Pectoral fin breeding tubercle clusters: a method to determine zebrafish sex.

Large breeding tubercle (BT) clusters, spike-like keratinized epidermal structures, have been identified along the middorsal surface of wild-type (AB, Ekkwill, Wik, Tuebingen) and long-fin mutant male zebrafish pectoral fin rays.1–4 The androgen-dependent growth of BT clusters occurs only in males and appears around the time of sexual maturation.1 Once present, these structures are maintained through the continuous renewal of the most superficial keratinized layer for the entire life of the fish, playing an essential role in stimulating egg release during spawning.2 The constant presence of these easily visualized structures only in males allows for the quick distinction of sex. Current guidelines available for distinguishing males from females rely on differences in the color, shape, and behavior of these fish. Generally, males appear pinkish-gold, possess a streamlined body, and chase females before mating. Females appear bluish-white and possess a protruding egg-filled abdomen.5–7 These guidelines have been proven subjective. For example, not all females possess an obvious distended abdomen, and there are reports of similar coloring in males and females.8 Although females are distinguishable from males by the presence of genital papilla,9 these ventrally located structures are small and difficult to observe in nonanesthetized fish. Overall, the ambiguities of previously set guidelines make the determination of zebrafish sex difficult, particularly for new students and staff members. In this study, we report the use of sexually dimorphic pectoral fin BTs as a means to facilitate in the identification of male and female zebrafish. The determination of sex using BTs requires that zebrafish are placed in a small fish tank that sits on a light-colored background. Examination of the dorsal surface of each spread pectoral fin will reveal the presence or absence of BTs. BTs are clearly visible from above as brown lines along the central fin rays of males (Fig. 1A). Females lack BTs and thus their pectoral fins are translucent (Fig. 1B). Together, these characteristics allow for the immediate distinction of zebrafish sex (Fig. 1C). BTs are easily visible in adult (AB, Ekkwill, Wik, Tuebingen, and long-fin mutant) zebrafish without any microscopic aids. However, a stereomicroscope can be used to identify small BTs on young, lightly anesthetized, fish as small as 1.3 cm or 2.5 months.1 A small bowl of 0.17 mg/mL of tricaine (ethyl-aminobenzoate) can be placed directly under the stereomicroscope.7 Fish immersed in tricaine solution are immobilized in seconds, spreading their pectoral fins laterally, enabling an immediate unobstructed view of the fin rays. Under the stereomicroscope, the spike-like BT structures are easily identifiable along the central fin rays1 (Fig. 1D–G). Fish exposed to tricaine will eventually flip upside down; however, dorsally located BTs are still visible from the ventral surface of pectoral fins due to inter-ray tissue transparency. Given the ease at which BTs are observed under the stereomicroscope, the determination of sex should take a maximum of 30 seconds. FIG. 1. Distinguishing male and female zebrafish based on large clusters of breeding tubercles (BTs). (A) Male zebrafish possess BT clusters, observed as brown lines, along the central pectoral fin rays (blue arrowheads). (B) Female pectoral fins are translucent, .