A Study on Testicular Development in Apteronotus albifrons

Apteronotus albifrons has its cloacal aperture located in the front of the body and on the back of the lower jaw. A. albifrons possess a pair of testes attached on the mesentery below its liver on the back of the abdominal cavity. In the early days of development, the testes are in translucent elongated thin strips in nude colour. As they develop, the testes will grow thicker and become white or creamy white in color. The rear end of the testes will split whereas the front end will merge to form the vas deferens which is connected to the external environment through the cloacal aperture. Testes of A. albifrons are lobular. There are six stages in its development. At 6 months of age, the testis structure shows clear medium with primary spermatocytes and abundant spermatogonia, suggesting that development is into the stageII. At 8 months of age, testes in male A. albifrons are in the shape of a thin rod in white with blood vessels in presence. Germ cells consist of spermatogonia, primary spermatocytes and spermatocytes, indicating stageIII development. At 11 months of age, testes in male A. albifrons grow fuller and are in creamy white with apparent blood vessels present. Primary spermatocytes, secondary spermatocytes, spermatids and a small amount of sperms are president, indicating stageIV of development. At 15 months of age, testes in male A. albifrons swells and consist of a large amount of sperms, reaching full maturity. This is the stageVIof development, after which is the stage featured by testes after spermiation.

Gonadal development is vital to the reproduction and the existence of fish population. It is also one of the most crucial factors in fish reproduction. Research on testis development in fish species have been the interest of scholars for years. By far, histological and molecular biological methods have been used to look into things such as gonadal differentiation in fish, morphological features of each testes development stage and its time phasing as well as the molecular regulation in testicular development (Kaneko et al., 2015;Fan et al., 2017;Hsu et al., 2018).
Apteronotus albifrons is of Gymnotiformes order and Apteronotidae family (Wan et al., 2002). A. albifrons has a mild temperament and they are amazingly beautiful swimmers which gained popularity among hobbyists. A. albifrons has no dorsal fin and is weakly electric. They are very sensitive to water quality and are extremely difficult to breed in captivity. Research on A. albifrons reproduction is very few. Morphological and histological observations in terms of A. albifrons testicular development in captive breeding are aimed at understanding the patterns in its testicular development to enrich studies on its biological productivity. It also helps to get a solid grasp on A. albifrons reproduction patterns, thus providing theoretical foundation for artificial breeding of the species, which will boast significant relevance to the protection and development of this species resource.

Sample Measurement and Morphological Observation of the Gonad
Measure individual body length, total length, body height, width and weight. Determine the sex via anatomy and observe gonad morphology. Take the gonad and keep it fixed in Bouin solution for 24 hours, then move to be preserved in 70% ethanol solution.

Data Analysis and Processing
Measure the biological parameters of the experimental fish, including body weight (BW), total length (TL), body length (BL) and gonadal weight (GW) etc. Calculate relevant gonadal development indices: Gonadosomatic index (GSI)= GW/BW x 100. Analyze the data with Microsoft Excel 2010. Mean ± SD is used for both measuring the data and calculating GSI. Conduct ANOVA analysis (SPSS software, V19.0) with the data above and check significance of difference. Note significance of difference (P) as 0.05. If P≤0.05 then there is statistical difference, if not, then no statistical difference.

Morphology and Structure of the Testes
A. albifrons has its cloacal aperture located in the front of the body and on the back of the lower jaw. A. albifrons possesses a pair of testes attached on the mesentery below its liver on the back of the abdominal cavity. In the early days of development, the testes are in translucent elongated strips in nude colour. As they develop, the testes will grow thicker and become white or creamy white in color. The rear end of the testes will split whereas the front end will merge to form the vas deferens which is connected to the external environment through the cloacal aperture.

Testicular Development Stages and Histological Observation
Referring to the testicular development staging methods by Yun Liu (Liu, 1933), Yaoguang Zhang (Zhang, Luo, & Zhong, 1992) and Yundong Lou (Lou, 1980) et al. and combined with the morphological features of A. albifrons testes and its germ cell distribution, six stages are identified during the development course of the testes: At stageⅠ, testes are in pairs and closely attached to the two sides of the abdominal cavity. They are in transparent and elongated thin strips which are hard to distinguish. Through histological studies, spermatogonia are randomly present and scattered among the connective tissues.
At stageⅡ, testes are in translucent and elongated thin strips with nude color. Blood vessels are inapparent. However, testes and ovaries can be distinguished at this stage with naked eyes. Early seminiferous lobules can be visible. Primary spermatocytes are present with the number of spermatogonia increasing and apparent medium among tissues.
At stageⅢ, testes are in the shape of a thin rod in white with blood vessels present. Seminiferous lobule becomes larger. At this stage, germ cells in the testes generally consist of spermatogonia, primary spermatocytes and secondary spermatocytes. Primary spermatocytes are round or oval shaped. They have a smaller diameter than the spermatogonia with rich chromosomes in the nuclei and a violet colour pigmentation. The nucleus is going through the first meiosis. Secondary spermatocytes are smaller than primary spermatocytes with its nucleus in violet colour and basophilia continuing to enhance. Compared to the second stage, primary spermatocytes count is significantly increased.
At stageⅣ sees further development of the testes. They become larger, fuller and creamy white with clear distribution of the blood vessels. Seminiferous lobules continue to grow and so is the seminiferous lobule cavity. Primary spermatocytes, secondary spermatocytes, spermatids and a small amount of sperms along with others of the same cell types are piled up. Secondary spermatocytes, after the second mitosis, develop into spermatids that are irregular in shape with no apparent cytoplasm. Only the round nucleus is visible which has high basophilia and a violet colour. Sperm counts in mid and later stages gradually go up.
At stageⅤ, testes are in creamy white blocks and reach maximum sizes. Blood vessels further develop, and the distribution is more distinctive. Testes are fully grown and reach full maturity. In this stage, the majority of germ cells in the testes are comprised of androcytes and sperms in metamorphosis. Sperms in great density swirled in and fulfilled the seminiferous lobule cavity, showing a deep blue colour. The sperm is the smallest cell in the At stageⅥ, testes become degenerated or have finished spermiation. Its size will decrease and the colour becomes light pink. Sperms in most seminiferous lobule cavities are discharged with a small amount of "old" sperms or undischarged sperms remaining to be absorbed. The rest are a very rare amount of spermatogonia and spermatocytes.  Changes in male A. albifrons GSIs can be seen from Figure 2. As the gonad starts to develop, testes increase in weight and size and parent GSI starts to climb. Development accelerates in the StageⅢ and peaks during the StageⅤ at 0.612±0.06 which is significantly higher than the 0.16±0.03 identified during the StageⅢ in ovaries.

Male A. albifrons Gonadal Development Features
Studies on fish testicular development are far less detailed than those on ovarian development. There is yet to be a standard set of demarcations on the staging of testicular development. Based on testicular anatomy and histological features, some researchers have divided testicular development into the following five stages, i.e. immature, developing, maturing, spawning and spent (Akayli et al., 2013). Chinese researchers normally divide the process into 6 stages. As spermatogonia gradually mature, they go through five major stages, i.e. spermatogonia, primary spermatocyte, secondary spermatocyte, spermatid and sperm. A. albifrons testicular development demonstrates a "catch-up growth" phenomenon that goes from synchronized development to non-sync development and then to synchronized development again. This phenomenon is also present in the testicular development in teleost such as Eleutheronema tetradactylum (Lan et al., n. d.) and Anguilla japonica (D. Lin & H. R. Lin, 1984). The key features are that during the At stageⅠand stageⅡ, spermatogonia and spermatocytes proliferate, and development is basically in sync. By the stageⅢ, the seminal vesicle near the seminiferous lobule cavity will first mature whereas the seminal vesicle near the intralobular interstitium