A fecal anaerobic coryneform bacteria strain CML199, a biological agent and its application in preparing a product for improving laying hen performance

Abstract

The present invention relates to the field of microorganisms, and in particular to a strain of anaerobic fecal coryneform bacteria CML199, a biological preparation and its application in preparing products for improving the performance of laying hens. The present invention has discovered a new anaerobic fecal coryneform bacteria (Anaerostipes caccae) CML199 without toxic side effects, and its preservation number is CGMCC No.31340. The anaerobic fecal coryneform bacteria can promote the bone development of laying hens, increase the level of bone formation markers in the femur, increase the level of bone formation markers in serum, improve the bone marrow immune microenvironment, and improve the eggshell quality. It is expected to be developed into a new and effective probiotic preparation, and has a very broad application prospect in the laying hen industry.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to a fecal anaerobic coryneform bacteria CML199, a biological agent and application thereof in preparing a product for improving laying hen performance. Background Art

[0002] The structure of my country's laying hen industry is changing from a quantitative growth model to a quality and efficiency model. Extending the egg-laying cycle is an effective solution to promote industrial transformation and achieve sustainable development. Extending the egg-laying cycle places higher demands on egg quality and the health of laying hens, especially on the long-term health of tissues and organs involved in egg production. Bones are one of the organs most relevant to egg-laying performance. Ensuring the full development of laying hen bones is of great significance to maintaining the health and egg-laying performance of laying hens. Bone development is regulated by many factors, such as nutrition (calcium, phosphorus, vitamin D), hormones (parathyroid hormone and estrogen), and immune cells.

[0003] At present, people use special laying hen feed to improve the bone development and eggshell quality of laying hens. However, the cost of special feed is high, which often causes a serious economic burden to breeders. In recent years, more and more studies have found that intestinal microorganisms and their metabolites can participate in the above-mentioned regulatory process and ultimately affect bone metabolism. At present, there are deficiencies in the research on the regulation of laying hen bone development by intestinal microorganisms, and the poultry intestinal microbial germplasm resources are in urgent need of development. Therefore, the field urgently screens new probiotics that have no side effects and can be used to regulate the bone development of laying hens. Summary of the invention

[0004] The purpose of the present invention is to provide a strain of anaerobic coryneform bacteria CML199, a biological preparation and its application in preparing a product for improving the performance of laying hens, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a strain of anaerobic coryneform bacteria CML199, which can promote the bone development of laying hens, increase the level of bone formation markers in the femur, increase the level of bone formation markers in serum, improve the bone marrow immune microenvironment, improve the eggshell quality and improve the intestinal environment.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The invention provides an anaerobic fecal coryneform bacteria (Anaerostipes caccae) CML199, and the preservation number of the anaerobic fecal coryneform bacteria CML199 is CGMCC No.31340.

[0007] The present invention provides the use of the above-mentioned fecal anaerobic coryneform bacteria CML199 in any one or more of the following items:

[0008] (1) Application in the preparation of products for promoting bone development in laying hens;

[0009] (2) Application in the preparation of a product for increasing the level of bone formation markers in laying hens' femurs;

[0010] (3) Application in the preparation of products for increasing the level of bone formation markers in laying hen serum;

[0011] (4) Application in the preparation of products for improving the immune microenvironment of laying hens’ bone marrow;

[0012] (5) Application in the preparation of products for improving eggshell quality;

[0013] (6) Application in the preparation of products for improving the intestinal environment of laying hens.

[0014] The present invention provides a microbial preparation, wherein the effective components of the microbial preparation include the above-mentioned fecal anaerobic coryneform bacteria CML199.

[0015] Further preferably, the active ingredients of the microbial preparation also include the culture (metabolites) of the fecal anaerobic Corynebacterium CML199.

[0016] Further preferably, the culture of anaerobic coryneform bacteria is a substance obtained by culturing anaerobic coryneform bacteria CML199 in a culture medium; such as a substance containing anaerobic coryneform bacteria CML199 and secreted into a liquid culture medium, i.e., a fermentation broth, or a substance containing anaerobic coryneform bacteria CML199 and secreted into a solid culture medium.

[0017] Further preferably, when the active ingredients of the microbial preparation include Anaerobic Corynebacterium feces CML199 and / or a culture of Anaerobic Corynebacterium feces CML199, the microbial preparation may be in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate.

[0018] Further preferably, the active ingredients of the microbial preparation may also contain other biological components or non-biological components; the other active ingredients can be determined by those skilled in the art according to the effects of the microbial preparation.

[0019] Preferably, the microbial preparation further comprises a carrier.

[0020] Further preferably, the carrier may be a solid carrier or a liquid carrier.

[0021] Further preferably, the solid carrier is a mineral material and / or a biological material; the mineral material may be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the biological material is at least one of various crop straws, pine husks, straw, peanut shells, corn flour, soybean flour, starch, peat and animal feces; the liquid carrier may be water.

[0022] Further preferably, surfactants (such as Tween 20, Tween 80, etc.), adhesives, stabilizers (such as antioxidants) and pH regulators, etc., may be added to the microbial preparation.

[0023] The present invention provides a method for preparing the above-mentioned microbial preparation, comprising the following steps:

[0024] The fecal anaerobic coryneform bacteria CML199 is inoculated into a culture medium for culturing to obtain the microbial preparation.

[0025] Preferably, the culture medium is a liquid culture medium or a solid culture medium.

[0026] Preferably, the culture time is 12 hours and the temperature is 37°C.

[0027] The present invention provides the use of the above-mentioned microbial preparation in any one or more of the following items:

[0028] (1) Application in the preparation of products for promoting bone development in laying hens;

[0029] (2) Application in the preparation of a product for increasing the level of bone formation markers in laying hens' femurs;

[0030] (3) Application in the preparation of products for increasing the level of bone formation markers in laying hen serum;

[0031] (4) Application in the preparation of products for improving the immune microenvironment of laying hens’ bone marrow;

[0032] (5) Application in the preparation of products for improving eggshell quality;

[0033] (6) Application in the preparation of products for improving the intestinal environment of laying hens.

[0034] The present invention discloses the following technical effects:

[0035] The present invention has discovered a new fecal anaerobic bacterium (Anaerostipes caccae) CML199 without toxic side effects. The fecal anaerobic bacterium species can promote the bone development of laying hens, increase the level of bone formation markers in the femur, increase the level of bone formation markers in serum, improve the bone marrow immune microenvironment, improve the eggshell quality and improve the intestinal environment. It is expected to be developed into a new and effective probiotic preparation and has a very broad application prospect in the laying hen industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 This is the result of comparing the PCR amplified sequence of the bacterial solution of anaerobic coryneformis CML199 on the EzBioCloud database;

[0038] Figure 2 The figure is the growth curve of fecal anaerobic Corynebacterium CML199 drawn under anaerobic conditions;

[0039] Figure 3 The results of anaerobic bacterium CML199 in fecal anaerobic bacteria promoting bone development in laying hens; the ordinate is the quantification of bone microstructure parameters, AF represents cortical bone volume, cortical bone thickness, cortical bone mineral content, trabecular volume fraction, trabecular thickness and trabecular bone density, respectively; * represents P<0.05, ** represents P<0.01; A.caccae represents anaerobic bacterium CML199 in fecal anaerobic bacteria.

[0040] Figure 4 The results of fecal anaerobic Corynebacterium CML199 increasing the level of bone formation markers in the femur; the ordinate is the alkaline phosphatase activity of laying hens' femurs, ** represents P<0.01; A.caccae is fecal anaerobic Corynebacterium CML199;

[0041] Figure 5 The results of fecal anaerobic Corynebacterium CML199 increasing the level of bone formation markers in serum; the ordinate is the serum osteocalcin concentration of laying hens, ** represents P < 0.01; A.caccae is fecal anaerobic Corynebacterium CML199;

[0042] Figure 6 The results of fecal anaerobic Corynebacterium CML199 improving the bone marrow immune microenvironment; A is CD25 positive cells (CD25 + cells), B is the percentage of KuL01 positive cells (KuL01 + The ordinate is the proportion of related immune cells in the bone marrow of laying hens, * represents P < 0.05, ** represents P < 0.01; A.caccae is fecal anaerobic rod-shaped bacteria CML199;

[0043] Figure 7The results of anaerobic bacterium CML199 improving eggshell quality; A is eggshell thickness, B is eggshell strength, and C is egg shape index; * represents P<0.05, and ** represents P<0.01; A.caccae represents anaerobic bacterium CML199.

[0044] Biological deposit information

[0045] Anaerostipes caccae CML199 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on July 17, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31340. DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1 Isolation, Culture and Identification of Anaerostipes caccae CML199

[0052] 1. Isolation and cultivation of fecal anaerobic coryneform bacteria

[0053] The isolated sample came from the cecal digesta of a healthy laying hen. The microorganisms were isolated and purified by 10-fold gradient dilution plate method. The modified GAM medium (Beijing Baiwangtongda Biotechnology Co., Ltd.) was used for anaerobic culture in a 37°C constant temperature anaerobic operation box (Whitley DG250Anaerobic Workstation, Bailai Puhui (Beijing) Technology Co., Ltd.) for 48 hours. The anaerobic gas composition was N2, CO2 and H2, and the volume ratio of N2, CO2 and H2 was 80:10:10. Then, a single colony was picked for plate streaking to obtain pure culture of each single strain. Refer to the kit ( The genomic DNA of the isolated strains was extracted by Blood & Tissue Kit (QIAGEN), and detected by 1% agarose gel electrophoresis, without diffusion or tailing; and the extracted DNA was tested for quality and concentration by UV spectrophotometer. DNA samples with A260 / 280 ratio of 1.8-2.0 and concentration not less than 20ng / μL were judged as qualified. Finally, the universal primers of 16S rDNA were used to amplify the genomic DNA of qualified strains by PCR. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the specific sequences are as follows:

[0054] Upstream primer 27F: 5′-AGAGTTTGATCATGGCTCAG-3′, SEQ ID NO. 1;

[0055] Downstream primer 1492R: 5'-TAGGGTTACCTTGTTACGACTT-3', SEQ ID NO.2.

[0056] PCR amplification system and program settings:

[0057] PCR reaction system: 30 μL system contains 1.5 μL of strain genomic DNA template, 1.5 μL of upstream and downstream primers, and 25.5 μL of Gold Mix (Beijing Qingke Xinye Biotechnology Co., Ltd.).

[0058] The PCR reaction program is shown in Table 1.

[0059] Table 1 PCR reaction procedure

[0060]

[0061] 2. Sequencing and identification of strain 16S rDNA

[0062] The obtained 16S rDNA amplification products were tested by 1.5% gel electrophoresis, and the qualified amplified sequences were sent to Shanghai Biotech Co., Ltd. for sequencing to obtain a 16S rDNA sequence of 911 bp (SEQID NO.3). The 16S rDNA sequence was compared with the Korean EzBioCloud database (https: / / www.ezbiocloud.net / identify), and the bacteria with the highest homology to the strain were Anaerostipes caccae DSM14662, with a similarity of 99.75% ( Figure 1 ). It was confirmed that the isolated strain belonged to Anaerostipes caccae, and was named Anaerostipes caccae CML199, abbreviated as Anaerostipes caccae CML199. Anaerostipes caccae CML199 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on July 17, 2024, with the deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 31340.

[0063] The 16S rDNA sequencing results are as follows:

[0064] 5’-ATAGTATGGCGGCGTGCTTACACATGCAAGTCGAACGAAGCATTTAGGATTGA AGTTTTCGGATGGATTTCCTATATGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGGAACCTGCCCTATACAGGGGGATAACAGCTGGAAACGGCTGCTAATACCGCATAAGCGCACAGAATCGCATGATTCAGTGTGAAAAGCCCTGGCAGTATAGGATGGTCCCGCGTCTGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCAGTAGCCGGCTTGAGAGAGTGAACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCGACGCCGCGTGAGTGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGAAAACAGACGGTACCTGACTAAGAAGCCCCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTATCCGGAATTACTGGGTGTAAAGGGTGCGTAGGTGGCATGGTAAGTCAGAAGTGAAAGCCCGGGGCTTAACCCCGGGACTGCTTTTGAAACTGTCATGCTGGAGTGCAGGAGAGGTAAGCGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATATTAGGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACTGTCACTGACACTGATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTTAGTCCACGTCGTTAAACGATGAATACTAGGTGTCGGGGCCGTAAGAAGGCTTCGGGTGCCGCAGCAAACGCAGTAAGTATTCCACCTGGGGAGTACGTTCGCAAGAATGAAGCTCAAAGGTATTGAACGGGGACCCCGAACAGCCGGA-3’(SEQ ID NO.3).

[0065] Growth curve of Anaerobaculum stercoricanum CML199 in Example 2

[0066] The formula of microbial culture medium is: Peptone 15g, tryptic peptone 10g, soy peptone 3g, yeast extract powder 5g, beef powder 2g, digested serum powder 13.5g, beef liver extract powder 1.2g, glucose 3g, potassium dihydrogen phosphate 2.5g, sodium chloride 3g, soluble starch 0.3g, L-cysteine ​​0.3g, sodium thioglycolate 0.15g and 1000mL of water, adjust the pH value to 7.0±0.1, add 2% agar to the liquid culture medium to prepare a solid culture medium.

[0067] The specific process is as follows:

[0068] 1) taking out the fecal anaerobic coryneform bacteria CML199 from a -80°C refrigerator, streaking on a solid culture medium, and placing the plate prepared above in an anaerobic operating box at 37°C for culture;

[0069] 2) After culturing for 24 hours, a single colony was picked from the solid culture medium and inoculated into 10 mL of modified GAM medium. The culture was anaerobically cultured at 37°C for 24 hours and the culture fluid was collected;

[0070] 3) The obtained culture solution was placed in an anaerobic operating box at 37°C and the OD was monitored every 2 hours using an Epoch2 microplate reader (BioTek, USA). 600nm Value until OD 600nm When the value stops rising and starts to drop, stop monitoring. With monitoring time as the horizontal axis, OD 600nm The value is the vertical axis, and the growth curve of fecal anaerobic rod-shaped bacteria CML199 is drawn, such as Figure 2 As shown. Figure 2 From the growth curve of monitoring fecal anaerobic coryneformis CML199 for 24 hours, it can be seen that fecal anaerobic coryneformis CML199 entered the logarithmic growth phase at 8 hours, entered the plateau phase at 12 hours, and began to decline at 14 hours.

[0071] Example 3: Anaerobic fecal bacteria CML199 have the function of promoting bone development in laying hens and improving eggshell quality

[0072] 1. Preparation of freeze-dried bacterial powder of anaerobic coryneformis CML199

[0073] The formula of microbial culture medium is: Peptone 15g, tryptic peptone 10g, soy peptone 3g, yeast extract powder 5g, beef powder 2g, digested serum powder 13.5g, beef liver extract powder 1.2g, glucose 3g, potassium dihydrogen phosphate 2.5g, sodium chloride 3g, soluble starch 0.3g, L-cysteine ​​0.3g, sodium thioglycolate 0.15g and 1000mL of water, adjust the pH value to 7.0±0.1.

[0074] The fecal anaerobic coryneform bacteria CML199 were inoculated into the microbial culture medium at an inoculum amount of 30%, and cultured at 37° C. for 12 hours to obtain a culture.

[0075] The obtained culture was centrifuged and freeze-dried to obtain freeze-dried bacterial powder of anaerobic coryneformis CML199. The effective viable bacterial count in the freeze-dried bacterial powder was 1×10 12 CFU / g.

[0076] 2. Experimental methods

[0077] Three hundred one-day-old Jingfen No. 6 laying hens were selected and divided into two treatments: a control group (Control group) and a freeze-dried bacterial powder of anaerobic coryneformis CML199 treatment group (ANA group). The control group was fed a basal diet, and the freeze-dried bacterial powder of anaerobic coryneformis CML199 treatment group (ANA group) was fed a basal diet containing anaerobic coryneformis CML199 at a content of 10 9 CFU / kg basal diet, the experimental chickens were free to eat and drink water. Each treatment had 10 replicates, each with 15 chickens. During the two weeks of the experiment, the laying hens in the ANA group received 10 CFU / kg every other day. 8 The laying hens were gavaged with CFU / mLANA live bacteria at a feeding volume of 1 mL / bird / day. The laying hens in the control group were gavaged with an equal amount of normal saline. The experiment lasted for 20 weeks.

[0078] 3. Research Results

[0079] 1) Fecal anaerobic Corynebacterium CML199 can promote bone development in laying hens

[0080] After the experiment, the femoral structural parameters were quantified by Micro-CT scanning. Figure 3 The results showed that fecal anaerobic Corynebacterium CML199 can increase the cortical bone volume, cortical bone thickness, cortical bone mineral content, trabecular volume fraction, trabecular thickness and trabecular bone density of laying hens in the growing period. It can be seen that fecal anaerobic Corynebacterium CML199 can promote the bone development of laying hens.

[0081] 2) Anaerobic Corynebacterium faecalis CML199 can increase the levels of bone formation markers in the femur

[0082] After the experiment, the alkaline phosphatase activity of the collected laying hen femurs was tested using the Biyuntian alkaline phosphatase detection kit. The results are as follows: Figure 4 The results showed that the alkaline phosphatase activity of laying hens in the ANA group was significantly higher than that in the normal control group. This shows that fecal anaerobic Corynebacterium CML199 can increase the level of bone formation markers in the femur.

[0083] 3) Anaerobic coryneformis CML199 can increase the level of bone formation markers in serum

[0084] After the experiment, the osteocalcin content of the collected laying hen serum was tested using the Aoqing Biotech Chicken Osteocalcin Detection Kit. The results are as follows: Figure 5 The results showed that the osteocalcin level of laying hens in the ANA group was significantly higher than that in the normal control group. This shows that fecal anaerobic Corynebacterium CML199 can increase the level of bone formation markers in serum.

[0085] 4) Fecal anaerobic Corynebacterium can improve the bone marrow immune microenvironment

[0086] After the experiment, the number of immune cells related to bone metabolism in the bone marrow was detected by flow cytometry. Figure 6 The results showed that CD25 related to bone metabolism + Cells and KuL01 + The number of cells in the ANA group increased significantly. This shows that anaerobic coryneformis CML199 has a significant improvement on the bone marrow immune microenvironment.

[0087] 5) Fecal anaerobic coryneformis CML199 can improve eggshell quality

[0088] After the experiment, the eggshell strength of the collected eggs was tested by Nabel DET-6000 egg quality analyzer, and the eggshell thickness and egg shape index of the collected eggs were tested by vernier calipers. The results are as follows: Figure 7 The results of eggshell quality test showed that the eggshell thickness, eggshell strength and egg shape index of the ANA group were significantly higher than those of the control group. This shows that fecal anaerobic Corynebacterium CML199 can improve eggshell quality.

[0089] In summary, the fecal anaerobic bacterium CML199 discovered in the present invention can not only promote the bone development of laying hens, but also increase the level of bone formation markers in the femur, improve the bone marrow immune microenvironment and improve the eggshell quality. It is a multifunctional strain and has a very broad application prospect in the laying hen industry.

[0090] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An anaerobic fecal bacterium (Anaerostipes caccae) CML199, characterized in that: The deposit number of the fecal anaerobic coryneform bacteria CML199 is CGMCC No.31340.

2. Use of the fecal anaerobic coryneform bacteria CML199 according to claim 1 in any one or more of the following: (1) Application in the preparation of microbial preparations for promoting bone development in laying hens; (2) Application in the preparation of a microbial preparation for increasing the level of bone formation markers in the femur of laying hens; the bone formation marker in the femur of laying hens is alkaline phosphatase; (3) Application in the preparation of a microbial preparation for increasing the level of bone formation markers in laying hen serum; the bone formation marker in the laying hen serum is osteocalcin; (4) Application in the preparation of microbial preparations for improving the immune microenvironment of laying hens’ bone marrow; (5) Application in the preparation of microbial preparations for improving eggshell quality.

3. A microbial preparation, characterized in that: The active ingredients of the microbial preparation include the fecal anaerobic Corynebacterium CML199 described in claim 1.

4. The microbial preparation according to claim 3, characterized in that The microbial preparation further comprises a carrier.

5. The method for preparing the microbial preparation according to claim 3, characterized in that: The following steps are involved: The fecal anaerobic coryneform bacteria CML199 is inoculated into a culture medium for culturing to obtain the microbial preparation.

6. The preparation method according to claim 5, characterized in that: The culture medium is a liquid culture medium or a solid culture medium.

7. The preparation method according to claim 5, characterized in that: The culture time is 12 hours and the temperature is 37°C.

8. Use of the microbial preparation according to claim 3 or 4 in any one or more of the following: (1) Application in the preparation of microbial preparations for promoting bone development in laying hens; (2) Application in the preparation of a microbial preparation for increasing the level of bone formation markers in the femur of laying hens; the bone formation marker in the femur of laying hens is alkaline phosphatase; (3) Application in the preparation of a microbial preparation for increasing the level of bone formation markers in laying hen serum; the bone formation marker in the laying hen serum is osteocalcin; (4) Application in the preparation of microbial preparations for improving the immune microenvironment of laying hens’ bone marrow; (5) Application in the preparation of microbial preparations for improving eggshell quality.

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