Six iso-nitrogenous (350 g protein kg )1 ) and iso-caloric (4100 kcal kg )1 ) diets with or without probiotics supplementation namely T 1 (Basal feed (BF) without probiotics; control), T 2 (BF + Bacillus subtilis and Lactococcus lactis), T 3 (BF + L. lactis and Saccharomyces cerevisiae), T 4 (BF + B. subtilis and S. cerevisiae), T 5 (BF + B. subtilis, L. lactis and S. cerevisiae) and T 6 (BF + heat-killed bacteria of B. subtilis, L. lactis and S. cerevisiae) were fed to Labeo rohita fingerlings (6.0 ± 0.06 g) for 60 days in triplicate tanks (30 fish per tank). In all probiotic-supplemented diets, the probiotic concentration was maintained at 10 11 cfu kg )1 feed. After 60 days of culture, the fish fed combination of three probiotics at equal proportion (T 5 ) had higher (P < 0.05) growth, protein efficiency ratio, nutrient retention and digestibility and lower (P > 0.05) feed conversion ratio over other treatment groups. Total heterotrophic bacterial population in intestine was drastically reduced on 15th and 30th days of sampling than the initial value (0 day of sampling) for T 3 , T 4 and T 5 groups. Except T 6 , the gut colonization of respective probiotics, which were supplemented through the diets, was also increased up to 30 days of culture of fish and thereafter remained constant. KEY WORDS

Summary To meet the ever‐increasing demand for animal protein, aquaculture continuously requires new techniques to increase the production yield. However, with every step towards intensification of aquaculture practices, there is an increase in stress level on the animal as well as on the environment. Feeding practices in aqua farming usually plays an important role, and the addition of various additives to a balanced feed formula to achieve better growth is a common practice among the fish and shrimp culturists. Probiotics, also known as ‘bio‐friendly agents’, such as LAB (Lactobacillus), yeasts and Bacillus sp., can be introduced into the culture environment to control and compete with pathogenic bacteria as well as to promote the growth of the cultured organisms. In addition, probiotics are non‐pathogenic and non‐toxic micro‐organisms, having no undesirable side effects when administered to aquatic organisms. Probiotics are also known to play an important role in developing innate immunity among the fishes, and hence help them to fight against any pathogenic bacterias as well as against environmental stressors. The present review is a brief but informative compilation of the different essential and desirable traits of probiotics, their mode of action and their useful effects on fishes. The review also highlights the role of probiotics in helping the fishes to combat against the different physical, chemical and biological stress.

A diverse array of sex determination (SD) mechanisms, encompassing environmental to genetic, have been found to exist among vertebrates, covering a spectrum from fixed SD mechanisms (mammals) to functional sex change in fishes (sequential hermaphroditic fishes). A major landmark in vertebrate SD was the discovery of the SRY gene in 1990. Since that time, many attempts to clone an SRY ortholog from non-mammalian vertebrates remained unsuccessful, until 2002, when DMY/DMRT1BY was discovered as the SD gene of a small fish, medaka. Surprisingly, however, DMY/DMRT1BYwas found in only two species among more than 20 species of medaka, suggesting a large diversity of SD genes among vertebrates. Considerable progress has been made over the last 3 decades, such that it is now possible to formulate reasonable paradigms of how SD and gonadal sex differentiation may work in some model vertebrate species. This review outlines our current understanding of vertebrate SD and gonadal sex differentiation, with a focus on the molecular and cellular mechanisms involved. An impressive number of genes and factors have been discovered that play important roles in testicular and ovarian differentiation. An antagonism between the male and female pathway genes exists in gonads during both sex differentiation and, surprisingly, even as adults, suggesting that, in addition to sex-changing fishes, gonochoristic vertebrates including mice maintain some degree of gonadal sexual plasticity into adulthood. Importantly, reviewing various SD mechanisms among vertebrates suggest that this is the ideal biological event that can make us understand the evolutionary conundrums underlying speciation and species diversity.

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Probiotics play an important role in growth increment, immune enhancement and stress mitigation in fish. Increasing temperature is a major concern in present aquaculture practices as it markedly deteriorates the health condition and reduces the growth in fish. In order to explore the possibilities of using probiotics as a counter measure for temperature associated problems, a 30 days feeding trial was conducted to study the hemato-immunological and apoptosis response of Labeo rohita (8.3±0.4 g) reared at different water temperatures, fed with or without dietary supplementation of a probiotic mixture (PM) consisting of Bacillus subtilis, Lactococcus lactis and Saccharomyces cerevisiae) (1011 cfu kg−1). Three hundred and sixty fish were randomly distributed into eight treatment groups in triplicates, namely, T1(28°C+BF(Basal feed)+PM), T2(31°C+BF+PM), T3(34°C+BF+PM), T4(37°C+BF+PM), T5(28°C+BF), T6(31°C+BF), T7(34°C+BF) and T8(37°C+BF). A significant increase (P<0.01) in weight gain percentage was observed in the probiotic fed fish even when reared at higher water temperature (34–37°C). Respiratory burst assay, blood glucose, erythrocyte count, total serum protein, albumin, alkaline phosphatase and acid phosphatase were significantly higher (P<0.01) in the probiotic fed groups compared to the non-probiotic fed groups. A significant (P<0.01) effect of rearing temperature and dietary probiotic mixture on serum myeloperoxidase activity, HSP70 level and immunoglobulin production was observed. Degree of apoptosis in different tissues was also significantly reduced in probiotic-supplemented groups. Hence, the present results show that a dietary PM could be beneficial in enhancing the immune status of the fish and also help in combating the stress caused to the organism by higher rearing water temperature.

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Despite identification of several sex-determining genes in non-mammalian vertebrates, their detailed molecular cascades of sex determination/differentiation are not known. Here, we used a novel RNAi to characterise the molecular mechanism of Dmy (the sex-determining gene of medaka)-mediated masculinity in XY fish. Dmy knockdown (Dmy-KD) suppressed male pathway (Gsdf, Sox9a2, etc.) and favoured female cascade (Rspo1, etc.) in embryonic XY gonads, resulting in a fertile male-to-female sex-reversal. Gsdf, Sox9a2, and Rspo1 directly interacted with Dmy, and co-injection of Gsdf and Sox9a2 re-established masculinity in XY-Dmy-KD transgenics, insinuating that Dmy initiates masculinity by stimulating and suppressing Gsdf/Sox9a2 and Rspo1 expression, respectively. Gonadal expression of Wt1a starts prior to Dmy and didn’t change upon Dmy-KD. Furthermore, Wt1a stimulated the promoter activity of Dmy, suggesting Wt1a as a regulator of Dmy. These findings provide new insights into the role of vertebrate sex-determining genes associated with the molecular interplay between the male and female pathways.

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