Foods and Raw Materials Foods and Raw Materials Foods and Raw Materials 2308-4057 2310-9599 65002 10.21603/2308-4057-2024-1-590 Research Article Research Article Research Article Effect of a new probiotic on Artemia cysts determined by a convolutional neural network Effect of a new probiotic on Artemia cysts determined by a convolutional neural network https://orcid.org/0000-0002-6218-3151 Evdokimov Ivan Yu. Evdokimov Ivan Yu. ivan.evdokimov.92@mail.ru https://orcid.org/0000-0002-4053-036X Malkova Angelina V. Malkova Angelina A. https://orcid.org/0000-0002-2664-1995 Irkitova Alena N. Irkitova Alena N. https://orcid.org/0000-0002-1628-2546 Shirmanov Maxim V. Shirmanov Maxim V. https://orcid.org/0000-0002-6954-8563 Dementev Dmitrii V. Dementev Dmitrii V. Altai State University Barnaul Россия Altai State University Barnaul Russian Federation Altai State University Barnaul Россия Altai State University Barnaul Russian Federation Altai State University Barnaul Россия Altai State University Barnaul Russian Federation Altai State University Barnaul Россия Altai State University Barnaul Russian Federation Arsal LLC Yarovoye Россия Arsal LLC Yarovoye Russian Federation 13 02 2024 13 02 2024 12 1 91 100 09 01 2023 https://jfrm.ru/en/issues/21683/21694/

One of the problems in sea farming is infections that cause mass mortality of crustaceans. To fight infections and improve sanitary conditions, farmers are actively using probiotic preparations. We aimed to study the effect of a new probiotic based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains on the incubation of Artemia franciscana cysts. Another purpose was to test a possibility of using a convolutional neural network for fast automatic counting of cysts, nauplii, and embryos. A pilot batch of the probiotic was prepared at the Prombiotech Engineering Center, Altai State University, from two strains of spore bacteria from the Center’s collection: B. toyonensis B-13249 and B. pumilus B-13250. The recommended amount of the probiotic was experimentally determined as 0.1 per 2 g of cysts. This concentration increased the number of hatched cysts by 1.4 and 10% in the batches from Lake Bolshoye Yarovoye (Z29.04) and from Lake Kuchuk (C9). It also increased the biomass yield to 7.40 ± 0.69 and 6.80 ± 0.43 g in these two batches, respectively, compared to the control samples where the yields were 5.30 ± 0.60 and 4.60 ± 0.50 g, respectively. The robot counter reduced the sample processing time 15 times and saved the data for further use. The probiotic based on B. toyonensis B-13249 and B. pumilus B-13250 had a positive effect on the hatching rate and biomass yield of A. franciscana. The new method for rapid counting of Artemia, which was based on the convolutional neural network and developed as an application of the Artemeter-1 robot, reduced the processing time and lowered labor costs.

One of the problems in sea farming is infections that cause mass mortality of crustaceans. To fight infections and improve sanitary conditions, farmers are actively using probiotic preparations. We aimed to study the effect of a new probiotic based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains on the incubation of Artemia franciscana cysts. Another purpose was to test a possibility of using a convolutional neural network for fast automatic counting of cysts, nauplii, and embryos. A pilot batch of the probiotic was prepared at the Prombiotech Engineering Center, Altai State University, from two strains of spore bacteria from the Center’s collection: B. toyonensis B-13249 and B. pumilus B-13250. The recommended amount of the probiotic was experimentally determined as 0.1 per 2 g of cysts. This concentration increased the number of hatched cysts by 1.4 and 10% in the batches from Lake Bolshoye Yarovoye (Z29.04) and from Lake Kuchuk (C9). It also increased the biomass yield to 7.40 ± 0.69 and 6.80 ± 0.43 g in these two batches, respectively, compared to the control samples where the yields were 5.30 ± 0.60 and 4.60 ± 0.50 g, respectively. The robot counter reduced the sample processing time 15 times and saved the data for further use. The probiotic based on B. toyonensis B-13249 and B. pumilus B-13250 had a positive effect on the hatching rate and biomass yield of A. franciscana. The new method for rapid counting of Artemia, which was based on the convolutional neural network and developed as an application of the Artemeter-1 robot, reduced the processing time and lowered labor costs.

 Bacillus pumilus Bacillus toyonensis Artemia franciscana aquaculture probiotics convolutional neural network bacteria counting Bacillus pumilus Bacillus toyonensis Artemia franciscana aquaculture probiotics convolutional neural network bacteria counting

Salunke M, Kalyankar A, Khedkar CD, Shingare M, Khedkar GD. A review on shrimp aquaculture in India: Historical perspective, constraints, status and future implications for impacts on aquatic ecosystem and biodiversity. Reviews in Fisheries Science and Aquaculture. 2020;28(3):283-302. https://doi.org/10.1080/23308249.2020.1723058 Salunke M, Kalyankar A, Khedkar CD, Shingare M, Khedkar GD. A review on shrimp aquaculture in India: Historical perspective, constraints, status and future implications for impacts on aquatic ecosystem and biodiversity. Reviews in Fisheries Science and Aquaculture. 2020;28(3):283-302. https://doi.org/10.1080/23308249.2020.1723058 Sampantamit T, Ho L, Lachat C, Sutummawong N, Sorgeloos P, Goethals P. aquaculture production and its environmental sustainability in Thailand: Challenges and potential solutions. Sustainability. 2020;12(5). https://doi.org/10.3390/su12052010 Sampantamit T, Ho L, Lachat C, Sutummawong N, Sorgeloos P, Goethals P. aquaculture production and its environmental sustainability in Thailand: Challenges and potential solutions. Sustainability. 2020;12(5). https://doi.org/10.3390/su12052010 Cortes A, Casillas-Hernandez R, Cambeses-Franco C, Borquez-Lopez R, Magallon-Barajas F, Quadros-Seiffert W, et al. Eco-efficiency assessment of shrimp aquaculture production in Mexico. Aquaculture. 2021;544. https://doi.org/10.1016/j.aquaculture.2021.737145 Cortes A, Casillas-Hernandez R, Cambeses-Franco C, Borquez-Lopez R, Magallon-Barajas F, Quadros-Seiffert W, et al. Eco-efficiency assessment of shrimp aquaculture production in Mexico. Aquaculture. 2021;544. https://doi.org/10.1016/j.aquaculture.2021.737145 Tepaamorndech S, Chantarasakha K, Kingcha Y, Chaiyapechara S, Phromson M, Sriariyanun M, et al. Effects of Bacillus aryabhattai TBRC8450 on vibriosis resistance and immune enhancement in Pacific white shrimp, Litopenaeus vannamei. Fish and Shellfish Immunology. 2019;86:4-13. https://doi.org/10.1016/j.fsi.2018.11.010 Tepaamorndech S, Chantarasakha K, Kingcha Y, Chaiyapechara S, Phromson M, Sriariyanun M, et al. Effects of Bacillus aryabhattai TBRC8450 on vibriosis resistance and immune enhancement in Pacific white shrimp, Litopenaeus vannamei. Fish and Shellfish Immunology. 2019;86:4-13. https://doi.org/10.1016/j.fsi.2018.11.010 Naylor RL, Hardy RW, Buschmann AH, Bush SR, Cao L, Klinger DH, et al. A 20-year retrospective review of global aquaculture. Nature. 2021;591:551-563. https://doi.org/10.1038/s41586-021-03308-6 Naylor RL, Hardy RW, Buschmann AH, Bush SR, Cao L, Klinger DH, et al. A 20-year retrospective review of global aquaculture. Nature. 2021;591:551-563. https://doi.org/10.1038/s41586-021-03308-6 Joshua WJ, Kamarudin MS, Ikhsan N, Yusoff FM, Zulperi Z. Development of enriched Artemia and Moina in larviculture of fish and crustaceans: A review. Latin American Journal of Aquatic Research. 2022;50(2):144-157. Joshua WJ, Kamarudin MS, Ikhsan N, Yusoff FM, Zulperi Z. Development of enriched Artemia and Moina in larviculture of fish and crustaceans: A review. Latin American Journal of Aquatic Research. 2022;50(2):144-157. Kovacheva NP, Litvinenko LI, Saenko EM, Zhigin AV, Kryahova NV, Semik AM. Current state and prospects of aquaculture artemia in Russia. Trudy VNIRO. 2019;178:150-171. (In Russ.). https://doi.org/10.36038/2307-3497-2019-178-150-171 Kovacheva NP, Litvinenko LI, Saenko EM, Zhigin AV, Kryahova NV, Semik AM. Current state and prospects of aquaculture artemia in Russia. Trudy VNIRO. 2019;178:150-171. (In Russ.). https://doi.org/10.36038/2307-3497-2019-178-150-171 Sellami I, Naceur HB, Kacem A. Study of cysts biometry and hatching percentage of the brine shrimp Artemia salina (Linnaeus, 1758) from the Sebkha of Sidi El Hani (Tunisia) according to successive generations. Aquaculture Studies. 2020;21(1):41-46. https://doi.org/10.4194/2618-6381-v21_1_05 Sellami I, Naceur HB, Kacem A. Study of cysts biometry and hatching percentage of the brine shrimp Artemia salina (Linnaeus, 1758) from the Sebkha of Sidi El Hani (Tunisia) according to successive generations. Aquaculture Studies. 2020;21(1):41-46. https://doi.org/10.4194/2618-6381-v21_1_05 Niu J, Chen P-F, Tian L-X, Liu Y-J, Lin H-Z, Yang H-J, et al. Excess dietary cholesterol may have an adverse effect on growth performance of early post-larval Litopenaeus vannamei. Journal of Animal Science and Biotechnology. 2012;3(1). https://doi.org/10.1186/2049-1891-3-19 Niu J, Chen P-F, Tian L-X, Liu Y-J, Lin H-Z, Yang H-J, et al. Excess dietary cholesterol may have an adverse effect on growth performance of early post-larval Litopenaeus vannamei. Journal of Animal Science and Biotechnology. 2012;3(1). https://doi.org/10.1186/2049-1891-3-19 Malkova A, Evdokimov I, Shirmanov M, Irkitova A, Dementyev D. New bacilli-based probiotic for aquaculture: Efficacy study on Macrobrachium rosenbergii. BIO Web of Conferences. 2022;42. https://doi.org/10.1051/bioconf/20224201011 Malkova A, Evdokimov I, Shirmanov M, Irkitova A, Dementyev D. New bacilli-based probiotic for aquaculture: Efficacy study on Macrobrachium rosenbergii. BIO Web of Conferences. 2022;42. https://doi.org/10.1051/bioconf/20224201011 Ofelio C, Planas M, Pintado J. Administration of the probiotic Lactobacillus rhamnosus IMC 501 as a strategy for the control of Vibrio bacteria in the brine shrimp Artemia. Letters in Applied Microbiology. 2021:73(3):336-342. https://doi.org/10.1111/lam.13518 Ofelio C, Planas M, Pintado J. Administration of the probiotic Lactobacillus rhamnosus IMC 501 as a strategy for the control of Vibrio bacteria in the brine shrimp Artemia. Letters in Applied Microbiology. 2021:73(3):336-342. https://doi.org/10.1111/lam.13518 Schar D, Zhao C, Wang Yu, Larsson DGJ, Gilbert M, Boeckel van TP. Twenty-year trends in antimicrobial resistance from aquaculture and fisheries in Asia. Nature communications. 2021:12(1). https://doi.org/10.1038/s41467-021-25655-8 Schar D, Zhao C, Wang Yu, Larsson DGJ, Gilbert M, Boeckel van TP. Twenty-year trends in antimicrobial resistance from aquaculture and fisheries in Asia. Nature communications. 2021:12(1). https://doi.org/10.1038/s41467-021-25655-8 Wall S. Prevention of antibiotic resistance - an epidemiological scoping review to identify research categories and knowledge gaps. Global Health Action. 2019;12. https://doi.org/10.1080/16549716.2020.1756191 Wall S. Prevention of antibiotic resistance - an epidemiological scoping review to identify research categories and knowledge gaps. Global Health Action. 2019;12. https://doi.org/10.1080/16549716.2020.1756191 Uddin TM, Chakraborty AJ, Khusro A, Zidan BMRM, Mitra S, Emran TB, et al. Antibiotic resistance in microbes: History, mechanisms, therapeutic strategies and future prospects. Journal of Infection and Public Health. 2021:14(12):1750-1766. https://doi.org/10.1016/j.jiph.2021.10.020 Uddin TM, Chakraborty AJ, Khusro A, Zidan BMRM, Mitra S, Emran TB, et al. Antibiotic resistance in microbes: History, mechanisms, therapeutic strategies and future prospects. Journal of Infection and Public Health. 2021:14(12):1750-1766. https://doi.org/10.1016/j.jiph.2021.10.020 Larsson DGJ, Flach C-F. Antibiotic resistance in the environment. Nature Reviews Microbiology. 2022:20:257-269. https://doi.org/10.1038/s41579-021-00649-x Larsson DGJ, Flach C-F. Antibiotic resistance in the environment. Nature Reviews Microbiology. 2022:20:257-269. https://doi.org/10.1038/s41579-021-00649-x Goh JXH, Tan LT-H, Law JW-F, Khaw K-Y, Zengin G, Chan K-G, et al. Probiotics: Comprehensive exploration of the growth promotion mechanisms in shrimps. Progress in Microbes and Molecular Biology. 2023:6(1). https://doi.org/a10.36877/pmmb.0000324 Goh JXH, Tan LT-H, Law JW-F, Khaw K-Y, Zengin G, Chan K-G, et al. Probiotics: Comprehensive exploration of the growth promotion mechanisms in shrimps. Progress in Microbes and Molecular Biology. 2023:6(1). https://doi.org/a10.36877/pmmb.0000324 da Costa Sousa N, do Couto MVS, Paixão PEG, dos Santos Medeiros E, de Souza JCN, Barros FAL, et al. Enriched Artemia nauplii with commercial probiotic in the larviculture of angelfish Pterophyllum scalare Lichtenstein (1823). Journal of Fisheries Science. 2020;2(1):17-21. https://doi.org/10.30564/jfsr.v2i1.1569 da Costa Sousa N, do Couto MVS, Paixão PEG, dos Santos Medeiros E, de Souza JCN, Barros FAL, et al. Enriched Artemia nauplii with commercial probiotic in the larviculture of angelfish Pterophyllum scalare Lichtenstein (1823). Journal of Fisheries Science. 2020;2(1):17-21. https://doi.org/10.30564/jfsr.v2i1.1569 Volkova GS, Serba EM. New Multistrain Bacterial Consortium for Feed Probiotics. Food Processing: Techniques and Technology. 2021;51(2):260-269. (In Russ.). https://doi.org/10.21603/2074-9414-2021-2-260-269. Volkova GS, Serba EM. New Multistrain Bacterial Consortium for Feed Probiotics. Food Processing: Techniques and Technology. 2021;51(2):260-269. (In Russ.). https://doi.org/10.21603/2074-9414-2021-2-260-269. Ahmadifard N, Aminlooi VR, Tukmechi A, Agh N. Evaluation of the impacts of long-term enriched Artemia with Bacillus subtilis on growth performance, reproduction, intestinal microflora, and resistance to Aeromonas hydrophila of ornamental fish Poecilia latipinna. Probiotics Antimicrobial Proteins. 2019:11:957-965. https://doi.org/10.1007/s12602-018-9453-4 Ahmadifard N, Aminlooi VR, Tukmechi A, Agh N. Evaluation of the impacts of long-term enriched Artemia with Bacillus subtilis on growth performance, reproduction, intestinal microflora, and resistance to Aeromonas hydrophila of ornamental fish Poecilia latipinna. Probiotics Antimicrobial Proteins. 2019:11:957-965. https://doi.org/10.1007/s12602-018-9453-4 Sekar A, Kima M, Jeonb H, Kima K. Screening and selection of bacteria inhibiting white spot syndrome virus infection to Litopenaeus vannamei. Biochemistry and Biophysics Reports. 2019:19. https://doi.org/10.1016/j.bbrep.2019.100663 Sekar A, Kima M, Jeonb H, Kima K. Screening and selection of bacteria inhibiting white spot syndrome virus infection to Litopenaeus vannamei. Biochemistry and Biophysics Reports. 2019:19. https://doi.org/10.1016/j.bbrep.2019.100663 Amoah K, Huang Q-C, Tan B-P, Zhang S, Chi S-Y, Yang Q-H, et al. Dietary supplementation of probiotic Bacillus coagulans ATCC 7050, improves the growth performance, intestinal morphology, microflora, immune response, and disease confrontation of Pacific white shrimp, Litopenaeus vannamei. Fish and Shellfish Immunology. 2019;87:796-808. https://doi.org/10.1016/j.fsi.2019.02.029 Amoah K, Huang Q-C, Tan B-P, Zhang S, Chi S-Y, Yang Q-H, et al. Dietary supplementation of probiotic Bacillus coagulans ATCC 7050, improves the growth performance, intestinal morphology, microflora, immune response, and disease confrontation of Pacific white shrimp, Litopenaeus vannamei. Fish and Shellfish Immunology. 2019;87:796-808. https://doi.org/10.1016/j.fsi.2019.02.029 Fernandes V, Sabu EA, Shivaramu MS, Gonsalves MJBD, Sreepada RA. Dynamics and succession of plankton communities with changing nutrient levels in tropical culture ponds of whiteleg shrimp. Aquaculture Environment Interactions. 2019;11:639-655. https://doi.org/10.3354/aei00341 Fernandes V, Sabu EA, Shivaramu MS, Gonsalves MJBD, Sreepada RA. Dynamics and succession of plankton communities with changing nutrient levels in tropical culture ponds of whiteleg shrimp. Aquaculture Environment Interactions. 2019;11:639-655. https://doi.org/10.3354/aei00341 Wang Y-C, Hu S-Y, Chiu C-S, Liu C-H. Multiple-strain probiotics appear to be more effective in improving the growth performance and health status of white shrimp, Litopenaeus vannamei, than single probiotic strains. Fish and Shellfish Immunology. 2019;84:1050-1058. https://doi.org/10.1016/j.fsi.2018.11.017 Wang Y-C, Hu S-Y, Chiu C-S, Liu C-H. Multiple-strain probiotics appear to be more effective in improving the growth performance and health status of white shrimp, Litopenaeus vannamei, than single probiotic strains. Fish and Shellfish Immunology. 2019;84:1050-1058. https://doi.org/10.1016/j.fsi.2018.11.017 Seenivasan C, Bhavan PS, Radhakrishnan S, Shanthi R. Enrichment of Artemia nauplii with Lactobacillus sporogenes for enhancing the survival, growth and levels of biochemical constituents in the post-larvae of the freshwater prawn Macrobrachium rosenbergii. Turkish Journal of Fisheries and Aquatic Sciences. 2012;12:23-31. Seenivasan C, Bhavan PS, Radhakrishnan S, Shanthi R. Enrichment of Artemia nauplii with Lactobacillus sporogenes for enhancing the survival, growth and levels of biochemical constituents in the post-larvae of the freshwater prawn Macrobrachium rosenbergii. Turkish Journal of Fisheries and Aquatic Sciences. 2012;12:23-31. Patra SK, Mohamed KS. Enrichment of Artemia nauplii with the probiotic yeast Saccharomyces boulardii and its resistance against a pathogenic Vibrio. Aquaculture International. 2003;11:505-514. https://doi.org/10.1023/b:aqui.0000004193.40039.54 Patra SK, Mohamed KS. Enrichment of Artemia nauplii with the probiotic yeast Saccharomyces boulardii and its resistance against a pathogenic Vibrio. Aquaculture International. 2003;11:505-514. https://doi.org/10.1023/b:aqui.0000004193.40039.54 Isamma A, Divya, KR, Ramasubramanian V, Arunjith TS, Sureshkumar S. Standartization of the bioencapsulation of probiotics and oil emulsion in Artemia parthenogenetica. International Journal of Research in Fisheries and Aquaculture. 2014;4(3):122-125. Isamma A, Divya, KR, Ramasubramanian V, Arunjith TS, Sureshkumar S. Standartization of the bioencapsulation of probiotics and oil emulsion in Artemia parthenogenetica. International Journal of Research in Fisheries and Aquaculture. 2014;4(3):122-125. Dementʹev DV, Komyshev GB, Semyonov PA. The convolutional neural network for determining the quality of Artemia. Patent RU 2021663036. 2021. https://www.elibrary.ru/ERHHLL Dementʹev DV, Komyshev GB, Semyonov PA. The convolutional neural network for determining the quality of Artemia. Patent RU 2021663036. 2021. https://www.elibrary.ru/ERHHLL Bochkovskiy A, Wang C-Y, Liao H-YM. YOLOv4: Optimal speed and accuracy of object detection. https://doi.org/10.48550/arXiv.2004.10934 Bochkovskiy A, Wang C-Y, Liao H-YM. YOLOv4: Optimal speed and accuracy of object detection. https://doi.org/10.48550/arXiv.2004.10934 Malkova AV, Evdokimov IYu, Shirmanov MV, Irkitova AN, Dudnik DE. Development of a probiotic for animals and aquaculture based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains. Proceedings of Universities. Applied Chemistry and Biotechnology. 2021;11(3):393-402. (In Russ.). https://doi.org/10.21285/2227-2925-2021-11-3-393-402 Malkova AV, Evdokimov IYu, Shirmanov MV, Irkitova AN, Dudnik DE. Development of a probiotic for animals and aquaculture based on Bacillus toyonensis B-13249 and Bacillus pumilus B-13250 strains. Proceedings of Universities. Applied Chemistry and Biotechnology. 2021;11(3):393-402. (In Russ.). https://doi.org/10.21285/2227-2925-2021-11-3-393-402 Baert P, Bosteels T, Sorgeloos P. Pond production. In: Lavens P, Sorgeloos P, editors. Manual on the production and use of live food for aquaculture. Rome: FAO; 1996. pp. 196-251. Baert P, Bosteels T, Sorgeloos P. Pond production. In: Lavens P, Sorgeloos P, editors. Manual on the production and use of live food for aquaculture. Rome: FAO; 1996. pp. 196-251. Jabir B, Rabhi L, Falih N. RNN- and CNN-based weed detection for crop improvement: An overview. Foods and Raw Materials. 2021;9(2):387-396. https://doi.org/10.21603/2308-4057-2021-2-387-396 Jabir B, Rabhi L, Falih N. RNN- and CNN-based weed detection for crop improvement: An overview. Foods and Raw Materials. 2021;9(2):387-396. https://doi.org/10.21603/2308-4057-2021-2-387-396 Hamsah, Widanarni, Alimuddin, Yuhana M, Zairin M. The nutritional value of Artemia sp. enriched with the probiotic Pseudoalteromonas piscicida and the prebiotic mannan-oligosaccharide. AACL Bioflux. 2017;10(1):8-17. Hamsah, Widanarni, Alimuddin, Yuhana M, Zairin M. The nutritional value of Artemia sp. enriched with the probiotic Pseudoalteromonas piscicida and the prebiotic mannan-oligosaccharide. AACL Bioflux. 2017;10(1):8-17.