3.1. will be determined as the length from

3.1. Research Design           One of the commonly used methods to determine annual fecundity among indeterminate spawning fishes is the Hydrated Oocyte Method developed by Hunter et al., (1985). In this method, the number of hydrated oocytes in tissue samples preserved in 4% formalin are counted. This estimation will then represent the numbers of hydrated oocytes in the entire ovary, and will be assumed to be equivalent to the fish’ annual fecundity.    This study involves different variables that represent the reproductive parameters of a female S. lemuru. It includes the body length and gonad weight at maturity (also known as Gonadosomatic index), the weight of individual ovarian tissue sample, the number of hydrated oocytes in each tissue sample, and the total number of hydrated oocytes in each gonad. The location of the oocytes extracted were also considered in fecundity estimation. 3.2 SamplingBecause of the various environmental factors that affect the spawning season of Sardinella lemuru, samples will be collected monthly starting from April 2017 to January 2018. The samples will be bought from New Lemery Public Market in Lemery, Batangas between 8:00AM to 12:00PM. Based from previous interviews with the local vendors of the market, it is during this time that fishermen deliver their fresh catch to the market.3.3 Data Gathering Procedure    A. MeasurementsUpon obtaining a substantial amount of the samples, the standard length, fork length, and total length of each fish sample will be determined using a digital vernier caliper. The standard length of the fish will be determined as the length from tip of the snout to the tip of the vertebra at the caudal peduncle. The fork length will be determined as the length from the tip of the snout to the center of the fork in the caudal fin. Finally, the total length of the fish will be determined as the length from the tip of the snout to the tip of the longest caudal lobe, with both lobes of the caudal fin compressed together (Kahn et al., 2004). After this, each fish will then be weighed using an analytical balance sensitive to 0.1mg. These measurements are essential (1) in order to identify the average length and weight range that Sardinella lemuru can be considered mature and (2) to compare these measurements obtained from previous studies on S. lemuru to see if its maturity length and weight has changed (Gaughan & Mitchell, 2000; Gina, 2006).    3.3.2. Estimation of Fecundity via Hydrated Oocyte Method (Hunter et al., 1985)    This study will follow the same procedure for Hydrated Oocyte Method used by Hunter et al., (1985) to estimate batch fecundity.    Prior to fecundity estimation, the ovaries will initially be dissected out from the previously weighed female fish, and then weighed separately as left and right lobes of the ovaries. Only the left lobe of the ovary will be used for fecundity estimation. Then, using the tip of the forceps or scalpel, the ovarian membrane will then be broken and three subsamples will be isolated from three different regions of the ovary, specifically from the anterior, middle, and posterior regions. This will only be done to verify if there is a significant difference in the location where the subsamples will be obtained. Furthermore, obtaining three subsamples from different regions of the ovary will be done on a random fish chosen to represent the collected population for the month. Subsamples from the rest of the fish samples will be obtained from the middle region of the left lobe of the ovary. The subsample to be obtained must weigh 30-50 mg, as this amount will contain about 100-200 oocytes. The weight of the subsample will be recorded to the nearest 0.1 mg, carefully adding or removing pieces of the ovary to obtain the desired weight. After which, the subsamples will be stored in separate vials and preserved in 4% phosphate buffered formalin overnight to a maximum of seven days (Hunter et al., 1985).     Following this, the tissue sample will be placed on a Sedgwick rafter counter slide and flooded with 3-4 drops of glycerin. After 10-15 minutes, the slide will be tapped gently with the forceps to loosen the oocytes. The sample will then be spread out over the slide and covered with a cover slip. Next, the slide will be observed under the microscope and the number of hydrated oocytes will be tallied using a hand counter tally. Hydrated oocytes can be distinguished by observing their large size, wrinkled appearance, and translucence (Hunter et al., 1985).    The final step in the method will be the estimation of batch fecundity. Batch fecundity for each female will be calculated using the formula Y = x(Z), wherein Y is the batch fecundity, x is the number of hydrated oocytes per unit weight in the tissue sample, and Z is the combined weight of the right and left lobes of the ovary (Hunter et al., 1985).3.4 Statistical Treatment of DataOne variable that may affect the estimation of fecundity is the location of the tissue sample taken from the ovary. In order to avoid bias, the three sub-samples should be taken in such a way that no two samples will come from the same portion of the ovary (Hunter et al., 1985). As such, one sub-sample will be taken from the center of the gonad (either the right or left ovarian lobe), while the other two will be taken from a point one-third of the distance from the each end of the ovary. The number of eggs per unit weight of the ovary (x) will then be calculated for each tissue sample, and the obtained values will then be grouped according to the location of the sub-sample. After which, the mean of each group will be calculated, and any differences between the means of these groups will be determined using analysis of variance (ANOVA).In addition to this, the study also aims to determine and establish separately the fecundity vs. body weight relationship, as well as the fecundity vs. body length relationship. This will be done to test the theory that the larger or longer the fish, the higher the estimates for its fecundity will be. To determine the relationship between fecundity and body weight, individual fecundities of each sub sample will be calculated using the formula F= (Gonad weight ×  Egg number in subsample)Sub-sample weight (Yeldan & Avsar, 2000). On the other hand, in forming a relationship between the fecundity and the body length, the data of total batch fecundity (FT) to Total length (TL), Fork Length (FL), Standard length (SL) and Ovary length (OL) will be determined. The relationship between total fecundity and S. lemuru’s body length as well as fecundity in relation with S. lemuru’s weight will then be evaluated using simple linear regression analyses which are based on natural-logarithmic transformation: LogW = Loga + b LogTL, where W is equivalent to the weight of S. lemuru, TL is the total length and a and b are constants (Lawson, 2011, & Ragade, 2015).    Lastly, the current spawning season of Sardinella lemuru will be established by computing for the Gonadosomatic Index (GSI) and Dobriyal index (DI), both of which are widely used values in determining the spawning season of various species. They only differ in that GSI involves the body weight of the fish, including the percentage of the body used for feeding, food storage, and those affected by environmental and physiological stress. This will be calculated using the formula GSI (%)= Gonad weight (in g)Total body weight (in g)×100. On the contrary, Dobriyal index does not involve body weight and will be calculated using the formula DI= sWg which is equal to the cube root of the average gonad weight in grams (Wg). The monthly variations of GSI and DI will be recorded, and their peaks will represent the spawning season of S. lemuru in the Philippines. (Almuktar et al., 2016).