Javier Linares-Casenave

Effect of ovarian stage on plasma vitellogenin and calcium in cultured white sturgeon

Abstract We developed an enzyme immunoassay for plasma vitellogenin (VTG) of white sturgeon Acipenser transmontanus , cultured in California. The assay, based on polyclonal rabbit antibody to purified VTG, was tested for specificity and applied on sturgeon broodstocks (age 4–12 years) reared at three locations. Plasma VTG concentrations were correlated with total plasma calcium and stage of ovarian maturity. Females in the previtellogenic ovarian stage and at the onset of yolk deposition had low VTG concentrations, but plasma VTG and calcium increased after onset of yolk deposition in the oocytes. Total plasma calcium exhibited a linear relationship with plasma VTG. Concentrations of both metabolites in plasma discriminate previtellogenic and vitellogenic females, but fail to discriminate specific stage of ovarian vitellogenesis. The immunoassay for plasma VTG or flame photometry for total plasma calcium can be utilized to segregate vitellogenic females in cultured sturgeon broodstocks.

Effects of thermal regime on ovarian maturation and plasma sex steroids in farmed white sturgeon, Acipenser transmontanus

Abstract Recently, commercial aquaculture farms in Northern California have exposed gravid, cultured white sturgeon females to cold water (12±1°C) throughout the late phase of vitellogenesis and ovarian follicle maturation resulting in improved ovulation rates and egg quality. However, the optimum timing for transfer of broodfish to the cold water and the capacity of transferred broodfish to maintain reproductive competence over an extended time in cold water had not been evaluated. Gravid white sturgeon females that have been raised at water temperatures of 16–20°C were transported to either cold water (12±1°C; Group 1) in November 1997 or maintained in ambient water temperatures (10–19°C; Group 2) until early spring. In March 1998, half of the fish in Group 2 had regressed ovaries, but the remaining females had intact ovarian follicles and were transported to the cold water. Ovarian follicles and blood were collected from females until they reached the stage of spawning readiness (determined by germinal vesicle position and an oocyte maturation assay) or underwent ovarian regression. Exposure of gravid sturgeon females to ambient water temperatures (14.5±2.3°C, mean±S.D.) from October to March led to a decrease in plasma sex steroids and a high incidence of ovarian regression in fish with a more advanced stage of oocyte development. Transfer of females with intact ovarian follicles to cold water (12±1°C) in the fall or early spring resulted in normal ovarian development in the majority of females. Holding females in cold water does not seem to override their endogenous reproductive rhythms but extends their capacity to maintain oocyte maturational competence over a longer period of time. A temperature-sensitive phase in ovarian development may occur during the transition from vitellogenic growth to oocyte maturation, and the degree and timing of sensitivity to environmental temperature are dependent on the females endogenous reproductive rhythm.

Effect of incubation temperature on green sturgeon embryos, Acipenser medirostris

Regulation of river flow and the amount of winter rainfall are the major factors affecting the water temperature of the spawning grounds, for green sturgeon in the Klamath River. During the primary spawning period of green sturgeon, mid-April to June, the water temperature may vary from 8 to 21°C. To estimate the potential implications of this modified thermal regime, we examined the survival and development in three progeny groups of green sturgeon embryos from zygote to hatch, at constant incubation temperatures (11–26°C). Temperatures 23–26°C affected cleavage and gastrulation and all died before hatch. Temperatures 17.5–22°C were suboptimal as an increasing number of embryos developed abnormally and hatching success decreased at 20.5–22°C, although the tolerance to these temperatures varied between progenies. The lower temperature limit was not evident from this study, although hatching rate decreased at 11°C and hatched embryos were shorter, compared to 14°C. The mean total length of hatched embryos decreased with increasing temperature, although their wet and dry weight remained relatively constant. We concluded that temperatures 17–18°C may be the upper limit of the thermal optima for green sturgeon embryos, and that the river thermal regime during dry years may affect green sturgeon reproduction.

A proteomic analysis of green and white sturgeon larvae exposed to heat stress and selenium.

Temperature and selenium are two environmental parameters that potentially affect reproduction and stock recruitment of sturgeon in the San Francisco Bay/Delta Estuary. To identify proteins whose expression is modified by these environmental stressors, we performed a proteomic analysis on larval green and white sturgeons exposed to 18 or 26 degrees C and micro-injected with Seleno-L-Methionine to reach 8microgg(-)(1) selenium body burden, with L-Methionine as a control. Selenium and high temperature induced mortalities and abnormal morphologies in both species, with a higher mortality in green sturgeon. Larval proteins were separated by two-dimensional gel electrophoresis and differential abundances were detected following spot quantitation and hierarchical cluster analysis. In green sturgeon, 34 of 551 protein spots detected on gels showed a variation in abundance whereas in white sturgeon only 9 of 580 protein spots were differentially expressed (P<0.01). Gel replicates were first grouped according to heat treatment. Fifteen of these spots were identified using MALDI TOF/TOF mass spectrometry. Proteins involved in protein folding, protein synthesis, protein degradation, ATP supply and structural proteins changed in abundance in response to heat and/or selenium. 40S ribosomal protein SA, FK506-binding protein 10, 65kDa regulatory subunit A of protein phosphatase 2, protein disulfide isomerase, stress-induced-phosphoprotein 1, suppression of tumorigenicity 13 and collagen type II alpha 1, were differentially expressed in high temperature treatment only. Serine/arginine repetitive matrix protein 1, creatine kinase, serine peptidase inhibitor Kazal type 5 and HSP90 were sensitive to combined temperature and selenium exposure. Valosin-containing protein, a protein involved in aggresome formation and in protein quality control decreased more than 50% in response to selenium treatment. Potential use of such proteins as biomarkers of environmental stressors in larval sturgeons could indicate early warning signals preceding population decline.

Distinguishing ovarian maturity of farmed white sturgeon (Acipenser transmontanus) by Fourier transform infrared spectroscopy: a potential tool for caviar production management.

Fourier transform infrared spectroscopy (FT-IR, 4000-400 cm(-1)) was applied to blood plasma of farmed white sturgeon (N = 40) to differentiate and predict the stages of ovarian maturity. Spectral features of sex steroids (approximately 3000 cm(-1)) and vitellogenin (approximately 1080 cm(-1)) were identified. Clear segregation of maturity stages (previtellogenesis, vitellogenesis, postvitellogenesis, and follicular atresia) was achieved using principal component analysis (PCA). Progression of oocyte development in the late phase of vitellogenesis was also monitored using PCA based on changes in plasma concentrations of sex steroid and lipid content. The observed oocyte polarization index (PI, a measure of nuclear migration) was correlated with changes in plasma sex steroid levels revealed by FT-IR PCA results. A partial least squares (PLS) model predicted PI values within the range 0.12-0.40 (R = 0.95, SEP = 2.18%) from differences in spectral features. These results suggest that FT-IR may be a good tool for assessing ovarian maturity in farmed sturgeon and will reduce the need for the invasive ovarian biopsy required for PI determination.

Induced Spawning, Artificial Fertilization, and Egg Incubation Techniques for Green Sturgeon

Abstract Establishment of hatchery breeding techniques for the threatened green sturgeon Acipenser medirostris is important for research and conservation hatcheries. Injections of either gonadotropin-releasing hormone analog (GnRHa) or GnRHa plus domperidone were used to induce ovulation in 13 female Klamath River green sturgeon and to induce spermiation in 19 males. Ovulated eggs were either rinsed in water or not rinsed before fertilization, and the eggs were fertilized with different milt dilutions and for different lengths of time. After fertilization, eggs either were allowed to adhere to the bottom of glass dishes or were silted for 1 h and then incubated in McDonald or upwelling jars. All broodfish ovulated or spermiated in all hormonal treatments, and the best treatment was GnRHa injected alone in a single dose of 10 μg/kg for males or in a 1-μg/kg priming dose and a 19-μg/kg resolving dose for females. Females were held at 12–13°C, and ovulation was observed 14 ± 3 h (mean ± SD) after the second ...

Selenium tissue burden compartmentalization in resident white sturgeon (Acipenser transmontanus) of the San Francisco Bay Delta estuary.

High selenium (Se) loads in the San Francisco Bay Delta are bioaccumulated and biomagnified in food webs and can impair the reproduction of resident oviparous animals such as white sturgeon. The objective of the present study was to determine the Se tissue burden in San Francisco Bay Delta-resident white sturgeon to assess Se bioaccumulation in different organs, including ovaries and liver where egg yolk precursor proteins are synthesized. The authors obtained 54 San Francisco Bay Delta-resident white sturgeon including 26 female and 28 male subadults with immature gonads, 8 females with vitellogenic eggs, and 13 males with maturing gonads. Length, weight, age, reproductive stage of development, and kidney, liver, gonad, and muscle Se concentrations were determined in all fish. Concentrations of Se in muscle, gonads, and liver significantly increased with fish size, whereas kidney Se was not correlated to body size and was at the highest level compared with other organs. There was no difference between the sexes (p > 0.05) in Se concentrations in kidney (12.83 ± 0.51 µg · g(-1) dry wt), liver (11.85 ± 1.04 µg · g(-1) dry wt), and muscle (7.09 ± 0.52 µg · g(-1) dry wt; mean ± standard error, n = 47); but Se concentration was higher in the ovary than in testis (p = 0.04). Females with vitellogenic eggs had higher Se concentrations in the ovaries (20.77 ± 4.11 µg · g(-1) dry wt vs 5.22 ± 2.50 µg · g(-1) dry wt), liver (21.84 ± 2.07 µg · g(-1) dry wt vs 8.03 ± 1.03 µg · g(-1) dry wt), and muscle (10.18 ± 1.93 µg · g(-1) dry wt vs 5.48 ± 0.64 µg · g(-1) dry wt) compared with less advanced, previtellogenic females (p < 0.05). The elevated Se concentrations in the ovaries and liver of vitellogenic San Francisco Bay Delta white sturgeon were comparable with levels previously shown to cause reproductive toxicity in dietary Se experiments with captive white sturgeon.