Effect of type and concentration of cryoprotectant on the motility, viability, and fertility of climbing perch Anabas testudineus Bloch, 1792 (Pisces: Anabantidae) sperm.

The climbing perch, Anabas testudineus is a freshwater fish that has economic value in Indonesia. It is cultured in the country, but the breeding technology, specifically sperm storage, is not well developed. Sperm cryopreservation is one of the preservation methods that need to be developed to support fish breeding technology. The type of cryoprotectants and its concentration are species-dependent and determines the success of this approach. Therefore, this study is aimed at determining the optimal type and concentration of cryoprotectant for sperm cryopreservation of A. testudineus. Four separate study series were performed, each of which evaluated one type of cryoprotectant at five concentration levels. The cryoprotectants used were DMSO, methanol, glycerol, and ethanol, and the tested concentrations were 0%, 5%, 10%, 15%, and 20%, which were combined with 5% egg yolks. Each treatment was conducted with three replications. The results showed that the type of cryoprotectant and its concentration significantly affected sperm motility, viability, and fertility of climbing perch (P < 0.05). The best outcome was obtained in DMSO, and methanol at a concentration of 10%, glycerol at 5%, and ethanol at 15%. However, the highest motility, viability, and fertility values were observed at 10% DMSO, indicating it is the best type and concentration for sperm cryopreservation of climbing perch A. testudineus.

Lernaea cyprinacea Linnaeus, 1758 (Copepoda: Lernaeidae) infection on Betta rubra Perugia, 1893 (Anabantiformes: Osphronemidae) from Aceh Province, Indonesia.

Betta rubra is an ornamental freshwater fish endemic to northern Sumatra, Indonesia. The B. rubra population has decreased in recent decades, and is classified as an endangered species in the IUCN Red List. This study aims to report for the first time infection by L. cyprinacea in B. rubra harvested from the Aceh Besar region of Indonesia. The fish samples were obtained from the Cot Bira tributaries, Aceh Besar District, Indonesia from January to December 2020. The results showed that the parasite infected 6 out of 499 samples in August and September, with a prevalence and intensity rate of 1% and 2 parasites/fish, respectively. The eyes and pectoral fins were the common infection sites. Despite B. rubra is not an optimal host (small size) for the parasite, this parasite might serve as additional threatening factors for the endangered B. rubra fish population.

Lernaea cyprinacea Linnaeus, 1758 (Copepoda: Lernaeidae) infection on Betta rubra Perugia, 1893 (Anabantiformes: Osphronemidae) from Aceh Province, Indonesia / Infecção por Lernaea cyprinacea Linnaeus, 1758 (Copepoda: Lernaeidae) em Betta rubra Perugia, 1893 (Anabantiformes: Osphronemidae) provenientes da província de Aceh, Indonésia

Abstract Betta rubra is an ornamental freshwater fish endemic to northern Sumatra, Indonesia. The B. rubra population has decreased in recent decades, and is classified as an endangered species in the IUCN Red List. This study aims to report for the first time infection by L. cyprinacea in B. rubra harvested from the Aceh Besar region of Indonesia. The fish samples were obtained from the Cot Bira tributaries, Aceh Besar District, Indonesia from January to December 2020. The results showed that the parasite infected 6 out of 499 samples in August and September, with a prevalence and intensity rate of 1% and 2 parasites/fish, respectively. The eyes and pectoral fins were the common infection sites. Despite B. rubra is not an optimal host (small size) for the parasite, this parasite might serve as additional threatening factors for the endangered B. rubra fish population.

Resumo Betta rubra é um peixe de água doce ornamental endemico da região norte Sumatra, Indonesia. A população de Betta rubra diminuiu ao longo dos anos, sendo classificada como espécie em extinção na Lista Vermelha da IUCN. Este estudo tem como objetivo relatar pela primeira vez infecção por L. cyprinacea em B. rubra coletados na região de Aceh Besar na Indonésia. As amostras de peixes foram obtidas nos afluentes Cot Bira, distrito de Aceh Besar, Indonésia de janeiro a dezembro de 2020. Os resultados mostraram que o parasito infectou 6 das 499 amostras em agosto e setembro, com uma prevalência e taxa de intensidade de 1% e 2 parasitas/peixes, respectivamente. Os olhos e as nadadeiras peitorais foram os sítios de infecção mais comuns. Apesar de B. rubra não ser um hospedeiro ideal (pequeno tamanho) para o parasita, este parasita pode servir como fator de ameaça adicional para a população de peixes B. rubra, ameaçada de extinção.

Immunohistochemical Expression of AQP2 and HSP70 in Broiler Kidney Tissue Treated with Salix tetrasperma Roxb. Extract under Heat Exposure.

The administration of plant extracts to broilers may be a way to mitigate the effects of heat stress. The importance of AQP2 and HSP70 compounds in maintaining the homeostasis of the chicken body when it is subjected to heat stress is well established. This study aims to determine the effect of giving the ethanolic extract of the leaves of Salix tetrasperma Roxb. on the immunohistochemical expression of AQP2 and HSP70 in exposed and unexposed broiler kidney tissue. This study used 36 samples of 28-day-old chicken kidneys. Chickens were kept in individual cages, provided with feed and drinking water ad libitum. The design used was a completely randomized design with 6 treatments and 6 replications: (a) chickens were reared in conditions exposed to heat (HS + 0); (b) chickens were reared in conditions exposed to heat and given Salix extract at a dose of 50 mg/L drinking water (HS + 50); (c) chickens were reared under heat-exposed conditions and given Salix extract at a dose of 100 mg/L drinking water (HS + 100); (d) chickens were reared in conditions without exposure to heat (n-HS + 0); (e) chickens were reared in conditions without exposure to heat and given Salix extract at a dose of 50 mg/L drinking water (nHS + 50); and (f) chickens were reared in conditions exposed without exposure to heat and given 100 mg/L drinking water (nHS + 100) of Salix extract. Salix extract was given for 24 hours and was renewed every 6 hours. The results showed that giving Salix extract 100 mg/L in drinking water to chickens exposed to heat (HS + 100) reduced the value of the H/L ratio. Giving Salix extract 50-100 mg/L in drinking water caused an upregulated AQP2 expression; on the other hand, it downregulated HSP-70 expression, in chicken kidney tubules both exposed to heat stress and nonexposed to heat stress. In conclusion, exposure to heat stress in broiler chickens and giving Salix extract can increase the formation of aquaporin 2 compounds and suppress the formation of HSP70.

Effect of cryoprotectant on the motility, viability, fertilization, and DNA integrity of naleh fish Barbonymus sp. (Cyprinidae) sperm / Efeito do crioprotetor na motilidade, viabilidade, fertilização e integridade do DNA no sêmen de peixes naleh Barbonymus sp. (Cyprinidae)

Naleh fish Barbonymus sp. is a commercial freshwater fish, which is indigenous to Aceh, Indonesia. The population of this species has declined over the years as a result of habitat perturbations and overfishing. Hence, the crucial need to develop a cryopreservation method to support breeding programs. This involved the use of a cryoprotectant as an important component. The objective of this study, therefore, was to explore the best cryoprotectant for naleh fish spermatozoa, and a total of five types were tested. These include the DMSO, Methanol, Ethanol, Glycerol, and Ethylene Glycol at a similar concentration of 10%, which were individually combined with 15% egg yolk, and every treatment was performed in three replications. Conversely, Ringer's solution was adopted as an extender, and the sperm was cryopreserved in liquid nitrogen for 15 days. The results showed significant influence on sperm motility and viability, as well as egg fertility of naleh fish (P <0.05), although the DMSO provided the best outcome, compared to others at 47.17%, 50.13%, and 45.67%, respectively. Furthermore, DNA fragmentation had not occurred in the fresh and cryopreserved sperm samples, indicating the protective effect of tested cryoprotectants. It is concluded that the 10% DMSO and 15% egg yolk is the best cryoprotectant for naleh fish spermatozoa.(AU)

O peixe naleh Barbonymus sp. é um peixe comercial de água doce, originário de Aceh, Indonésia. Durante vários anos, as perturbações provocadas no seu habitat e a pesca predatória determinaram o declínio da sua população, cuja preservação deve apoiar-se em um programa de reprodução controlada, com o emprego de espermatozoides criopreservados. O presente trabalho realizou um estudo comparativo de cinco crioprotetores: dimetilsultóxido, metanol, etanol, glicerol e etileno glicol. Todos os crioprotetores foram testados na concentração de 10%, combinados a 15% de gema de ovo. Cada tratamento foi efetuado em triplicatas. A solução de ringer foi utilizada como extensor e o esperma foi criopreservado em nitrogênio líquido por 15 dias. Os resultados obtidos revelaram a existência de influência significante (P<0,05) na viabilidade e motilidade espermática bem como na fertilidade dos ovos do peixe naleh, em que o dimetilsulfóxido apresentou o melhor resultado com os valores de 47,17%, 50,13% e 45,67%, respectivamente. Por outro lado, a fragmentação do DNA não ocorreu nas amostras de esperma fresco e criopreservado, indicando o efeito protetor dos crioprotetores testados. A conclusão obtida foi que o dimetilsulfóxido e 15% de gema de ovo foram o melhor crioprotetor para os espermatozoides do peixe naleh.(AU)

Length-weight relationships and condition factors of mullets Liza macrolepis and Moolgarda engeli (Pisces: Mugilidae) harvested from Lambada Lhok waters in Aceh Besar, Indonesia.

Background: The mullets fish Liza macrolepis and Moolgarda engeli are predominant in the Lambada Lhok waters in Aceh province. At present, no scientific report on this species in Aceh waters is available. Therefore, the objective of the present study is to examine the growth pattern and condition factor of the species of mullets L. macrolepis and M. engeli harvested from the aforementioned coastal waters. Methods: The sampling was done in three locations in the Lambada Lhok waters from July to November 2018. The fish were captured using gillnets from 6:00 AM to 3:00 PM four times a month for five months.  A total of 242 L. macrolepis and 109 M. engeli were used for the analysis. The growth pattern was analyzed using linear allometric model; then, two condition factors, Fulton's and relative weight, were calculated. Results: The study revealed a b value of 2.49 for the male L. macrolepis and 1.81 for the female. The b value was 3.22 for the male M. engeli and 3.41 for the female. The b value of the fish was higher during the dry season. The Fulton's condition factor of the male L. macrolepis was 1.19, and that of the female was 1.19. The relative condition factor of this species was 100.11 and 100.01 for males and females, respectively. The Fulton condition factor of male M. engeli was 1.05 and that of the female was 1.06. The relative weight condition factors were 101.08 and 100.61 for the male and female, respectively.   Conclusions: The growth pattern of  M. engeli tends to be isometric, whereas that of  L. macrolepis has a negative allometric growth pattern. The condition factors indicate that the Lambada Lhok waters are still in good condition and support the growth of the mullets, but  M. engeli is more adaptable than  L. macrolepis.

Disentangling the taxonomy of the subfamily Rasborinae (Cypriniformes, Danionidae) in Sundaland using DNA barcodes.

Sundaland constitutes one of the largest and most threatened biodiversity hotspots; however, our understanding of its biodiversity is afflicted by knowledge gaps in taxonomy and distribution patterns. The subfamily Rasborinae is the most diversified group of freshwater fishes in Sundaland. Uncertainties in their taxonomy and systematics have constrained its use as a model in evolutionary studies. Here, we established a DNA barcode reference library of the Rasborinae in Sundaland to examine species boundaries and range distributions through DNA-based species delimitation methods. A checklist of the Rasborinae of Sundaland was compiled based on online catalogs and used to estimate the taxonomic coverage of the present study. We generated a total of 991 DNA barcodes from 189 sampling sites in Sundaland. Together with 106 previously published sequences, we subsequently assembled a reference library of 1097 sequences that covers 65 taxa, including 61 of the 79 known Rasborinae species of Sundaland. Our library indicates that Rasborinae species are defined by distinct molecular lineages that are captured by species delimitation methods. A large overlap between intraspecific and interspecific genetic distance is observed that can be explained by the large amounts of cryptic diversity as evidenced by the 166 Operational Taxonomic Units detected. Implications for the evolutionary dynamics of species diversification are discussed.

Antibacterial activity in secondary metabolite extracts of heterotrophic bacteria against Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa.

Background: Disease causing bacteria such as Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa present a problem for fish farming. Treatment to remove them are generally carried out using antibiotics which have side effects on fish, the environment and humans. However, the use of antibacterial compounds derived from heterotrophic bacteria serve as a good alternative for antibiotics. Therefore, this study aimed to explore antibacterial activity in the secondary metabolite extracts of heterotrophic bacteria against Vibrio alginolyticus, Aeromonas hydrophila, and Pseudomonas aeruginosa. Methods: Heterotrophic bacteria namely Bacillus sp. JS04 MT102913.1, Bacillus toyonensis JS08 MT102920.1, Bacillus cereus JS10 MT102922.1, Bacillus sp. JS11 MT102923.1, Pseudoalteromonas sp. JS19 MT102924.1, Bacillus cereus JS22 MT102926.1, and Bacillus sp. strain JS25 MT102927.1 were used in this study. The sequences of these bacteria have been deposited and are available from NCBI GenBank. Each heterotrophic bacterium was cultured on 6L nutrient broth for 8 days, and extracts produced using ethyl acetate to obtain their secondary metabolites. These extracts were tested for their phytochemical contents using FT-IR and also tested for their inhibitory property in pathogenic bacteria by agar diffusion method. Results: Phytochemical test results showed that the seven heterotrophic bacterial isolates produced terpenoid compounds. Based on the inhibitory test, the secondary metabolite extracts from Bacillus sp strain JS04 had the highest inhibitory effect on the growth of pathogenic bacteria namely, V. alginolyticus (17.5 mm), A. hydrophila (16.8 mm), and P. aeruginosa (17.3 mm). Conclusion: It was concluded that the secondary metabolite extracts of heterotrophic bacteria inhibit the growth of V. alginolyticus, A. hydrophila, and P. aeruginosa.

Potential of bacteriocins produced by probiotic bacteria isolated from tiger shrimp and prawns as antibacterial to Vibrio, Pseudomonas, and Aeromonas species on fish.

Backgrounds: Bacteriocin has been used widely in industry as a biopreservative agent. The objective of the present study was to investigate the potency of Bacteriocin isolated from tiger prawn Penaeus monodon and freshwater shrimp Macrobrachium rosenbergii as an anti-bacterial on fish. Methods: A total of ten candidates of probiotic bacteria consisted of five isolates from tiger shrimps (H1, H2, H3, H4, H5) and five isolates from freshwater prawns (W1, W2, W3, W4, W5) were evaluated. Bacteriocin wasBacteriocin was produced by centrifugation at a speed of 150 rpm and at 37 °C for 24 hours. The bacteriocin extract was purified by adding sulphate ammonium salt {(NH4) 2SO4} at 80% of the saturation level. Bacteriocin activity was determined using a diffusion method against pathogenic bacteria Vibrio alginolyticus, Aeromonas hydrophillaAeromonas hydrophilla and Pseudomonas stutzeri. Bacteriocins were analyzed usinganalyzedusing High Performance Liquid Chromatography (HPLC) and Fourier Transform Infra-Red (FTIR). The data were subjected to analysis of variance (ANOVA) and followed with Duncans multiple range test. Results: Bacteriocins produced by bacteria isolate H4 from tiger prawn indicated the highest bacteriocin activity againstbacteriocin activity against Pseudomonas stutzeri at stutzeri at the diameter of inhibition zone of 887.10 ± 409.24 mm 2/mL. While isolate W2 from freshwater shrimp indicated inhibition zone of 1466.96 ± 127.62 mm 2/mL. Both bacteriocins were purified by chromatography column using Sephadex LH-20. Conclusions: This finding showed that bacterial isolates H4 and W2 have the potential to produce bacteriocins which inhibit the pathogenic bacteria. FTIR analysis showed an amide group at wave number 1652cm -1 contained in the bacteriocins of isolates H4 and W2.

Exploring hidden diversity in Southeast Asia's Dermogenys spp. (Beloniformes: Zenarchopteridae) through DNA barcoding.

Members of the freshwater halfbeak genus Dermogenys are hard to identify to the species level, despite several previous attempts to isolate fixed meristic, morphometric and colour pattern differences. This has led to ongoing confusion in scientific literature, records of species occurrence, and entries in museum collections. Here, a DNA barcoding study was conducted on the genus to gain further understanding of its taxonomic status across the Southeast Asian region. Fish were collected from 33 localities, spanning freshwater and brackish habitats in Malaysia, Western Indonesia, Thailand and Vietnam. In total, 290 samples of Dermogenys spp. were amplified for a 651 base pair fragment of the mitochondrial cytochrome oxidase c subunit I (COI) gene. Analysis was able to successfully differentiate the three species: D. collettei, D. siamensis, D. sumatrana; reveal the presence of a new putative species, Dermogenys sp., that was sampled in sympatry with D. collettei at three locations; as well as uncovering two genetic lineages of a fifth species, D. bispina, that display non-overlapping geographical distributions in drainages of northern Borneo; Kudat and Sandakan. This study expands the barcode library for Zenarchopteridae, demonstrates the efficacy of DNA barcoding techniques for differentiating Dermogenys species, and the potential thereof in species discovery.

Induced spawning of siban fish Cyclocheilichthys apogon using Ovaprim.

Background: The objective of the present study was to examine the effect of Ovaprim dosage on the latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates of the siban fish, Cyclocheilichthys apogon. Methods: Three dosages of Ovaprim were tested in this study, namely 0.3 ml kg-1 of broodfish body weight, 0.5 ml kg-1 body weight, and 0.7 ml kg-1 body weight, plus control (without Ovaprim).  Results: The results showed that the best latency period, relative number of ovulated eggs, fertilization, hatching, and survival rates were obtained at a dosage of 0.7 ml kg -1 body weight. Conclusions: The best dosage of Ovaprim for siban fish from the dosages tested, was determined to be 0.7 ml kg-1 body weight.

Phenotype and genotype of lactic acid bacteria (LAB) isolated from the tiger grouper Epinephelus fuscoguttatus alimentary tract.

Lactic acid bacteria (LAB) have been isolated successfully from the tiger grouper Epinephelusfuscoguttatus intestine. However, their genus or species have not been identified. Therefore, the objective of the present study was to characterize the three isolated LAB (KSBU-12C, KSBU-5Da, and KSBU-9) based on their phenotype and genotype. The LAB phenotype was examined by observing morphological features including cell morphology, spore production and motility. The physiological tests were performed in 6.5% NaCl at the  temperatures of 10 oC and 45 oC, and the biochemical tests were evaluated based on the production of enzymes catalase, oxidase and arginine dehydrolase, following  the Standard Analytical Profile Index, API 50 CH kit.  The genotype was examined based on 16S rDNA gene sequence analysis , and the products were analyzed using the BLAST (Basic Local Alignment Search Tool) NCBI database. The three isolates (KSBU-5Da, KSBU-12C, and KSBU-9) were categorized into the genus Enterococcus. 16S rDNA sequence analysis indicated that the isolates had 99% similarity to E. hirae ATCC 9790, registered in GenBank with accession number NR_075022.1. It was concluded that the three LAB isolates taken from the tiger grouper Epinephelus fuscoguttatus are E. hirae.

Growth performance and feed utilization of keureling fish Tor tambra (Cyprinidae) fed formulated diet supplemented with enhanced probiotic.

Background The objective of the present study was to determine the optimum dosage of probiotic in the diet of keureling fish ( Tor tambra) fry. MethodsLactobacillus casei from Yakult® was used as a starter, and enhanced with Curcuma xanthorrhiza, Kaempferia galanga and molasses. The mixture was fermented for 7 days prior to use as probiotic in a formulated diet containing 30% crude protein. Four levels of probiotic dosage; 0 ml kg -1 (control), 5 ml kg -1, 10 ml kg -1 and 15 ml kg -1 were tested in this study. The fish was fed twice a day at 08.00 AM and 06.00 PM at the ration of 5% body weight for 80 days. Results The results showed that growth performance and feed efficiency increased with increasing probiotic dosage in the diet from control (no probiotic) to 10 ml kg -1 of probiotic dosage and then decreased when the dosage was increased up to 15 ml kg -1. Conclusions The best values for all measured parameters were recorded at the dosage of 10 ml kg -1. Therefore, it was concluded that the optimum dosage of enhanced probiotic for T. tambra fry was 10 ml kg -1 of feed.