Determination of Age and Size at Sexual Maturation of Yellowfin Tuna (Thunnus albacares) in the NW Atlantic Ocean

This report examines sexual maturation of yellowfin tuna (Thunnus albacares; YFT) above 35° North in the western Atlantic Ocean. The majority of samples were collected near Oregon Inlet, North Carolina with additional samples from New England to determine if there is a difference in maturation with latitude. Maturation was determined by gross classification of gonads as well as histological examination. Data were compiled based on sex and fish length to elucidate trends. Females collected at lower latitudes were found to have a lower GSI and gonad weight than those from higher latitudes. Males in both locations had a similar GSI and gonad weight based on the length of the fish. At higher latitudes, female fish were immature up to the largest fish collected (124 cm straight fork length (SFL)). Despite fish at lower latitudes having a lower gonadosomatic index (GSI) and gonad weight, beginning stages of oocyte development were present only in females ranging from 110-116 cm SFL in these locations. Overall, female fish were found to be in different stages of immaturity whereas a number of male fish were ready to spawn, suggesting that males may mature at a smaller size than females in both locations. In North Carolina, males ranging from 96-121 cm SFL were found mature, but only males ranging from 127-130 cm SFL were mature in New England. Results of this study are consistent with other studies that show size selectivity by angling gear and a delay in YFT maturation at higher latitudes.


Determination of Age and Size at Sexual Maturation of Yellowfin Tuna (Thunnus Albacares) in the NW Atlantic Ocean
Chelsea Roy Fisheries, Animal, and Veterinary Sciences, University of Rhode Island, Kingston, RI,

USA INTRODUCTION
Yellowfin tuna (Thunnus albacares; YFT) are a highly active pelagic fish found worldwide mainly between 45° N and 40° S in temperate and tropical waters (Collette and Nauen, 1983). YFT became a target commercial species in the 1950s, when a large-scale longline fishery began to proliferate, and remain a target species with the development of purse seines to capture fish near the surface. YFT annual catches in the Atlantic increased from 60,000 metric tons (MT) in the 1950s to a peak of 210,000 MT in the early 1990s. Since then, it has decreased to about 150,000 MT (Miyake et al., 2004). YFT are valuable in wild-caught fisheries and have generated interest as a potential aquaculture species. Critical to astute management of fisheries and aquaculture ventures is determination of when fish attain sexual maturity and spawn. Knowledge of sexual maturation is key in developing catch size limits for sustainable fishing and broodstock development for egg production for aquaculture ventures.
Research on the sexual maturation of YFT has been undertaken in the Pacific Ocean (Itano, 2000;Sun et al., 2005), Indian Ocean (Nootmorn et al., 2005;Marsac et al. 2006;Zhu et al., 2008;Zudaire et al., 2010;), and in the mid-Atlantic Ocean (Arocha et al., 2000, Arocha et al., 2001 YFT spawning occurs throughout the year in the center of its geographical distribution, i.e., between 15° N and 15° S, but spawning may occur in other areas at specific times of the year (ICCAT, 1992). YFT in the Eastern Pacific have spawned at temperatures as low as 22° C, but most spawning activity takes place between 26° C and 30° C (Shaefer, 1998). Spawning is often inferred by the presence of YFT larvae or maturation stage of gonads. YFT larvae have been found in the Central and Western Pacific at temperatures above 24° C (Ueyanagi, 1969).
Major spawning grounds in the Atlantic are in the Gulf of Guinea (off West Africa; ICCAT, 1991) and the Gulf of Mexico (Arocha et al., 2000). Thousands of YFT tagged in the Northwest Atlantic have been recaptured in the Gulf of Guinea (Ortiz, 2001). Reproductively active females also have been found in other areas, including the western tropical Atlantic and the southwestern Caribbean off the coast of Venezuela (Arocha et al., 2000). YFT were found to spawn between February and November in the Western Central Atlantic (Arocha et al., 2000). Tagging studies of Atlantic YFT suggest a single stock with limited information on the extent of fish spawning range (ICCAT, 1997). As YFT that are the size of sexually mature fish are caught in the Atlantic in areas outside of known spawning locations during the spring and summer months by recreational fishermen, spawning activity may be occurring.
This study looks at YFT maturation in the Western North Atlantic to determine if spawning activity is occurring.
Many factors may affect maturation stage and rate of development. There is a large degree of variation among studies attempting to determine sexual maturation by examining captured fish (Table 1). Factors include location, gear type, method of determining, and whether both sexes were collected. One method that is often used to determine maturation is when 50% of fish reach maturity. To calculate the length at which 50% of sampled fish reach maturity, the sample population is split into 5 cm length classes and percentage of mature fish in each length class is calculated. Female YFT in the Western Pacific are 107.8 cm (Sun et al., 2005) when 50% of females are mature, and 92 cm in the Eastern Pacific (Schaefer, 1998). In the Eastern Indian Ocean, 50% of fish are mature at 95.4 cm for females and 99.6 cm mature for males (Nootmorn et al., 2005). Tuna, like many species of fish, may exhibit gear bias or avoidance as a result of vertical stratification of individuals in different reproductive states (Suzuki, 1994). Juvenile YFT are typically found near the surface and closer to the coastline, whereas larger fish may move offshore and remain near the surface waters (Miyake et al., 2004). Itano (2000) suggests a delay in maturation of Pacific YFT in higher latitudes compared to fish caught near the equator.
Many factors influence the growth and maturation of YFT. Nutrition, photoperiod, and water temperature may impact the rate at which YFT grow, and may have an impact on sexual maturation or a hormonal response of the fish to begin maturation ( Figure 1). Decreased water temperature leads to decreased feeding activity in captive YFT (Wexler et al., 2003). Water temperature also impacts the occurrence and timing of spawning activity, while an increase in food ration leads to an increase in egg production of female YFT (Margulies et al. 2005). Decreasing photoperiod in captive YFT also lead to cessation in spawning activity (Margulies et al., 2005). In the wild, water temperature has an impact on feed availability, growth rate, and sexual maturation (Lehodey and Leroy, 1999). Finally, time of year dictates photoperiod and water temperature for different locations, which may influence development of YFT.
Some YFT spawn year-round and others spawn seasonally (Itano, 2000). These factors may also come into play with YFT in the NW Atlantic Ocean. In order for fish to spawn, both female and male sexually mature individuals need to meet under the right conditions.
YFT do not exhibit sexual dimorphism or external maturation characteristics, so the only means to determine sex and maturation stage at this time is to excise and examine the gonads. DNA sex-linked markers are currently available for Pacific bluefin but not yet for YFT (Agawa et al., 2014).
GMI classification is one way of determining sex and maturation stage of YFT tuna. This method of classification is based on the work by Schaefer (1987) and Nootmorn et al. (2005) and describes the gonads based on sight and physical characteristics of whole, uncut gonad for male, female, and immature fish. This method can be done in the field, as opposed to fixation and staining of tissue sections, but is not as accurate as histology (Schaefer, 2001).
Histological examination of gonad tissue is required to definitively determine specific maturation stages of YFT. Gross maturation indices may not be as effective for staging YFT, particularly females. In female YFT, the gonad diameter can be similar in both the vitellogenic phase and in postspawning, atretic fish (Schaefer, 2001). Fish are classified as immature when females have unyolked or early yolked oocytes and mature when advanced yolked oocytes or atresia are present. Males are classified as mature if there is histological evidence of the presence of sperm (Schaefer, 2001).
In the current report, fork length measurements and gonad samples were obtained from the recreational charter and private angling fishery. In North Carolina, samples were collected from charter fishing vessels at Oregon Inlet Fishing Center in Nags Head. In New England, all samples came from volunteer fishermen who measured the fish and excised the gonads to store on ice for the trip back to port. As maturation is linked to the size of the fish, the goal was to ascertain the size that fish begin to mature and at various stages of the process.
Information on the reproductive development of YFT is of value to fishery managers in the formulation of plans to protect stocks from overfishing and to facilitate maintenance of spawning populations. This study focused on a single species over a narrow geographic range, that is further north than other spawning studies in the equatorial Atlantic region. The size and weight at maturity of wild caught YFT may also be beneficial to YFT aquaculture research programs. Knowledge of these characteristics of tuna at different maturation stages would assist in collection and selection of potential broodstock that have attained or are approaching maturation.
This report also helps fill in the gaps of YFT tuna maturation characterization across the Atlantic Ocean. Of interest, previous investigations suggest that spawning size also varies with location (McPherson, 1991;Shaefer, 1998;Itano, 2000;Miyake et al., 2004;Nootmorn et al., 2005). Most studies on sexual maturation of tunas are limited due to the difficulty of collecting tissue from enough individuals to encompass fully immature to 100% mature fish during a single period of reproductive activity (Schaefer, 2001 Center were brought intact on ice to the dock within 4 to 6 hours of capture. Upon return to the harbor, fish were immediately transferred to a fish processing facility located at the dock. Prior to processing, straight fork length (SFL) was measured to the nearest 0.2 cm using a flexible measuring tape and recorded. Curved fork length (CFL) was also measured. When possible, fish were weighed individually in 200-l barrels using a Toledo Model 700 floor scale with accuracy to 0.5 kg. Due to the nature of fish processing in this location, it was not possible to obtain weight for all fish.
In some cases, loins were removed from whole fish prior to the SFL being measured and gonads collected. After the loins were taken, fish were stored vertically in 200-l barrels in a room maintained at 4° C prior to measurements and collection of tissue. Storing them in this manner minimized bending or compression of the fish for accurate length measurement and preserved integrity of gonads. All samples were processed and tissue preserved within eight hours of capture.
To collect gonad tissue from intact fish, a ventral incision was made using a fillet knife to gain access to the peritoneal cavity. The gonads were excised and placed into a 1-l Ziploc bag marked with a unique identifier and stored on ice in a cooler.
Fish collected in NE were measured using the same methodology, where fish were brought to the dock whole. Curved fork length was recorded prior to the fish being gutted or decapitated; gonads were also collected at this time as above. Straight fork length was also measured. The fishermen were provided data sheets to record location of capture, sea surface temperature (if known), name, and the date. If the fishermen did not know the sea surface temperature, but were able to provide an estimation of the area fished, Satellite Imagery via the Rutgers University Coastal Ocean Observation Lab (Rutgers University) was used to estimate the sea surface temperature at point of capture. Gonads were weighed and sliced into thin sections of gonads placed into 10% neutral buffered formalin (NBF) as quickly as possible. NBF fixative was composed of 4.0 g monobasic sodium phosphate, 6.5 g dibasic sodium phosphate, 100 ml of 37% formaldehyde, and 900 ml of distilled water (Carson, 1992). In some instances, fishermen excised gonads and stored them on ice. These samples were collected immediately from fishermen for processing. Gonads were weighed to the nearest 0.1 g using an A&D EK-2000 electronic balance for calculation of GSI. Visual inspection of gonads took place immediately after gonads were weighed and before subsections were cut for fixation.
Four tuna mortalities from the recirculating aquaculture system at the University of Rhode Island were measured and tissue collected as above. Three fish were collected prior to initiation of this study and maturation stage was determined histologically, since gross classification and/or gonad weight were not available. For the one additional mortality, gonads were visually classified and weighed.
For fish collected in all locations, weight of fish, gonadosomatic index (GSI), and the age of the fish were calculated. When use of a scale to weigh fish was not possible, the total weight of the fish (Wf) was estimated using the equation = * where is1.886 x 10 -5 , SFL is the straight fork length of the fish, and is 3.0195 based on a sample of 6,752 YFT caught in the Indian Ocean (Marsac et al., 2006). GSI was calculated using the formula = / * 100, where is the weight of gonads in grams, and is the total weight of the fish including gonads (Stequert et al., 2001). Age of fish was estimated using the equation = (245.541 * (1 − (−0.281 * ( − 0.0423)), where t is time in years (Shuford et al., 2007). This growth equation provides an estimate, but does not take into account growth rate as water temperature-dependent.
Sex of fish was assessed by visual inspection and GMI when possible for samples from all locations. Undeveloped ovaries are round in cross-section and roll easily between fingers due to a lumen (Schaefer 2001). Developed ovaries are fusiform or spindle-shaped and circular in cross-section and tend to have a yellow hue due to presence of yolk in the oocytes (Schaefer, 2001). Developed testes have a flattened appearance (in cross-section) and are usually white in color due to the presence of milt. Undeveloped testes are solid and cannot be rolled easily through the fingers (Schaefer, 2001). When illuminated, a longitudinal sperm duct is visible medially (Schaefer, 2001).
For GMI classification, gonads were staged using a visual index of 1 to 5 based on the work by Nootmorn et al., (2005) (Tables 2 and 3). The approach allows classification of maturation of male and female gonad samples using the same numerical scale of 1) immature, 2) early developing, 3) later developing, 4) mature, and 5) spawned. Classification characteristics include color, presence of a visible blood vessel, size, and shape of the gonad.

TISSUE FIXATION
Two sections of tissue were excised from the central section of the ovary or testis and sliced approximately 0.5 cm in thickness to facilitate penetration of formalin, perpendicular to the lumen. Depending on the diameter of the gonad, multiple 1-cm 2 sections were taken to obtain a sample of the entire cross section of the gonad. Gonad samples were placed into 15-ml conical tubes containing 10% NBF (Carson, 1992).
Tissue samples were fixed for at least 24 hours in 10% NBF and transferred to histology cassettes and 70% ethanol. Tissues were embedded and sectioned by a commercial laboratory (Mass Histology Services, Worcester, Massachusetts, USA); sections were stained with Harris' hematoxylin and eosin counterstain (Prophet, 1992

OVARIES
Classification of maturation of ovaries using histology was based on the work by Itano (2000) and Schaefer (2001) on YFT in Hawaiian waters and in the Western Tropical Pacific Ocean. Ten different maturity stages were used to define the degree of oocyte development, condition of oocytes, spawning stage, and degree of atresia (Table 4). Stages 1-3 include immature ovaries and stages 4-10 represent mature samples with advancing stages of spawning activity (Table 4).
Samples collected included ovaries at the primary growth phase of development. Thus, stages 1 and 2 of maturation (Table 4; Itano, 2000) have been classified into more detailed developmental stages based on physical description and diameter of the oocytes (Table 5; Zudaire et al., 2013). For example, a stage 1 by Itano (2000), could then be a stage 1.1 or 1.2 by the descriptions from Zudaire (2013). A stage 2 by the descriptions from Itano (2000) would be a stage 2.1 or 2.2 by Zudaire (2013). As each oocyte develops, it increases in diameter due to the development of yolk plates and the uptake of fluids. Mature oocytes can be up to 750 µm in diameter.

TESTES
Maturation stage of the testes using histology was based on the degree of spermatogenesis within the germ cells using the system developed by Schaefer (2001).
All germ cells within a cyst were approximately at the same level of development.
Stages of maturation of sperm were classified as primary spermatogonia, primary spermatocytes, secondary spermatocytes, spermatids, and spermatozoa (Table 6).
To further characterize testis, the diameter of the largest cyst or duct was measured and presence or absence of milt in the lumen of the testis was recorded. The percentage of the testis comprised of cysts and ducts was estimated across the area of the sample. As testes develop, the cysts lyse and form ducts which move milt toward the lumen. A large number of cysts suggest testes are less developed (Schaefer, 2001).

NC SAMPLES
A total of 385 samples were collected from Oregon Inlet from June 15-17, 2014. Fish were caught less than 65 km offshore where sea surface temperature was approximately 26° C. All samples were visually classified as male, female or unknown and given a GMI classification of 1-5.
Of the samples from Oregon Inlet, weight was measured for 29 fish ( Figure   2a). The heaviest fish was a male weighing 29.0 kg, and the lightest was a female weighing 5.9 kg. The equation relating SFL in centimeters to whole body weight (Wf) in kilograms for male fish is Wf=0.4484(SFL)-25.6 and for female fish is Wf=0.4603(SFL)-27.7. This equation was not used to calculate GSI due to the low number of samples (n=19). Fish whose gender was not able to be determined were not included. Female fish had a higher gonad weight per total body weight than male fish ( Figure 2b).
Based on GMI classification, 83 fish were male, 205 were female, and 97 were unknown ( Table 7). Maturation of female and male fish was considered at GMI classification stage 4. Eight of the male fish and 63 of the female fish were found to be at stage 4 (Table 7). No fish were found to be at stage 5, i.e., spawning or post-spawn.
In stage 4 females the gonad is pale orange and rounded, when the oocytes are ready to be released from the follicle layer and ovulated. The smallest fish to reach this stage was 71 cm or approximately 1.3 years of age ( Figure 3c). In males, stage 4 was characterized by testis which contain milt and are white or reddish in color. The smallest male to reach stage 4 was 96 cm long, or 1.8 years of age ( Figure 3d). All samples where sex could not be determined by GMI classification were at GMI stage 1.
In total, 105 samples from NC fish were processed for histology. The initial batch of samples for histology included 35 females, 35 males, and 35 unknown sex based on GMI classification. Histological examination showed that 45 were female, 58 were male, and 2 could not be classified.
The 45 females ranged in length from 65 cm to 116 cm, which equates to an approximate age of 1.14 to 2.32 years (Table 8; Figure 4c). The GSI for fish determined to be female by histological classification ranged from 0.054 to 1.062 ( Figure 5c). Overall gonad weight ranged from 3.5g to 123.2g. As SFL of NC female YFT increases, gonad weight does as well (Figure 6c). For NC samples, gonad weight and GSI are closely related.
Based on histology, ovary samples were more immature than expected given the size of the fish. As oocytes develop, they increase in diameter. All female samples were stage 1 out of 10 except 5 that were stage 2 (Table 4). Because all female samples were immature, a more precise staging was used to classify immature oocytes into more specific categories (Table 5). The more precise staging took into account degree of development and oocyte diameter. Of the samples that were immature, twenty-two samples were stage 1.1 (perinucleolar), 18 samples were stage 1.2 (chromatin), 4 were stage 2.1 (cortical alveoli), and one was stage 2.2 (early stage of vitellogenic, vtg 1) (Table 9; Figure 7c). No fully yolked oocytes were present, suggesting no female fish were ready to spawn.
The diameter of the largest oocytes ranged from a maximum single oocyte diameter of 40 µm in one female to 270 µm in another (Figure 8). The ovary with the smallest oocytes was classified stage 1.1 and the ovary with the largest oocytes was stage 2.2 (Table 5). This suggests that the most developed ovaries were still in the primary growth phase and just beginning to develop yolk vesicles.
To be considered approaching maturation, females had to be at above a stage one, i.e., at stage two (Table 4) or at stage 2.1 or 2.2 the early stage of vitellogenic (Table 5). Five of the females were classified as beginning to mature (Table 9) or 11.1% of the females collected. The smallest female that reached stage 2 was 110 cm.
Similar SFL fish do not all mature at the same rate and therefore may be in different maturation stages.
The 58 males as determined by histology ranged in length from 62 cm to 121 cm, which is an estimated age span of 1.1 to 2.5 years (Table 10; Figure 4d). The two unknown samples were 67 cm and 102 cm SFL, which is an estimated age of 1.2 years and 1.9 years, respectively. The GSI for these males ranged from 0.027 to 1.022 ( Figure 5d) with a gonad weight of 1.1g to 114.5g. As SFL of male YFT increases, gonad weight does as well (Figure 6d).
Overall, there were fourteen males at stage 5 (24%), the most mature stage present with spermatozoa present in the testes ( Females and males were considered mature at a GMI classification of stage 4 when gonads are in active spawning condition. Based on this classification, the smallest female fish at stage 4 was 82 cm ( Figure 3a); a single male was categorized as stage 4 (130 cm SFL; Figure 3b).
Thirty samples were assessed by histology including 10 identified as males from gross classification, 10 identified as females, and 10 of unknown sex. These samples were chosen to reflect all the male and unknown sex fish, and ten females that included the fish with the maximum straight fork length, minimum straight fork length, mean fork length, one standard deviation above and below the mean. The samples also included the minimum gonad weight, maximum gonad weight, mean, and one standard deviation above and below. The sex of all samples could be identified by histological examination with 11 determined to be females, and 19 males.
The eleven females ranged from 69 cm to 124 cm SFL, an estimated age range of 1.2 to 2.5 years (Table 12a; Figure 4a). GSI ranged from 0.22 to 6.28 (Figure 5a), and gonad weight from 4.1g to 73.8g. SFL and gonad weight does not have a strong relation in NE female YFT (Figure 6a). Males ranged from 78 cm to 130 cm SFL, with an estimated age range of 1.4 to 2.7 years (Table 13a; Figure 4b). GSI ranged from 0.20 to 0.85 ( Figure 5b) and gonad weight from 2.3g to 38.1g. For NE male YFT, as SFL increases, gonad weight does as well (Figure 6b). The GSI (r square= 0.13) and gonad weight (r square= 0.00) for females from NE was less related to SFL than it was for GSI (r square= 0.47) and gonad weight (r square= 0.49) for females from NC.
Histological examination showed that six ovaries were in the chromatin nuclear stage and 5 in the perinucleolar, with none at more developed stages (Table  12b; Figure 7a). The diameter of the largest oocytes measured for each fish ranged from 55 µm to 100 µm. The ovary with the smallest oocytes was at stage 1.1 and the ovary with the largest oocytes was stage 1.2. Based on histology, none of the female samples from NE were mature. Females in NE were less developed compared to NC, where 11% of females were beginning to mature. Both stage 1.1 (chromatin nuclear) and 1.2 (perinucleolar) reflect stages of primary growth of the ovary, rather than maturation.
The maturation stage of male gonads was also determined by histological examination. Two males were at the most advanced stage (5; spermatozoa), none at stage 4 (spermatids), five at stage 3 (secondary spermatocytes), eight at stage 2 (primary spermatocytes), and four at stage 1 (primary spermatogonia) (  The range of gonad weights for both male and female fish was larger in NC than in NE. In NC, females had gonad weights from 3.5 g to 123.2 g and males from 1.1 g to 114.5 g. Females in NE had gonad weights from 4.1 g to 73.8 g and males from 2.3 g to 38.1 g. The GSI for females and males in NC was similar, with a range of 0.05 to 1.06 for females and 0.03 to 1.02 for males. In NE, females (0.22 to 6.28) had larger GSIs than males (0.20 to 0.85). In NE the seasonality present may force fish to physiologically decide between putting energy toward growth or sexual development. In NC, where the water temperature and photoperiod are more stable, the GSI and gonad weight appeared to increase relative to SFL at a more predictable rate. It is interesting that female fish in both locations had higher GSIs and gonad weights than male fish as the females were just beginning to mature but the males were at higher maturation stages. Higher GSI in females has been evidenced in other species, including dolphinfish (Castro et al. 1999), tilapia (Mahomoud et al. 2011), and marine catfishes (Gomes and Araujo, 2004). Two of the male fish in NE were mature with gonad weights of only 11.9 g and 38.1 g.
In the present study, microscopic examination proved to be a much more accurate method for maturity staging than GMI visual classification. Microscopic examination was used to definitively identify samples that were first classified visually. For fish caught in NC, 78% of the fish that had been grossly identified as female and 48% of male fish were corroborated by histological examination. Three percent of samples could not be identified using the gross maturation index. For fish caught in NE, visual classification was accurate for 63.6% of female samples, and 47.3% of male samples. The remaining percentages of fish samples that were incorrectly identified visually were either the opposite sex or could not be identified by histology. All samples from NE were able to be sexed using histology. In contrast, in NC 98% of samples were correctly identified by histological examination, where 43% were definitively identified as females and 55% as males. Only 2% of samples could not be identified. In NE, 100% of samples were able to be sexed and staged using histological examination, with 37% females and 63% males. Histological examination is a more accurate and definitive, though time consuming, method of sexual maturation staging than gross classification of gonads for species that exhibit no outward sexual dimorphism. The use of histological examination to assess maturation stage of gonads is the most precise staging method (Schaefer, 1998).
This study supports previous research that suggests fish at higher latitudes may mature at larger sizes as compared to fish closer to the equator (Itano, 2000). Tagging studies of Atlantic YFT suggest a single stock (ICCAT, 1997) so the fish may be travelling through different latitudes and maturing at different rates based on water temperature and food availability. Higher latitudes have a lower annual average sea surface temperature. While the theory that there is a single stock of YFT in the Atlantic is currently accepted, future research should be done on genetics of YFT in this area, as recent genetic work shows there are at least three genetically discrete populations of YFT in the Pacific (Grewe et al., 2015).
Results of this study show a difference in the timing of maturation of female fish as compared to male fish in the same location. This has been witnessed in other species, such as salmonids (Barson et al., 2015) and tilapia (Bhatta et al., 2013). A difference in timing of maturation of females and males has also been evidenced in YFT the Eastern Indian Ocean (Nootmorn et al., 2005) and the Eastern Pacific (Schaefer, 1998 Life stage sexual dimorphism has been observed in YFT in the eastern Pacific where Wild (1986) found that initially females are larger than males of the same age, growth curves cross around age 2 (where fish are approximately 95 cm FL) and then males are larger than females of the same age. Further investigation showed growth rate of males is higher than females (Wild, 1994). This may be due to the amount of energy required for oocyte production and ovulation. In the Western Atlantic, with appropriate seasonal temperature and light requirements, tuna spawn every 1.47-3.35 days and produce 54.2 oocytes per gram of body weight per spawn event (Arocha et al., 2000).
The results from the current study are consistent with a previous year-long study on immature female YFT caught in the tropical Western Atlantic Ocean which showed actively spawning females to be larger than those sampled in this study.
Closest in geographic location to this study, Arocha et al. (2000)  Size at first maturity for males and females in this study is greater than that for fish caught in the central and western Indian Ocean. Zudaire et al. (2010) found female fish caught by purse seines in the equatorial area of the Indian Ocean first mature at 77.8 cm FL. More commonly reported than size at first maturity is size that 50% of the sample population reaches maturity. Itano (2000) focused on females caught by purse seine, longline, and handline in the central and western Pacific Ocean and found that fish were 50% mature when they were 107 to 120 cm in length. Sun et al. (2005) found that female YFT caught by longline were 107.77 cm in length when they are 50% mature in the western Pacific Ocean, or 2.4 years old. In the eastern Pacific Ocean, Schaefer (1998) found that females were 92.1 cm when 50% of the fish were mature. If more larger and reproductively mature fish were obtained during sample collection for this study, an attempt at determination of length at 50% maturity would have been attempted.
Findings of this study are consistent with previous work that shows size selectivity of fishing gear. Fish caught in NC ranged from 62 cm to 121 cm with an average of 96 cm and fish caught in NE were 69 cm to 132 cm with an average of 105 cm. This is in the mid-range of YFT sizes caught by purse seine and by longlines. Arocha et al. (2000) found that fish caught by purse seine ranged from 35 to 145 cm but were predominantly 60 to 75 cm, whereas the longline caught fish ranged from 40 cm to 140 cm but are predominantly 135 to 140 cm FL. In the Japanese YFT fishery, purse seines are used to capture fish smaller than 70 cm and longline gear is used to harvest fish larger than 90 cm (Suzuki, 1988). Actively feeding yellowfin schools are often made up of reproductively active fish; these tuna aggregations where spawning occurs daily or near-daily influences the vulnerability of the tuna to purse seine gear (Itano, 2000). Mature but reproductively inactive fish are more likely to be captured with deep-set longline gear (Itano, 2000).
The results of this study are of value to fisheries managers. The size limit for retention of YFT in both commercial and recreational fisheries in the US Atlantic is 68 cm (27 inches) CFL. SFL and CFL are closely related. For bluefin tuna, SFL= 0.972*CFL (r square=0.999, p<0.001, n=1,308; Salz et al., 2007). Fish at this size were found to be immature in both locations. If fish are continuously caught at this small size, these fish are unable to spawn and replace themselves in the wild prior to being caught. The largest female fish from NC, at 116 cm, was just beginning the yolk development stage. None of the female fish collected in NC or NE were ready to spawn so a larger minimum size limit is recommended.
Additionally, knowledge about sexual maturation of YFT would be beneficial to aquaculturists for developing methods to culture large pelagic species. Three of the four samples taken from broodstock mortalities showed immature gonads, at fork lengths up to 98 cm. Capturing broodstock that are already mature can decrease holding times for fish to reach maturity. Tuna aquaculture has increased globally, with tuna farming and ranching, both inshore and offshore in net pens. YFT aquaculture is gaining popularity, with research centers in Panama, Hawaii, Indonesia, Australia, and Rhode Island. YFT are asynchronous spawners, producing up to several million eggs daily given the right temperature and photoperiod.
The Achotines Lab in Panama has been successful in spawning YFT tuna for a period of nine months when the temperature and light were manipulated (Margulies et al., 2007). These broodstock were 51 cm to 78 cm in length, with an average of 62 cm FL and were collected from waters near the laboratory in Panama. The YFT collected for broodstock are a Pacific strain of YFT found close to the equator. The fish at Achotines attained maturity at a smaller size than the fish collected in this study. The smallest male collected in NC that attained maturity was 96 cm, with an average length of 109.6 cm for mature males. None of the females were mature in this study, up to the largest female at 116 cm. The results from the University of Rhode Island broodstock samples show a delay in maturation as compared to wild caught fish, despite being in a tank with manipulatable photoperiod and seawater temperature. The gonads were undeveloped in three out of the four broodstock fish sampled, up to 98 cm. A 122 cm long fish was a fully mature male. It is possible that the broodstock maturation in the tank was inhibited by stress response to captivity or a lack of social cues. Stress response has been observed to impair many functions of fish species (Harper et al., 2009) including oocyte maturation in common carp (Wang et al., 2008) and decreased GSI and gametogenesis (Thomas et al., 2007). As three of the four samples were unable to be sexed, the gender ratio of broodstock in the tank could not be determined.
There is evidence that YFT spawn in the Gulf of Mexico, Caribbean, and Gulf of Guinea. Arocha et al. (2001)  for approximately one year (Richards, 1969). After that, they move south to Angola, and then north to warmer water. YFT older than two years of age then travel to the central tropical Atlantic, but they do return to the Gulf of Guinea in the warm months to spawn. It appears likely that fish collected for the present study were spawned in the Gulf of Mexico, as it is the nearest spawning location to the region of collection.  (Lehodey and Leroy, 1999). In this study, both male and female fish were found to mature at larger sizes than in the equatorial Pacific, and at larger sizes the further from the equator they were collected. In contrast, populations of bluefin tuna caught in the eastern Atlantic Ocean are 135 cm or 3 years of age when 50% mature, and can take 4 to 5 years for the entire population to reach maturity (Corriero et al., 2005). Atlantic bluefin can live upwards of 35 years (Corriero et al., 2003). Bigeye (Thunnus obesus) caught in the tropical Atlantic also mature at a larger size, as they attain first maturity at 104 cm for females and 108 cm for males (Guoping et al., 2011). YFT may be a more ideal tuna species for captive broodstock type scenarios.
One of the limitations of this study is that only females at early stages of maturation were collected. Broadening the study to incorporate larger sized YFT may have helped better characterize maturation in female fish. Tagging fish caught recreationally in NC and NE to determine migration patterns as fish grow and mature may also provide information about size selectivity and maturation stage selectivity of fishing gear. At present, there is one study which shows sportfish, longline, and purseseine caught fish tagged in the western Atlantic recaptured near the Gulf of Guinea (Ortiz, 2001). It would be of interest to tag small males and females and track their whereabouts throughout their maturation rather than simply a tagged location and recapture location. If location and sea surface temperature could be obtained throughout the summer months when most YFT spawn, alternative spawning grounds might be detected. Tagging studies in the Gulf of Mexico show YFT dive deeper during the day and stay near the surface at night, but the short duration of the study (80 days) did not show lateral movement outside of a bias against areas that were more than 6°C cooler than surface water temperature (Weng et al., 2009). Tagging studies in the northeastern Pacific show YFT remained within 1,445 km of their tagging location even after 1,161 days had passed (Schaefer et al., 2007).
To conclude, YFT in the western North Atlantic mature at a different rate as compared to YFT caught in tropical waters, with more mature fish at lower latitudes.
With YFT being migratory, it is possible that YFT in the western North Atlantic may move south or offshore as they become sexually mature. Males were found to mature at smaller sizes than females in NC and NE. More research should be on broodstock YFT maturation due to the low sample size included in this study. A future research direction could be to tag recreationally caught fish to see where they aggregate when they are reproductively mature, or to determine if there is a place further north in the Atlantic Ocean than the Gulf of Mexico where spawning activity occurs. Genetic testing would also provide insight as to how distinct the stocks of YFT in the Atlantic are-do they have predictable migration patterns, remain dissociated, or do they intermingle? By further developing the current understanding of this incredible species, YFT can continue to be researched and harvested sustainably for conservation efforts, captivity for aquaculture, and consumption worldwide.               NE male, c) NC female and d) NC male yellowfin tuna. One-way ANOVA between maturation stages was not significant for NE females (p=0.64) and NE males (p=0.52), but it was significant (p<0.05) for NC females (p= 0.02) and NC males (p <0.001).
Figure 4: Straight fork length in cm versus age in years. Age is determined by , where t is time in years (Shuford et al., 2007). Locations and sexes are a) NE female YFT, b) NE male YFT, c) NC female YFT, d) NC male YFT. Y = -0.03633*X + 5.451 Y = -0.001056*X + 0.4536 Y = 0.01077*X -0.7472 Figure 5: Straight fork length in cm versus gonadosomatic index (GSI). GSI is based on gonad weight and total weight of the fish. Weight is calculated by where is1.886 x 10 -5 , SFL is the straight fork length of the fish, and is 3.0195 based on a sample of 6,752 YFT caught in the Indian Ocean (Marsac et al., 2006) and GSI is calculated by , where is the weight of gonads in grams, and is the total weight of the fish including gonads (Stequert et al., 2001). Locations and sexes are a) NE female YFT, b) NE male YFT, c) NC female YFT, d) NC male YFT. NC male YFT. One-way ANOVA analyses were run on each of the maturation stages in each location for each sex. Differences between mean sizes for female YFT from NE were not statistically significant, but they were statistically significant for males from NE, females from NC, and males from NC (P <0.05).