Epigenetics and Probiotics Application toward the Modulation of Fish Reproductive Performance

Abstract

Fish represent an excellent source of animal protein as well as a biomedical research model as a result of their evolutionary relatedness and similarity with the human genome. Commercial and ornamental fish culture has achieved popularity, but reproductive dysfunctions act as a limiting factor for quality fry production, interfering with the sustainability of the aquaculture industry. Fish reproduction is crucial for any species’ existence, and reproductive performance can potentially be improved through applications of epigenetics and probiotics. Epigenetics is a highly sensitive molecular approach that includes chromatin structure and function alteration, DNA methylation, and modification of non-coding RNA molecules for the transfer of desired information from parents to offspring. DNA methyltransferase improves reproductive cyp11a1, esr2b, and figla gene expression and feminizes zebrafish (Danio rerio). Moreover, epigenetics also contributes to genome stability, environmental plasticity, and embryonic development. However, methylation of specific genes can negatively affect sperm quality, resulting in poor fertilization. Probiotic administration is able to induce responsiveness of incompetent follicles to maturation-inducing hormones and can change oocyte chemical composition during vitellogenic development. The positive role of probiotics on testicular cells is validated by upregulating the transcription levels of leptin, bdnf, and dmrt1 genes facilitating the spermatogenesis. This review not only discusses the effects and mechanism of epigenetics and probiotics for improving fish reproduction, but also presents an overview of the causal factors and current techniques used to eradicate dysfunction. Moreover, key genes and hormones related to fish reproduction along with research gaps and future prospects are also considered. This review provides an overview of necessary information for students, scientists, researchers, and breeders to resolve fish reproduction-related problems to ensure profitable and sustainable aquaculture.

1. Introduction

Aquaculture has been known for millennia, when the trend of captive fish rearing began, and it is now playing a crucial role in solving the world food crisis, particularly in meeting the protein demand [1]. However, broodstock management, which includes the optimization of critical reproductive processes, such as nutrition, maturation, egg and sperm production, and spawning, remains a major obstacle to the advancement of the aquaculture industry. Captive broodstocks are most vulnerable to the disruption of reproductive activities by hormonal imbalance and unfavorable environmental parameters [2] resulting in reproductive dysfunction. Such reproductive impairments include no or poor-quality egg or sperm production, defective or weak spawn, less growth and high mortality of fry, and sometimes mortality of the brood. Reproductive dysfunctions can be triggered by direct or indirect variations of the gametes and endocrine system, captivity-induced stress, unsuitability of the spawning environment, and deficiencies in the nutritional profiles of feeds [3,4]. Some taxonomic groups of fishes are far more sensitive to such inhibitory influences than others. Reproductive dysfunction is common in Anguilla spp. (catadromous eels), Seriola spp. (greater amberjack and Japanese yellowtail), Caranx ignobilis (giant trevally), Epinephelus spp. (groupers) and Thunnus spp. (bluefin tuna) [5], Anoplopoma fimbria (Sablefish) [6], and Oncorhynchus mykiss (Rainbow trout) [2].

To avoid reproductive dysfunction, scientific management of broodstock animals ensures proper physiology, immunology, reproductive enzymes pathways activation, and transcription of specific reproductive genes. Many commercial farms seek to maintain healthy broodstocks by supplementation with probiotics to increase reproductive enzymes activities and gene transcription, but dysfunction remains fairly common during breeding seasons [7,8]. Moreover, epigenetic mechanisms include DNA methylation, histone modification, and noncoding RNA action underlying various processes have recently received considerable attention in aquaculture and offer some promise in the amelioration of breeding performance [9].

The term epigenetics literally means “above” or “on top of” genetics, and epigenetic traits refer to stable heritable phenotypes resulting from changes in the status of chromosomes without alteration of DNA sequences [10]. Epigenetic reprogramming underpins many developmental processes such as gametogenesis and embryogenesis, foundation of environment regulating events of fish sex differentiation, providing a linkage between phenotypic and metabolic changes during domestication [11]. In several studies, epigenetic inheritance was found to be more feasible in fish as compared with terrestrial animals [12,13,14]. Epigenetic information has the potential to contribute to lower disease prevalence and the potential eradication of the use of antibiotics in commercial aquaculture [15]. Sperm quality has been linked to DNA methylation in spermatozoa and in Morone saxatilis (striped bass) sperm DNA methylation has a positive relationship with male reproductive capacity [16]. Epigenetics influence fish sex determination and differentiation, facilitating interactions between these processes with other surroundings [17]. When it comes to valuable fish species, such as grouper, it is essential to know sex patterns, especially when their sex is changed. In aquaculture, a stable and predictable mating system is critical for healthy fry production [9] and alterations in DNA methylation patterns triggered by higher temperatures can lead to more masculine traits in females [18,19]. Although epigenetics has huge potential, studies on economically important aquaculture species remain preliminary, and many unanswered questions remain [12,20]. An understanding of epigenetic mechanisms for commercial species could contribute to expansions and improvements in the economic viability of large-scale aquaculture.

Aquaculture technology has developed a positive view of probiotics application as an alternative to the application of synthetic antibiotics or chemicals [21,22,23]. Recognition of advantages of practical use of probiotics has grown in light of evidence of upregulation of fish growth, stress adaptation, immune modulation, and disease resistance [24,25]. Probiotics are defined as “live microorganisms administrated at an appropriate concentration that exert beneficial effects on host health and immune parameters” [26]. Ghosh, et al. [27] initially reported probiotics capacity to restore viable nutrients in female live-bearing ornamental fishes such as Poecilia reticulata (Guppy), P. sphenops (molly), Xiphophorus helleri (Swordtail) and X. maculatus (Platyfish). A later investigation was also carried out in X. helleri [28], Danio rerio (Zebrafish) [29,30,31,32,33,34], Carassius auratus (Goldfish) [35], and Fundulus heteroclitus (Killifish) [36]. Limited probiotics studies were also conducted to improve reproductive performance in commercial fishes such as Oreochromis niloticus (Nile Tilapia) [37], O. mykiss [38], Ompok pabda (Butter catfish) [39], Clarias gariepinus (African catfish) [40] and A. anguilla (European eel) [41] on the subject of reproductive indicators such as fecundity (Fec), hatching rate (HR), gamete quality, gonadosomatic index (GSI), fertilization rate (FR), and survival rate (SR). Pathogenic infections often leading to mortality of brood or offspring may be unnoticed by hatchery managers. Synthetic antibiotics or chemical applications to control infection may lead to mass environmental bacterial killing, antibiotic deposition in fish body and the generation of antibiotic-resistant pathogens [42,43]. As a result, probiotics may be a favorable option for broodstocks and fry for the control of infections and for improved reproductive success [44]. The use of probiotics helps to stabilize and diversify the intestinal microbial community, leading to improved reproduction processes through activation of different hormones, enzymes, and genes transcription resulting better FR, HR, SR, and larval growth [7,38].

In this study, recent research on the effects of epigenetics and probiotics in relation to the improvement of broodstock reproductive performance as a means of reproductive dysfunction eradication are extensively reviewed. Moreover, factors causing reproductive dysfunctions are also carefully enumerated. Finally, summarization of the key findings of epigenetics and probiotics in commercial and ornamental fish reproduction along with research gaps and future prospects for commercial aquaculture development are evaluated.

2. Fish Reproductive Dysfunctions

Globally, aquaculture is continually striving for the consistency of physiological integrity of fingerlings through standardized reproductive programmes, which are a key objective for sustainable aquaculture [45]. Brood fish rearing and their management strategies have been considered as vital for aquaculture production for the last three decades. Generally, many species of captive fishes do not perform normal reproduction (especially females), a phenomenon potentially caused by a lack of natural spawning stimuli, specifically failure of oocyte maturation [46,47]. Supplied protein, fatty acids, lipid, vitamins, especially E and C, and carotenoids also influence fish Fec, FR, HR, and larval development [48,49]. Moreover, presence of chemical fertilizers, antibiotics, hormones and industrial effluents, environmental degradation, and frequent fluctuation of temperature in nature and captivity suppress the immunity and beneficial microbial activity in broodstock fishes, making them more susceptible to infectious disease [27,50,51,52]. Hypothalamus, pituitary, and gonads form the hypothalamic-pituitary-gonadal (HPG) axis, which regulates reproductive function in most fishes [53]. The hypothalamus and the pituitary glands modulate the production of pituitary gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH), and gonadal sex steroids (SS) regulate vitellogenesis and oocyte maturation. FSH and LH have been confirmed to figure importantly in oocyte growth and maturation [54]. Gioacchini et al. [7] have categorized three types of female broodstocks reproductive dysfunctions based on the affected pattern of reproductive cycle whereas, Selvaraj, et al. [55] documented two modes of dysfunction of teleost fishes (Figure 1). Both groups of investigators associated the most disruptive reproductive dysfunctions occurred with the vitellogenic phase, as exemplified by failed vitellogenesis in Anguilla spp. and Seriola spp. [56] and Mugil cephalus (Grey Mullet) [57]. The second mode is categorized as the inability to reach final oocyte maturation to ovulation as seen in farmed white and striped bass [58,59,60] and in Cyprinidae [61]. The third set of dysfunctions leads to failures to spawn in the breeding season; sometimes E. aeneus, M. saxatilis, and Dentex dentex (common dentex) females may release eggs after ovulations without exhibiting characteristic breeding behavior [7,62]. Under some circumstances, cultured salmonids fail to complete the reproductive cycle and eggs in the abdominal cavity are reabsorbed over the following months [63]. These are examples of failures among female broodstocks that do not appear to be accompanied by reproductive problems that can be attributed to male fishes.

One common problem faced by cultured male fishes is low quantity or quality of milt production during spermiation [60]. The presence of both male and female germ cells in the testis tissue is referred to as an intersex state caused by endocrine disruption through chemical exposure and hormonal imbalance [64]. The histopathological assessment of gonadal tissue during the development stage allows the detection of intersex reproductive dysfunction in male Japanese medaka (Oryzias latipes) [65].

Figure 1. Schematic representation of the reproductive system and gonadal dysfunctions of fish. The broken line indicates the reproductive developmental stages. These dysfunctions may occur: (a) Lack of effectivity of luteinizing hormone (LH) for oocyte maturation; (b) outreaching of follicle-stimulating hormone (FSH) for oocyte development; (c) lower or no production of vitellogenin from the liver; (d) malformation of sperm and its motility, lowered testosterone to the testis. GnRH: gonadotrophin releasing hormone, MIS: maturation-inducing steroid.

Figure 1. Schematic representation of the reproductive system and gonadal dysfunctions of fish. The broken line indicates the reproductive developmental stages. These dysfunctions may occur: (a) Lack of effectivity of luteinizing hormone (LH) for oocyte maturation; (b) outreaching of follicle-stimulating hormone (FSH) for oocyte development; (c) lower or no production of vitellogenin from the liver; (d) malformation of sperm and its motility, lowered testosterone to the testis. GnRH: gonadotrophin releasing hormone, MIS: maturation-inducing steroid.

3. Reproductive Dysfunction Caused by Endocrine Disruptors

Endocrine disruption occurs in fish from the several sources including water, sediment, chemical or fertilizer, and diet (Figure 2). However, the toxic effects of endocrine disruptors on fish depend on exposure period and duration, chemical properties of the substances, or whether the exposed substance is single or mixed with other substances [66]. The causes of reproductive dysfunctions and examples of specific endocrine disruption are discussed below.

3.1. Chemical Stressors

Chemicals in the environment such as fungicides, herbicides, insecticides, and endocrine disruptor compounds (EDC) can inhibit fish from reproducing normally and are highly toxic to fishes and to other organisms in the food web [67]. Moreover, in aquatic systems pesticides have adverse impacts on fish reproductive activities, such as Fec, FR, GSI, oocyte maturation, ovulation, spawning, and spermiation [68,69,70,71]. Endocrine-disrupting effects may transpire during the early developmental stages of fish; for example, low concentrations of pesticides can act as mimics or blockers of sex hormones, causing abnormal sexual development, feminization of males, abnormal sex ratios, and unusual behavioral patterns associated with mating [72,73].

Fish reproductive activities are affected through numerous pesticides such as organochlorines (1,2,3,4,5,6-hexachlorocyclohexane, c-BHC; dichloro-diphenyl-trichloroethane, DDT; Kepone, and Toxaphene) and second and third-generation organophosphates (fenitrothion, Malathion, carbaryl, carbofuran, endosulfan, aldrin, onocrotophos, methylparathion, phenthoate, sumithion, profenofos) [70,74,75]. Pesticides not only impair morphological status of testes and ovaries of fishes, but they also cause delays in oocyte development and inhibition of steroid hormone synthesis [76]. McAllister and Kime [77] reported that tributyltin exposure of zebra fish in early life stage resulted in sperm deformity (lacking flagellae) during adulthood. The uptake of pesticides (lindane, pentachlorophenol and propoxur) had adverse effects on GSI as well as overall growth and reproductive performance of O. niloticus, C. gariepinus, and Bagrid catfishes [78]. Contamination of surface water by insecticides (organophosphate, carbamates, organochlorine, pyrethroids, and necotenoides) may lead to reduced growth coupled with reproductive dysfunctions of fishes (Figure 2). Moreover, malathion and dimethoate have adverse effects on the steroidogenesis, milt quality, hatchability, and survivability of fishes [75]. Water contaminated with tetramethrin, γ-cyhalothrin, permethrin, deltamethrin, cypermethrin, tamoxifen and resmethrin has been known to produce toxic impacts on reproduction, and survivability of fish and shellfish [79,80]. Similarly, pyrethroids provoke disorders of reproductive behavior in male brown trout (Salmo trutta) and Atlantic salmon (S. salar) [81]. It has been reported recently that a mixture of pyrethroid insecticides and fungicides (carbendazim and fenpropimorph) can cause direct sublethal effects on fish larvae at environmental concentrations in Swiss water [82]. Early exposure to carbaryl and carbamate insecticide on fathead minnow (Pimephales promela) impacted general growth and impaired reproductive performance in later life [83]. Vinclozolin and bis(2-ethylhexyl)phthalate are toxic to male fishes and cause androgen-dependent reproductive disorders [84]. Exposure to herbicides, such as atrazine and glyphosate, negatively affects GSI, steroid hormone synthesis, and embryonic development of zebra fish [82].

Figure 2. Presentation of different factors induced reproductive dysfunctions and effects in fish. (a) different chemical stressors mixed with the waterbody through surface runoff and uptake by gills resulted in bioaccumulation in the body; (b,c) alteration of temperature and photoperiod produce stress on the fish impact on physiology, immunology, and reproductive performance; (d) chemical and environmental stressors causing reproductive dysfunction in fish.

Figure 2. Presentation of different factors induced reproductive dysfunctions and effects in fish. (a) different chemical stressors mixed with the waterbody through surface runoff and uptake by gills resulted in bioaccumulation in the body; (b,c) alteration of temperature and photoperiod produce stress on the fish impact on physiology, immunology, and reproductive performance; (d) chemical and environmental stressors causing reproductive dysfunction in fish.

3.2. Stress, Temperature, and Photoperiod

Disruption of fish reproductive performances is a common phenomenon associated with stress. The detrimental impact of stress on vitellogenesis process (for example, follicular atresia) has been recorded in salmonids such as rainbow trout, brown trout and brook trout in the last few decades [85,86,87,88]. Some fishes are far more sensitive to stress-related inhibition of their reproductive axes than others, and in such cases even mild stress or captivity can result in complete reproductive failure [89]. Water temperature and photoperiod are primary environmental cues that trigger oocyte maturation and spawning in fish [60,90,91]. Moreover, unusual changes in water temperatures can cause acute stress, resulting in extensive follicular atresia in mature fish ovaries [92]. In addition, photoperiod plays a significant role in accelerating maturation and spawning of Scophthalmus maximus (Turbot) [90]. Many commercially important fishes, such as O. mykiss [93], Dicentrarchus labrax (sea bass) [94], O. niloticus [95], D. rerio [96], Platichthys stellatus (starry flounder) [97], and C. magur [98], respond to the changes of photoperiod, leading to the onset and completion of gonadal maturation and spawning.

4. Existing Methods for Eradicating Reproductive Dysfunctions of Brood Fish

4.1. Broodstock Management

Broodstock management is necessary for the optimization of reproductive processes in cultured fishes. Maternal health, nutrition, and endocrine status are determinants of larval capacity for survival [89]. In captive conditions, confinement under suboptimal conditions has been associated with the production of reduced milt quality in male fishes, leading to difficulties in breeding on farms [99]. For this reason, it can be necessary to use increased numbers of males to produce a sufficient quantity of spermatozoa for fertilization. Furthermore, some female fishes exhibit systemic reproductive deficiencies during yolk deposition and ovulation, in some cases leading to barren female fish [100]. Improper domestication and an unfavorable environment during oogenesis contribute to such failures.

4.2. Manipulation of the Reproductive System in Fish

In recent decades, artificial manipulation of reproductive seasonality has been widely used in aquaculture, as manifested in various new and emerging technologies [101]. Temperature, salinity, dissolved oxygen (DO), pH, and nutritional status are the governing factors of ovarian development and maturation [102]. Exogenous steroid treatment is an established approach for sex reversal and the induction of sexual maturation for artificial breeding. Steroid hormones have both positive and negative influences on fish reproductive status, which can impact patterns of profit and loss [103].

Sex reversal by steroid application can improve the growth/production of a certain fish gender (male or female) but can also induce skeletal abnormalities [104]. On the other hand, off-season hormone applications can contribute to the production of value-added fingerlings, at some risk of reduced breeding performance over the long run [105]. One approach to controlling the age at first maturity in farmed fish is through the manipulation of puberty. Alteration of the onset of puberty in potential broodstock that typically attain sexual maturity late (e.g., sturgeon and striped bass) is possible, as is the delay of the pubertal program before harvest in gilthead seabream, salmon, and many others.

The gut microbiota contributes to the gonadal maturation and subsequent reproductive success of the fish. Application of Lactobacillus rhamnosus alters the gut microbiome and can accelerate the sex differentiation through the larval development of zebrafish [106]. These authors also reported that L. rhamnosus can influence the timing of sex differentiation, resulting in significantly different sex ratios. Six days post fertilization (dpf) transgenic zebrafish larvae (GnRH3-GFP) were supplemented with L. rhamnosus; probiotics significantly accelerated growth rate, resulting in faster spinal calcification, earlier gonadal maturation and sex differentiation. In addition, fish also exhibited higher insulin-like growth factors -I and -II (igf1 and 2) mRNA levels, peroxisome proliferator-activated receptors -α and -β (PPAR-α and -β), vitamin D receptor-α (VDR-α), and retinoic acid receptor γ (RAR-γ) gene expressions. Adult female zebrafish treated with Lab. rhamnosus upregulate vitellogenic follicles, GSI, number of ovulated eggs, and ensure optimal activities of reproductive hormones (kisspeptins, gonadotropin-releasing hormone (GnRH)-3, and leptin) resulting in increased reproductive success [29]. Fish culture practices are threatened by at least eighty microbial pathogens, adding challenges during brood management. Antibiotics have been used for protection of broodfish from infectious bacterial diseases following artificial propagation [107]. The low antibiotic concentrations have reduced effects on fish reproductive systems but can eliminate beneficial microbial species [108]. Moreover, antibiotics have been found responsible for fish reproductive disorder as well as demonstrated immunosuppressive and genotoxic impacts, essentially the opposite of the desirable actions of probiotics.

Herbal products enhance growth, appetite, immune response, antioxidant, and hepatoprotective activity and their bioactive compounds, such as saponin, phenols, flavonoids, and polysaccharides, influenced the reproductive performances [109]. Fish reproduction is regulated through the coordination of hormones and genes at the hypothalamus, pituitary, and gonad of the HPG axis. The experimental provision of selected herbal extracts and plant secondary metabolites led to alterations in fish fertility through endocrine regulatory changes, either upgrading physiological or pathologically suppressing reproductive status [110]. Some phytochemicals downregulate the estradiol concentration through inhibition of aromatase (cyp19a) or reduce the bioconversion of testosterone to estradiol, with stimulatory effects on reproductive output [111]. On the other hand, some of these phytochemicals can also inhibit vitellogenesis through binding to the estrogen receptor, effectively blocking estradiol. The application of phytoestrogen to the diet of male Cyprinus carpio for two months increased the concentration of vitellogenin [112], also altering transport protein and hepatic lipid generation for the growing oocytes [113]. However, upregulation of the estrogen receptor gene and the high estradiol concentration are responsible for genistein that induced vitellogenesis in adult male fishes [114]. During the estrogenic responses in adult male C. carpio, testosterone levels rise and the steroidogenic enzyme activity decreases, which indicates a possible delay of maturation. The transcription of hepatic estrogen receptors is closely related to the regulation of vitellogenin synthesis in most teleosts. The vitellogenin synthesized and secreted from the liver in response to estrogenic stimulation reaches the ovary through the blood, is absorbed by oocytes, and is then converted into phospholipid-rich yolk proteins [115].

Certain nutrients individually or in combination can be key factors for increasing fish growth and reproductive maturation [116]. Nutrient deficiencies can delay or disrupt seasonal spawning migrations and reproductive cycles [117]. The endocrine system is sensitive to external and internal factors such as size, age, supplies of sugars, amino acids, and lipids required for reproduction. Increasing dietary lipid level up to 180 g kg⁻¹ significantly improved total PUFA (LA and LNA), long chain-PUFA (EPA, DHA, and ArA), and n-3 and n-6 series in ovary and liver of female C. striatus effective for breeding activities [118]. Broodfishes are likely to complete reproductive development and spawning under optimal conditions or delay/abort in adverse situations. Malnutrition in females can result in serious reproductive problems such as inhibition of vitellogenesis, oocyte maturation and spawning, and males exhibit the decreased sperm volume and diminished milt fluidity, resulting in reduced rates of fertilization [116]. Artificial feed formulas during maturation and breeding period influence growth and reproductive performance and the mortality rate of offspring. Nevertheless, nutrients used from living organisms, such as probiotics, plants, and some algae [44], exert both direct and indirect effects on fish reproductive programmes.

5. Key Genes and Hormones Related to Reproductive Function

Genes transmit hereditary information through the generation of structural or regulatory proteins. Some genes generate proteins with signaling activity to modify cellular events in the reproductive system [119]. Reproduction in fish is regulated primarily by the HPG axis, which is conserved from fish to humans, with the release, both centrally and peripherally, of ovarian hormones [120]. Among higher-level regulatory compounds, GnRH is a hypothalamic product with anterior pituitary stimulatory actions encoded by the GnRH receptor gene [121]. The GnRH is a member of the seven-transmembrane, G-protein coupled receptor (GPCR) family that can be highly expressed on the surface of pituitary gonadotrophs during the fish reproductive season and promotes the release of gonadotropins LH and FSH [122] (Figure 3). Similarly, kisspeptin encoded by KISS1 gene is also a member of the GPCR family that modulates the activity of GnRH signaling and sex hormone synthesis in the HPG axis [123,124]. Isotocin is a nine amino acid nonapeptide homologous to oxytocin released from the anterior pituitary of bony or teleost fish, with an important role in modulating social as well as the reproductive behavior of fishes [125].

Gonadal steroids have direct effects on fish reproduction function and low levels of 17 β estradiol does not only accelerate vitellogenesis, as these compounds also decline with a concomitant increase in 17α-20β-dihydroxy 4-pregnen-3-one (DHP) during oocyte maturation and ovulation [126]. Vitellogenin is a critically important component of oocytes, that contains lipid, phospholipids, triglycerides, and cholesterol transported into developing ova for the formation of the vitelline membrane.

On the other hand, gamete maturation is a critical stage for the male regulated by the progestational steroids, such as maturation-inducing hormone (MIH) besides two other mediators viz. LH and maturational promoting factor (MPF) [127]. In cyprinids, DHP triggers final oocyte maturation and initiate the meiotic division of spermatogonia and controls the spermatozoa maturation in male fish [128]. In males, MIH activates the synthesis of T to 11-KT from interstitial cell and initiates spermatogenesis as well as final sperm maturation [129]. As a result, in both male and female fish of S. trutta, S. gairdneri, O. mykiss, perch (Perca fluviatilis) and O. masou have exhibited a higher density in plasma cortisol levels during the pre-spawning or spawning period than in immature fishes [130]. Temporal changes in plasma cortisol level in male rainbow trout resulted in altered physiological functions favoring reproduction, as opposed to stress. Defects or loss of the activity of these genes and hormones are associated with reproductive dysfunction in fish, with negative economic implications.

Figure 3. Demonstrating the specific genes or hormones associated with fish reproduction. (A) The left side represents, specific organs of fish from which the associated hormones/enzymes and genes are released. (B) The right portion demonstrates the posterior and anterior pituitary glands and genes of fish associated with fish reproduction. GnRH: gonadotrophin releasing hormone; FSH: follicle stimulating hormone; LH: luteinizing hormone; KISS: Kisspeptin-1.

Figure 3. Demonstrating the specific genes or hormones associated with fish reproduction. (A) The left side represents, specific organs of fish from which the associated hormones/enzymes and genes are released. (B) The right portion demonstrates the posterior and anterior pituitary glands and genes of fish associated with fish reproduction. GnRH: gonadotrophin releasing hormone; FSH: follicle stimulating hormone; LH: luteinizing hormone; KISS: Kisspeptin-1.

6. Epigenetics Mechanism and Modifications of Fish Reproductive Performance

Considerable attention is focused in aquaculture on brood rearing, breeding, feeding, sex control, and disease management. The study of epigenetic mechanisms underlying various molecular mechanisms has the potential to contribute to favorable hatchery productivity. Environmental cues can cause phenotypic changes in organisms via epigenetic mechanisms (Figure 4). Moreover, environmental temperature, dissolved oxygen, water, pH, pollutants, and other factors influenced epigenetic changes in fish gonad and sex-dependent behavior [11,131,132]. Epigenetic modifications include DNA methylation, histone modification, chromatin re-modelling, and the action of noncoding RNAs (ncRNAs), attracting interest for their practical potential in fish reproduction [12].

The structure and consequently the function of chromatin can be altered by epigenetic mechanisms, resulting in the modulation of patterns of gene expression [133]. DNA methylation is the most well-known and well-understood epigenetic mechanism, in response to variable environmental factors, such as photoperiod, toxins, temperature, nutrients, etc. [134]. This entails the addition or removal of a methyl group to DNA, which alters gene function and transcription level as well. The methyl group is covalently added to the 5-carbon location of the cytosine ring, resulting in 5-methylcytosine (5-mC), known colloquially as the “fifth base” of DNA. DNA methylation takes place at CpG doublets either within CpG islands (dinucleotide CG), intergenic regions, or the gene body [135]. It has been reported that CpG islands influence gene expression by regulating transcription factor binding with chromatin structure. De novo DNA methylation and its maintenance are carried out by a family of DNA methyltransferase enzymes (DNMTs) and during embryogenesis, DNMT3A and DNMT3B are responsible for de novo methylation [136]. DNA demethylation is also equally important and accompanied by DNA methylation which is necessary for epigenetic reprogramming of genes. Through the incorporation of histone variants and post-translational modification of histones, chromatin structure can be altered to enhance or repress transcription. These altered chromatin states can be inherited both mitotically and meiotically, suggesting that they may transmit epigenetic information to the next generation. Evidence suggests that certain modified histones are retained non-randomly during spermatogenesis in both mammals and zebrafish, and these marks are thought to play a role in transferring epigenetic information to embryos [137,138]. The ncRNAs, which are made up of small and long RNA molecules, can influence gene expression [139]. Investigations reveal that ncRNAs play important roles in genome stability, environmental plasticity, and embryonic development [140,141]. The majority of research on ncRNAs in fish and shellfish, including important aquaculture species, has been conducted in Atlantic salmon and rainbow trout [142,143]. Aside from developmental programming, broodstock holding/conditioning is an important consideration for the potential transmission of epigenetic information transfer from parents to offspring [144]. Importantly, epigenetic transmission can occur on both the maternal and paternal sides [145] and the study of transgenerational plasticity in fish has grown in popularity [146].

A summary of key findings of studies addressing epigenetic modifications to improve the reproductive performance of commercially important farmed fishes is presented in Table 1. Recent investigations have analyzed the impact of epigenetics, specifically DNA methylation on breeding dynamics and productivity in finfish aquaculture. DNA methylation patterns have been found to change in response to temperature increases, leading to the masculinization of genetically female D. labrax [147], Cynoglossus semilaevis (Halfsmooth tongue sole) [18,19]; a trait which can be passed to offspring. In another study, Campos, et al. [148] found that Solea senegalensis (Senegalese sole) larvae undergoing metamorphosis had higher methylation levels on the myog promoter in skeletal muscle when reared at lower temperatures (15 °C). Hatchery offspring grown in captivity acquired epigenetic alterations in sperm which may explain rapid genetic and phenotypic alterations in the hybrid fishes. The differential methylation in hatchery salmon displayed the presence of their TATA-binding protein (a transcription factor that binds specifically to a DNA sequence) during spermiogenesis and embryonic development [149,150,151]. Epigenetics and broodstock nutrition also influence sperm production and quality in the aquaculture industry. Diet-induced methylation influence freshwater and saltwater trout and higher salt-containing diets caused dramatic changes in global methylation patterns [152]. Moreover, hydrogen sulfide stimulates DNA methylation in environments that can be continued generationally through the germline even after withdrawal of this toxic chemical [14]. Hypoxia-induced reproductive impairment of gonadal development, low sperm count, and motility through different methylation in sperm genes are inherited by the next generation [153]. Transgenerational epigenetic alteration in spermatozoa of aquatic animals resulted in phenotypic variation and methylation of specific gene groups, with potential negative effects on sperm quality and fertilization in male striped bass [16].

Global and gene-specific methylation in spermatozoa significantly affect the fertilization performance of C. carpio, in which methylation at CpG sites markedly increased and decreased after 24 and 96 h of post stripping, respectively [154]. In Nile tilapia, the gonadal transcriptomic study demonstrated that all of the DNMTs were expressed in both male and female reproductive organs while specific DNMTs were more highly expressed in the testis. Incubation of gonads with DNMTs inhibitor showed downregulated DNMTs with increased expression of male and female sex determinant gene dmrt1 and cyp19a1a, respectively [155].

It has also been revealed that cyp19a1a expression in Nile tilapia was critically controlled by environmental factors like temperature [156]. Similarly in zebrafish, DNMTs treatment feminizes the fish, stimulating long-term expression of key reproduction-related genes (e.g., cyp11a1, esr2b and figla) [157]. However, integrative transcriptome of Atlantic salmon testis revealed the involvement of differentially expressed micro (mi) RNA, thereby resulting in early puberty. This was the first study to link specific groups miRNA involvement in testis maturation in an inversely correlated relationship with targets [158]. Moreover, the endogenous non-coding RNAs (MicroRNAome) of sperm are affected by overexpression of growth hormone and consequently reduced sperm quality and fertilization potentiality of transgenic zebrafish [159]. Finally, the insufficiency of miR-202 compromised oogenesis or folliculogenesis, and significantly decreased the number of follicles [160].

Figure 4. A diagram depicting the potential applications of epigenetics for modulation of reproductive performance. (a) Environmental cues can alter gene transcription via epigenetic mechanisms; (b) DNA methylation, histone modifications, and non-coding RNA action resulting in phenotypic changes; (c) outcomes of epigenetics in phenotypic traits from early to later life stage.

Figure 4. A diagram depicting the potential applications of epigenetics for modulation of reproductive performance. (a) Environmental cues can alter gene transcription via epigenetic mechanisms; (b) DNA methylation, histone modifications, and non-coding RNA action resulting in phenotypic changes; (c) outcomes of epigenetics in phenotypic traits from early to later life stage.

Table 1. Epigenetic modifications effects on the reproductive performances of commercially important farmed fishes. Symbol: no change (→); increase (↑); decrease (↓) versus controls.

Species	Conditions of Exposure and Stressors	Epigenetics Marker	Methodology	Epigenetics Results	Phenotypic Outcomes	References
Dicentrarchus labrax(European sea bass)	High temperature during the thermosensitive phase	Methylation of gonadal promoters of cyp19a and β-actin	Bisulphite sequencing (BS Seq)	Methylation of cyp19a ↑	Masculinization	[147]
Cynoglossus semilaevis(Halfsmooth tongue sole)	Exposure to higher temperatures during early developmental period	DNA methylation of gonadal tissue and methylation status of dmrt1	BS-Seq	Genes involved in sex determination during sexual reversal ↑	Masculinization	[18,19]
Solea senegalensis (Senegalese sole)	Larvae undergoing transformation were raised at various temperatures	Cytosine methylation of the Myogenin (myog) promoter	BS-seq	Methylation of myog gene promoterat ↑, Myog transcription ↓ and dnmt1 and dnmt3s ↑ at 15 °C	Plasticity of myogenesis	[148]
Gadus morhua (Atlantic cod)	Juvenile exposure to two distinct photoperiods	Expression of DNA methyltransferases (dnmts) in fast muscle tissue	Semi quantitative and Quantitative real-time PCR (qRT-PCR)	Expression of dnmt1 and dnmt3a in muscle ↑	Muscle growth and final weight	[161]
Salmo trutta (Brown trout)	Freshwater fish are exposed to seawater after consuming a salt-enriched diet	DNA methylation at CpG sites in gill tissue samples	Methyl sensitive amplification and polymorphism (msAP)	Salt-induced reversible and temporary changes in global DNA methylation	Physiological adaptation of freshwater to seawater	[152]
Anguillla anguilla (European eel)	Chronic cadmium (Cd) exposure of immature organisms	Global and site-specific CpG methylation in liver tissue	Enzyme-linked immunosorbent assay (ELISA) and msAP-PCR	Global CpG methylation↑; Hypermethylation of genes related to intracellular trafficking and phospholipid biosynthesis ↑		[162]
Danio rerio (Zebrafish)	Copper and heat stress exposure during embryogenesis	Global DNA methylation; stress-related geneexpression	Colorimetric technique—MethylFlash Methylated DNA Quantification Kit; qRT-PCR	Global cytosine methylation →, Expression of dnmt3 ↑; mRNA expression of mt2 and hsp70 ↑	High mortality, late embryo hatching, and low hatching rate	[163]
Oryzias melastigma (Medaka fish)	Exposure to hypoxia in mature stage	DNA methylation of sperm cells (F0 F2)	qRT-PCR and Methylated DNA	DNA methylation in sperm (F2 fish) ↑; Hypomethylation of ehmt2 and ptk2b ↑; Hypermethylation of foxp2 exonic regions ↑	Aberrant sperm motility and decreased spermatid and fertility rate	[153]
D. rerio	Temperature and Cd exposure during whole life	DNA methylation, environmental sex determination	-	Methylation of cyp19a1a gene ↑; foxl2a/dmrt1 methylation levels ↑	Masculinization	[131]
D. rerio	Tris (1,3-dichloro-2-propyl) phosphate exposure during early development	Global DNA methylation	ELISA and qRT-PCR	Transcription mbpa, gap43 and syn2a ↑	Sex-dependent behavior in adults	[132]
Cyprinus carpio(Common carp)	-	Global and gene specific DNA methylation in sperm aging	Whole-genome BS-Seq	Global CpG methylation ↑	Sperm motility, velocity, concentration, and viability	[154]
Oryzias latipes(Medaka)	-	microRNAs (miRNA) expression in gonad	qRT-PCR and Agilent 8x60K microarray	The knockout of mir-202 gene resulted higher fecundity ↑; vitellogenic follicles number ↓	Number and quality of eggs	[160]
Menidia beryllina(Inland silverside)	Exposure to endocrine-disrupting chemicals like Bifenthrin, EE2, levonorgestrel, trenbolone	DNA methylation patterns for F0, F1, and and F2 generation	BS-seq	Promoter and/or gene body methylation multigenerational (F1) and transgenerational (F2) ↑	Fish phenotypic variation in future generations	[164]
Oncorhynchus mykiss(Steelhead trout)	Nutrition and stress in captive and natural conditions, and exposure to toxicants	DNA methylation in sperm and RBC DNA	qRT-PCR -Seq	DNA methylation in sperm and RBC ↑; Epigenetic transgenerational and phenotypic variation of next generations ↑	Growth/maturation	[165]
D. rerio	Early exposure of 5-aza-2′-deoxycytidine	DNA methylation during sexual development	Microarray and qRT-PCR	Expression of cyp11a1, esr2b and fgla ↑, Fanconi anemia or the Wnt signaling pathways ↓	Altered embryonic development, delayed hatching and increased teratology and mortality	[157]
Salmo salar(Atlantic salmon)	-	miRNAs related to the immature, prepubertal, and pubertal testis	miRNA and mRNA-seq	Expression of miRNAs and their mRNA targets during early puberty ↑	Pubertal maturation	[158]
Oreochromis niloticus(Nile Tilapia)	Exposure to Aromatase inhibitors	DNA methylation in the head kidney, testis, and ovary	qRT-PCR	The expression of dnmts ↓and cyp19a1a and dmrt1 ↑	Gonadal development	[155]
O. niloticus	Exposure to higher temperature	DNA methylation in three age groups of fish	BS-seq and qRT-PCR	cyp19a1a promoter DNA methylation levels ↑; cyp19a1a mRNA expression ↓	Masculinization	[156]
Morone saxatilis(Striped bass)	In vitro fertilization	DNA methylation in sperm fertility	Methyl-CpG-binding domain sequencing	WDR3/UTP12 and GPCR ↑ involved in sperm flagella formation, hormonal signaling and tissue development regulation	Sperm fertility	[166]

Table 1. Epigenetic modifications effects on the reproductive performances of commercially important farmed fishes. Symbol: no change (→); increase (↑); decrease (↓) versus controls.

Species	Conditions of Exposure and Stressors	Epigenetics Marker	Methodology	Epigenetics Results	Phenotypic Outcomes	References
Dicentrarchus labrax(European sea bass)	High temperature during the thermosensitive phase	Methylation of gonadal promoters of cyp19a and β-actin	Bisulphite sequencing (BS Seq)	Methylation of cyp19a ↑	Masculinization	[147]
Cynoglossus semilaevis(Halfsmooth tongue sole)	Exposure to higher temperatures during early developmental period	DNA methylation of gonadal tissue and methylation status of dmrt1	BS-Seq	Genes involved in sex determination during sexual reversal ↑	Masculinization	[18,19]
Solea senegalensis (Senegalese sole)	Larvae undergoing transformation were raised at various temperatures	Cytosine methylation of the Myogenin (myog) promoter	BS-seq	Methylation of myog gene promoterat ↑, Myog transcription ↓ and dnmt1 and dnmt3s ↑ at 15 °C	Plasticity of myogenesis	[148]
Gadus morhua (Atlantic cod)	Juvenile exposure to two distinct photoperiods	Expression of DNA methyltransferases (dnmts) in fast muscle tissue	Semi quantitative and Quantitative real-time PCR (qRT-PCR)	Expression of dnmt1 and dnmt3a in muscle ↑	Muscle growth and final weight	[161]
Salmo trutta (Brown trout)	Freshwater fish are exposed to seawater after consuming a salt-enriched diet	DNA methylation at CpG sites in gill tissue samples	Methyl sensitive amplification and polymorphism (msAP)	Salt-induced reversible and temporary changes in global DNA methylation	Physiological adaptation of freshwater to seawater	[152]
Anguillla anguilla (European eel)	Chronic cadmium (Cd) exposure of immature organisms	Global and site-specific CpG methylation in liver tissue	Enzyme-linked immunosorbent assay (ELISA) and msAP-PCR	Global CpG methylation↑; Hypermethylation of genes related to intracellular trafficking and phospholipid biosynthesis ↑		[162]
Danio rerio (Zebrafish)	Copper and heat stress exposure during embryogenesis	Global DNA methylation; stress-related geneexpression	Colorimetric technique—MethylFlash Methylated DNA Quantification Kit; qRT-PCR	Global cytosine methylation →, Expression of dnmt3 ↑; mRNA expression of mt2 and hsp70 ↑	High mortality, late embryo hatching, and low hatching rate	[163]
Oryzias melastigma (Medaka fish)	Exposure to hypoxia in mature stage	DNA methylation of sperm cells (F0 F2)	qRT-PCR and Methylated DNA	DNA methylation in sperm (F2 fish) ↑; Hypomethylation of ehmt2 and ptk2b ↑; Hypermethylation of foxp2 exonic regions ↑	Aberrant sperm motility and decreased spermatid and fertility rate	[153]
D. rerio	Temperature and Cd exposure during whole life	DNA methylation, environmental sex determination	-	Methylation of cyp19a1a gene ↑; foxl2a/dmrt1 methylation levels ↑	Masculinization	[131]
D. rerio	Tris (1,3-dichloro-2-propyl) phosphate exposure during early development	Global DNA methylation	ELISA and qRT-PCR	Transcription mbpa, gap43 and syn2a ↑	Sex-dependent behavior in adults	[132]
Cyprinus carpio(Common carp)	-	Global and gene specific DNA methylation in sperm aging	Whole-genome BS-Seq	Global CpG methylation ↑	Sperm motility, velocity, concentration, and viability	[154]
Oryzias latipes(Medaka)	-	microRNAs (miRNA) expression in gonad	qRT-PCR and Agilent 8x60K microarray	The knockout of mir-202 gene resulted higher fecundity ↑; vitellogenic follicles number ↓	Number and quality of eggs	[160]
Menidia beryllina(Inland silverside)	Exposure to endocrine-disrupting chemicals like Bifenthrin, EE2, levonorgestrel, trenbolone	DNA methylation patterns for F0, F1, and and F2 generation	BS-seq	Promoter and/or gene body methylation multigenerational (F1) and transgenerational (F2) ↑	Fish phenotypic variation in future generations	[164]
Oncorhynchus mykiss(Steelhead trout)	Nutrition and stress in captive and natural conditions, and exposure to toxicants	DNA methylation in sperm and RBC DNA	qRT-PCR -Seq	DNA methylation in sperm and RBC ↑; Epigenetic transgenerational and phenotypic variation of next generations ↑	Growth/maturation	[165]
D. rerio	Early exposure of 5-aza-2′-deoxycytidine	DNA methylation during sexual development	Microarray and qRT-PCR	Expression of cyp11a1, esr2b and fgla ↑, Fanconi anemia or the Wnt signaling pathways ↓	Altered embryonic development, delayed hatching and increased teratology and mortality	[157]
Salmo salar(Atlantic salmon)	-	miRNAs related to the immature, prepubertal, and pubertal testis	miRNA and mRNA-seq	Expression of miRNAs and their mRNA targets during early puberty ↑	Pubertal maturation	[158]
Oreochromis niloticus(Nile Tilapia)	Exposure to Aromatase inhibitors	DNA methylation in the head kidney, testis, and ovary	qRT-PCR	The expression of dnmts ↓and cyp19a1a and dmrt1 ↑	Gonadal development	[155]
O. niloticus	Exposure to higher temperature	DNA methylation in three age groups of fish	BS-seq and qRT-PCR	cyp19a1a promoter DNA methylation levels ↑; cyp19a1a mRNA expression ↓	Masculinization	[156]
Morone saxatilis(Striped bass)	In vitro fertilization	DNA methylation in sperm fertility	Methyl-CpG-binding domain sequencing	WDR3/UTP12 and GPCR ↑ involved in sperm flagella formation, hormonal signaling and tissue development regulation	Sperm fertility	[166]

7. Influence of Probiotics on Reproductive Performance of Fish

Host-derived probiotic bacteria are being used increasingly in the aquaculture sector. The host-associated probiotics have not only improved the reproductivity of fish [167] but also helped to improve the endocrine and reproductive signaling of fish. Isolated Enterococcus xiangfangensis, Citrobacter freundii, Pseudomonas aeruginosa, P. stutzeri, and B. subtilis from fish resulted in growth, hematological, and reproductive performance upregulation among host fishes [168].

The action mechanism of probiotics on reproductive function is normally indicated by increasing egg production along with improvements in the vitellogenic follicles and GSI [166]. However, the lipidic and glucidic components elevated during pre-vitellogenesis result in oocytes maturation, modification of the secondary structure of protein, and impacts on hydration and phosphorylation [169]. Probiotic administration can induce the responsiveness of incompetent follicles (stage IIIa) to MIH in the maturation period and changes oocyte chemical composition, fostering the vitellogenic development [170]. Modifications of the electrophoretic pattern at maturation, and, to a lesser extent, at yolk protein levels have changed the ooplasma components [171]. In fish, probiotics inhibit apoptosis and increase the rate of follicular survival during the developmental stage. Furthermore, effects of probiotics also increase the production of sperm from the testis, with stimulatory actions on two components known as intertubular (or interstitial) and tubular sections [34]. Some probiotics have been shown to stimulate steroidogenic Leydig cells, blood/lymphatic vessels, macrophages and mast cells, neural, and connective tissue cells. This is the case although the somatic Sertoli cells and the germ cells are found at different stages of development and Sertoli cells function in the determination of spermatogenic capacity [172]. The close and continuous interaction with Sertoli cells may contribute to germ cell survival. Consequently, the positive role of probiotics in molecular parameters in testicular cells is validated by upregulating the transcription level of leptin, bdnf and dmrt1 genes facilitated the potentialities of spermatogenesis [41]. Moreover, increased transcription levels of activin, arα, arβ, pr1, and fshr contribute to sperm quality improvement during spermatogenesis [41].

Probiotics are widely used as feed enrichment for farming aquatic organisms especially fish. The initial application of probiotics in aquaculture was for growth promoters and fish health. However, new areas of research, such as their effect on reproduction, maturation, and fecundity, have been found, though these require more comprehensive development [167]. Several studies have found that probiotics improve host microbial balance and thus improve health, disease resistance, growth performance, feed utilization, and reproductive performance [167,173,174]. Nutrient supplements for broodstock are critical for aquaculture success. Major nutrients such as lipid, protein, fatty acids, vitamins E and C, and carotenoids are essential for various reproduction processes such as fecundity, fertilization, hatching, and larval development [49]. In general, studies in fish show that decreased food availability or starvation causes gonad regression and a decrease in female spawning and egg production, whereas increased food availability and supplemented adequate nutrients promote growth and larger body sizes, causing earlier maturation and higher fecundity in some species [116]. However, the administration of different growth promoters (hormones, antibiotics, nutrient mixtures) can cause suppression of the beneficial microbial activity in the intestinal tract of the broodfish. In such conditions, probiotic supplements can be used to repair these deficiencies and improve the fish health conditions in breeding time.

Additionally, supplemented probiotics such as lactic acid bacteria improved feed utilization and reproductive function of fish [175]. The administration of probiotics through feed supplementation could regulate and modify the expression patterns of genes or hormones responsible for the regulation of fish reproduction [176]. Therefore, using probiotics as feed supplements can potentially improve reproductive function and activate reproductive genes to correct reproductive dysfunction.

7.1. Ornamental Fish

Ornamental fish have popularity for their unique colors and their importance is increasing not only for hobby purposes, but also for their use as animal models for scientific research. A diverse array of probiotics induces beneficial effects on ornamental fish reproductive performance, as presented in Table 2.

7.1.1. Male

Ornamental fish especially, zebrafish were used over the last 20 years for studying genetics and gonadal development [177,178]. Probiotics have been used in zebrafish trials to observe the transcription of the genes related to reproductive maturity and reproduction [179,180]. Transcription of KiSS1, KiSS2, and gnrh3 genes in the brain can trigger the reproductive system of male fish by the direct or indirect secretion of many hormones in response to probiotics, especially leptin [124]. Valcarce et al. [34] first reported P. acidilactici supplementation correlates with and upregulates male reproductive performance of zebrafishes. This probiotic triggered five genes, including brain-derived neurotrophic factor (bdnf), BCL2-interacting killer (bik), double-sex and mab-3 related transcription factor 1 (dmrt1), and FSH beta subunit, and leptin a (lepa) transcription, as an indication of good sperm quality (SQ) marker. These genes all produce positive effects on testicular cells, potentially improving reproductive performance [34,181]. The blended dietary administration P. acidilactici (0.2%) and nucleotide (0.5%) demonstrated positive effects on SQ, sperm motility (SM) and sperm density (SDn) in goldfish (C. auratus) [35]. Furthermore, Lab. rhamnosus and Bifidobacterium longum have been showing antioxidant and anti-inflammatory features to assist zebrafish SQ and male reproductive behavior [182]. These probiotics demonstrated a positive effect on SQ, SDn, total and progressive SM, and fast spermatozoa subpopulations.

7.1.2. Female

Probiotic Lab. rhamnosus IMC 501 has striking effects on the ovarian development of female zebrafish [7,31]. Moreover, in this fish, dietary administration of L. rhamnosus at 10⁶ CFU g⁻¹ improved Fec, GSI, and oocyte maturation (FD and FM), supporting reproductive performance by improving fecundity [29,171,177. This probiotic increases the transcription level of transforming growth factor b1 (tgfb1), growth differentiation factor9 (gdf9), and bone morphogenetic protein15 (bmp15) contributing to oocyte development of that fish. Carnevali, et al. [183] also monitored the long-term effects of the same probiotic on zebrafish and reported dietary effectiveness on FD; ovulated oocytes quantification; embryo quality and larval growth performance. This probiotic stimulates sexual maturation of this species by improving the expression of aromatase cytochrome p 19 (cyp19a), vitellogenin (vtg), an isoform of the E2 receptor (era), LH receptor, 20-b hydroxysteroid dehydrogenase (20b-hsd), membrane progesterone receptors a and b, cyclin B, activinbA1, smad2, tgfb1, gdf9 and bmp15, which are responsible for regulating reproductive hormone secretion. Autophagy was observed during follicle development in the ovarian tissue and L. rhamnosus has a key role in follicle maturation as confirmed by focal plane array analysis [30]. Miccoli et al. [33] conducted an experiment with the same probiotic including similar dose, duration, and species as reported by Gioacchini et al. [30], and reported that probiotics promote embryonic development by changing both maternal and zygotic mRNA levels. Similarly, the use of L. rhamnosus CICC 6141 and L. casei BL23 probiotic effects on D. rerio were documented and later probiotic markedly improved Fec, OVr, HR, and FR [184].

Probiotic, L. rhamnosus IMC 501 treatment has profound effects on killifish (Fundulus heteroclitus) GSI, Fec, and embryo SR [36]. Dietary supplementation of probiotic positively improves egg and ovum diameter, absolute fecundity, and some other properties in goldfish [35]. Probiotic administration caused a substantial impact on reproductive performance in four live-bearing ornamental species: Poecilia reticulata, P. sphenops, X. helleri, and X. maculatus [27]. In these fishes, administration of B. subtilis for 1-year improved GSI, Fec, and fry production of spawning females. Additionally, probiotic could synthesize vitamin B1 and vitamin B12 that controlled the mortality or body deformities of fry. The live-bearing ornamental female swordtails displayed improved GSI, Fec, and fry production after taking commercial probiotic (PrimaLac) (see Table 2) as a feed additive for 182 days [28].

Table 2. Dietary supplemented probiotics effects on ornamental fish reproduction. Symbol: no change (→); increase (↑); decrease (↓) versus controls.

Supplemented Probiotics	Fish Species	Fish Number	Duration	Concentration	Effects on Fish	References
Bacillus subtilis	Poecilia reticulata (Guppy), P.sphenops (Valenciennes), Xiphophorus helleri (Swordtail fish) and X. maculatus (Platyfish)	60 virgin females of each species	365 days	5 × 107–5 × 108 CFU g−1 and 5 × 105–5 × 106 CFU g−1	EP Fec and GSI ↑; SR (fry) ↑; Fry death and deformities ↓	[27]
Lactobacillus rhamnosus IMC 501	Danio rerio (Zebrafish)	10 females	10 days	106 CFU g−1	EP Fec, GSI, and Ovolution rate ↑; Oocyte maturation G and FD ↑;	[29,31,176,185]
					Oocyte maturation FD and FM ↑	[170]
					Follicular survival ↑ and apoptosis ↓	[30]
Lab. rhamnosus IMC 501	D. rerio	40 males and females	10 days	106 CFU g−1	Embryo development ↑; HR ↑	[134]
Pediococcus acidilactici (Bactocell®)	D. rerio	5 wild males	10 days	106 CFU g−1	SP testicular cells ↑	[164]
Lab. rhamnosus CECT8361 and Bifidobacterium longum CECT7347	D. rerio	36 Males	21 days	109 CFU g−1	SP SQ, SDn, SM ↑	[182]
PrimaLac® (Lab. acidophilus, Lab. casei, Enterococcus faecium, Bifidobacterium thermophilum)	X. helleri	10 females and 3 males	182 days	0.04%, 0.09% and 0.14%	EP Fec and GSI ↑; SR (fry) ↑	[28]
P. acidilactici	Carassius auratus (Goldfish)	720 fishes	180 days	0.1, 0.2, and 0.3%	EP GSI, HSI, AF, RF, ED, OD, FR, and HR ↑; SP SM, SD, SDn, and Stc ↑	[133]
Lab. rhamnosus IMC 501	Fundulus heteroclitus (Killifish)	10 females and 10 males	8 days	106 CFU mL−1	EP GSI, Fec ↑ and HR →; SR (fry) ↑; GP L and W ↑	[36]

AF: absolute fecundity; EP: egg parameters; ED: egg diameter; Fec: fecundity; FD: follicle development; FM: follicle maturation; FR: fertilization rate; G: growth; GP: growth parameters; GSI: gonadosomatic index; HR: hatching rate; HSI: hepatosomatic index; OD: ovum diameter; RF; relative fecundity; SR: survival rate; SP: sperm parameters; SM: sperm motility (%); SD: sperm duration; SDn: sperm density; Stc: spermatocriet (%); W: weight.

Table 2. Dietary supplemented probiotics effects on ornamental fish reproduction. Symbol: no change (→); increase (↑); decrease (↓) versus controls.

Supplemented Probiotics	Fish Species	Fish Number	Duration	Concentration	Effects on Fish	References
Bacillus subtilis	Poecilia reticulata (Guppy), P.sphenops (Valenciennes), Xiphophorus helleri (Swordtail fish) and X. maculatus (Platyfish)	60 virgin females of each species	365 days	5 × 107–5 × 108 CFU g−1 and 5 × 105–5 × 106 CFU g−1	EP Fec and GSI ↑; SR (fry) ↑; Fry death and deformities ↓	[27]
Lactobacillus rhamnosus IMC 501	Danio rerio (Zebrafish)	10 females	10 days	106 CFU g−1	EP Fec, GSI, and Ovolution rate ↑; Oocyte maturation G and FD ↑;	[29,31,176,185]
					Oocyte maturation FD and FM ↑	[170]
					Follicular survival ↑ and apoptosis ↓	[30]
Lab. rhamnosus IMC 501	D. rerio	40 males and females	10 days	106 CFU g−1	Embryo development ↑; HR ↑	[134]
Pediococcus acidilactici (Bactocell®)	D. rerio	5 wild males	10 days	106 CFU g−1	SP testicular cells ↑	[164]
Lab. rhamnosus CECT8361 and Bifidobacterium longum CECT7347	D. rerio	36 Males	21 days	109 CFU g−1	SP SQ, SDn, SM ↑	[182]
PrimaLac® (Lab. acidophilus, Lab. casei, Enterococcus faecium, Bifidobacterium thermophilum)	X. helleri	10 females and 3 males	182 days	0.04%, 0.09% and 0.14%	EP Fec and GSI ↑; SR (fry) ↑	[28]
P. acidilactici	Carassius auratus (Goldfish)	720 fishes	180 days	0.1, 0.2, and 0.3%	EP GSI, HSI, AF, RF, ED, OD, FR, and HR ↑; SP SM, SD, SDn, and Stc ↑	[133]
Lab. rhamnosus IMC 501	Fundulus heteroclitus (Killifish)	10 females and 10 males	8 days	106 CFU mL−1	EP GSI, Fec ↑ and HR →; SR (fry) ↑; GP L and W ↑	[36]

AF: absolute fecundity; EP: egg parameters; ED: egg diameter; Fec: fecundity; FD: follicle development; FM: follicle maturation; FR: fertilization rate; G: growth; GP: growth parameters; GSI: gonadosomatic index; HR: hatching rate; HSI: hepatosomatic index; OD: ovum diameter; RF; relative fecundity; SR: survival rate; SP: sperm parameters; SM: sperm motility (%); SD: sperm duration; SDn: sperm density; Stc: spermatocriet (%); W: weight.

7.2. Commercial Fish

Comparatively less information is available on the effects of probiotics on reproduction regulating gene and hormone activities modulation in commercial fish despite the prevalence of reproductive dysfunctions among important farmed species. To date, the various beneficial bacteria used as probiotics in commercial fish for the enhancement of reproductive performance are presented in Table 3.

7.2.1. Male

The reproductive process is influenced by a variety of factors, the most important of which are fish species, the degree of domestication, nutrition, and environmental parameters. In the last two decades, nutrition was the foremost among factors which can be influenced by beneficial bacteria in the gastrointestinal tract of fishes [186]. Nutrients alter physiological functions in fish, thereby influencing the sperm quality and subsequent fertility rates [187]. Nutritional deficiencies and functional activities of beneficial bacteria in body and aquatic environment may lead to reduced fry quality with higher mortality rates [188]. Administration of B. subtilis probiotic not only contributes to immunonutrition but also improves sperm quality, the viability of gametes as well as the FR in Nile tilapia [189]. Secretion of hormones is influenced by the absorption of amino and fatty acids, generally improving metabolism and reproduction. Probiotic bacteria Lab. acidophilus, Bif. longhum, Bif. thermophylu and Streptococcus faecium were responsible for increased serum total testosterone and progesterone levels, which can serve as predictors of sperm quality [190]. The use of molecular techniques reveals that Bacillus sp. and Bacteroides sp. supplementation improves the sperm quality of Tor tambroides [191]. Application of Lab. rhamnosus IMC 501 probiotic in A. anguilla improved spermatogenesis by increasing the expression of signaling genes. Addition of this probiotic in aquaria water at 10⁵ CFU mL⁻¹ increased sperm production along with SM% and improved the percentage of straight-swimming spermatozoa in this species [41].

7.2.2. Female

Dietary additives containing probiotic bacteria offer an attractive option for inducing overall health benefits in the host [192]. Recent studies have focused on the role of probiotics in the reproductive process and new progeny, with a particular focus on commercial marine species [193]; comparable reproductive enhancements have also been reported in some brackish water fish species [194].

Dias et al. [37] administered dietary B. subtilis at 10¹⁰ CFU g⁻¹ for 90 days resulting in improved egg production, fry SR, and an increased number of O. niloticus spawning females led to economic benefits. In another study, Rahman et al. [39] reported that dietary supplementation of Lab. Rhamnosus results in significant improvements in GSI, Fec, FR, and SR in butter catfish (Ompok pabda). Through 16S rRNA gene sequencing, it has been confirmed that Enterococcus xiangfangensis, Pseudomonas stutzeri, B. subtilis, Citrobacter freundii, and P. aeruginosa were capable of enhancing the reproduction parameters of Silver barb (Barbonymus gonionotus) [168]. Findings validate that the probiotic strains consortium (1.35 × 10⁹ CFU kg⁻¹) increased GSI, ova per gram of body weight, FR and HR. Female rainbow trout that ingested commercial probiotic ‘Bio-Aqua’ (containing 8 bacterial strains or species) at 4 × 10⁹ CFU kg⁻¹ diet twice daily demonstrated higher absolute and relative Fec, FR, HR, and egg survival after 8 weeks [38]. Similarly, combined probiotics (5 bacteria and 2 yeasts) administered to female Clarias gariepinus (African catfish) broodstock fishes at dose of 5 mL kg⁻¹ for 31–41 days and laser firing treatment at 1.125 Joule revealed that the probiotics treatment accelerated gonadal maturation [195].

Table 3. Probiotics supplementation effects on commercial fish reproduction. Symbol: increase (↑) versus controls.

Probiotics Used	Fish Species	Fish Number	Duration	Application Mode	Effects on Fish	References
Enterococcus xiangfangensis, Pseudomonas stutzeri, Bacillus subtilis, Citrobacter freundii, and Pseudomonas aeruginos	Barbonymus gonionotus (Silver barb)	96 males and females	60 days	Dietary supplementation at 1.35 × 109 CFU kg−1	EP GSI, Fec, FR, and HR ↑	[168]
B. subtilis	Oreochromis niloticus (Nile tilapia)	118 females and 48 males	14 days	Dietary supplementation at 1010 CFU g−1	EP Fec ↑; SR (fry) ↑; EFi TcN, GR and ToP ↑	[22]
Bio-Aqua® (P. acidilactici, Enterococcus faecium, B. subtilis, Lactobacillus acidophilus, Lab. plantarum, Lab. casei, Lab. rhamnosus, Bifidobacterium bifidum and Saccharomyces cerevisiae)	Oncorhynchus mykiss (Rainbow trout)	60 females	8 weeks	Dietary supplementation at 4 × 109 CFU g−1	EP AF, RF, FR, HR, and ES ↑; SR (fry) ↑	[38]
Lab. rhamnosus	Ompok pabda (Butter catfish)	240 males and females	60 days	Dietary supplementation at 5 × 106−8 CFU g−1	EP GSI, Fec, FR, and HR ↑; SR (fry) ↑	[39]
Probio-7 (Saccharomyces cerevisiae, Aspergillus oryzae, Lab. acidophilus, B. subtilis, Rhodopseudomonas, Actinomycetes, and Nitrobacter)	Clarias gariepinus (African catfish)	-	80 days	Fermented dose at 5 mL kg−1	EP FR, HR, and SR ↑; Maturity time ↑	[195]
Lab. rhamnosus IMC 501	Anguilla anguilla (European eel)	40 males	63 days	Added water at 103, 105 and 106 CFU mL−1	SP SM, SDn, and Spermatogenesis ↑	[41]

AF: absolute fecundity; EP: egg parameters; ES: egg survival rate. EFi: economic feasibility indexes; Fec: fecundity; FR: fertilization rate; GR: gross revenue; GSI: gonadosomatic index; HR: hatching rate; RF; relative fecundity; SR: survival rate; SP: sperm parameters; SM: sperm motility (%); SDn: sperm density; TcN: total cost of nutrition; ToP: total operational profit.

Table 3. Probiotics supplementation effects on commercial fish reproduction. Symbol: increase (↑) versus controls.

Probiotics Used	Fish Species	Fish Number	Duration	Application Mode	Effects on Fish	References
Enterococcus xiangfangensis, Pseudomonas stutzeri, Bacillus subtilis, Citrobacter freundii, and Pseudomonas aeruginos	Barbonymus gonionotus (Silver barb)	96 males and females	60 days	Dietary supplementation at 1.35 × 109 CFU kg−1	EP GSI, Fec, FR, and HR ↑	[168]
B. subtilis	Oreochromis niloticus (Nile tilapia)	118 females and 48 males	14 days	Dietary supplementation at 1010 CFU g−1	EP Fec ↑; SR (fry) ↑; EFi TcN, GR and ToP ↑	[22]
Bio-Aqua® (P. acidilactici, Enterococcus faecium, B. subtilis, Lactobacillus acidophilus, Lab. plantarum, Lab. casei, Lab. rhamnosus, Bifidobacterium bifidum and Saccharomyces cerevisiae)	Oncorhynchus mykiss (Rainbow trout)	60 females	8 weeks	Dietary supplementation at 4 × 109 CFU g−1	EP AF, RF, FR, HR, and ES ↑; SR (fry) ↑	[38]
Lab. rhamnosus	Ompok pabda (Butter catfish)	240 males and females	60 days	Dietary supplementation at 5 × 106−8 CFU g−1	EP GSI, Fec, FR, and HR ↑; SR (fry) ↑	[39]
Probio-7 (Saccharomyces cerevisiae, Aspergillus oryzae, Lab. acidophilus, B. subtilis, Rhodopseudomonas, Actinomycetes, and Nitrobacter)	Clarias gariepinus (African catfish)	-	80 days	Fermented dose at 5 mL kg−1	EP FR, HR, and SR ↑; Maturity time ↑	[195]
Lab. rhamnosus IMC 501	Anguilla anguilla (European eel)	40 males	63 days	Added water at 103, 105 and 106 CFU mL−1	SP SM, SDn, and Spermatogenesis ↑	[41]

AF: absolute fecundity; EP: egg parameters; ES: egg survival rate. EFi: economic feasibility indexes; Fec: fecundity; FR: fertilization rate; GR: gross revenue; GSI: gonadosomatic index; HR: hatching rate; RF; relative fecundity; SR: survival rate; SP: sperm parameters; SM: sperm motility (%); SDn: sperm density; TcN: total cost of nutrition; ToP: total operational profit.

8. Concluding Remarks and Future Directions

Fish reproductive performance plays a crucial role in the growth, development, and reproductive behavior of progeny, and is a determinant of the success of captive reproduction. However, several factors are also responsible for the modulation of offspring growth, mortality, and reproduction for the generation of progeny from cultured parents. Probiotics such as S. faecalis, Clostridium butyricum, B. mesentericus, and Lactobacillus sp. have already shown positive outcomes on sperm, gonadal development, fecundity, hatching, and survival percentages [196]. The probiotic efficacy and detailed mechanism of approach need further analysis and optimization based on research and practical application, since the large majority of studies are conducted in laboratories under artificial conditions. Probiotics research was mostly conducted on the reproductive performance of females rather than the gonadal development and spermatogenesis of male fishes. In addition, relatively few studies have reported positive effects of probiotics on marine and freshwater commercial fish reproductive performance.

The study of genetic and physiological mechanisms responsible for controlling beneficial traits is vital for maintaining and improving aquaculture sustainability. Meanwhile, the application of epigenetics will significantly benefit the aquaculture sector [9], potentially establishing a reliable link between phenotype and environment. A better understanding of epigenetic mechanisms would also help researchers explain the extent that intergenerational inheritance can be passed from parents to offspring via gametes. The ability to control these parameters is termed sex determination and differentiation, reproductive maturation, morphologic plasticity, and environmental adaptation. These activities are likely to promote breeding programs’ accuracy and increase the productivity of farmed fish populations in the near future. However, epigenetics in aquaculture is not sufficiently studied to increase production levels in commercial aquaculture industries [197]. Bioinformatic tools and methods for assessing the epigenetic status of organisms should be pursued and improved in order to fill research knowledge gaps and quantify the functional relationship between epigenetic changes and alteration of reproduction related genes activities. These activities can potentially open a new window of innovative and promising research in the field of aquaculture.

Now, none of the studies have been performed on a large scale on marine commercial species affected with reproductive dysfunctions. Numerous probiotics were supplemented in aquaculture, but clear mechanisms of action are yet to be ascribed to the alteration of reproductive performance. Moreover, future research should be conducted to identify and quantify epigenetics heredity percentages from F0 to next successive generation (F1–F4-5). Highly sophisticated molecular biology like gene transcriptions, transcriptomic study, gene editing and epigenetics modulation will become most widely applied technology to ensure species-specific sustainable aquaculture practices for higher profitability. Future research could be also performed to ascertain how the genome, environment, and gut microbiome interact to influence breeding behavior, performance, and heredity resulting the phenotypes.