Next Article in Journal
SELENOF Mitigates Bovine Mastitis by Preserving Mitochondrial Homeostasis and Suppressing NLRP3-Mediated Pyroptosis
Previous Article in Journal
Increased Intake and Use of Euthanasia in Dog Shelters in the UK and Republic of Ireland 2021–2023
Previous Article in Special Issue
Effects of Water-Delivered Probiotics on Performance, Carcass Traits, Immunity, Blood Biochemistry, and Ileal Morphology of Broilers Reared at High Stocking Density Under Warm Ambient Temperature
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Animals
Volume 16
Issue 5
10.3390/ani16050792
animals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Effects of Probiotic Lacticaseibacillus paracasei NSMJ27 on Laying Performance and Gut Health Indicators in Aged Laying Hens

by
Viet Anh Vu
1,2,
Yoo-Bhin Kim
1,
Soo-Ki Kim
1,2,
Ji Young Jung
3,
Sang Seok Joo
4,
Byeongcheol Ban
4,
Myunghoo Kim
5,6,
Minji Kim
7 and
Kyung-Woo Lee
1,*
1
Department of Animal Science and Technology, College of Life Sciences, Konkuk University, 120 Neungdong-ro, Gwangjin-gu, Seoul 05029, Republic of Korea
2
Department of Animal Breeding and Genetics, Vietnam National University of Agriculture, Hanoi 12406, Vietnam
3
Microbial Research Department, Nakdonggang National Institute of Biological Resources (NNIBR), 137, Donam 2-gil, Sangju 37242, Republic of Korea
4
Department of Animal Science, College of Natural Resource & Life Sciences, Pusan National University, Miryang 50463, Republic of Korea
5
Department of Agricultural Biotechnology, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea
6
The Institute of Animal Science and Technology, Research Institute of Agriculture and Life Sciences, College of Agriculture and Life Sciences, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea
7
Animal Nutrition and Physiology Division, National Institute of Animal Science, Rural Development Administration (NIAS-RDA), 1500, Kongjwipatjwi-ro, Iseo-myeon, Wanju 55365, Republic of Korea
*
Author to whom correspondence should be addressed.
Animals 2026, 16(5), 792; https://doi.org/10.3390/ani16050792
Submission received: 26 January 2026 / Revised: 26 February 2026 / Accepted: 27 February 2026 / Published: 3 March 2026
(This article belongs to the Collection Application of Antibiotic Alternatives in the Poultry Industry)
Browse Figures
Versions Notes

Simple Summary

Probiotics are live beneficial microbes that are known to improve the performance, health and welfare of laying hens. In our attempts to develop novel probiotic strains for poultry, Lacticaseibacillus paracasei NSMJ27, isolated from Korean fermented foods, gained our attention for its probiotic potential. It is known that dietary probiotic strains, although they exert beneficial potentials in vitro, require in vivo validation with chickens to verify biological effectiveness. In the present research, we attempted to evaluate the effect of a novel probiotic strain on gut health in aged laying hens. Our study shows that L. paracasei NSMJ27 enhanced antioxidant capacity and improved gut health, suggesting its potential use as a feed additive for laying hens.

Abstract

This experiment was designed to determine the effect of the Lacticaseibacillus paracasei (paracasei) strain NSMJ27, isolated from Korean fermented vegetable food (home-made kimchi), on laying performance, egg quality, intestinal histology, cecal short-chain fatty acids, and ileal antioxidant/immunity indicators of laying hens. Ninety-six 55-week-old Hy-Line Brown hens were randomly assigned to two dietary treatments with each treatment comprising eight replicates of six hens each. Experimental diets were prepared by mixing corn and soybean meal basal diets without or with L. paracasei NSMJ27 at 2.5 × 109 CFU/kg. The experiment lasted 4 weeks. Laying hens fed with the NSMJ27-supplemented diet were not affected (p > 0.05) in their laying performance or egg quality. With respect to ileal morphology, villus height: crypt depth ratio tended to be higher (p = 0.067) in laying hens fed with L. paracasei NSMJ27 vs. control diets. Dietary L. paracasei did not affect (p > 0.05) the activities of glutathione peroxidase and catalase, malondialdehyde contents, or secretory immunoglobulin A in ileal mucosa, but increased (p = 0.048) the activity of superoxide dismutase compared with the control diet-fed laying hens. Dietary L. paracasei elevated (p = 0.016) the relative percentage of butyrate but lowered (p = 0.057) that of isovalerate in cecal digesta. Dietary L. paracasei did not affect the percentages of cells expressing macrophages, B cells, CD4+ T cells, CD8+ T cells, or TCRγδ T cell surface markers (p > 0.05). Overall, these results suggest that dietary L. paracasei NSMJ27 could enhance gut health via increasing gut antioxidant capacity and butyrate production in the cecal digesta of laying hens.

1. Introduction

The avian gastrointestinal tract fulfills dual critical functions, serving as both a primary digestive organ and the largest immune barrier within a bird’s body [1], and harbors a well-balanced gut microbiota crucial for maintaining optimal gut health [2]. In aged laying hens, physiological aging leads to gut dysbiosis, impaired intestinal barrier function, weakened immunity, and reductions in egg production and quality due to oxidative stress and microbiota shifts [3,4,5,6]. To maintain a balanced intestinal ecosystem in chickens, dietary probiotics are frequently supplemented [7], providing essential health benefits through the administration of adequate viable microbes [8]. In addition, probiotic inclusion in layer diets has been known to improve egg quality, feed efficiency, nutrient utilization, and overall flock health with less dependence on in-feed antibiotics [9,10,11,12].
Lactobacillus species are known to possess antibacterial properties against various enteric pathogens and potentially improve gut microbiota composition [10]. Among them, Lacticaseibacillus paracasei (L. paracasei) is a Gram-positive, lactic-acid bacterium naturally found in fermented dairy products and the animal intestinal tract [11]. L. paracasei has been shown to possess several probiotic properties that could be beneficial for animal health. These properties include the suppression of mucosal inflammatory cytokines such as tumor necrosis factor alpha and interleukin 12, the mitigation of intestinal inflammation, the enhancement of secretory immunoglobulin A (sIgA) levels in the small intestine, and the modulation of gut microbiota [12]. It has been reported that dietary L. paracasei subsp. paracasei L1 enhanced growth performance, feed intake, and cecal microbiota diversity in chickens [13], while unclassified L. paracasei improved feed conversion ratio, body weight gain, and antioxidant status in E. coli-challenged broilers [14]. The beneficial health or growth-promoting effects of L. paracasei strains were also reported in murine models [15] and pigs [16].
L. paracasei strain NSMJ27, recently isolated from Korean fermented foods, appears to be a particularly promising candidate due to its tolerance to the harsh gut environment (e.g., at low pH values and high bile salt concentrations) characteristics commonly found in the gut [17]. In our ongoing attempts to find a novel probiotic candidate for chickens, we reported the beneficial effect of the L. paracasei NSMJ56 strain, which enhanced egg quality, exhibited antioxidants, and modulated cecal microbiota composition in young laying hens [18]. Although L. paracasei species has been used in poultry, no studies have been tested with recently isolated L. paracasei NSMJ27 strain on cecal short-chain fatty acid (SCFA) profiles, ileal antioxidant status, or immune responses in aged laying hens. Therefore, the purpose of this communication was to elucidate the potential benefits of probiotic L. paracasei strain NSMJ27 supplementation with emphasis on cecal short-chain fatty acids and ileal antioxidant/immune status in aged laying hens.

2. Materials and Methods

2.1. Ethics Approval Statement

All experiment protocols received approval from the Institutional Animal Care and Use Committee in Konkuk university (KU 21083), Republic of Korea.

2.2. Animals Experimental Design

Initially, 12-week-old Hy-Line Brown pullets were obtained from a local commercial farm (Eunha farm, Cheonan-si, Chungcheongnam-do, South Korea) and reared at a KU research farm (Chungju-si, Chungcheongbuk-do, South Korea). In this study, laying hens that had been raised at the KU research farm were selected at 53 weeks of age from a flock of 300 hens to have similar laying performance. Before the initiation of the experiment, egg weights and egg production were monitored for 2 weeks to ensure that all hens were producing and treatment groups had equal laying performance. Ninety-six 55-week-old Hy-Line Brown laying hens were randomly allocated to two dietary treatments (eight replicates of 6 hens each) in the 3-tier multi-layer battery, evenly distributed across all tiers and rows with an alternating arrangement. A mash-type diet based on corn–soybean meal was prepared (Table 1). Experimental diets were prepared by mixing a basal diet supplemented without or with lyophilized L. paracasei NSMJ27 at the content of 2.5 × 109 CFU/kg. This inclusion level was determined based on a prior trial with L. paracasei NSMJ56 strain [18], which reported to influence immunity and microbiota in 21-week-old laying hens fed on a diet containing 5 × 108 CFU/kg of NSMJ56 strain. In addition, aged layers typically exhibit compromised gut health requiring higher probiotic doses to achieve improvements in gut function and subsequently enhance laying performance. According to Alaqil et al. [19] and Wang et al. [20], dietary supplementation at 2 × 109 or 3 × 109 CFU/kg probiotics showed positive effects on the laying performance and egg quality of aged laying hens. L. paracasei NSMJ27, isolated from Korean fermented vegetable food (home-made kimchi), was shown to be viable even at low pH or bile salt environment [17]. The L. paracasei NSMJ27 strain was prepared in a lyophilized form which had been cultured in MRS medium, centrifuged, resuspending in 10% skim milk, and freeze-dried as described [21]. The basal diet devoid of feed additives was considered the control diet. Each cage (45 cm × 45 cm × 45 cm) housed two hens, and three adjacent cages were considered replicates. This setup aligns with optimized industry housing systems, safely accommodating up to 2 laying hens per cage without welfare compromise. Feed and water were supplied ad libitum for 4 weeks. The chicken room maintained a 24 ± 2 °C ambient temperature and 60% relative humidity, and a 16L:8D light schedule was applied.

2.3. Measurements of Laying Performance and Egg Quality

The feed intake of each replicate was measured at the beginning and end of the experiment to calculate the average daily feed consumption per bird. Egg production and egg weight were recorded daily and used to calculate the egg mass. Feed-to-egg ratio was determined as total feed consumption divided by egg mass. During the final three days of the trial, six unbroken eggs were sampled from each replicate. The TSS QCR reflectometer (Technical Services and Supplies, York, UK) was used to measure eggshell color. A digital egg tester (DET-6000, Nabel, Kyoto, Japan) was employed to assess Haugh unit, eggshell strength, eggshell thickness (excluding the shell membrane) and yolk color score.

2.4. Intestinal Sample Collection

At 28 days, one hen per replicate was chosen at random and euthanized using a carbon dioxide overdose. Laying hens that were not used for sampling in this study were not culled but raised at the KU research farm. The small intestine and paired ceca were sampled immediately after euthanasia. The jejunal segment was also sampled for immune cell subpopulation analysis. For histological analysis, an approximately 1 cm mid-segment of the ileum was fixed in 10% neutral buffered formalin solution for 48 h. The remaining distal ileal segment was processed for the measurement of antioxidant/immune markers. A pair of ceca were kept on ice and processed to assess the concentrations of short-chain fatty acids (SCFAs), which serve as an important indicator of microbial fermentation activity in the cecal digesta, on the day of sampling.

2.5. Measurements of Ileal Histology

Hematoxylin and eosin staining was applied to 5 μm paraffin sections, per standard protocols [22]. The mucosal layer was evaluated under a light microscope (Olympus BX43, Tokyo, Japan) and captured with a digital camera (eXcope T500, DIXI Science, Daejeon, Republic of Korea). Villus height and crypt depth were quantified by analyzing ten properly sectioned villi and crypts. The villus height-to-crypt depth ratio was subsequently determined.

2.6. Measurements of Antioxidant and Immune Markers in Ileal Mucosa

The ileal mucosa samples were obtained by carefully scraping the distal ileal segment with a tissue-culture scraper. Next, the samples were then centrifuged at 1000× g for 10 min at 4 °C and stored at −20 °C until analysis. The activities of glutathione peroxidase, superoxide dismutase, and catalase, and the concentrations of malondialdehyde and secretory immunoglobulin A were determined using commercial assay kits. These analyses were conducted using the following commercially available kits in order: EnzyChrom™ Glutathione Peroxidase Assay Kit, EnzyChrom™ Superoxide Dismutase Assay Kit (BioAssay Systems, Hayward, CA, USA), OxiSelect™ Catalase Activity Assay Kit, OxiSelect™ TBARS Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA), and Chicken IgA ELISA Kit (Bethyl Laboratories, Montgomery, TX, USA). The concentrations of total protein in ileal mucosa samples were measured with Bradford assay reagent (Sigma-Aldrich, St. Louis, MO, USA).

2.7. Measurements of Short-Chain Fatty Acids (SCFAs) in Cecal Digesta

Approximately 1 g of cecal digesta was homogenized in 4 mL of ice-cold distilled water and added with 0.05 mL of saturated HgCl2, 1.00 mL of 25% H3PO4, and 0.20 mL of 2% pivalic acid. The mixture was then centrifuged at 1000× g for 20 min at 4 °C. Following centrifugation, 1 mL of the supernatant was collected for SCFA analysis using gas chromatography (6890 Series GC System, HP, Palo Alto, CA, USA), as previously detailed [23].

2.8. Measurements of Jejunal Immune Cell Subpopulation Using Flow Cytometer

Immune cells in jejunal mucosa samples were isolated by density gradient centrifugation, as outlined by [24], and analyzed using the FACS Canto II system (BD, Franklin Lakes, NJ, USA). Viable cells were identified by excluding dead cells through live/dead fixable dead cell stain (Thermo Fisher Scientific, Waltham, MA, USA). For staining, the anti-chicken antibodies, including anti-CD3 (CT-3), anti-CD4 (CT-4), anti-CD8a (CT-8), anti-TCR (TCR-1), anti-MHC II (2G11), anti-Bu-1 (AV20), and anti-Monocyte/Macrophage (KUL01), were utilized (Southern Biotech, Birmingham, AL, USA).

2.9. Statistical Analysis

Data was analyzed with SAS software (SAS 9.4, SAS Institute Inc., Cary, NC, USA). PROC UNIVARIATE (SAS 9.4, SAS Institute Inc., USA) was applied to test for outliers and normal distribution across all variables. All experimental results are expressed as means and pooled standard error of the means. Each experimental unit consisted of three adjacent cages. Student’s t-test was employed to compare between dietary treatments. Statistical significance was declared at p < 0.05.

3. Results

3.1. Laying Performance and Egg Quality

Dietary supplementation with L. paracasei NSMJ27 did not affect (p > 0.05) laying performance, including feed intake, egg production, egg mass, egg weight, or feed-to-egg ratio in laying hens (Table 2). No supplemental effects of L. paracasei were observed on yolk color, Haugh unit, eggshell strength, or eggshell thickness. Eggshell color tended to be darker (p = 0.057) in laying hens fed diets containing L. paracasei vs. control diets (Table 3).

3.2. Ileal Histology

Dietary L. paracasei NSMJ27 did not affect (p > 0.05) ileal morphology, including villus height, crypt depth, and villus height-to-crypt depth ratio (Table 4). Nonetheless, probiotic-fed laying hens had a higher villus height: crypt depth ratio (p = 0.067) by on average 15.2% compared with the control diet-fed treatment.

3.3. Short-Chain Fatty Acids in Cecal Digesta

Acetate was the dominant short-chain fatty acid, followed by propionate and butyrate in cecal digesta (Table 5). Dietary L. paracasei NSMJ27 did not affect the relative percentages of acetate, propionate, isobutyrate, valerate, branched-chain fatty acids or short-chain fatty acids. However, dietary L. paracasei NSMJ27 increased (p = 0.016) the relative percentage of butyrate by on average 19.7% but decreased (p = 0.057) that of isovalerate in cecal digesta by on average 26.0% compared with the control group.

3.4. Antioxidant and Immune Markers in Ileal Mucosa

Dietary L. paracasei NSMJ27 increased (p = 0.048) the superoxide dismutase activity in ileal mucosa compared with the control group. However, no effect by dietary L. paracasei was noted (p > 0.05) in the activities of catalase and glutathione peroxidase, or the contents of malondialdehyde of ileal mucosa. In addition to antioxidant markers, secretory immunoglobulin A, a key marker of the gut mucosal immune system, was assessed to evaluate the effect of dietary L. paracasei on gut health. It was found that dietary L. paracasei did not affect (p = 0.622) the concentration of secretory immunoglobulin A in the ileal mucosa of laying hens (Table 6).

3.5. Jejunal Immune Cell Subpopulations

Dietary L. paracasei NSMJ27 did not affect (p > 0.05) the subpopulations of immune cells expressing the macrophage, B cell and T cell surface markers (CD4+, CD8+, and TCRγδ+ T cells) (Figure 1) in jejunal mucosa samples.

4. Discussion

The interplay between small intestinal morphology, antioxidant defense systems, and mucosal immunity is integral in optimizing nutrient utilization, promoting growth, and enhancing egg production in laying hens, which allowed us to analyze ileal morphology and gut health indicators, including ileal antioxidant/immune markers and cecal short-chain fatty acids. It is clear from this study that dietary L. paracasei NSMJ27, recently isolated from fermented foods with probiotic potentials, did not affect the laying performance or egg quality parameters of aged laying hens. These findings are consistent with previous studies in laying hens fed combinations of L. paracasei [25] and Bacillus subtilis [26]. A trend toward darker eggshell color was observed in the L. paracasei diet treatment, which may be attributed to a healthier intestinal environment leading to enhanced absorption of mineral or pigment precursors necessary for eggshell pigmentation [27,28,29]. Indeed, dietary L. paracasei NSMJ27 displayed beneficial properties in improving gut health (ileal villus height: crypt depth ratio, butyrate in cecal digesta and superoxide dismutase activity in ileal mucosa) in aged laying hens. It should be pointed out, however, that the feeding trial lasted 4 weeks in this study, although the effectiveness (e.g., antioxidant/immune markers) of dietary probiotics, if any, can be disclosed in laying hens. In this sense, caution is needed to conclude the lack of probiotic effect on laying performance and egg quality, which may need a longer duration of feeding (i.e., more than 12 weeks).
The beneficial role of dietary probiotics on ileal morphology (e.g., villus height: crypt depth ratio) has been well reported in broilers [30,31] and laying hens [32,33]. It is postulated that probiotic-mediated improvement in ileal morphology is enacted by either balancing gut microbiota [34] or by augmenting local immunity [3], leading to improved nutrient absorption. In this study, laying performance (e.g., feed-to-egg ratio), secretory immunoglobulin A in ileal mucosa, and jejunal immune cell subpopulations including macrophages, B cells and T cells were not altered by dietary L. paracasei. Thus, it is likely that dietary L. paracasei NSMJ27 might improve ileal villus height: crypt depth ratio via balancing the composition of gut microbiota in ileal digesta. Indeed, we found that dietary L. paracasei NSMJ27 altered short-chain fatty acids (increased butyrate and decreased isovalerate in cecal digesta), which are regulated by beneficial gut microbiota. In line with our study, a higher villus height: crypt depth ratio was noted in laying hens fed L. salivarius SNK-6 [35], L. crispatus [36], Bacillus coagulans [37], and B. subtilis [38].
Our results demonstrated that dietary L. paracasei markedly enhanced the superoxide dismutase activity in the ileal mucosa of aged laying hens, suggesting a significant improvement in local antioxidant defense mechanisms. Our finding is consistent with previous reports [14,39,40] which documented the increased antioxidant enzyme activities in various tissues of poultry following supplementation with Lactobacillus and Lacticaseibacillus species. This enhanced antioxidant status likely stems from L. paracasei’s capacity to modulate mucosal immune responses [21,41] or produce bioactive metabolites [42] that reduce reactive oxygen species damaging epithelial tissues. The present study also demonstrated that dietary L. paracasei NSMJ27 enhanced the relative percentage of butyrate in the cecal digesta of laying hens. In addition to the function of short-chain fatty acids (i.e., butyrate) as the primary energy source for colonocytes, it is known that they can contribute to improving gut health and to modulating immune responses [43]. Kim et al. [18] reported that laying hens fed a diet containing L. paracasei NSMJ56 had an altered cecal microbial community via increasing short-chain fatty acid-producing bacteria including Flintibacter, Dielma, and Coprobacter. In addition, Thananimit et al. [44] identified specific L. paracasei strains exhibiting high butyrate production potential. Apart from the increase in butyrate concentrations by dietary L. paracasei NSMJ27, it lowered the relative percentage of isovalerate in cecal digesta. It is known that branched-chain fatty acids, including isovalerate, are produced from bacteria-mediated fermentation on undigested proteins [45]. This is significant, as elevated concentrations of branched-chain fatty acids are associated with increased protein degradation and potential pathogenic bacterial activity [45,46]. Tentatively, our findings demonstrate that L. paracasei NSMJ27 favors a beneficial shift in the cecal bacterial community, increasing carbohydrate fermentation but lowering protein fermentation in the ceca. Taken together, our findings demonstrate that L. paracasei NSMJ27 supplementation strengthens intestinal health through a coordinated enhancement of local antioxidant defenses, the promotion of beneficial bacterial metabolism, and a reduction in metabolic byproducts associated with unfavorable microbiota composition.

5. Conclusions

In summary, L. paracasei NSMJ27 supplementation did not affect laying performance but improved ileal villus heigh: crypt depth ratio, superoxide dismutase in ileal mucosa, and the relative percentage of butyrate in cecal digesta. This study shows that dietary L. paracasei NSMJ27 can be used as a feed additive to enhance the gut health of aged laying hens.

Author Contributions

Conceptualization, V.A.V., S.-K.K. and Y.-B.K.; methodology, Y.-B.K., J.Y.J. and S.S.J.; validation, Y.-B.K. and S.S.J.; formal analysis, Y.-B.K., B.B. and M.K. (Minji Kim); investigation, V.A.V. and Y.-B.K.; data curation, V.A.V. and M.K. (Minji Kim); writing—original draft preparation, V.A.V. and Y.-B.K.; writing—review and editing, M.K. (Minji Kim), M.K. (Myunghoo Kim) and K.-W.L.; supervision, M.K. (Myunghoo Kim) and K.-W.L.; funding acquisition, M.K. (Myunghoo Kim) and K.-W.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with the support of “The Cooperative Research Program for Agriculture, Science and Technology Development (Project No. PJ01621002)” Rural Development Administration, Republic of Korea.

Institutional Review Board Statement

The experimental procedures were approved by the Institutional Animal Care and Use Committee of Konkuk University (KU21083) on 17 May 2021.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

Vu Viet Anh was partially supported by the KUVEC-VNUA Joint Research Project, funded by Korea Institutional Cooperation Agency (KOICA).

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Yegani, M.; Korver, D.R. Factors Affecting Intestinal Health in Poultry. Poult. Sci. 2008, 87, 2052–2063. [Google Scholar] [CrossRef]
  2. Abdelqader, A.; Irshaid, R.; Al-Fataftah, A.-R. Effects of Dietary Probiotic Inclusion on Performance, Eggshell Quality, Cecal Microflora Composition, and Tibia Traits of Laying Hens in the Late Phase of Production. Trop. Anim. Health Prod. 2013, 45, 1017–1024. [Google Scholar] [CrossRef]
  3. Choi, S.; Kim, E.B. A Longitudinal Investigation of Gut Microbiota Dynamics in Laying Hens from Birth to Egg-Laying Stages. Anim. Biosci. 2025, 38, 1773–1783. [Google Scholar] [CrossRef] [PubMed]
  4. Kuroda, A.; Jaisue, J.; Isobe, N.; Nii, T. Age-Related Changes in Intestinal Mucosal Barrier Function in Laying Hens during the Mid-to-Late Production Stages. Poult. Sci. 2026, 105, 106241. [Google Scholar] [CrossRef] [PubMed]
  5. Liu, X.; Lin, X.; Zhang, S.; Guo, C.; Li, J.; Mi, Y.; Zhang, C. Lycopene Ameliorates Oxidative Stress in the Aging Chicken Ovary via Activation of Nrf2/HO-1 Pathway. Aging 2018, 10, 2016–2036. [Google Scholar] [CrossRef] [PubMed]
  6. Kim, C.-H.; Song, J.-H.; Lee, J.-C.; Lee, K.-W. Age-Related Changes in Egg Quality of Hy-Line Brown Hens. Int. J. Poult. Sci. 2014, 13, 510–514. [Google Scholar] [CrossRef]
  7. Naeem, M.; Bourassa, D. Probiotics in Poultry: Unlocking Productivity Through Microbiome Modulation and Gut Health. Microorganisms 2025, 13, 257. [Google Scholar] [CrossRef]
  8. Jha, R.; Das, R.; Oak, S.; Mishra, P. Probiotics (Direct-Fed Microbials) in Poultry Nutrition and Their Effects on Nutrient Utilization, Growth and Laying Performance, and Gut Health: A Systematic Review. Animals 2020, 10, 1863. [Google Scholar] [CrossRef]
  9. Dibakoane, S.R.; Mhlongo, G.; Moonsamy, G.; Wokadala, O.C.; Mnisi, C.M.; Mlambo, V. Phenomenological and Mechanistic Insights into Potential Dietary Nucleotide—Probiotic Synergies in Layer Chickens: A Review. Poult. Sci. 2025, 104, 105049. [Google Scholar] [CrossRef]
  10. Liu, X.; Ma, Z.; Wang, Y.; Li, L.; Jia, H.; Zhang, L. Compound Probiotics Can Improve Intestinal Health by Affecting the Gut Microbiota of Broilers. J. Anim. Sci. 2023, 101, skad388. [Google Scholar] [CrossRef]
  11. Alizadeh Behbahani, B.; Jooyandeh, H.; Taki, M.; Falah, F. Evaluation of the Probiotic, Anti-Bacterial, Anti-Biofilm, and Safety Properties of Lacticaseibacillus paracasei B31-2. LWT 2024, 207, 116676. [Google Scholar] [CrossRef]
  12. Dempsey, E.; Corr, S.C. Lactobacillus spp. for Gastrointestinal Health: Current and Future Perspectives. Front. Immunol. 2022, 13, 840245. [Google Scholar] [CrossRef]
  13. Xu, Y.; Tian, Y.; Cao, Y.; Li, J.; Guo, H.; Su, Y.; Tian, Y.; Wang, C.; Wang, T.; Zhang, L. Probiotic Properties of Lactobacillus Paracasei Subsp. Paracasei L1 and Its Growth Performance-Promotion in Chicken by Improving the Intestinal Microflora. Front. Physiol. 2019, 10, 937. [Google Scholar] [CrossRef] [PubMed]
  14. Lokapirnasari, W.P.; Maslachah, L.; Saputro, A.L.; Rokana, E.; Yulianto, A.B.; Rosyada, Z.N.A.; Firdaus, M.A.; Silfia, H.I.; Lisnanti, E.F.; Baihaqi, Z.A.; et al. Potency of Lacticaseibacillus paracasei as an Alternative to Antibiotic Growth Promoter in Broiler Chicken Challenged with Avian Pathogenic Escherichia coli. Vet. World 2025, 18, 1180–1189. [Google Scholar] [CrossRef] [PubMed]
  15. Wang, F.; Chen, L.; Li, Y.; Gao, G.; Wang, Y.; Shi, R.; Zhang, H.; Chen, Y.; Cao, R.; Shi, Q.; et al. Lacticaseibacillus paracasei subsp. Paracasei Q- 1 Exhibits Good Safety and Effectively Prevents Escherichia coli K99-Induced Diarrhea in Mice. Probiotics Antimicrob. Proteins 2025. Online ahead of print. [Google Scholar] [CrossRef]
  16. Tufarelli, V.; Rossi, G.; Laudadio, V.; Crovace, A. Effect of a Dietary Probiotic Blend on Performance, Blood Characteristics, Meat Quality and Faecal Microbial Shedding in Growing-Finishing Pigs. S. Afr. J. Anim. Sci. 2017, 47, 875–882. [Google Scholar] [CrossRef]
  17. Jung, J.Y.; Han, S.-S.; Kim, Z.-H.; Kim, M.H.; Kang, H.K.; Jin, H.M.; Lee, M.H. In-Vitro Characterization of Growth Inhibition against the Gut Pathogen of Potentially Probiotic Lactic Acid Bacteria Strains Isolated from Fermented Products. Microorganisms 2021, 9, 2141. [Google Scholar] [CrossRef]
  18. Kim, Y.B.; Park, J.; Lee, H.-G.; Song, J.-Y.; Kim, D.-H.; Ji, W.; Joo, S.S.; Kim, M.; Jung, J.Y.; Kim, M.; et al. Dietary Probiotic Lacticaseibacillus paracasei NSMJ56 Modulates Gut Immunity and Microbiota in Laying Hens. Poult. Sci. 2024, 103, 103505. [Google Scholar] [CrossRef]
  19. Alaqil, A.A.; Abbas, A.O.; El-Beltagi, H.S.; El-Atty, H.K.A.; Mehaisen, G.M.K.; Moustafa, E.S. Dietary Supplementation of Probiotic Lactobacillus acidophilus Modulates Cholesterol Levels, Immune Response, and Productive Performance of Laying Hens. Animals 2020, 10, 1588. [Google Scholar] [CrossRef]
  20. Wang, J.; Wang, W.; Qi, G.; Cui, C.; Wu, S.; Zhang, H.; Xu, L.; Wang, J. Effects of Dietary Bacillus Subtilis Supplementation and Calcium Levels on Performance and Eggshell Quality of Laying Hens in the Late Phase of Production. Poult. Sci. 2020, 100, 100970. [Google Scholar] [CrossRef]
  21. Joo, S.S.; Yoon, J.H.; Jung, J.Y.; Joo, S.Y.; An, S.H.; Ban, B.C.; Kong, C.; Kim, M. The Modulatory Effects of Lacticaseibacillus paracasei Strain NSMJ56 on Gut Immunity and Microbiome in Early-Age Broiler Chickens. Animals 2022, 12, 3413. [Google Scholar] [CrossRef] [PubMed]
  22. Kim, D.-H.; Yang, H.M.; Song, J.-Y.; Park, J.; Kwon, B.-Y.; Vu, A.V.; Lee, D.S.; Lee, K.-W. Effects of Dietary Mangosteen Peel Powder and Extract on the Growth Performance, Meat Quality and Indicators for Immunity, Gut Health and Antioxidant Activity in Broiler Chicks. Poult. Sci. 2024, 103, 104477. [Google Scholar] [CrossRef] [PubMed]
  23. Heo, Y.-J.; Park, J.; Kim, Y.-B.; Kwon, B.-Y.; Kim, D.-H.; Song, J.-Y.; Lee, K.-W. Effects of Dietary Protein Levels on Performance, Nitrogen Excretion, and Odor Emission of Growing Pullets and Laying Hens. Poult. Sci. 2023, 102, 102798. [Google Scholar] [CrossRef] [PubMed]
  24. Bucková, B.; Revajová, V. Immunophenotyping of Intraepithelial (IEL) and Lamina Propria Lymphocytes (LPL) in the Chicken Intestine by Flow Cytometry. Folia Vet. 2014, 75–77. [Google Scholar]
  25. Naseem, S.; Willits, N.; King, A.J. Varying Combinations of Lactobacillus Species: Impact on Laying Hens’ Performance, Nitrogenous Compounds in Manure, Serum Profile, and Uric Acid in the Liver. Transl. Anim. Sci. 2021, 5, txab018. [Google Scholar] [CrossRef]
  26. Zhang, G.; Wang, H.; Zhang, J.; Tang, X.; Raheem, A.; Wang, M.; Lin, W.; Liang, L.; Qi, Y.; Zhu, Y.; et al. Modulatory Effects of Bacillus Subtilis on the Performance, Morphology, Cecal Microbiota and Gut Barrier Function of Laying Hens. Animals 2021, 11, 1523. [Google Scholar] [CrossRef]
  27. Samiullah, S.; Roberts, J.R.; Chousalkar, K. Eggshell Color in Brown-Egg Laying Hens—A Review. Poult. Sci. 2015, 94, 2566–2575. [Google Scholar] [CrossRef]
  28. Dai, D.; Gao, L.; Pan, Y.; Chen, C.; Ma, K.; Zhang, H.; Wu, S.; Qi, G.; Wang, J. Eggshell Depigmentation in the Late Phase of Production Is Associated with Altered Microbiota and Metabolism of the Uterus in Laying Hens. Poult. Sci. 2025, 104, 105258. [Google Scholar] [CrossRef]
  29. Lu, Y.; Xu, H.; Hu, Z.; Li, D.; Rustempasic, A.; Zhou, Y.; Deng, Q.; Pu, J.; Zhao, X.; Zhang, Y.; et al. Probiotics Improve Eggshell Quality via Regulating Microbial Composition in the Uterine and Cecum. Poult. Sci. 2025, 104, 104849. [Google Scholar] [CrossRef]
  30. Awad, W.A.; Ghareeb, K.; Abdel-Raheem, S.; Böhm, J. Effects of Dietary Inclusion of Probiotic and Synbiotic on Growth Performance, Organ Weights, and Intestinal Histomorphology of Broiler Chickens. Poult. Sci. 2009, 88, 49–56. [Google Scholar] [CrossRef]
  31. Hashemitabar, S.H.; Hosseinian, S.A. The Comparative Effects of Probiotics on Growth, Antioxidant Indices and Intestinal Histomorphology of Broilers under Heat Stress Condition. Sci. Rep. 2024, 14, 23471. [Google Scholar] [CrossRef] [PubMed]
  32. Onbaşılar, E.E.; Yalçın, S.; Bakıcı, C.; Batur, B.; Kartal, Y.K.; Ahlat, O.; Kılıçlı, İ.B.; Yalçın, S. Comprehensive Evaluation of Probiotic Effects on Laying Hen Physiology: From Performance to Bone and Gut Morphology. Animals 2025, 15, 2408. [Google Scholar] [CrossRef] [PubMed]
  33. Wang, Y.; Zhang, C.; Chen, X.; Zheng, A.; Liu, G.; Ren, Y.; Chen, Z. Dietary Supplementation of Compound Probiotics to Improve Performance, Egg Quality, Biochemical Parameters and Intestinal Morphology of Laying Hens. Front. Vet. Sci. 2024, 11, 1505151. [Google Scholar] [CrossRef]
  34. Zhang, S.; Yu, M.; Zhao, T.; Geng, Y.; Liu, Z.; Zhang, X.; Yu, L. Effects of Probiotics on Gut Microbiota in Poultry. AIMS Microbiol. 2025, 11, 754–768. [Google Scholar] [CrossRef] [PubMed]
  35. Liu, W.; Liu, J.; Li, D.; Han, H.; Yan, H.; Sun, Y.; Lei, Q.; Wang, J.; Zhou, Y.; Cao, D.; et al. Effect of Lactobacillus Salivarius SNK-6 on Egg Quality, Intestinal Morphology, and Cecal Microbial Community of Laying Hens. Poult. Sci. 2024, 103, 103224. [Google Scholar] [CrossRef]
  36. Jing, L.; Guo, L.; Chen, B.; Hao, K.; Ma, H.; Liu, Y.; Min, Y. Effects of Different Probiotic Fermented Feeds on Production Performance and Intestinal Health of Laying Hens. Poult. Sci. 2022, 101, 101570. [Google Scholar]
  37. Wang, X.; Jian, H.; Zhao, W.; Li, J.; Zou, X.; Dong, X. Effects of Dietary Bacillus Coagulans on the Productive Performance, Egg Quality, Serum Parameters, and Intestinal Morphology of Laying Hens during the Late Laying Period. Ital. J. Anim. Sci. 2023, 22, 95–105. [Google Scholar] [CrossRef]
  38. Gao, H.; Xiao, J.; Zhang, Q.; Man, H.; Tang, X.; Wang, Z.; Fang, R.; Jiang, S. Effect of Supplementation Continuously in Pullet and Early Lay Period with Bacillus Subtilis and Yeast Cell Wall on the Intestinal Morphology, Bone Parameters, and Egg Quality of Hens. Front. Vet. Sci. 2025, 12, 1584627. [Google Scholar] [CrossRef]
  39. Kang, X.; Li, X.-D.; Zhou, H.-Y.; Wang, F.; Lin, L.-B. Genome-Wide and 16S rRNA Sequencing-Based Analysis on the Health Effects of Lacticaseibacillus paracasei XLK401 on Chicks. Microorganisms 2023, 11, 2140. [Google Scholar] [CrossRef]
  40. Chang, H.M.; Loh, T.C.; Foo, H.L.; Lim, E.T.C. Lactiplantibacillus plantarum Postbiotics: Alternative of Antibiotic Growth Promoter to Ameliorate Gut Health in Broiler Chickens. Front. Vet. Sci. 2022, 9, 883324. [Google Scholar] [CrossRef]
  41. Yoon, J.H.; Joo, S.S.; An, S.H.; Ban, B.C.; Jung, M.; Ji, W.; Jung, J.Y.; Kim, M.; Kong, C. Inclusion of Lacticaseibacillus paracasei NSMJ15 in Broiler Diets Induces Changes in Jejunal Immune Cell Population and Cecal Microbiota. Anim. Biosci. 2024, 37, 2155–2166. [Google Scholar] [CrossRef]
  42. Sirisopapong, M.; Shimosato, T.; Okrathok, S.; Khempaka, S. Assessment of Lactic Acid Bacteria Isolated from the Chicken Digestive Tract for Potential Use as Poultry Probiotics. Anim. Biosci. 2023, 36, 1209–1220. [Google Scholar] [CrossRef]
  43. Liu, L.; Li, Q.; Yang, Y.; Guo, A. Biological Function of Short-Chain Fatty Acids and Its Regulation on Intestinal Health of Poultry. Front. Vet. Sci. 2021, 8, 736739. [Google Scholar] [CrossRef]
  44. Thananimit, S.; Pahumunto, N.; Teanpaisan, R. Characterization of Short Chain Fatty Acids Produced by Selected Potential Probiotic Lactobacillus Strains. Biomolecules 2022, 12, 1829. [Google Scholar] [CrossRef]
  45. Diether, N.E.; Willing, B.P. Microbial Fermentation of Dietary Protein: An Important Factor in Diet–Microbe–Host Interaction. Microorganisms 2019, 7, 19. [Google Scholar] [CrossRef]
  46. Apajalahti, J.; Vienola, K.; Raatikainen, K.; Holder, V.; Moran, C.A. Conversion of Branched-Chain Amino Acids to Corresponding Isoacids—An in Vitro Tool for Estimating Ruminal Protein Degradability. Front. Vet. Sci. 2019, 6, 311. [Google Scholar] [CrossRef]
Animals 16 00792 g001aAnimals 16 00792 g001b
Figure 1. Effect of dietary Lacticaseibacillus paracasei NSMJ27 on jejunal lymphocyte subpopulations in laying hens. The percentages of cells expressing the indicated surface markers (B cells, macrophages, CD4, CD8, TCRγδ) assessed by flow cytometry. Filled circle and triangle symbols indicate individual data per treatment. Data is presented as mean and standard deviation. Abbreviations: CON, control diet; NSMJ27, diet supplemented with 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27.
Figure 1. Effect of dietary Lacticaseibacillus paracasei NSMJ27 on jejunal lymphocyte subpopulations in laying hens. The percentages of cells expressing the indicated surface markers (B cells, macrophages, CD4, CD8, TCRγδ) assessed by flow cytometry. Filled circle and triangle symbols indicate individual data per treatment. Data is presented as mean and standard deviation. Abbreviations: CON, control diet; NSMJ27, diet supplemented with 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27.
Animals 16 00792 g001aAnimals 16 00792 g001b
Table 1. Ingredients and nutrient composition of the basal diet.
Table 1. Ingredients and nutrient composition of the basal diet.
Ingredientsg per 100 g of Diet
Corn61.65
Soybean meal, 47% crude protein17.63
Tallow2.69
Corn gluten meal3.42
Limestone11.53
Monocalcium phosphate1.82
Sodium bicarbonate0.21
Sodium chloride0.27
Choline chloride0.05
L-Lysine HCl-56%0.20
DL-Methionine-99%0.13
L-Threonine-99%0.09
L-Valine-99%0.11
Vitamin premix 10.10
Mineral premix 20.10
Calculated chemical composition
Nitrogen-corrected apparent metabolizable energy, kcal/kg2700
Dry matter, %88.53
Crude protein, %15.0
Ether extract, %4.89
Ash, %15.5
Calcium, %4.40
Available phosphorus, %0.40
Lysine, %0.79
Methionine + Cysteine, %0.64
Threonine, %0.65
Valine, %0.81
1 Vitamin premix provided the following nutrients per kg of diet: vitamin A, 20,000 IU; vitamin D3, 4600 IU; vitamin E, 40 mg; vitamin K3, 4 mg; thiamin, 3.6 mg; riboflavin, 8 mg; pyridoxine, 5.798 mg; cobalamin, 0.04 mg; biotin, 0.24 mg; D-pantothenic acid, 18.355 mg; niacin, 6 mg; folic acid, 0.14 mg. 2 Mineral premix provided the following nutrients per kg of diet: Fe, 70 mg; Mn, 7.5 mg; Zn, 60 mg; Cu, 7.5 mg; I, 1 mg; Co, 0.13 mg; Se, 0.20 mg.
Table 2. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on laying performance in laying hens 1.
Table 2. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on laying performance in laying hens 1.
ItemsCONNSMJ27Pooled SEMp-Value
Feed intake, g/d/bird99.6998.041.2460.206
Egg production, %84.5285.801.1390.289
Egg weight, g/egg61.1861.580.2670.164
Egg mass, g/d52.2452.331.2620.944
Feed-to-egg ratio, kg/kg1.8971.8610.0250.174
1 CON, control diet; NSJM27, CON + 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27; SEM, standard error of the means.
Table 3. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on egg quality in laying hens 1.
Table 3. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on egg quality in laying hens 1.
ItemsCONNSMJ27Pooled SEMp-Value
Yolk color7.5177.6700.1620.368
Haugh unit87.1387.210.7160.912
Eggshell strength, kg/cm24.8704.4790.2200.106
Eggshell thickness, mm0.3900.3830.0110.596
Eggshell color, unit24.9823.220.8430.057
1 CON, control diet; NSJM27, CON + 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27; SEM, standard error of the means.
Table 4. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on ileal histology in laying hens 1.
Table 4. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on ileal histology in laying hens 1.
ItemsCONNSMJ27Pooled SEMp-Value
Villus height (VH), µm824.7814.051.300.839
Crypt depth (CD), µm134.4127.14.1590.114
VH:CD ratio5.5436.3860.4190.067
1 CON, control diet; NSJM27, CON + 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27; SEM, standard error of the means.
Table 5. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on the relative percentage (% of total) of short-chain fatty acids in cecal digesta of laying hens 1.
Table 5. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on the relative percentage (% of total) of short-chain fatty acids in cecal digesta of laying hens 1.
ItemsCONNSMJ27Pooled SEMp-Value
Acetate60.1858.933.1890.701
Propionate16.6216.521.7120.951
Isobutyrate3.6134.1571.1830.653
Butyrate10.90 b13.05 a0.7820.016
Isovalerate3.7352.7630.4690.057
Valerate4.9484.5820.4970.474
BCFA12.3011.501.6600.640
SCFA87.7088.501.6600.640
1 CON, control diet; NSJM27, CON + 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27; SEM, standard error of the means; SCFAs, short-chain fatty acids (acetate + propionate + butyrate); BCFAs, branched-chain fatty acids (isobutyrate + isovalerate + valerate). a,b Values having a different superscript within a row differ significantly (p < 0.05).
Table 6. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on antioxidant and immune markers of ileal mucosa in laying hens 1.
Table 6. Effect of dietary Lacticaseibacillus paracasei strain NSMJ27 on antioxidant and immune markers of ileal mucosa in laying hens 1.
ItemsCONNSMJ27Pooled SEMp-Value
SOD, U/mg protein1.494 b1.921 a0.1920.048
MDA, nmol/mg protein3.2812.6900.7170.424
CAT, U/mg protein8.5678.6900.9780.902
GPX, U/mg protein0.1640.1420.0430.608
sIgA, ug/mg protein34.3038.969.1850.622
1 CON, control diet; NSJM27, CON + 2.5 × 109 CFU/kg L. paracasei subsp. paracasei NSMJ27; SEM, standard error of the means; SOD, superoxide dismutase; MDA, malondialdehyde; CAT, catalase; GPX, glutathione peroxidase; sIgA, secretory immunoglobulin A. a,b Values having a different superscript within a row differ significantly (p < 0.05).
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Vu, V.A.; Kim, Y.-B.; Kim, S.-K.; Jung, J.Y.; Joo, S.S.; Ban, B.; Kim, M.; Kim, M.; Lee, K.-W. Effects of Probiotic Lacticaseibacillus paracasei NSMJ27 on Laying Performance and Gut Health Indicators in Aged Laying Hens. Animals 2026, 16, 792. https://doi.org/10.3390/ani16050792

AMA Style

Vu VA, Kim Y-B, Kim S-K, Jung JY, Joo SS, Ban B, Kim M, Kim M, Lee K-W. Effects of Probiotic Lacticaseibacillus paracasei NSMJ27 on Laying Performance and Gut Health Indicators in Aged Laying Hens. Animals. 2026; 16(5):792. https://doi.org/10.3390/ani16050792

Chicago/Turabian Style

Vu, Viet Anh, Yoo-Bhin Kim, Soo-Ki Kim, Ji Young Jung, Sang Seok Joo, Byeongcheol Ban, Myunghoo Kim, Minji Kim, and Kyung-Woo Lee. 2026. "Effects of Probiotic Lacticaseibacillus paracasei NSMJ27 on Laying Performance and Gut Health Indicators in Aged Laying Hens" Animals 16, no. 5: 792. https://doi.org/10.3390/ani16050792

APA Style

Vu, V. A., Kim, Y.-B., Kim, S.-K., Jung, J. Y., Joo, S. S., Ban, B., Kim, M., Kim, M., & Lee, K.-W. (2026). Effects of Probiotic Lacticaseibacillus paracasei NSMJ27 on Laying Performance and Gut Health Indicators in Aged Laying Hens. Animals, 16(5), 792. https://doi.org/10.3390/ani16050792

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop