Reproductive parameters and oocyte fatty acid compositions in European sprat Sprattus sprattus sampled in the Baltic Sea

Fecundity of marine fish species is highly variable, but trade-offs between fecundity and egg quality have rarely been observed at the individual level. We investigated spatial differences in reproductive investment of individual European sprat Sprattus sprattus (Linnaeus 1758) females by determining batch fecundity, condition indices (somatic condition index and gonadosomatic index) as well as oocyte dry weight, protein content, lipid content, spawning batch energy content, and fatty acid composition. Sampling was conducted in five different spawning areas within the Baltic Sea between March and May 2012. Sampling was conducted in the Baltic Sea during three cruises of the German RV “Alkor” in March (https://www2.bsh.de/aktdat/dod/fahrtergebnis/2012/20120331.htm), April (http://dx.doi.org/10.3289/CR_AL390), and May (http://dx.doi.org/10.3289/CR_AL392) 2012. Five different areas were sampled: KB, AB, Bornholm Basin (BB), Gdansk Deep (GD), and Gotland Basin (GB). Fish were caught with a pelagic trawl. Trawling time was in general 30 minutes per haul. The total lengths (TL, ±0.1 cm) of at least 200 sprat per haul were measured for length frequency analysis. Only female sprat with ovaries containing fully hydrated oocytes were sampled, running ripe females were rejected to avoid possible loss of oocytes, as this would lead to an underestimation of batch fecundity. Sprat were sampled immediately after the haul was on deck and stored on crushed ice. The sampled fish were weighed (wet mass WM, ±0.1 g) and measured (TL, ±0.1 cm), and their ovaries were dissected carefully. Oocytes were extracted from a single ovary lobe, rinsed with deionized water, and counted under a stereo microscope (Leica MZ 8). A counted number of oocytes (around 50 oocytes per fish) were transferred to pre-weighed tin-caps (8 x 8 x 15 mm). These samples were used to determine the oocyte dry weight, lipid content, and fatty acid composition. In addition, a counted number of oocytes (around 10 oocytes per fish) were sampled in Eppendorf caps for determination of protein content. Oocyte samples were stored at -80 °C for subsequent fatty acid and protein analysis in the laboratory. Finally, both ovary lobes were stored in 4% buffered formaldehyde solution for further fecundity analysis. Ovary free body mass (OFBM, ±0.1 g) of sampled frozen fish and fixed ovary mass (OM, ±0.1 g) were measured (Sartorius, 0.01 g) in the laboratory on land, to avoid imprecise measurements due to the ship's motion at sea. Absolute batch fecundity (ABF) was determined gravimetrically using the hydrated oocyte method suggested by Hunter et al. (1985) for indeterminate batch spawners. For ascertainment of the relative batch fecundity per unit body weight (RBF), ABF was divided by OFBM. Further, a condition index (CI) was determined: CI = (OFBM/〖TL〗^3 )× 100. A gonadosomatic index (GSI) was calculated with the following formula: GSI = (OM/OFBM)× 100. Oocyte dry weight was determined to the nearest 0.1 µg (Sartorius SC 2 micro-scale), using the samples stored in pre-weighed tin caps, after freeze-drying (Christ Alpha 1-4) for at least 24 hours. After subtracting the weight of the empty tin cap, the average oocyte dry mass (ODM) was then calculated by dividing the total weight by the number of oocytes contained in the tin cap. The fatty acid signature of oocytes was determined by gas chromatography (GC). Lipid extraction of the dried oocytes was performed using a 1:1:1 solvent mix of dichloromethane:methanol:chloroform. A five component fatty acid methyl ester Mix (13:0 - 21:0, Restek, Bad Homburg, Germany; c = 8.5 ng component µl-1) was added as an internal standard and a 23:0 fatty acid standard (Restek, Bad Homburg, Germany, c = 25.1 ng µl-1) was added as an esterification efficiency control. Esterification was performed over night at 50 °C in 200 µl 1% H2SO4 and 100 µl toluene. The solvent phase was transferred to 100 µl n-hexane and a 1 µl aliquot measured in a Thermo Fisher Trace GC Ultra with a Thermo Fisher TRACETM TR-FAME column (10 m*0.1 mm*0.2 µm). For more details on sample preparation and GC settings, see Hauss et al. (2012). The total lipid content per oocyte was determined by adding up the weights of all detected fatty acids. To ensure comparability with past studies, results for FA are given as a percentage of the combined weights of all detected FA. An average of 10 oocytes were transferred to 5*9 mm tin cups (Hekatech) and dried at 50 °C for >24 h. Total organic carbon (C) and nitrogen (N) content was measured using a Thermo Fisher Scientific Elemental Analyzer Flash 2000. From the total amount of N in the sample, the oocyte protein content was calculated according to Kjeldahl (Bradstreet, 1954), using a factor of 6.25. The oocyte gross energy content was calculated on the basis of measured protein and lipid content, which were multiplied with corresponding energy values from literature. The measured amount of proteins per given oocyte (P, mg) was multiplied by a factor of 23.66 J mg-1 and was added to the total amount of lipids per oocyte (L, mg) multiplied by 39.57 J mg-1 (Henken et al. 1986). Consequently, the oocyte energy content of each individual female sprat was multiplied by its relative batch fecundity in order to obtain a standardized estimate of the total amount of energy invested into a single spawning batch (SBEC, J g-1 OFBM): SBEC = [(P × 23.66 (J )/mg)+(L × 39.57 (J )/mg)]× RBF