Ultrasonography Is a Valuable Tool for Assisting in Marine Fish Reproduction: Applications in Brazilian Sardine (Sardinella brasiliensis) and Lebranche Mullet (Mugil liza)

Abstract

Urogenital cannulation is a traditional method used in aquaculture to achieve sexual differentiation, but it is considered invasive. Ultrasonography is a valuable non-invasive tool for determining sex and gonadal development in fish species like mullet (Mugil liza) and Brazilian sardine (Sardinella brasiliensis) that lack sexual dimorphism. The methodology involves emitting high-frequency sound waves (20 MHz to 20,000 MHz) above the human hearing range. These waves interact with the tissues of the body, producing echoes that are detected by a transducer. The echoes are then processed by computer graphics to generate detailed images of the internal structures of the organism. This allows for the determination of the sex of fish based on the sonographic features of the tissues. For instance, in male fish, hypoechogenic structures reflect fewer sound waves, leading to darker images. Conversely, in female fish, hyperechogenic tissues reflect more sound waves, resulting in lighter images. It is possible to classify the gonadal maturation stage based on differences in image texture. This non-invasive method eliminates the need for specimen dissection. It is especially valuable when the goal is to preserve the spawners’ life and integrity. This review emphasizes the application of this technology in aquaculture, specifically targeting fish from the Clupeidae and Mugilidae families.

1. Introduction

In Brazil, the Lebranche mullet (Mugil liza) and the Brazilian sardine (Sardinella brasiliensis) are valuable fishing resources and have become increasingly important in Brazilian aquaculture because they are considered near-threatened species [1]. Multiple studies have highlighted the potential of aquacultured species, covering topics such as reproductive and feeding management [2,3,4], disease prevention and treatment [5,6], transportation [7], and fillet quality of aquacultured sardines [8]. In this way, the farming of low-trophic-level species has been shown to be a feasible option in Brazilian marine aquaculture because of their biological traits and economic and ecological significance. Despite this advantageous scenario, research involving these animals necessitates regular handling for biometric, sanitary, and reproductive evaluations. Additionally, on a global scale, ethics committees for animal experimentation agencies advocate and mandate ethical treatment of animals, minimizing animal suffering during procedures, providing alternative methods when possible, and ensuring animal welfare. Therefore, ultrasonography is recognized as a valuable tool in aquaculture.

Ultrasonography is a commonly used diagnostic imaging tool in both human [9] and veterinary medicine [10]. It is a non-invasive and safe procedure that uses high-frequency sound waves to create real-time images of the soft tissues and internal organs of animals. This technique has revolutionized emergency, clinical, and veterinary care by enabling quick and accurate diagnoses with increased convenience for patients [11,12,13,14,15].

Ultrasound involves the use of a transducer that emits sound waves to penetrate the tissues of the animal’s body. As the waves encounter structures of varying densities, such as organs, fluids, and bones, some are reflected back to the transducer. The device then interprets these reflected waves and generates real-time two-dimensional (or three-dimensional in more advanced systems) images [9,10].

In the field of veterinary medicine, key uses of ultrasound include diagnosing conditions in the abdominal cavity, which contains vital organs like the liver, kidneys, spleen, bladder, stomach, and intestines. It helps identify abnormalities such as ascites, tumors, inflammation, and structural changes [11,12,14,15].

Ultrasonography is widely utilized for monitoring pregnancy and reproduction in mammalian animals, enabling the confirmation of pregnancy, fetal monitoring, and birth planning [16]. The effectiveness, precision, and non-invasiveness of this technology have led to its adoption in other areas of animal production, including aquaculture. In aquaculture, ultrasonography can help minimize unnecessary handling, such as during assisted reproduction of fish [17,18,19,20,21,22].

Mali et al. [13] state that reproductive ultrasound has revolutionized animal reproduction by enabling visualization of the reproductive tract, early pregnancy diagnosis, monitoring of embryonic or fetal development, and detection of the estrous cycle phase. This highlights the value of reproductive ultrasound as a valuable tool for fertility management in production animals.

Sex identification in immature Eurasian perch (Perca fluviatilis) using ultrasonography was conducted by Ledoré et al. [23]. Determining the sex of individuals was crucial for effective management of the breeding population, and ultrasonography proved to be a rapid, non-lethal, and non-invasive method with 98.9% accuracy in identifying females (n = 180) and 98.8% accuracy in identifying males (n = 92). Similarly, Leng et al. [24] documented the effective application of ultrasound in assisted artificial reproduction of the critically endangered Sichuan taimen (Hucho bleekeri). The study involved assessing gamete maturity in 43 wild adult specimens. Ultrasonography proved to be a valuable tool for monitoring the gonad status of adult Sichuan taimen during artificial reproduction, facilitating further research on the large-scale cultivation and successful restoration of Sichuan taimen resources. Furthermore, ultrasonography can play a crucial role in identifying the gender of fish species that lack sexual dimorphism or visible gametes. Chiotti et al. [19] demonstrated this by accurately determining the sex (with an accuracy range of 88–96%) and reproductive stage (with an accuracy of 89–100%) of adult sturgeon (Acipenser fulvescens) using this method. The image collection process took approximately 2 to 3 min.

Ultrasonography is a well-established technique in fish reproduction. However, to ensure consistent results, standardization is necessary. This includes maintaining uniformity in equipment and control settings to prevent variations within the same species, as well as using standardized fish selection procedures during examinations [18]. Additionally, a skilled operator is required for accurate interpretation of the images.

Understanding ultrasound results requires knowledge of anatomy and image planes. Examinations are done in transverse, sagittal, and frontal planes. Evaluation includes size, shape, and echotexture of organs. Echotexture helps differentiate normal from abnormal findings but can be misinterpreted due to errors. Sonographers use terms like hyperechoic, hypoechoic, anechoic, and isoechoic. Incorrect gain settings can lead to inaccurate diagnoses. Veterinary ultrasound may require adjusting settings beyond defaults due to animal size variations. Artifacts can result from ultrasound wave or energy beam phenomena [10,15].

Ultrasound has become an essential tool in clinics, veterinary hospitals, and diagnostic centers in recent years. The importance of diagnostic imaging is increasing significantly, making ultrasound a key component in modern aquaculture practices. Proper and strategic use of ultrasound can enhance reproductive management and enable early diagnoses without the necessity of invasive procedures. Therefore, this review focused on the principles of ultrasonography and its applications in fish research, with a specific emphasis on its utility in aquaculture. The review includes studies that utilized ultrasonography to determine sex and/or gonadal development stage in various commercially important fish species, as well as in emerging species in Brazilian aquaculture like Brazilian sardine (Sardinella brasiliensis) and Lebranche mullet (Mugil liza).

2. Principles of Ultrasound Image Formation

Ultrasound, also known as ultrasonography or sonography, involves the use of sound waves with frequencies above the human hearing range, typically between 20 MHz and 20,000 MHz. These sound waves are emitted into tissues or internal structures, generating echoes that are processed in real time by computer graphics to create images of the organism’s interior. High-precision studies, such as microimaging of tissues, research on small fish, and experiments with biological samples, utilize frequencies higher than 20 MHz (up to 50 MHz or more). Frequencies above 30 MHz to 70 MHz are employed to observe minute structures, such as embryos or details of very small organisms [10,25].

Ultrasound was initially utilized for medical diagnosis in the 1940s [26]. The use of ultrasound in animals was first recorded in 1956 in the United States, where researchers at the University of Colorado measured loin thickness in beef cattle. Concurrently, European studies concentrated on assessing pig carcasses [27]. The official commencement of ultrasound as a veterinary diagnostic tool occurred in 1966, when it was employed to detect pregnancy in goats in a study carried out by the Agriculture and Livestock Service in Beltsville, MD, USA [26].

The advantages of ultrasound as a diagnostic imaging tool in veterinary medicine are plentiful. Regular examinations show that ultrasound is a safe method, with no adverse biological effects on the patient or the operator. This procedure can be conducted in any setting, without the need for specific safety precautions [28]. Being a non-invasive technique, it is well received by animals and enables not only disease diagnosis but also treatment progress monitoring [15].

Ultrasound is a commonly used tool in veterinary medicine for diagnostic and monitoring purposes in animals. It is utilized for evaluating the heart, lungs, eyes, muscles, bones, abdomen, and reproductive organs [15,29]. Sound waves pass through the body cavity and interact with tissues, experiencing attenuation until they reach a reflective surface that sends the echo back to the probe. Probes or transducers are essential components of ultrasound equipment, producing and receiving echoes from different interfaces. Real-time images allow for dynamic examination of various structures in any spatial orientation [30]. Ultrasound can visualize bones, muscles, tendons, ligaments, joints, ovaries, testes, and internal materials within organs [9,10,31].

3. Reproductive Assessment Techniques in Marine Fish

Classical techniques, such as hormone analysis in blood plasma, are used to assess gonadal development in fish [32,33]. This method, which involves advanced devices to determine hormone levels, provides valuable information. Urogenital cannulation is the most commonly used technique for sex identification and selection in both marine and freshwater species [34,35]. This method, primarily used for females during gonadal development for reproduction, involves inserting a urethral collection probe into the genital pore to collect material and sample ovarian tissue in females and semen in males. In males, gametes can also be identified and collected by applying abdominal pressure, depending on the characteristics of the testicles and the degree of gonadal maturation [34].

Overall, these techniques are typically viewed as invasive and harmful to the health and reproductive capabilities of animals. They can result in negative outcomes, including increased stress for breeders, introduction of pathogens, suppression of ovulation, and reduced efficiency, particularly outside of reproductive seasons. Furthermore, these methods do not facilitate quick selection of animals for breeding and spawning [18,36,37].

3.1. Ultrasonography in Aquaculture

In recent years, ultrasonography has emerged as a valuable tool for researching marine and diadromous species (Table 1). In the field of aquaculture, ultrasound technology is a relatively new but highly significant tool. It is utilized for tasks such as diagnosing diseases, evaluating carcasses, and studying fish reproduction. Ultrasound is increasingly being employed to identify sex and assess gonadal maturation in a non-invasive way, particularly for fish species lacking obvious sexual differences [18,22,38].

Using ultrasonography to evaluate and determine the stage of gonadal maturation could enhance the effectiveness of induced spawning procedures in fish by pinpointing the optimal timing to start the induction protocol [34,39,40,41]. Early detection of the reproductive status of fish offers benefits for aquaculture, including enhanced larval production rates and tailored nutritional strategies for specific fish species [42]. Additionally, it helps regulate the sale of fish at different stages of gonadal development [42]. This is important because mature gonads can take up to 75% of the coelomic cavity, leading to reduced carcass yield and lower market prices in the industry [36].

Karlsen and Holm [43] found that ultrasonography was successful in determining the sex of cod (Gadus morhua) aged one to six years with 95% accuracy during the early stages of growth and gonadal development. Recent studies have shown that ultrasonography is nearly 100% effective in sex differentiation and has benefits such as improved post-examination fish survival, reduced invasiveness, and faster procedures in sturgeon (Acipenser ruthenus) [44], lamprey (Lampetra fluviatilis) [45], and Lebranche mullet [22].

Table 1. Compilation of research studies focusing on the application of ultrasound in the reproductive processes of marine and diadromous fish. The assemblage showcases the key findings and progress made for various species.

Common Name	Application Area	Species	Main Target	Key References
Sturgeons	Aquaculture	Acipenser gueldenstaedtii	Determination of sex and gonad maturity.	Memiş et al. [46]
Chinese sturgeon	Biology	A. sinensis	Gender and gonadal maturity stage.	Du et al. [39]
White Sturgeon	Ichthyology	A. transmontanus	Describing the reproductive structure of hatchery-origin and wild fish.	Maskill et al. [47]
Persian Sturgeon	Aquaculture	A. persicus	Findings of ovary and testis in adult during artificial propagation.	Vajhi et al. [48]
Beluga sturgeon	Aquaculture	Huso huso	Early sex identification of 18-month cultured sturgeon.	Esmailnia et al. [49]
European eel	Aquaculture	Anguilla anguilla	Determine sex and to perform gonad biopsy.	Kucharczyk et al. [50]
Silver eel	Aquaculture and Fisheries	A. anguilla	Sex determination and maturation monitoring.	Bureau du Colombier et al. [51]
European eel	Aquaculture	A. anguilla	Ultrasound predictors of ovarian response following hormonal treatment for assisted reproduction.	Müller et al. [52]
European eel	Aquaculture	A. anguilla	Indicators of oocyte maturation.	Jéhannet et al. [53]
Pufferfish	Aquaculture	Arothron manilensis	Sex determination to artificial breeding.	Doi et al. [54]
Lumpfish	Aquaculture	Cyclopterus lumpus	Monitoring gonadal development.	Mlingi et al. [40]
Lebranche mullet	Aquaculture	Mugil liza	Sex determination.	Santarosa et al. [22]
Manta rays and Devil ray	Biology	Mobula kuhlii, M. thurstoni, M. mobular, M. tarapacana, M. birostris, and M. alfredi	Internal anatomy and reproductive activity.	Froman et al. [55]
Sockeye Salmon	Aquaculture	Oncorhynchus nerka	Accuracy of sexing for captive broodstock management.	Frost et al. [56]
Hapuku	Aquaculture	Polyprion oxygeneios	Sex identification.	Kohn et al. [57]
Thornback ray	Biology	Raja clavata	Assessment of maturity in oviparous elasmobranchs.	Whittamore et al. [58]
Atlantic salmon	Aquaculture	Salmo salar	Monitoring reproductive physiology in female.	Næve et al. [59]
Bonnethead shark	Biology	Sphyrna tiburo	Determine pregnancy status and fecundity.	Anderson et al. [60]
Small-spotted catshark	Biology	Scyliorhinus canicula	Assessment of maturity in oviparous elasmobranchs.	Whittamore et al. [58]
Hilsa herring	Aquaculture and Fisheries	Tenualosa ilisha	Assessment of sex, gonad volume, and reproductive maturation status.	Dasgupta et al. [41]
Whitetip Reef Shark	Biology	Triaenodon obesus	Description of the late embryonic developmental stage.	Santos et al. [61]

Table 1. Compilation of research studies focusing on the application of ultrasound in the reproductive processes of marine and diadromous fish. The assemblage showcases the key findings and progress made for various species.

Common Name	Application Area	Species	Main Target	Key References
Sturgeons	Aquaculture	Acipenser gueldenstaedtii	Determination of sex and gonad maturity.	Memiş et al. [46]
Chinese sturgeon	Biology	A. sinensis	Gender and gonadal maturity stage.	Du et al. [39]
White Sturgeon	Ichthyology	A. transmontanus	Describing the reproductive structure of hatchery-origin and wild fish.	Maskill et al. [47]
Persian Sturgeon	Aquaculture	A. persicus	Findings of ovary and testis in adult during artificial propagation.	Vajhi et al. [48]
Beluga sturgeon	Aquaculture	Huso huso	Early sex identification of 18-month cultured sturgeon.	Esmailnia et al. [49]
European eel	Aquaculture	Anguilla anguilla	Determine sex and to perform gonad biopsy.	Kucharczyk et al. [50]
Silver eel	Aquaculture and Fisheries	A. anguilla	Sex determination and maturation monitoring.	Bureau du Colombier et al. [51]
European eel	Aquaculture	A. anguilla	Ultrasound predictors of ovarian response following hormonal treatment for assisted reproduction.	Müller et al. [52]
European eel	Aquaculture	A. anguilla	Indicators of oocyte maturation.	Jéhannet et al. [53]
Pufferfish	Aquaculture	Arothron manilensis	Sex determination to artificial breeding.	Doi et al. [54]
Lumpfish	Aquaculture	Cyclopterus lumpus	Monitoring gonadal development.	Mlingi et al. [40]
Lebranche mullet	Aquaculture	Mugil liza	Sex determination.	Santarosa et al. [22]
Manta rays and Devil ray	Biology	Mobula kuhlii, M. thurstoni, M. mobular, M. tarapacana, M. birostris, and M. alfredi	Internal anatomy and reproductive activity.	Froman et al. [55]
Sockeye Salmon	Aquaculture	Oncorhynchus nerka	Accuracy of sexing for captive broodstock management.	Frost et al. [56]
Hapuku	Aquaculture	Polyprion oxygeneios	Sex identification.	Kohn et al. [57]
Thornback ray	Biology	Raja clavata	Assessment of maturity in oviparous elasmobranchs.	Whittamore et al. [58]
Atlantic salmon	Aquaculture	Salmo salar	Monitoring reproductive physiology in female.	Næve et al. [59]
Bonnethead shark	Biology	Sphyrna tiburo	Determine pregnancy status and fecundity.	Anderson et al. [60]
Small-spotted catshark	Biology	Scyliorhinus canicula	Assessment of maturity in oviparous elasmobranchs.	Whittamore et al. [58]
Hilsa herring	Aquaculture and Fisheries	Tenualosa ilisha	Assessment of sex, gonad volume, and reproductive maturation status.	Dasgupta et al. [41]
Whitetip Reef Shark	Biology	Triaenodon obesus	Description of the late embryonic developmental stage.	Santos et al. [61]

Similarly, Memiş et al. [46] effectively studied the sex and gonadal stage of Russian sturgeon (Acipenser gueldenstaedtii) in concrete tanks under natural water conditions using ultrasound technology. They concluded that ultrasound examination is crucial during the breeding season for identifying females with mature eggs to facilitate successful fertilization. Esmailnia et al. [49] found that ultrasonography is a simpler and more cost-effective method for determining the sex of cultured beluga sturgeon (Huso huso) compared to other methods. They also noted that it is the least invasive method for determining the sex of 18-month-old fish, with an overall accuracy rate of 80.95%.

In a recent study, Doi et al. [54] successfully implemented artificial reproduction techniques for pufferfish (Arothron manilensis) following ultrasonographic sex determination. The hatching process took place five days post-fertilization, resulting in larvae with an average total length of 2.23 ± 0.15 mm and notochord length of 2.08 ± 0.14 mm. Nevertheless, the researchers noted that further refinements in hormonal injection timing are necessary to enhance the artificial reproduction process for A. manilensis.

Unconventional but highly significant species in aquaculture, such as low-trophic-level species from the Clupeidae family, are crucial for the sustainable development of the aquaculture industry [1]. Dasgupta et al. [41] effectively used ultrasonography to assess the sex, gonad volume, and reproductive maturation status of Indian shad (T. ilisha). This non-invasive and rapid tool provided accurate information on the maturity stages in both genders, demonstrating its reliability for monitoring gender and gonadal development and predicting the spawning patterns of the species [41]. Given the small size of Clupeidae species, ultrasonography could enhance the reproductive management and welfare of these animals.

The efficiency of ultrasound in determining the sex of Lebranche mullet was investigated in terms of analysis time and accuracy. Santarosa et al. [22] found that there was no significant difference in scanning time for sex determination between male and female specimens, with times of 2.58 ± 1.68 s for males and 2.40 ± 1.05 s for females. This indicates that the method was equally effective for both sexes.

Due to the difficulties in some species reproduction in worldwide fish farming, ultrasound emerges as a hopeful solution, enabling the evaluation of fish gender and reproductive condition in a non-intrusive and quick way. By utilizing this technology, fish farms can recognize the various phases of gonadal development, aiding in determining the optimal spawning time and consequently enhancing the success rate of fish reproduction. Despite being a promising method, its adoption encounters obstacles, including the requirement for specialized equipment and trained professionals to analyze the images. However, its application in scientific research has been crucial in understanding fish reproduction.

3.2. Ultrasonography for Fish Reproductive Assessment

Ultrasonography has become a popular choice for non-invasive and simpler sex identification in fish, replacing invasive and complicated methods. Martin et al. [62] were the first to use ultrasound to determine the sex of live fish, starting with Coho salmon (Oncorhynchus kisutch). This method has since become a valuable tool in many areas of fisheries research, including contemporary aquaculture. The effectiveness of ultrasound in fish reproduction has been consistently confirmed [47,48,52,53,59,63,64], and new non-invasive indices have been created [18].

In the early 1990s, Mattson [36] characterized ultrasonography as a non-invasive technique for determining the sex and size of Atlantic salmon (Salmo salar) gonads. While identifying the gonads of immature males could be challenging, those of females were consistently visible, enabling deduction-based sexing. On the other hand, Preston and Shaw [28] found that fish sex can be identified based on ultrasound textures. In males, areas with lower tissue density show up as darker images due to reflecting fewer ultrasound waves, while in females, areas with higher density appear lighter in the images because they reflect more ultrasound waves. Martin-Robichaud and Rommens [17] used ultrasonography to determine the sex and ovarian maturation of juvenile and adult halibut (Hippoglossus hippoglossus), winter flounder (Pleuronectes americanus), yellowtail flounder (Pleuronectes ferruginea), and adult haddock (Melanogrammus aeglefinus). They were able to easily distinguish between immature and mature ovaries in females, while in males, only mature testes were consistently identifiable. Additionally, they could assess ovarian maturation in haddock and ovulatory cycles in halibut using ultrasound imaging.

Ultrasonography has been utilized to evaluate emblematic species as well. Bureau du Colombier et al. [51] employed ultrasonography as a non-invasive method for determining sex and monitoring maturation in silver eels (Anguilla anguilla). Given the decrease in natural stocks, this technique could enhance the comprehension and management of eel maturation for both scientific and commercial objectives. The gonads of 96 silver eels were examined using portable equipment paired with a 6–15 MHz sonar, achieving a 100% success rate in sex determination, demonstrating the significant potential of the technology for both conservation and aquaculture.

Interestingly, Maskill et al. [47] conducted a study using a combination of techniques, including ultrasonography, to analyze the reproductive structure of hatchery-origin and wild White Sturgeon (Acipenser transmontanus) populations. The study evaluated 332 hatchery-origin and 75 wild individuals over a period of 2 years. Results showed that all hatchery-origin fish were either premeiotic males (n = 158) or previtellogenic females (n = 174) and had not reached puberty. Sex assignment accuracy using histology was 97% for hatchery-origin fish and 94% for wild fish. Endoscopy (an otoscope) was found to have the highest sex assignment accuracy for both hatchery (98%) and wild (100%) fish, while plasma sex steroid assessment had lower accuracy rates of 69% for hatchery fish and 74% for wild fish. Ultrasonography provided 57% accuracy for hatchery fish and 70% accuracy for wild fish. The authors concluded that endoscopy is a more reliable tool for sex assignment in prepubertal and postpubertal individuals of this species. The effectiveness of ultrasonography in fish reproduction has been consistently confirmed, and new non-invasive indices have been created. Nevertheless, there is a noticeable inconsistency in the reporting of instrument settings and handling procedures, which may impact the outcomes [18]. In most of the research presented here, the success of ultrasound is confirmed by the accuracy in the gametogenesis stage, followed by the successful reproduction of species.

Over the years, numerous studies have documented the use of ultrasound in various fish studies, particularly in reproductive biology over the past three decades [18]. Currently, ultrasound is widely utilized in aquaculture research for tasks such as sex determination [64], assessing egg presence and maturity in species like common carp (Cyprinus carpio) [20], examining the effects of carrageenin on gilthead seabream (Sparus aurata) muscle [65], identifying sex and evaluating reproduction in Nile tilapia (Oreochromis niloticus) [21], studying gonadal development in striped catfish (Pangasianodon hypophthalmus) [66], and determining the sex of Lebranche mullet [22]. Ultrasound holds significant potential in aquaculture by facilitating research on the crucial reproductive stages of various aquacultured species.

3.3. Phantoms as a Training and Research Tool

The use of phantoms, which are synthetic models that mimic biological tissues, has become a valuable alternative to using live animals for training professionals and students in ultrasound. These models are created to replicate the characteristics of biological tissues and are commonly used in imaging techniques. Phantoms are easy to produce and can be easily adjusted, making them adaptable for various experiments and training purposes [67,68].

The model created for M. liza aimed to replicate the sonographic appearance of aquacultured marine fish tissues, simulating the physical characteristics of biological tissue. The phantom was made using a mixture of colorless edible gelatin and warm water, with a ratio of 24 g of gelatin to 250 mL of water. Initially, a layer of the gelatin solution was used to house cavity organs like the stomach, intestine, liver, gallbladder, spleen, kidneys, testes, and ovaries from one male and one female specimen of the species. The organs were placed in the gelatin as it solidified, and a second layer was added to cover them completely, creating a structure that mimics the desired sonographic properties (Figure 1).

4. Ultrasonography Applied to Brazilian Sardine (S. brasiliensis) and Lebranche Mullet (M. liza)

S. brasiliensis, commonly known as the Brazilian sardine, is a member of the Clupeidae family and is found extensively along the Brazilian coast from Rio de Janeiro (Cabo de São Tomé, 22º S) to Santa Catarina (south of Cabo de Santa Marta Grande, 28º S). This migratory species thrives in coastal waters and exhibits remarkable physiological adaptability to navigate between bays and estuaries. With its slender, elongated, silvery body, S. brasiliensis plays a crucial role in the economy of the Southeast and South regions of Brazil, particularly in the canned food and fish (fresh and frozen) industries. Additionally, it is a key player in the live bait market, primarily used for skipjack tuna (Katsuwonus pelamis) fishing [1,69].

On the other hand, the Lebranche mullet can grow up to one meter in length and is found along the Atlantic coast of South America, from the Caribbean to Argentina [70,71]. According to the Ministry of Fisheries and Aquaculture report [72], this species is the most important for commercial fishing of Mugilids in Brazil. Due to their commercial value and significance, they are extensively researched to support their reproduction and cultivation in Brazilian aquaculture [2,3,4,73,74,75,76].

Both species, S. brasiliensis and M. liza, do not show obvious sexual dimorphism, making it impossible to visually determine the sex of the specimens. Therefore, ultrasonography has become a crucial tool for advancements in the artificial reproduction of these species in Brazilian aquaculture.

Sexual differentiation in mugilids is typically only possible during the advanced stage of vitellogenesis using the urogenital cannulation technique for ovarian sampling in females or by applying abdominal pressure to confirm spermiation in males. In sardines, the small size of the fish makes cannulation impractical, limiting the applicability of this method to larger species. While effective, the procedure is invasive and carries the risk of causing internal injuries to the oviduct and gonads. It is also a slow process and can potentially stress the animals, leading to delays or even prevention of ovulation [37,50]. In this context, ultrasonography may be a promising alternative for the differentiation and visualization of gonads in Mugilidae, with strategic applicability in smaller species such as Clupeidae.

4.1. Ultrasound Equipment and Frequency Settings

Regarding the Lebranche mullet, according to Magnotti et al. [75], male Lebranche mullet in captivity need to be at least 25 cm long and weigh over 205 g to be capable of reproduction, while female specimens should be at least 47 cm long and weigh over 1200 g to be reproductive. These zootechnical parameters are essential for evaluating gonadal development in mullet. In contrast, Brazilian sardines were included in the analysis regardless of their reproductive stage, as they do not have the same size and weight requirements. To determine the sex and degree of gonad maturation in sardines, careful handling is necessary to ensure their well-being and facilitate the process. Anesthesia by immersion in a benzocaine solution at 50 mg L⁻¹ is recommended as a safe method to reduce stress and prevent fighting behavior, allowing for accurate analysis with minimal risks to the fish [77].

The ultrasound analysis of mullets and sardines utilized a portable GE device, model Logiq and Veterinary, with a multifrequency linear probe known for its practicality and versatility in various frequencies. The selection of frequency is crucial in the analysis [18]. For mullets, a frequency of 10.0 MHz is optimal for detailed imaging of male gonads, while 8.0 MHz provides better clarity for female gonads in both longitudinal and transverse planes. In sardines, using the same frequency range for both males and females ensures consistent visualization of the gonads. The linear probe captures rectangular images with a depth of 3.0 cm and a horizontal field of view of 3.86 cm, allowing for freeze frames for measurements and annotations. As mentioned previously, ultrasound technology utilizes high-frequency sound waves to generate images of the body’s internal organs. Higher frequencies offer enhanced resolution and detail, making them well-suited for imaging superficial structures, whereas lower frequencies can penetrate deeper [11,13]. This accounts for the use of varying high-frequency (MHz) sound waves to visualize male and female gonads in detail.

To achieve high-quality images, the operator must be proficient in performing various movements with the transducer, including sliding, rotating, back-and-forth or sweeping, and applying pressure. These maneuvers are crucial for adjusting the angle and position of the transducer to ensure clear visualization of the internal structures of the fish. Accurate interpretation of ultrasound images requires differentiation between normal and abnormal anatomy, considering parameters such as size, shape, echogenicity, position, and architecture of the structures analyzed [30,78].

Measuring the length, area, and shape of different parts of the organism is vital for documenting abnormalities and monitoring changes over time. Image optimization involves meticulous adjustments, with the overall gain (Gain) typically set between 65 and 90 dB to regulate brightness and contrast. Temporal gain compensation (TGC) is essential for adjusting the attenuation of sound waves, with specific knobs providing greater control in more distant areas, such as the most distal regions of the ovaries, ensuring uniform visualization. Adjusting the position of the main focus and the depth maximizes the visibility of internal structures, accurately capturing the characteristics of the reproductive organs [11,13,14,30,60,79]. The ultrasound images will be saved on the device and can be transferred to a computer using a USB connection for secure storage and in-depth analysis at a later time. This method, which is non-invasive and emphasizes quality and detail, offers a precise look at the internal structures of the mullet, enabling highly accurate reproductive monitoring.

To conduct the ultrasound, the fish are placed in dorsal recumbency. An assistant supports the animal’s head and tail while keeping them partially submerged in a container with seawater (mullet) or on a damp cloth (sardine). A water-based ultrasound gel (Condu Gel, Contato Industrial LTDA.) is then applied to the fish’s skin, without requiring the probe to be submerged. Initially, the probe is placed longitudinally in the midline of the ventral abdomen. The cranial end of the probe should face towards the fish’s head, while the caudal end aligns with the animal’s anal orifice, which serves as an external reference point. Longitudinal, caudal, and cranial scans are performed from this position, as well as in the sagittal and transverse planes. The fish is then positioned in right and left lateral decubitus for a comprehensive scan of the body cavity and detailed structure identification (Figure 2 and Figure 3).

4.2. Ultrasound Characterization of Gonadal Echogenicity

At the beginning of the ultrasound examination, the probe is positioned over the anal orifice, allowing identification of the final portion of the intestine, which is located longitudinally in the body cavity, between the male and female reproductive organs. During this reproductive stage, the ovaries and testicles appear as elongated structures. They extend from the cranial portion—adjacent to the pectoral fins externally and the liver and gastric cavity internally—to the caudal region close to the terminal segment of the intestine. In both sexes, the reproductive organs are located side by side, occupying the right and left sides of the body cavity, with their width gradually decreasing towards the genital orifice.

In the Lebranche mullet, the echogenicity of the gonads is determined by their brightness compared to surrounding tissues, while echotexture refers to their granular appearance. Homogeneity indicates tissue uniformity. In both sexes, the gonads are bordered by a thin hyperechoic layer. Testicles appear hypoechoic and homogeneous with a fine echotexture (Figure 4A–C). Ovaries exhibit a hyperechoic appearance with a granular echotexture (Figure 4D–F), leading to increased sound wave attenuation in the organ’s distal region.

In the ultrasound examination of sardines, the ovaries show increased echogenicity, appearing hyperechogenic compared to the surrounding environment, and have a heterogeneous and coarse echotexture (Figure 5). In males, the gonads have reduced thickness compared to the ovaries, with a predominantly hypoechogenic, homogeneous sonographic appearance and fine echotexture in relation to the surrounding environment. In some cases, an even thinner thickness is evident (Figure 6A,C). Furthermore, the gonads may display an increased size, with an area of mixed echogenicity consisting of hyperechogenic and heterogeneous zones interspersed with hypoechogenic and homogeneous areas (Figure 6B). In certain instances, the testicles show rounded contours and variable echogenicity in different regions (Figure 6D). Similar to the ovaries, the boundaries of the male gonads are delineated by a thin, hyperechogenic line with slightly irregular contours.

5. Final Considerations

In Lebranche mullet, ultrasound enables a thorough reproductive assessment when the specimens are in the gonadal maturation stage, making it easier to visualize the gonads clearly, as seen in previous studies on mature individuals [42,80,81]. For Brazilian sardines, ultrasound can also detect gonads in less advanced stages, providing versatility for monitoring at various developmental stages.

In order to enhance the precision of maturation stage classification, it would be beneficial to implement a standardized method for measuring gonads using ultrasound [26]. This would involve establishing reference values for each stage of development. These pioneering studies are crucial in utilizing ultrasound for sexing and monitoring gonadal development in mullet and sardines. While there is room for improvement, this foundational information will lay the groundwork for future research. Subsequent research could concentrate on determining typical measurements for immature, developing, and mature gonads, thereby enabling a more consistent and replicable classification system. Such standardization would be valuable for captive care, as well as for cross-species comparisons and scientific investigations.

Despite being a costly technique (with equipment priced at over USD 26,170.00) and still in the development stage, ultrasound is valued for its efficiency and accuracy. It provides immediate sex identification, saving time and costs. The high level of accuracy in sex identification and minimal contact with the fish’s body are key advantages of this technique. During ultrasonography, the fish remains still in the water with the use of an anesthetic, and the procedure is carried out with minimal physical contact, which has a significant positive impact on the health and well-being of the fish. This approach eliminates the need for catheterization and biopsy methods for sexing in species without dimorphism [18,38].

6. Conclusions

Ultrasonography is recognized as a valuable, quick, and non-invasive technique for sex determination in fish, reducing stress and maintaining fish welfare. Its use in aquaculture enhances reproductive management by enabling ongoing monitoring of gonads. Furthermore, this method establishes a reliable foundation for standardization and future research, potentially broadening its utility across various species and environments. Therefore, ultrasound is a crucial and cutting-edge tool for promoting sustainable practices in aquaculture, facilitating reproductive control, and advancing understanding of gonadal development in studied species.