Invertebrate Reproduction and
Development, 35~3
(1999) 251-259
251
Balaban, PhiladelphiaRehovot 0168-8170/99/$05.00 0 1999 Balaban
Comparison of ovary histology between different-sized wild and pond-reared shrimp Litopenaeus vannamei (=Penaeus vannarnei) E. PALACIOS, C. RODRíGUEZ-JARAMILLO
and LS. RACOTTA’
División de Biología Marina, Centro de Investigaciones Biológicas del Noroeste, Apdo. Postal 128, La Paz, Baja Calgornia Sur 23000, Mexico Tel. +52 (112) 53633, ext. 144; Fax +52 (112) 54715; email:
[email protected]
Received 10 July 1998; Accepted 22 Aprill999
Summary Ovarian maturation of pond-reared and wild Litopenaetu vannamei (= Penaeus vannamei) broodstock was compared by analyzing morpho-histological characteristics. The number of spawns by each female was also considered to determine if successive rematurations can affect ovarian development. Individually tagged 42 wild and 47 pond-reared females were sampled at the end of a production period in a commercial hatchery. Morphological variables were recorded and ovaries were histologically evaluated for oocyte type and diameter. Although wild shrimp were bigger than pond-reared, no differences were seen in gonadosomatic indexes, either considered as a group or within stages of maturation. Differences between pond-reared and wild sluimp were observed in previtellogenic and early-vitellogenic oocyte distribution in vitellogenic and cortical stages. However, no differences were obtained in relation to the proportion of late vitellogenic oocytes and mature oocytes with cortical rods, or oocyte diameter in each maturation stage. Occurrence of atresia was negatively correlated to the number of spawns of each female, whereas gonadosomatic index and postovulatory follicles had a positive correlation with the number of spawns. Pond-reared broodstock appeared to have a normal maturation compared to wild Litopenaeus vannamei (= Penaeus vannameQ in our conditions. Successive spawnings did not seem to produce substantial alterations in the rematuration capacities of individual spawners in terms of the variables measured and the conditions of this experiment. Kq words: Oocyte frequency, gonadosomatic index, multiple spawns
In troduction Shrimp reproduction in captivity is important for seed supply for grow-out in farms. Optimization strategies to improve nauplii production and quality are needed to achieve independence from wild seed stock. Today, though nauplii production is achieved in captivity, a large reliance on wild broodstock is still
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common practice in many countries. The use of pondreared broodstock could representan adequate strategy to avoid uncertain availability of wiid spawners. The reproductive performance between wild and pondreared shrimp has been compared, and some reported a greater quantity and quality of nauplii from wild stocks (Menasveta et al., 1993; Cavalli et al., 1997; Mendoza,
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E. Palacios et al. / IRLI 35 (1999) 25 I-259
1997). However, others report practically no differences between wild and captive stocks (Simon, 1982; Browdy et al., 1986; Menasveta et al., 1994; Palacios et al., 1999). The production of nauplii depends on an adequate maturation process. One way to evaluate differences in maturation between shrimp of different origins is through histological analyses. For shrimp matured in captivity, histology has been used to analyze the effect of eyestalk ablation and as a comparison between wild and pond-reared broodstock (Ramos and Torras, 1986; Tan-Fermin, 1991; Medina et al.. 1996). Using histology, no substantial differences in oocyte distribution for pond-reared Penaeus monodon w e r e o b s e r v e d (Tan-Fennin, 1991). However, an inability to attain complete mature oocytes was observed in pond-reared P. kerathurus (Medina et al., 1996). It is necessary to evaluate the maturation of wild VS. pond-reared shrimp matured ideally under identical hatchery conditions. If the conditions are adequate, shrimp that are put into production under hatchery conditions usually remature severa1 times (Wyban and Sweeney, 1991; Palacios et al., 1999). The effect of the subsequent rematurations on the physiological condition of the female and on nauplii quality is still controversial (Simon, 1982; Ottogalli et al., 1988; Bray et al., 1990; Hansford and Marsden, 1995; Palacios et al., 1999). Lumare (1979) observed that females with several spawnings had ovaries that did not fully enlarge and in which the development was not uniform. He suggested this to represent reproductive exhaustion. However, no histological evaluation of the effect of several maturations under hatchery conditions has been made. In a previous study we evaluated the productive data of a batch of pond-reared and wild broodstock, taking into account the number of spawns, and we observed a higher spawning frequency and number of nauplii/spawn in wild shrimp. However, fertilization and hatching rates were higher for pond-reared shrimp (Palacios, et al., 1999). In the present work, we anal@ the histology of ovaries from wild and pondreared females to determine if there is a difference in the reproductive process. Females of both origins with dif’ferent sizes and with different number of spawns were sampled to evaluate the effect of multiple spawns on the histology. Material and Methods G¿mrd hatchery condiíions H’ild and pond-reared adult shrimp were brought to matumtion at the commercial hatchery, Acuacultores
de La Paz, SA (APSA). Wild shrimp were captured on the coast of Sinaloa-Nayarit (México) and averaged 50g. Pond-reared broodstock was selected during the initial grow-out in 1995 when Gl juveniles (first generation of captive shrimp produced from wild spawners “GO” from the coast of Sinaloa-Nayarit) were subjected to accidental oxygen depletion in the growout ponds of Centro de Investigaciones Biologicas del Noroeste (CIBNOR), which caused massive mortalities among smaller shrimp (4g), leaving mostly large shrimp (24g) alive (F. Magallón, pers. comm.). Survivors were raised ti11 20g for 4 months in tidal earthem ponds at a density of five shrimplm’ and with pellets containing 35% protein of commercial grow-out diet (PIASA, La Paz, BCS, México). Food was administered with the use of feeding trays; when food was completely consumed, the ratio was increased by 5% (Martínez-Cordova et al., 1998). At 2Og, shrimp were further grown to spawner size (approximately 35g) for 5 months at CIBNOR, at a density of l/m’. Fresh squid was offered twice a week (approximately 10% of biomass on a fresh weight basis) in addition to pellets. These shrimp (Gl) were used as spawners by APSA to obtain a second generation (G2) of domesticated shrimp. Grow-out of G2 was made under similar conditions as G 1. When an average weight of ll .5 g was obtained, a selection for weight of the 1,000 individuals from the top 20% was made (> 14g). The selected shrimp were grown to spawner size (35 g, age 12 months) under similar conditions as G 1. Both wild and pond-reared (G2) shrimp were acclimated in the hatchery for 2 weeks before eyestaIk ablation. Shrimp were stocked and matured under normal hatchery conditions; 25m2 black fiberglass maturation tanks, six animalslm’, 1: 1 male to female ratio, 200% daily water exchange, and 28°C and 36960 (average temperature and salinity). The diet was composed (on a fresh weight basis) of 40% squid, 15% polychaetes, 40% clams, and 5% of a commercial maturation diet (Rangen Inc., Idaho, USA) divided into five daily rations accounting for a total daily supply of 10% of biomass. Ablation, tagging of females, and daily inspection for mature females have been previously described (Palacios et al., 1999). For spawning, mature females with an attached spermatophore were placed in individual 160 L spawning tanks. Females with a mature gonad, but which were not mated, were retumed to the maturation tank. In the moming the females were retumed to the maturation tank whether they spawned or not.
E. Palacios et al. / IRLI 35 (1999) 251-259
Femak sampLing At the end of the production cycle (approximately 3 months), 42 wild and 47 pond-reared females were sampled. Total weight of the females was deterrnined after blotting them dry, and total length and width of the first abdominal segrnent were recorded. The gonad fiom the abdominal region was dissected and fixed in Davidson solution for 24 h, embedded in a paraffmparaplast mixture, sectioned (6-8 Pm), and stained with Harris hematoxylin-eosin (Humanson, 1972). The hepatopancreas and gonad from the cephalothoracic region were dissected and weighed. The gonadosomatic index (GSI) and hepatopancreatic index (HPI) were calculated as their proportion relative to total body weight.
Gonad histology Four stages of gonadal maturation were determined based on the classification proposed by Tan-Fermin and Pudadera (1989): the previtellogenic stage (P), vitellogenic stage (V), cortical stage (C), and spent stage (S). The oocyte fiequency was estimated in three different regions of an ovary, counting the total number of oocytes appearing in a predetermined test area (Briarty, 1975) and determining the percentage of each type of oocyte per area. The diameter range of the oocytes was determined in three different regions of an ovary by use of an objective micrometer, and only oocytes showing nuclei sectioned approximately at the equatorial plane were measured. The average and maximum oocyte diameter for each female was calculated. Depending on the stage of maturation and individual variations per female, the number of oocytes considered ranged from 30 to 200. The oocytes were classified according to their vitellogenic stage, as described by Mohamed and Diwan (1994): previtellogenic oocytes (PVO), early or endogenous vitellogenic oocytes (EVO), late or exogenous vitellogenic ooc*ytes (LVO), and mature oocytes with cortical rods (MO). The oocyte frequency was used to calculate the proportion of oocyte types (Tan-Fermin, 1991; Medina et al., 1996). Besides the oocyte proportion, the occurrence of postovulatory follicles (POF) and atresia (A) was recorded and reported as number of occurrences in a determined test area. Oocytes with vacuolated cytoplasm were also considered atresic, as suggested by Tan-Fermin and Pudadera (1989).
Statibtical analysis 1. Two-way analyses of variance followed by a Tukey test for unequal N post-hoc mean comparisons
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(Statistica Version 4.3) were done to assess significant differences in each maturation stage (factor MS with four levels-previtellogenic, vitellogenic, cortical and spent) and between the origin of the shrimp (factor 0 with two levels - wild or pond-reared). When there was a significant effect of the shrimp body weight, it was included as a covariable (Grant and Tyler, 1983) 2. Correlations were made between number of spawns and oocyte frequency or GSI to determine the effect of consecutive spawns on the maturation process. The number of spawns each female had was determined during the production period in the hatchery using the colored number-ceded tag. Females were grouped according to their origin (wild or pondreared) and to the stage of maturation (three groups: previtellogenic, vitel!ogenic-spent or cortical). Oocyte frequencies and GSI were transformed to arcsine for the analyses (Sokal and Rohlf, 1981), but data are presented in percentage. Data are reported as mean*s. The leve1 of significance was preset at PCO.05.
Results Wild shrimp were larger than pond-reared animals when stocked in the maturation tank (see methods), a difference that was still apparent when organisms where sampled at the end of the production period (Table 1). The GSI was not signifìcantly diflerent between origins, but the HP1 was higher for pondreared females (P~0.01). When the weight was used as a covariable in the ANOVA analyses, there were no significant differences in length between wild and pond-reared shrimp, but the first abdominal segment was wider for pond-reared females. At the termination of the experiment, the GSI and the average and maximum diameter of oocytes had a similar distribution among the different maturation stages (Table 2). No significant differences between wild and pond-reared shrimp were observed for GSI or oocyte diameter. There were significant correlations between the GSI and oocyte average diameter (r=0.72; P
