Antonio Medina

Preparation and Administration of Gonadotropin-Releasing Hormone Agonist (GnRHa) Implants for the Artificial Control of Reproductive Maturation in Captive-Reared Atlantic Bluefin Tuna (Thunnus thynnus thynnus)

Abstract Mature migrating Atlantic bluefin tuna (Thunnus thynnus thynnus) were captured in the Mediterranean Sea with a purse seine and reared in floating cages for 2 to 3 years. During the natural spawning period (June–July) of two consecutive years, fish were randomly implanted underwater with a controlled-release delivery system (implant) loaded with gonadotropin-releasing hormone agonist (GnRHa), in order to induce final oocyte maturation (FOM), ovulation/spermiation, and spawning. At the time of sampling, males were significantly larger than females (ANOVA, P < 0.001), having a mean (± SE) fork length and body weight of 190 ± 3 cm and 122 ± 5 kg, compared to 176 ± 3 cm and 94 ± 4 kg of females, respectively. All fish were reproductively mature, with their age ranging between 5 and 12 years and males being a year older, on average. After GnRHa implantation, fish were monitored for spawning and the release of eggs, and were sacrificed at different times after hormone treatment in order to examine the progressive effect of the treatment on gonad maturation. The in vitro GnRHa release from the produced implants was maximal during the first 2 d, with a mean (± SE) release of 525 ± 166 μ g GnRHa implant−1 day−1. The plasma GnRHa profile in vivo reflected the release in vitro, and statistically significant elevations in plasma GnRHa levels were measured until 7 d after treatment (ANOVA, P < 0.01). The underwater implantation procedure was improved between 2004 and 2005, requiring an average (± SD) of 3.1 ± 1.4 min for each fish, and was 64 and 84% successful in 2004 and 2005, respectively. There were no differences between the histological appearance of the testes of GnRHa-treated and control males, and almost all of them contained intra-testicular spermatozoa. However, the proportion of spermiating control males (n = 17) was only 12% compared to 26% for the GnRHa-implanted males (n = 19). Also, there were no differences between controls and GnRHa-implanted fish in sperm concentration, initial spermatozoa motility, or duration of forward motility, which ranged between 29.02–48.54 × 1010 spermatozoa ml−1, 58–63% and 8–9 min, respectively. Final oocyte maturation (FOM) and post-ovulatory follicles (POFs) occurred in 63% and 88%, respectively, of the GnRHa implanted females (n = 16), compared to 0% and 21%, respectively, of the control females (n = 14). In addition, two GnRHa-implanted females in 2005 were found to be ovulated at the time of sacrifice, and their eggs were fertilized in vitro with sperm from spermiating males, which resulted in viable embryos and larvae. Finally, although spawning was not observed, fertilized eggs were collected from the cages. Larvae produced from these eggs were identified as Atlantic bluefin tuna, demonstrating that the present GnRHa implantation method can be used to induce FOM, ovulation/spermiation, and spawning in captive-reared Atlantic bluefin tuna.

A comparative study of the ovarian development in wild and pond-reared shrimp, Penaeus kerathurus (Forskål, 1775)

Abstract Ovarian maturation was studied in pond-reared and wild-caught specimens of the shrimp Penaeus kerathurus in order to evaluate the influence of extensive culture conditions on the reproductive capacity of this species. Monthly samples of five female shrimp were taken from the wild and the pond from late winter (February–March) to mid-summer (July). For each specimen sampled, the body length, body weight and ovarian weight were recorded, and the gonadosomatic index (GSI) calculated. Once dissected out and weighed, the ovaries were processed for histological examination. The size reached by the shrimp reared in the pond was comparable to that recorded in the wild. However, significant differences were found in the GSI and the frequency (expressed as percentage) of postvitellogenic (i.e. vitellogenic plus mature) oocytes, which may be considered as good indicators of the sexual maturation rate. On the basis of the overall gonad histology, five distinct stages have been identified throughout the process of ovarian maturation. A major histological anomaly detected in all pond-reared shrimp examined was the absence of fully mature oocytes, which is due to the inability of late vitellogenic oocytes to synthesize cortical rods. Consequently, stage IV ovaries (characterized by the presence of mature oocytes i.e., those bearing cortical rods) were not found in captive animals. As the cortical rods are believed to play an important role at early development in penaeid eggs, their absence is probably one major constraint resulting in a broodstock population with reduced reproductive potential under culture conditions.

Changes in lipid class and fatty acid contents in the ovary and midgut gland of the female fiddler crab Uca tangeri (Decapoda, Ocypodiadae) during maturation

Changes in biochemical composition, lipid class and fatty acid contents were studied in the ovaries and midgut glands of the fiddler crabs Uca tangeri Eydoux during maturation. Wild females were caught during spring and early summer of 1992 in the Bay of Cádiz (southwest Spain), near the mouth of the San Pedro river. Protein and total lipid contents in the ovaries increased significantly from Stages III to IV, at the expense of total carbohydrate, which showed a large decrease during the same period. In the midgut gland, the protein content did not present any significant variation, whereas total lipids and total carbohydrates presented opposite up and down trends during maturation. In the ovary, total polar lipids increased significantly during the final phase of maturation (Stages III to IV), mainly due to the significant contribution of the phosphatidylcholine and phosphatidylethanolamine fractions. In contrast, total neutral lipids showed an upward trend throughout the whole maturation period, mainly due to significant increases of the triacylglycerol fraction. In the midgut gland, total polar lipids (mainly phosphatidylcholine) and total neutral lipids (mainly triacylglycerol) presented significant decreases from Stages II to III, the phase which preceded major increases in both polar and neutral lipids in the ovaries. Cholesterol content did not vary during maturation in either organ, in the ovary or midgut gland. Major fatty acids in the ovaries [16:0, 16:1 (n-7), 18:1 (n-9), 18:1 (n-7), 18:2 (n-6), 18:3 (n-3), 20:4 (n-6), 20:5 (n-3) and 22:6 (n-3)] did, however, accumulate significantly at later stages of maturation. It is noteworthy that arachidonic acid [20:4 (n-6)] content remained constant during all stages of maturation but decreased significantly in total polar lipids in the later phases of maturation. In contrast, eicosapentaenoic acid [20:5 (n-3)] increased significantly in all lipid fractions in the later stages, and docosahexaenoic acid [22:6 (n-3)] remained constant in the polar lipids and increased during later stages in the triacylglycerol fraction. Major fatty acids in the midgut gland lipids showed significant decreases from Stages II to III, just before the final period of maturation.

Spawning Behaviour and Post-Spawning Migration Patterns of Atlantic Bluefin Tuna (Thunnus thynnus) Ascertained from Satellite Archival Tags

Spawning behaviour of Atlantic bluefin tuna (Thunnus thynnus) was investigated using electronic satellite tags deployed in the western Mediterranean spawning ground, around the Balearic Islands (years 2009-2011). All the fish were tagged underwater and released within schools. In general, the fish tagged in the same year/school displayed common migratory trends. Following extended residency around the Balearic Islands, most tagged tuna crossed the Strait of Gibraltar heading for the North Atlantic. Discrepancies between the migratory tracks reconstructed from this and previous electronic tagging studies suggest that the bluefin tuna Mediterranean population may comprise distinct units exhibiting differing migratory behaviours. The diving behaviour varied between oceanic regions throughout the migratory pathways, the shallowest distribution taking place in the spawning ground and the deepest at the Strait of Gibraltar. A unique diving pattern was found on the majority of nights while the fish stayed at the spawning ground; it consisted of frequent and brief oscillatory movements up and down through the mixed layer, resulting in thermal profiles characterized by oscillations about the thermocline. Such a pattern is believed to reflect recent courtship and spawning activity. Reproductive parameters inferred from the analysis of vertical profiles are consistent with those estimated in previous studies based on biological samples.

Quantification of the age-pigment lipofuscin in brains of known-age, pond-reared prawns Penaeus japonicus (Crustacea, decapoda).

A quantitative study of the lipofuscin content was carried out by image analysis in brains of known-age, pond-reared Penaeus japonicus (Crustacea, Decapoda) with the aim of assessing the applicability of the lipofuscin technique as an estimator of the physiological age in penaeids. With this purpose, three distinct measurements of lipofuscin levels (% area fraction, granule density and mean granule size) were recorded in ten sections of the olfactory lobe cell mass (OLCM) per animal. The image analysis was based on the autofluorescence emitted by the pigment, which accentuates the contrast between the lipofuscin granules and the background tissue. The concentration of lipofuscin increased significantly with age and was independent of sex. The relationship between age and lipofuscin concentration (area fraction and granule density) was best described by a seasonalized von Bertalanffy function, since the accumulation rate of the pigment dramatically slowed down in fall-winter, probably as a result of reduced seasonal metabolism. The present results confirm the potential of the lipofuscin method in the estimation of physiological age in penaeids and suggest that the application of this methodology can be useful in studies of age structure in wild populations and in the assessment of natural resources. J. Exp. Zool. 286:120-130, 2000.

Variations in lipid content and nutritional status during larval development of the marine shrimp Penaeus kerathurus

Lipid class and fatty acid contents, survival and nutritional status of Penaeus kerathurus larvae, reared on a mixture of the marine microalgae Tetraselmis chuii and Isochrysis galbana (clone T-ISO), the rotifer Brachionus plicatilis and Artemia (parthenogenetic strain from Cadiz, S.W. Spain), were studied throughout development. Dry weight and free sterol contents increased and correlated positively (r = 0.91, P < 0.05) during development, whereas the ratio of triacylglycerol/free sterol (an indicator of the nutritional status of the larvae) decreased as did survival. Fatty acid contents at different larval stages reflected fatty acid content of foods. The content of docosahexaenoic acid (22:6n - 3) remained constant during the mysis and first postlarval stage and a low bioconversion rate from its precursor, eicosapentaenoic acid (20:5n - 3) (very abundant in Artemia food) was observed.

Spermiogenesis and sperm structure in the shrimp Parapenaeus longirostris (Crustacea: Dendrobranchiata): comparative aspects among decapods

Early spermatids of the dendrobranchiate shrimp Parapenaeus longirostris (Lucas, 1846) have a spherical nucleus with large patches of heterochromatin, surrounded by a cytoplasmic mass that contains the conspicuous proacrosomal vesicle. The highly polarized mid spermatid mainly consists of the nuclear region, displaying a discontinuous nuclear envelope, and a large proacrosomal vesicle located at the opposite side of the cell. The most recent spermiogenic transformations primarily concern elongation of the proacrosomal vesicle to form a tapering spike. This results in the typically tack-shaped sperm of natantian decapods. The initial steps of spermiogenesis in the two studied dendrobranchiates prove to be parallel to reptant spermiogenesis in some respects, namely rupture of the nuclear envelope, chromatin decondensation and differentiation of electron-dense regions within the proacrosomal vesicle content. Specifically, whereas the anteriormost condensation gives rise to the operculum in brachyurans, in dendrobranchiates it becomes the apical portion of the spike. Despite an unquestionable morphological similarity between the sperm of carideans and dendrobranchiates, spermiogenesis in both groups displays meaningful differences. Spermatids of caridean shrimps lack a distinct proacrosomal vesicle. In the course of spermiogenesis, the spike arises from aggregated cytosolic materials; hence it is not membrane-bound. Unlike in other decapods, caridean sperm do not undergo a conventional acrosome reaction, since exocytotic events are not involved in this process. The above arguments suggest that, in the Decapoda, separation into three sperm classes is more suitable than the two traditionally accepted classes. The dendrobranchiate and reptant sperm types share a number of spermiogenic and functional features, while the caridean sperm type appears to represent an independent evolutive line with regard to sperm development and function.

Spermiogenesis and sperm structure in the crabUca tangeri (Crustacea, Brachyura), with special reference to the acrosome differentiation

SummaryEarly spermatids of the crabUca tangeri consists of the nucleus of granular chromatin and the cytoplasm, which contains a proacrosomal vesicle in close association with membrane lamellae. In the mid spermatids an invagination of the acrosomal vesicle membrane gives rise to the formation of the perforatorium, a spindle-shaped tubule which encloses tubular membranous structures. The pair of centrioles located at the base of the acrosome is not directly involved in perforatorial differentiation. The acrosomal vesicle shows a heterogeneous content composed of the operculum, the thickened ring, and three layers of different materials concentrically arranged around the perforatorium. During the late spermatid stage the nuclear profile differentiates numerous slender arms and the chromatin arranges into fibers. Membranous tubules from the cytoplasm become incorporated into the tubular structures of the perforatorium. The mature spermatozoon has the typical structure of the branchyuran sperm, with a complex acrosome, cupped by the nucleus, and a thin cytoplasmic band intervening between the former main elements. The centrioles are degenerate. The nuclear arms are unusually numerous (more than 20) and lack microtubules or microtubular derivatives.

Structural Modifications of Sperm from the Fiddler Crab Uca tangeri (Decapoda) during Early Stages of Fertilization

ABSTRACT The spermatozoan of Uca tangeri consists of a large spherical acrosome, a cup-shaped filamentous nucleus, which extends into numerous radiating arms, and a thin cytoplasmic band. Oocytes from ripe ovaries and sperm from seminal receptacles were mixed in filtered sea water, fixed at different times, and examined under a scanning electron microscope. Several spermatozoa became attached by the arms to the surface of each oocyte. The sperm bound to oocytes underwent the acrosome reaction, which started with swelling of the acrosomal vesicle followed by extrusion of its contents. Subsequently, the perforatorium projected forward ∼ 1 µm beyond the apical end of the extruded acrosomal vesicle contents. As the ejected perforatorium becomes the leading edge of the reacted sperm, it is thought to be the constituent at which level membrane fusion between both gametes must occur.

Comparative assessment of the reproductive status of female Atlantic bluefin tuna from the Gulf of Mexico and the Mediterranean Sea.

Despite attention focused on the population status and rebuilding trajectory of Atlantic bluefin tuna (Thunnus thynnus), the reproduction and spawning biology remains poorly understood, especially in the NW Atlantic. At present, the eastern and western spawning populations are believed to exhibit different reproductive characteristics and, consequently, stock productivity. However, our study suggests that the two spawning populations, the Gulf of Mexico and the Mediterranean Sea, could show similar reproductive features and spawning strategies. Between 2007 and 2009, gonad samples from female Atlantic bluefin tuna were collected in the northern Gulf of Mexico (n = 147) and in the western Mediterranean Sea (n = 40). The histological and stereological analysis confirmed that sampled eastern and western bluefin tuna exhibit the same spawning duration (three months) but the spawning in the Gulf of Mexico begins one month earlier than in the Mediterranean Sea. Western bluefin tuna caught in the peak of the spawning season (May) showed a similar spawning frequency (60%) to the spawning peak observed in the Mediterranean Sea (June). Fecundity for the Gulf of Mexico fish () was lower but not significantly different than for fish sampled in the Mediterranean Sea (). Our study represents the first comparative histological analysis of the eastern and western spawning stocks whose findings, combined with new determinations of size/age at maturity and possible alternative spawning areas, might suggest basic life history attributes warrant further scientific and management attention.