Testicular development, reproducing references and length at first maturity of Acentrogobius viridipunctatus (Actinopteri: Gobiiformes) in the southwest Viet Nam.

This study provided knowledge on testicular development, spawning reference and length at first maturity (L m ) of Acentrogobius viridipunctatus-a target catching fish for food supply in the southwest Viet Nam. A total of 528 individuals were collected using trawl nets from January to December 2020 at Long Huu-Tra Vinh (LHTV), Trung Binh-Soc Trang (TBST), Dien Hai-Bac Lieu (DHBL) and Tan Thuan-Ca Mau (TTCM). Five developmental stages of testis were found, and testis diameter increased from ∼1.07 (stage I) to ∼4.82 mm (stage V). Testis stage I consisted of spermatogonia (S) which shifted to primary spermatocytes (SC1) in stage II. In testis stage III, SC1 was divided into secondary spermatocytes (SC2). At this stage, testis consisted of S, SC1, SC2 and some spermatid (ST). At stages IV and V, testis comprised mainly spermatozoa (SZ) besides S, SC1, SC2 and ST, showing that this goby was a multi-spawner. Testis frequency and gonadosomatic index analyses showed that this species released sperms yearly, with a prominent peak in the wet season. The L m decreased from 9.3 ± 0.3 cm at TBST to 9.0 ± 0.3 cm at LHTV, 8.9 ± 0.2 cm at TTCM and 6.5 ± 0.5 cm at DHBL. Findings provided fundamental data to set up suitable fishing and conserving strategies for this species.

Introduction

Acentrogobius, a fish genus of the Gobiidae [14], plays a fundamental role in fish food supply due to its high nutritional content in the southwest Viet Nam known as the Vietnamese Mekong Delta (VMD) [27]. Acentrogobius consists of 22 species globally living from the Indian Ocean to the western Pacific Ocean [14]. In VMD, there are three species, including Acentrogobius globiceps (Hora, 1923), Acentrogobius caninus (Valenciennes, 1837) and Acentrogobius viridipunctatus (Valenciennes, 1837), are recorded [4, 33, 35, 36]. Among them, A. viridipunctatus is the most common species caught for food supply [27, 34]. This fish displays a dark curved line under the eyes and many bright blue spots on the head and body described by Tran et al. [33]. This species reaches a maximum total length of 16.5 cm in Israel [3] and feeds mainly on small fish, algae and organic matter in Hue, Centre of Viet Nam [21]. In VMD, the number of individuals of fish species, including this goby, has decreased in recent years [32]; therefore, there is a need to find a suitable strategy for conserving and exploiting fish resource there.

Reproduction of fish and other organisms is crucial to ensure the survival and development of the species [23]. Information on the maturation and degeneration of the gonads helps us better understand and determine the changes and development of fish populations in the wild [29]. Nikolsky [28] indicates that the testis in most fishes is divided into six stages based on their external morphological characteristics. The fish length first maturity (L) is also an important indicator in managing fish stocks [13, 31]. The L is species-specific and varies accordingly to location and reproductive behaviour [6]. Reproducing reference is closely related to producing and capturing aquatic species [19, 24]. On the other hand, A. viridipunctatus and other gobies are being overexploited in VMD [37]. Therefore, studying reproductive biology, i.e., testicular development, type and time of sperm releasing, and size at first maturity in A. viridipunctatus is essential. The findings will help set up a suitable strategy for conserving and exploiting the fish resources.

Material and methods

Fish samples were collected from four sites, including Long Huu-Tra Vinh (LHTV), Trung Binh-Soc Trang (TBST), Dien Hai-Bac Lieu (DHBL) and Tan Thuan-Ca Mau (TTCM) (Figure 1). In these sites, the precipitation varies significantly with the season, ranging from ∼0 in the dry season (January–May) to ∼400 mm monthly precipitation in the wet season (June–December). The tide of these sites was semi-diurnal tide with a range of ∼1.2 m, the annual temperature was ∼27 °C temperature, and the pH was ∼8 pH, as documented by Le et al. [20].

Figure 1

Allocation map of sampling sites (1: Long Huu-Tra Vinh; 2: Trung Binh-Soc Trang; 3: Dien Hai-Bac Lieu; 4: Tan Thuan-Ca Mau; Dinh, 2018).

A total of 960 individuals (528 males and 432 females) were sampled at four sites from January to December 2020. Five hundred twenty-eight males were used in the present study (Table 1), whereas the number of females was used in another study. Samples were collected once a month using trawl nets and were anaesthetized with MS222 before fixing in 5% formalin and shifting to the laboratory for analysis. Herein, fish samples were classified based on their external morphology, documented by Tran et al. [33] and sex determination based on genital spines (male: triangle and female: oval) [5]. Then the total length (TL) and weight (W) of fish were measured to the nearest 0.1 cm and 0.01 g, respectively. After dissecting, testis was visually classified into five-stage development using criteria description for Parapocryptes serperaster, documented by Dinh et al. [5]. After this, the testis was weighed (GW) to the nearest 0.01 mg using an analytical balance and measured diameter to the nearest 0.01 mm using Motic Image software v2.0 integrated with microscopy.

Table 1

The weight (W) and total length (TL) of male Acentrogobius viridipunctatus collected at the four sites in 2020.

Month	Long Huu-Tra Vinh			Trung Binh-Soc Trang			Dien Hai-Bac Lieu			Tan Thuan-Ca Mau
	No.	W range (g)	TL range (cm)	No.	W range (g)	TL range (cm)	No.	W range (g)	TL range (cm)	No.	W range (g)	TL range (cm)
January	19	6.18–20.68	8.1–12.4	15	4.44–17.73	6.5–10.5	16	5.97–33.26	7.1–13.3	15	5.73–18.79	7.2–11.1
February	7	5.87–8.84	7.5–8.8	24	6.25–19.18	7.9–11.7	7	9.69–27.80	8.9–11.9	7	9.00–39.13	9.9–15.5
March	12	3.33–14.65	6.3–10.3	18	6.25–18.44	7.9–11.1	8	3.82–26.19	6.4–12.6	13	7.02–37.16	8.4–15.7
April	12	6.84–54.07	8.7–17.2	14	3.88–37.88	6.7–15.0	8	6.56–40.24	7.5–15.4	7	11.4–28.69	10.5–14.0
May	11	6.87–24.16	8.6–12.9	5	3.16–25.80	6.5–13.5	8	6.76–24.50	7.4–12.6	11	4.49–47.34	7.0–16.2
June	8	6.74–19.90	7.8–12.0	16	4.85–33.13	7.7–14.5	13	4.57–24.59	7.3–12.0	9	6.23–20.54	7.8–11.9
July	11	5.2–17.38	7.7–11.3	11	9.04–29.15	8.8–12.8	7	4.11–44.56	7.1–15.9	6	9.08–22.64	9.4–13.0
August	8	4.86–22.24	7.3–13.0	19	7.79–24.33	8.9–14.4	12	6.93–20.86	8.8–12.3	12	5.87–17.23	7.4–12.0
September	14	3.02–16.89	6.7–10.8	10	3.50–31.21	6.3–14.2	10	7.79–22.56	9.9–13.8	14	2.71–28.82	6.5–12.8
October	17	2.38–41.24	7.0–15.6	5	6.91–10.40	9.4–10.4	7	7.49–14.79	8.6–11.0	4	7.72–13.75	8.8–10.5
November	10	6.16–17.37	8.1–11.3	6	7.15–9.33	8.6–10.1	18	7.35–22.53	9.3–13.4	5	7.67–13.95	8.9–10.5
December	14	2.74–18.37	6.2–11.2	11	3.92–17.23	6.6–12.1	12	7.35–21.69	9.3–13.0	2	7.95–13.27	8.7–10.6
Total	143	2.38–54.07	6.2–17.2	154	3.16–37.88	6.3–15.0	126	3.82–44.56	6.4–15.9	105	2.71–47.34	6.5–16.2

Twenty-five testis (five per stage) was examined histologically using the staining procedure (i.e., fixing, dehydrating, paraffin wax embedding impregnating, 6-μm thick cutting and staining with Hematoxylin and Eosin-Y) of Ho et al. [15] for gamete developmental determination.

The length at first mature (L) was the length in which 50% of fish reached sexual maturity and determined from the formula of King [18]: P = 1/(1+e-r(), where P was the percentage of mature fish in a given length, TL was fish total length, and r was a model parameter. The fish reached sexual maturity when its testis was in stage III onward. The relationship between P and TL was qualified using the non-linear regression of the SPSS v21 to obtain the L.

The reproducing pattern was estimated from the appearance of the gamete in the testis histological slides [28]. The sperm releasing time was determined based on the apparent frequency of testis and gonadosomatic index (GSI) [6, 30]. The GSI was determined from the formula: GSI = GW∗100/W [22]. One-way ANOVA determined the change over the months of the GSI at a 5% significance level. SPSS v.21 software was used for statistical processing.

Results

Testicular development

The testes of A. viridipunctatus consisted of two chambers located close to the spine in the fish's abdominal cavity fixed by various connective tissue types. The testis was long tubular sacs that contained sperm. The testes developed from a slender form in stage I to swollen form in stage V. As follows,

Testis was a white, small, and almost triangular cross-section (Figure 2a). The diameter of this stage testis was ∼1.07 mm. Histology of the testis at this stage only observed spermatogonia (S) that were dark purple with Hematoxylin dye and densely distributed throughout the testis (Figure 2f).

Figure 2

The morphology and histological in testes of A. viridipunctatus (a, b, c, d, and e: stage I–V of testis; f, g, h, i, and j: histology of the testis in stages I–V; S: spermatogonia, SC1: primary spermatocytes, SC2: secondary spermatocytes, ST: spermatid, SZ: spermatozoa).

Testis was opaque white and increased to 1.75 mm in diameter (Figure 2b). The S began to divide within the testis to primary spermatocytes (SC1). The SC1 had nuclei that were strongly stained with dye and were abundant in the testis. Two different types of gamete, i.e., S (large cells) and SC1 (small cells), were found in the testis (Figure 2g).

Testis in this stage was milky white, rough and wavy surface, increasing to ∼2.05 mm in diameter (Figure 2c). The S began to decline sharply in number, with only a few small clusters scattered in the testis. Besides S, SC1 also decreased in number because they divided into secondary spermatocytes (SC2). The SC2 were smaller than SC1 and lighter in colour with dyes. In the testis at this stage, the SC1 and SC2 were observed. Besides, spermatid (ST) also appeared in this stage. The vas deferens were increasingly evident and easier to find (Figure 2h).

Testis occupied ½ of the abdominal cavity volume, with a diameter of ∼3.98 mm, and was milky white (Figure 2d). ST was differentiated from spermatozoa (SZ) in the testis. The size of SZ the very small and was stained bright with Hematoxylin (Figure 2i).

Testis reached the maximum diameter of ∼4.82 mm, and the surface was distended (Figure 2e). Spermatozoa could come out when pressing on the fish belly from the outside. In the testis, there were clusters of SC1 and SC2 (Figure 2j). It can be seen that, after the ejaculatory phase, the testis had a recovery and returned stage similar to stage II.

Stage VI was not detected during the study period. This stage's duration might have been relatively short, so it was not seen. Besides, stage VI was also the period when fish rest after a spawning cycle, so catching fish at this stage was unlikely.

Reproducing reference and length at first maturity

The testis stage composition and gonadosomatic index (GSI) could determine the spawning season in A. viridipunctatus. The testis stage composition provided information about the period during which fish were most fertile. Unlike the testis stage composition, the GSI provided specific months of the fish spawning season, i.e., fish released sperms mainly in the highest value of the GSI period. Testis stage IV appeared in four study sites in most of the months, and testis stage V appeared from April to July (Figure 3a–d), indicating that it released sperms year-round, with a peak in the April–July period.

Figure 3

The testicular stage composition of Acentrogobius viridipunctatus in 2020 (a: Long Huu-Tra Vinh, b: Trung Binh-Soc Trang, c: Dien Hai-Bac Lieu, d: Tan Thuan-Ca Mau; number in the column: number of individuals).

The results of the analysis of GSI at LHTV showed that GSI displayed a low value in the dry season and a higher value in the wet season, especially in December (One-way ANOVA, F = 2.33, p < 0.001, Figure 4a). This showed that although these fish spawn here all year round, the primary reproducing season was in the wet season, from June to December. This change occurred similarly at TBST; however, the main sperm releasing period at TBST began later than LHTV. The GSI in TBST was low from January to August but gradually increased from September to December (F = 3.66, p < 0.001, Figure 4b). At DHBL, the reproducing season of fish occurred mainly from April to July due to the high GSI in these months (F = 2.64, p < 0.001, Figure 4c). At TTCM, the GSI remained low in the dry season and increased in the wet season from July to October (F = 2.87, p < 0.001, Figure 4d), which was the main sperm releasing of this fish.

Figure 4

The gonadosomatic index (GSI) of Acentrogobius viridipunctatus in 2020 (a: Long Huu-Tra Vinh, b: Trung Binh-Soc Trang, c: Dien Hai-Bac Lieu, d: Tan Thuan-Ca Mau; Different letters (a, b, c, and d) showed the monthly variation of GSI at each site).

The analysis results of L in A. viridipunctatus in four study areas showed that L at LHTV, TBST, and TTCM had similar values (Figure 5a–d). Specifically, this value was 9.0 ± 0.3 cm at LHTV (Figure 5a), 9.3 ± 0.3 cm at TBST (Figure 5b), and 8.9 ± 0.2 cm at TTCM (Figure 5d). Meanwhile, at DHBL, L exhibited the lowest value of 6.5 ± 0.5 cm (Figure 5c).

Figure 5

Length at first maturity of Acentrogobius viridipunctatus (a: Long Huu-Tra Vinh, n = 143; b: Trung Binh-Soc Trang, n = 154; c: Dien Hai-Bac Lieu, n = 126; d: Tan Thuan-Ca Mau, n = 105; Numbers in the figure: percentage of individuals in each length group with testis reaching stage III onward).

Discussion

The histological analysis of A. viridipunctatus showed that this fish could release sperms many times during the spawning season because different stages of spermatocytes were detected in the testis in stages IV and V [6]. After the SZ was released, SC2 and ST continued developing and forming SZ for the next cluster of sperm-releasing cycles. The testicular developmental and sperm releasing patterns of A. viridipunctatus were also found in some gobies living in and out of VMD, i.e., Eleotris melanosoma [38], Oxyeleotris urophthalmus [39], Glossogobius giuris [12, 16], Butis butis [6], Stigmatogobius pleurostigma [8], G. sparsipapillus [25], and G. aureus [11].

The sperm-releasing season of A. viridipunctatus in the four study sites varied between months, but the sperm-releasing season of this species occurred mainly in the wet season. It could be that more favourable environmental conditions in the wet season (low ambient temperature, abundant water) helped these fish spawn mainly in the wet season. The main sperm releasing period in the wet season was also found in G. giuris (August to November) [12, 16], E. melanosoma (April to June) [38], O. urophthalmus (May to June) [39], Trypauchen vagina (June to August) [7], S. pleurostigma (March to November) [8], G. sparsipapillus (September to November) [26], B. koilomatodon (August to December) [10], and G. aureus (August to October) [11]. The A. viridipunctatus showed spatial changes in sperm-releasing due to variation of GSI among these sites, which was found in its congener, A. pflaumi, and some fishes like Chasmichthys dolichognathus and Tridentiger spp., in Korea—a temporary region where they spawned in the spring-summer season from May to July [1, 2, 17]

The L shared a similar pattern at LHTV, TBST, and TTCM and was higher than DHBL. The environment in this area might have influenced the L of this species. According to Dinh et al. [10], the salinity displayed the highest at DHBL (23.5 ‰), followed by TTCM (23.1 ‰), and was higher than at TBST (14.0 ‰), and LHTV (12.3 ‰). This showed that when salinity exceeded the tolerance threshold of fish, L tended to decrease. Variation of L with salinity was also found in several other fish species in VMD. In G. aureus, L tends to decrease from freshwater (12.45 ± 1.48 cm) to saltwater (10.45 ± 0.34 cm) [11]. In addition, there was a similar change in L in Periophthalmodon septemradiatus—a fish of the family Oxudercidae in VMD. Specifically, P. septemradiatus exhibited an increase of L from saltwater (8.2 cm) to freshwater (9.2 cm) [9]. To sustain the source of A. viridipunctatus in the study region, the local government should ask fishermen to catch fish with a total length higher than L.

Conclusion

Acentrogobius viridipunctatus could release sperms year-round, with a prominent peak in the wet season. This species had the length at first mature in males depending on environmental changes. Capturing the L changes of this fish in the study areas was the basis for local authorities to determine the appropriate size of the catch. Since then, there have been measures to conserve and develop this fish species. This information is also beneficial for further studies on the artificial reproduction of this fish.

Declarations

Author contribution statement

Ngon Trong Truong: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.

Ton Huu Duc Nguyen: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Quang Minh Dinh: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

Quang, M. Dinh was supported by Can Tho University.

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.