initiation period. Supplementary 2: Scanning electron microscopy images of mammillary knobs of expelled eggs. MK: mammillary knobs. The figure shows the density of mammillary knobs. Supplementary 3: Scanning electron microscopy images of mammillary knobs of normal eggs. MK: mammillary knobs. The figure shows the density of mammillary knobs. Ethics approval The experiment was approved by the Scientific Ethics Committee of Huazhong Agricultural University. The ethical approval code is HZAUCH-2018-007. Conflict of interest The authors declare no competing financial interest. References Ahmed, A. M. H., Rodriguez-Navarro, A. B., Vidal, M. L., Gautron, J., García-Ruiz, J. M., & Nys, Y., 2005. Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens. Br. Poult. Sci. 46, 268-279. Arias, J. L., Cataldo, M., Fernandez, M. S., Kessi, E., 1997. Effect of betaaminoproprionitrile on eggshell formation. Br. Poult. Sci. 38, 349-354. Bain, M. M., 2005. Recent advances in the assessment of eggshell quality and their future application. Worlds Poult. Sci. J. 61, 268-277.
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differences of gonadal hormones and uterine transcriptome during shell calcification of hens laying hard or weakshelled eggs. BMC Genomics. 20, 707. Zhang, Y. N., Zhang, H. J., Wu, S. G., Wang, J., Qi, G. H., 2017. Dietary manganese supplementation modulated mechanical and ultrastructural changes during eggshell formation in laying hens. Poult. Sci. 96, 2699-2707. Figure Legends Figure 1：The eggshell appearance of expelled and normal eggs. A: control; B: oil-2 h; C: oil-5 h; D: P4-2 h; E: P4-5 h. Figure 2. The effects of progesterone on eggshell quality and the interval between ovipositions. All data were expressed as mean ± SD (n=8). Different symbols among groups indicate significant difference. **(P < 0.01) significant differences compared to the oil-2 h group and ##(P < 0.01) significant differences compared to the oil-5 h group. A: Expelled eggs; B, C, D, E: Normal eggs. Figure 3: Scanning electron microscopy images showing the transverse view of the eggshell ultrastructure from Expelled eggs. MK: mammillary knob; EL: effective layer; ML: mammillary layer. Figure 4: Scanning electron microscopy images showing the transverse view of the eggshell ultrastructure from normal eggs. MK: mammillary knob; EL: effective layer; ML: mammillary layer. Figure 5. Parameters of the mechanical properties of eggshell ultrastructure from expelled eggs and normal eggs. Results are reported as the mean ± SD (n=8). Different symbols among groups indicate significant difference. *,**Means in P4-
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2 h group differ significantly compared to oil-2 h group (*P < 0.05; **P < 0.01) and #,##Means in P4-2 h group differ significantly compared to oil-2 h group (#P < 0.05; ##P < 0.01). A: Expelled eggs; B: Normal eggs. MK: mammillary knob; EL: effective layer; ML: mammillary layer. Figure 6. The concentrations of amino acids in eggshell membranes from normal eggs. Results are reported as the mean ± SD (n=8). Different symbols among groups indicate significant difference. *Means in P4-2h group differ significantly compared to oil-2h group (*P < 0.05) and #Means in P4-5h group differ significantly compared to oil-5h group (#P < 0.05).
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Progesterone shown the paradoxical effects on eggshell quality.
The paradoxical effects depended on injected time of progesterone after oviposition.
Progesterone injected 2 h post-oviposition promoted the fusion of mammillary knobs.
Progesterone injected 2 h post-oviposition improved eggshell quality.
Progesterone injected 5 h post-oviposition reduced eggshell quality.
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