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Abstract—Testis histological structure
was studied in bluefin tuna (Thunnus thynnus) from the eastern Atlantic and Mediterranean during the reproductive season (from late April to early June). Testicular maturation was investigated by comparing samples from bluefin tuna caught on their eastward reproductive migration off Barbate (Strait of Gibraltar area) with samples of bluefin tuna fished in spawning grounds around the Balearic Islands. Histological evaluations of cross sections showed that the testis consists of two structurally different regions, an outer proliferative region where germ cells develop synchronously in cysts, and a central region made up of a welldeveloped system of ducts that convey the spermatozoa produced in the proliferative region to the main sperm duct. Ultrastructural features of the different stages of the male germ cell line are very similar to those described in other teleost species. The bluefin tuna testis is of the unrestricted spermatogonial testicular type, where primary spermatogonia are present all along the germinative portion of the lobules. All stages of spermatogenesis were present in the gonad tissue of migrant and spawning bluefin tuna, although spermatids were more abundant in spawning fish. The testis size was found to increase by a factor of four (on average) during migration to the Mediterranean spawning grounds, whereas the fat bodies (mesenteric lipid stores associated with the gonads) became reduced to half their weight, and the liver mass did not change significantly with sexual maturation. Linear regression analysis of the pooled data of migrant and spawning bluefin tuna revealed a significant negative correlation between the gonad index (IG) and the fat tissue index (IF), and a weaker positive correlation between the gonad index (IG) and the liver index (IL). Our analyses indicate that the liver does not play a significant role in the storage of lipids and that mesenteric lipid reserves constitute an important energy source for gametogenesis in bluefin tuna.
Manuscript submitted 27 April 2003 to Scientific Editor’s Office. Manuscript approved for publication 25 March 2004 by the Scientific Editor. Fish. Bull. 102:407–417 (2004).
Testicular development in migrant and spawning bluefin tuna (Thunnus thynnus (L.)) from the eastern Atlantic and Mediterranean Francisco J. Abascal César Megina Antonio Medina Departamento de Biología Facultad de Ciencias del Mar y Ambientales Universidad de Cádiz Av. República Saharaui 11510 Puerto Real Cádiz, Spain E-mail address (for A. Medina, contact author):
[email protected]
The Atlantic northern bluefin tuna (Thunnus thynnus thynnus (L.)), is one of the most commercially valuable wild animals in the world. In the last two decades this species has been subject to intense over-fishing, which has caused a decline in both the eastern and western populations because of lowered recruitment (Mather et al., 1995; Sissenwine et al., 1998). The bluefin tunas (T. thynnus and T. maccoyii) are unique among tuna species in that they live mainly in cold waters and move into warmer waters to spawn (Olson, 1980; Lee, 1998; Schaefer, 2001); therefore the migratory pattern of these species depends substantially on reproduction. The eastern stock of Atlantic bluefin tuna spawns from June through August in the Mediterranean Sea, where natural conditions are apparently optimal for the survival of offspring. From late April to mid June, bluefin tuna breeding stocks migrate from the North Atlantic to spawning grounds in the Mediterranean (Mather et al., 1995; Ravier and Fromentin, 2001). A good understanding of the reproductive parameters (especially sexual maturation, fecundity, and spawning) of tunas is of paramount importance for population dynamics studies and the management of fisheries that target tunas. Nevertheless, “a very limited amount of scientifically useful infor-
mation is available on the reproductive biology for most tunas” (Schaefer, 2001). Recent work has increased our knowledge on the reproductive biology of female Thunnus thynnus in the eastern Atlantic and the Mediterranean (Susca et al., 2000, 2001a, 2001b; Hattour and Macías, 2002; Medina et al., 2002; Mourente et al., 2002), but many questions remain still to be answered regarding male reproductive activity in this and other tuna species. Histological examination of gonads is a useful tool for assessing the maturity state of fish. However, very few light-microscopy studies have been published on bluefin tuna and no ultrastructural studies of reproductive organs are yet available. The male reproductive cycle of T. thynnus has been characterized histologically by Santamaria et al. (2003), and Ratty et al. (1990) and Schaefer (1996, 1998) have reported valuable histological descriptions on male and female gonads of the Pacific albacore (Thunnus alalunga) and the yellowfin tuna (Thunnus albacares), respectively. In this article we report biometric and histological data on male T. thynnus caught during their reproductive migration and spawning period in order to provide further information on the biological aspects of reproduction for this species.
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Materials and methods
Statistical analysis
Samples and condition indices
The bluefin tuna specimens used in this study showed considerable variability in size, especially those caught by purse seine in Balearic waters, where weight ranged between 12 and 349 kg. The purse-seine fishery is, in fact, much less size-selective than are traps, which seldom catch small bluefin tuna (Rodríguez-Roda, 1964; Mather et al., 1995). Analysis of covariance (ANCOVA), with body weight as covariate, was used as the most suitable method (García-Berthou, 2001) to test interannual differences in the weight of the organs within the two sampling sites. ANCOVA was likewise applied to compare the weights of the three organs between both areas. All data were previously log-transformed to meet the prerequisites of normality and homoscedasticity (Zar, 1996). Linear least-squares regression analyses were performed to test possible correlations between IG and the two other indices (I L and I F ) by using the pooled data of Barbate and the Balearic Islands. In the regression between IG and I F, the Balearic samples corresponding to year 2001 were excluded because the reduced fat-body size (adipose tissue was almost nonexistent in the mesentery) of these small bluefin tuna did not permit an accurate weight measurements on board. The values of the indices were arcsine-transformed prior to the statistical analysis (Zar, 1996). A P-value ≤0.05 was considered statistically significant for all tests.
During the eastward migration, 62 adult male bluefin tuna weighing between 71 and 273 kg (mean 195.17 kg) were obtained from the trap fishery in the area of the Strait of Gibraltar (Barbate, Cádiz, southwestern Spain) from late April to early June 1999, 2000, and 2001. Thirty-four mature males, weighing between 19 and 349 kg (mean 115.11 kg), were sampled in June−July 1999−2001 from the purse-seine fleet operating in the Mediterranean spawning grounds of bluefin tuna off the Balearic Islands. Whenever possible, the total body weight (W) was recorded to the nearest kg. When individual body weights were not available, W was estimated from the fork length (L F) measurements (recorded to the nearest cm), according to the formula: W = 0.000019 × L F 3 (Table VIII in Rodríguez-Roda, 1964). Following dissection, the liver, testes, and the fat bodies associated with the gonads were removed and weighed to the nearest g. The condition of the fish was assessed by three different indices. The gonad index (gonadosomatic index) (IG ) is indicative of the maturation state and was calculated as: IG = (WG / W) × 100, where WG = gonad weight. The liver index (hepatosomatic index) (I L) and fat-body index (I F) were calculated as IL = (W L / W) × 100, and IF = (W F / W) × 100 (where W L and W F represent liver and fat-body weights), respectively, and are considered as good indicators of the metabolic condition and energy reserves of the fish. All measurements are expressed as means ±SD.
Results
Histology
Condition indices
For light microscopy, tissue samples from the central part of the testes were fixed for 48−96 hours in 10% formalin in phosphate buffer, 0.1 M, pH 7.2. After dehydration in ascending concentrations of ethanol, a part of each sample was embedded in paraffin wax and the remainder was embedded in plastic medium (2-hydroxyethyl-methacrylate). Paraffin sections (6 µ m thick) were stained with haematoxylin-eosin, and plastic sections (3 µ m thick) were stained with toluidine blue. These were examined and photographed on a Leitz DMR BE light microscope. For electron microscopy, small fragments of testis were fixed for 3−4 hours in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate buffer (pH 7.2). Following two 30-min washes in cacodylate buffer, they were postfixed for 1 hour at 4°C in cacodylate-buffered 1% osmium tetroxide, rinsed several times in buffer, dehydrated in ascending concentrations of acetone, and embedded in epoxy resin (either Epon 812 or Spurr). Thin sections (∼80 nm thick) were picked up on copper grids, stained with uranyl acetate and lead citrate, and examined in a Jeol 1200 EX transmission electron microscope. Approximate dimensions provided for germ cells are measurements (means ±SD) of the largest cell diameters on electron micrographs.
ANCOVA did not reveal significant interannual differences in gonad, liver, and fat-body weight in the samples of Barbate as well as in those of the Balearic Islands. In contrast, a strongly significant difference in testicular size (P
