Fatty acid and proximate composition of wild male and female king angelfish ( Holacanthus passer ) gonads during the ripe and spent developmental stages

Overfishing of king angelfish Holacanthus passer reduced populations in the Gulf of California. This study aimed to determine the fatty acid and proximate composition of male and female gonads, and the E2 concentration in plasma of wild organisms with different stages of gonadal development captured during early and mid-summer at El Datil, near Kino Bay, Sonora, Mexico, to help establish basic reproductive aspects and possibly to help develop the aquaculture of this ornamental species. Out of the 45 adult specimens analyzed, 66.7% were females and 33.3% were males; 16.7% of the females had ripe gonads while 83.3% had spent gonads, and 20% of males had ripe gonads while 80% had spent gonads, indicating they were reproducing earlier, likely in the spring. Females had smaller size but numerically greater gonadosomatic index (GSI) and condition factor (K) than males. Ripe and spent females showed no statistical differences in weight, length or K; however, ripe females showed significantly higher GSI (P = 0.0005) than spent females, 3.43 vs. 0.87%. Lipid content was higher in ripe gonads, with higher content of fatty acids. In both sexes, DHA was quantitatively the major fatty acid, (10.83-16.28 mg of DHA g of gonad wet weight). The n-3/n-6 ratios varied from 1.99 to 3.54, lower for ripe organisms due to a higher content of ARA and n-6 derivatives. Gonad DHA content changed in relation to its developmental stage and it might serve as an additional maturation index. Information on the chemical composition and fatty acid profile of the gonads can be used as indicators of dietary lipid and essential fatty acid requirements of broodfish, which may contribute to the formulation of a balanced diet for the culture of this ornamental species.


Introduction
The growing demand of coral reef species for exhibition in aquariums around the world has made the marine ornamental aquaculture a very profitable economic activity (Wabnitz et al., 2003;Bruckner, 2005;Livengood and Chapman, 2007).The supply of organisms that populate the exhibits however, is questionable, since it relies on extraction from their natural habitats, quite often captured illegally (Lango-Reynoso et al., 2012).The king angelfish, Holacanthus passer (Valenciennes, 1846), also known as Passer angelfish, is among the highly valued species in the international ornamental market.A member of the Pomacanthidae family, this non-migratory tropical species is distributed in the Pacific Ocean, from the north coast of Baja California, Mexico, to the north coast of Peru, and the Galapagos Islands in Ecuador (Arellano-Martínez et al., 1999;Aburto-Oropeza et al., 2000;Pyle et al., 2010).Adults have an attractive dark blue color with a lateral vertical white stripe behind the pectoral fin, while juveniles are characterized by their yellow or orange coloration, shimmering-blue vertical stripes and blue rimming of the dorsal and anal fins, although they undergo changes in coloration as they mature.Females and males are isomorphic, except for their pelvic fins, which are white in males and yellow in females (Thomson et al., 2000;Aquarium of the Pacific, 2016).This fascinating and contrasting coloration of H. passer is what drives the commercial interest and the reason why it is an economically important species for aquaria exhibition, particularly for Mexico and Costa Rica, where juveniles are captured to be exported to the United States of America and other countries around the world, fetching prices of over US$200.00dollars per individual (LiveAquaria, 2016).
Overfishing has caused a decline in king angelfish populations (Sánchez-Alcántara et al., 2006), thus, in the Gulf of California, the species was included in the list of protected species by the Mexican norm NOM-059- SEMARNAT-2001(Mexico, 2002) as the first conservation effort of the government.Additionally, it is well known that the reproduction of marine finfish under controlled conditions also reduces the impact of heavy fishing pressure on wild populations (Mehdi and Ehsan, 2011;Arantes et al., 2012), but successful reproduction in captivity requires the knowledge of several aspects of the adult life stage of an organism (Izquierdo et al., 2001;Olivotto et al., 2011).For H. passer, Arellano-Martínez et al. (1999) established that it is a partial spawner with asynchronous development of the gonads; its reproductive cycle is dictated by five phases of gonadal development, with sexual maturity and reproductive activity attained during the period of April to November.
Seasonal variations in the reproductive cycle of teleost fish are influenced by exposure to environmental stimuli acting on the hypothalamus-pituitary-gonad axis (Mylonas and Zohar, 2001;Zohar et al., 2010); when sexual maturity has been attained and under the adequate environmental conditions, two types of gonadotropins (GtH) are produced by the pituitary gland and released into the bloodstream, namely, the follicle stimulating hormone (FSH or GtH I), which controls both, vitellogenesis and spermatogenesis, and the luteinizing hormone (LH or GtH II), which controls the final oocyte maturation and spermiation.In the female gonad during vitellogenesis, the FSH and LH promote the synthesis of sexual steroids such as testosterone, which subsequently, will be aromatized into 17βestradiol (E2; Nagahama, 1994;Mylonas et al., 2000;Mylonas and Zohar, 2001).This steroid hormone induces the biosynthesis of vitellogenin in the liver of mature female fish, which is subsequently incorporated into the developing oocyte (Collier et al., 2003;Swanson et al., 2003;Guerriero, 2007).A correspondence between plasma concentration of E2 and the size or weight of the gonads has been established (Aruke and Goksoyr, 2003;Moncaut et al., 2003;Berg et al., 2004), therefore, it is considered as an indicator of female gonadal maturation or final oocyte maturation in female fish (Scott et al., 1998;Kokokiris et al., 2000).
Reproductive success in fish requires the physiological allocation of lipid resources for storage, usually in liver, muscle and intraperitoneal fat, for later use in gonadal maturation, namely ovogenesis and spermiogenesis (Abdel-Aziz and El-Nady, 1993;Sargent, 1995;Luzzana et al., 1996).Polyunsaturated and highly unsaturated fatty acids (PUFA and HUFA, respectively) play an important role in gonadal maturation, egg quality, and larval growth (Izquierdo et al., 2001).Eicosapentaenoic acid (20:5n-3; EPA) and arachidonic acid (20:4n-6; ARA) are precursors of thromboxanes, prostaglandins, and leukotrienes (Wolfe and Horrocks, 1994), hormone-like molecules that play a role in immune and inflammatory responses, haematological and cardiovascular activity, renal and neural function, as well as in reproduction, specifically in ovarian and testicular steroidogenesis (Tocher, 2003).ARA is also thought to be involved in the embryonic development of the immune system, as well as in hatching and early larval performance (Sorbera et al., 1998).Docosahexaenoic acid (22:6n-3, DHA) on the other hand, plays a major role as a constituent of neural and visual tissues (Sargent el al., 2002), in consequence, mostly saturated and monounsaturated fatty acids are preferentially catabolized to meet energy demands, while HUFA are transferred to the eggs (Henderson et al., 1995), therefore, the concentration of specific fatty acids such as DHA in gonads perhaps could be used as an additional indicator of gonadal maturation, although this requires further investigation.The aim of this study was to determine the fatty acid and proximate composition of male and female king angelfish (H.passer) gonads, and the concentration of E2 in plasma of wild organisms with different stages of gonadal development captured during the summer time in the area of El Datil, a location near Kino Bay, Sonora, Mexico.

Animals
A scientific collection permit (SGPA/DGVS/ 00809/14, issued on January 30th, 2014) was obtained through the Mexican Agency for the Environment and Natural Resources (SEMARNAT) to retrieve wild adult female and male king angelfish, which were acquired by SCUBA diving at El Datil, latitude 28°42 ʹ N and longitude 112°17ʹ W, near Kino Bay, Sonora, Mexico.A total of 45 organisms were captured during July and August 2014 using fish landing nets.They were kept in a submersible cage and transported live in a circular tank of 200 L, provided with aeration and clean seawater, to the Kino Bay Experiment Station of the Department of Scientific and Technological Research of the University of Sonora, in Kino Bay, Sonora, Mexico.Upon arrival blood was collected from the caudal vein of all fish using 3 ml syringes, plasma was then separated by centrifuging (Heraeus Fresco 21, Thermo Scientific, Osterode am Harz, Germany) the blood at 850 g for 15 min at 4°C, and then stored in microcentrifuge tubes at -20°C until E2 was determined.Fish were then euthanized with an overdose of tricaine methanesulfonate (MS-222) prior to collection of biological indexes: individual weight (IW, g) and length (IL, cm) were measured to determine the condition factor (K) = (wet body weight × 100) / total length 3 , cm;  Ricker, 1975).Gonads were then dissected from all fish and weighed to calculate the gonadosomatic index (GSI, %) = (gonad wet weight, g /total wet weight, g) × 100; after this, each gonad was divided into two similar sections, one of them was used for the determination of the fatty acid and proximate composition and stored in an ultrafreezer (ThermoFisher Scientific, Isotemp Basic U86-13A41, Waltham, MA, USA) at -84.0ºC, and the other one for the histological determination of the developmental stage of the gonad and stored in Davidson's fixative solution (acetic acid:95% ethanol:formaldehyde:H 2 O at a 1:3:2:3 ratio).

Proximate composition and fatty acid analysis
Crude protein (N factor = 6.25) was analyzed (method 968.06;Association of Official Analytical Chemists -AOAC, 2005) via combustion by the Dumas method with a Dumas Nitrogen Analyzer (Model NDA 702, VELP® Scientifica, Usmate, Italy).Moisture (method 930.15;AOAC, 2005) and ash (method 942.05;AOAC, 2005) were determined by standard official methods.Crude fat (CF) was extracted by the method of Folch et al. (1957) and quantified gravimetrically after drying a 5 ml aliquot under nitrogen.The remaining lipid extracted after the CF determination was used for fatty acid analysis.Fatty acids were transesterified with boron trifluoride and fatty acid methyl esters (FAME) were analyzed with a Varian 3800 gas chromatograph equipped with a 30 m × 0.25 mm fused silica capillary column and a flame ionization detector as previously described (Lochmann and Gatlin, 1993).Fatty acids were identified by comparison of retention times to those of known standards, and quantified by using an internal standard (heptadecanoic acid, 17:0); they were expressed as mg g -1 of gonad wet weight.

Histological and plasma 17β-estradiol (E2) analyses
Gonads from each fish stored in Davidson's fixative solution were fixed during 24 h, after which they were transferred to a 70% ethanol solution for long-term storage.Three segments of each gonad (∼1 cm length) were sliced, embedded in paraffin, and transverse sections were processed to 4 μm slides and stained with hematoxylin and eosin.The slides were examined under a light microscope (Leica Microsystems, DM1000, Wetzlar, Germany) equipped with a digital camera (Moticam Pro, 282A) and the Motic Images Plus 2.0 software (Motic, Richmond, BC, Canada) was used for image acquisition.Gonads were further analyzed for assessing their developmental stage according to the description provided by Arellano-Martínez et al. (1999).Additionally, plasma E2 was measured by Enzyme-Linked Immune Sorbent Assay (ELISA) according to the procedure described by Semenkova et al. (2002) using a commercial kit (MBS044518, My Biosource, San Diego, CA, USA).

Statistical analyses
Data presented here are means and standard error of the mean (S.E.) of biological parameters such as individual weight and length, GSI, K, E2, in addition to proximate compositions and fatty acid concentrations of female and male gonads of the identified developmental stages detected in H. passer captured during this season, which were ripe and spent.The non-parametric Kruskal-Wallis analysis was used to test differences among ripe and spent female and male gonads for these variables (P < 0.05) using the Statistical Analysis System (SAS Institute Inc., 2013, Software Release 9.4, Cary, NC, USA) software package.

Gonad histology
A total of 45 adult specimens of H. passer were captured during July and August 2014.Of the captured king angelfish, a total of 30 organisms were females and 15 were males.Through the histological analyses of the gonads and the description provided by Arellano-Martinez et al. (1999), it was established that 16.7% of the females had ripe gonads, while 83.3% had spent gonads; in ripe ovaries, mature oocytes were evident and predominant, some oocytes were in a hydrated or pre-hydrated state and the lumen was not visible, whereas in the spent ovaries early oocytes were abundant and atresia was evident in some of them (Fig. 1).In the case of males, 20% had ripe gonads while 80% had spent gonads.The ripe testis showed tubules filled with spermatozoa, while in spent testis the tubules were practically empty, with residual spermatozoa in lumen and ducts (Fig. 2).

Biological parameters
Females had notably smaller size, but numerically greater GSI and K values than males.Ripe and spent females showed no statistical differences in weight, length or K; however, ripe females showed a significantly higher GSI (P = 0.0005) than spent females (3.43 vs. 0.87%, respectively).On the other side, ripe males were significantly heavier (P = 0.0141), larger (P = 0.0208), and they also had a significantly higher GSI (P = 0.0440) than spent males, but there were no significant differences (P = 0.0604) in their K (Table 1).

Proximate composition and E2 concentration
The proximate composition of the gonads of both sexes showed that moisture was the major component, ripe and spent females having a higher content than males, and ash followed the same trend but it was the minor component (Table 2).On the other hand, crude protein and crude fat content of ripe gonads, as expected, were higher in mature females and males than in the spent gonads; however, none of the components of the proximate composition were significantly different in ripe or spent fish, except for the ash content of male gonads, which was significantly higher (P = 0.0126) in spent males than in ripe ones.E2 plasma concentration was analyzed only in ripe and spent females.In ripe females values ranged from 6.47 to 6.93 ng ml -1 , with a mean of 6.68 ng ml -1 (S.E.= 0.13).In spent females, values ranged from 3.52 to 4.10 ng ml -1 , with a mean of 3.84 ng ml -1 (S.E.= 0.15), which was significantly lower (P = 0.0323) than that of ripe females.

A) B)
C) D)

Discussion
The aquaculture of ornamental fish represents an opportunity to reduce the impact that the extraction of organisms for commercialization has on the natural populations and, at the same time, it also represents the opportunity to provide jobs and a source of income for people.In order to achieve a closed cycle in aquaculture, that is, to become independent of the wild organisms that the environment provides, which are mostly broodstock, a method to keep the broodfish in an ideal setting needs to be developed.This method needs to include the provision of an adequate culture environment that would promote the development of the gonads, as well as a diet that would meet the nutritional requirements for the optimal allocation of nutrients to the maturation of the gonad.
The determination of dietary requirements may follow a traditional approach, such as feeding fish graded levels of a particular nutrient for a period of time and establishing the level that promotes the best growth response as the dietary requirement, although nowadays this approach may include other parameters into consideration, such as the maximum tissue protein retention, a nitrogen balance, or other more suitable parameters (Ogino, 1980a, b;Wilson and Poe, 1985).Another traditional approach is to determine the concentration of essential nutrients in tissues, which helps drawing conclusions about the nutrient needs (Kaushik, 1979;Tacon and Cowey, 1985).For instance, when the established essential amino acids requirements of Ictalurus punctatus were regressed against the essential amino acids present in its body, a regression coefficient of 0.96 was obtained (Wilson and Poe, 1985), indicating that the composition of the tissues in the body may very well be used as a pattern of the requirement for essential nutrients.Thus, this last approach was used in this study for the male and female king angelfish gonads in an effort to determine the requirement for essential fatty acids drawn from their fatty acid profile.
Out of the 45 adult specimens of H. passer analyzed, 66.7% were females and 33.3% were males; 16.7% of the females had ripe gonads while 83.3% had spent gonads, and 20% of the males had ripe gonads while 80% had spent gonads.These wild organisms were captured during July and August at the location of El Datil, near Kino Bay, Sonora, Mexico.During this time of the year Arellano-Martinez et al. (1999) reported that in king angelfish collected from Cueva de Leon (24°02ʹ N, 110°24ʹ W) in South Baja California, most of the female gonads were ripe and a small portion were still developing; their reproductive activity took place from June to November, and was well correlated with their GSI.For males they observed that some gonads were developing or ripe in July and August, and some males also had spawning and spent gonads, but no correlation was established between the GSI and the reproductive activity of males because of the low weight the male gonads reach during maturation.Cueva de Leon and El Datil are different geographical areas with different temperatures and conditions; local temperatures Anim.Reprod., v.13, n.4, p.820-829, Oct./Dec.2016 at El Datil and the Kino Bay area are higher than those observed at Cueva de Leon during the same period of time, and seawater generally reaches temperatures above 25ºC in late April to early May at El Datil.It is well known that, among the environmental parameters, temperature plays one of the leading roles in gonad maturation and spawning of fish (Zohar, 1989;Bye, 1990), which partly explains why a large percentage of female and male gonads in this study were already spent and a small percentage was still ripe, indicating that fish were reproducing earlier than populations in southern areas, most likely in the spring, but a yearly study is required to confirm this.
One could expect that, as the gonad develops into a fully ripe gonad and its weight increases, the GSI percentages also increase.In this study, the GSI in H. passer females with ripe gonads was 3.42%, significantly higher than that of females with spent gonads with 0.87%.For the same gender Arellano-Martínez and Ceballos-Vázquez (2001) reported the largest GSI value of 2.45% in June, decreasing to 2.0% in July at Cueva de Leon, which are lower values than the ones reported in this study.The GSI values they reported are means of all females captured in that period, whereas in this study, GSI are means for females with a similar state of gonadal development; in addition, females in their study averaged 16.19 cm whereas in this study, both, ripe and spent females were larger, averaging 24.12 and 24.22 cm, respectively, which explains the larger gonad size and thus, the larger GSI.The GSI values observed in this study are also consistent with values reported for other species, for example, Dentex dentex females with a GSI of 4.1 and 0.7% in mature and resting females, respectively (Chatzifotis et al., 2004).On the other hand, ripe males were significantly heavier and larger than spent males in this study, and the GSI for ripe gonads (0.15%) also was significantly higher than in spent gonads (0.08%).Both ripe (33.03 cm) and spent (30.27 cm) males, also were larger than males reported by Arellano-Martínez and Ceballos-Vázquez (2001), with a mean length of 20.12 cm and a GSI of 0.10% in June and 0.12% in July, very similar GSI values to the ones recorded in this study, which confirms their observation of a very low weight of the male gonads, even during the mature stage.Conversely, for D. dentex, GSI values of 3.4 and 0.2% were in mature and spent males, respectively (Chatzifotis et al., 2004), which makes evident the wide variety of morphological features characterizing the gonads of teleost fish.The morphology of this species is characterized by a small visceral cavity, which may partly explain the small GSI values observed in males.
The Fulton's condition factor or K is an indirect estimate of the robustness of fish, common values reported for marine fish are usually above 1.0, lower values indicate less robust fish and higher values more robust fish (González-Félix et al., 2015, 2016;Perez-Velazquez et al., 2016).In this study, K values for H. passer ranged from 2.76 to 3.42, an indication of the overall robustness of all organisms.Although not statistically significant, females and males with ripe gonads showed slightly higher K values than their spent counterparts, which is explained by the fact that this index is an estimate of the body weight-to-length ratio, and decreases sensibly as body weight decreases or length increases.Hence, smaller K values in this study corresponded to spent females that had smaller gonad/body weight, and to spent males that also had smaller gonad/body weight and smaller body length.For this species, Arellano-Martínez and Ceballos-Vázquez (2001) reported K values close to 4.3 for females, and close to 4.0 and 4.3 for males during June and July.These authors suggested that for this species, the reproductive activity is inversely related to the nutritional status, thus higher K values were observed during the inactive reproductive period that coincided with smaller GSI values.In this study, wild organisms were sampled only for two months in the summer, and K values of mature females and males, 3.42 and 2.95, respectively, as well as of spent females and males, 3.05 and 2.76, respectively, were smaller than K values reported by Arellano-Martínez and Ceballos-Vázquez (2001).These lower values are explained by the smaller lengths and weights of the organisms sampled in their study, compared to the ones in our study; but, since a limited period of time was monitored here, the verification of the inverse relationship between GSI and K could not be performed for this work.The E2 analysis could be performed only in plasma samples of ripe and spent females, and their mean values of 6.68 and 3.84 ng ml -1 , respectively, are comparable, though slightly higher, to values reported for three different stages of ovarian development of wild female Persian sturgeon, Acipenser persicus, they were the vitellogenic stage III (E2: 5.33 ng ml -1 ), the ripe stage IV (E2: 1.98 ng ml -1 ) and the ovulation stage V (E2: 2.31 ng ml -1 ; Hosseinzadeh et al., 2013), while Semenkova et al. (2002) reported that E2 concentration in plasma of stellate sturgeon (Acipenser stellatus) ranged from 1.22 to 2.05 ng ml -1 and decreased to 0.16 ng ml -1 after ovulation, a similar trend for the E2 values observed in this study.
The proximate composition of H. passer male gonads showed that, in spite of their low weight, their lipid content was proportionally higher than in the female gonads.Numerically, but not statistically significant, the lipid content was higher in ripe gonads, which coincided with the higher content of fatty acids.In both sexes of this species, DHA was quantitatively the major fatty acid, ranging from 10.83 up to 16.28 mg of DHA g -1 of gonad wet weight.The n-3 fatty acids were quantitatively higher than n-6 fatty acids, and the n-3/n-6 ratios ranged from 1.99 up to 3.54.The lower ratios observed in ripe organisms (Tables 3 and 4) can be attributed not to the lower content of n-3 fatty acids, but to the higher content of ARA and derivatives of this fatty acid from the n-6 family, which would reduce the ratio, and, as previously suggested, ARA is the second major component of gonads in many fish species (Suloma and Ogata, 2011).Similar DHA contents have been reported in mature male (35.97% of total FAME) and female (21.65% of total FAME) gonads of spawning herring, Clupea harengus pallasi (Huynh et al., 2007), and in the male (22.0% of total FAME) and female (25.0% of total FAME) gonads of Atlantic bluefin tuna (Thunnus thynnus) broodstock during reproductive migration, where n-3/n-6 ratios of 5.4 and 6.4, respectively, were observed (Sprague et al., 2012).In other species, consistently high DHA contents have been observed, but not necessarily the highest, such as in Salmo trutta labrax, where DHA content in female gonads was reported to be 15.55% of total FAME with a 5.23 n-3/n-6 ratio (Aras et al., 2003).D. dentex also had comparable n-3/n-6 ratios, ranging from 2.56 up to 7.50 in females caught in different seasons of the year, with DHA contents ranging from 7.4 to 25.9 mol%, while males had ratios ranging from 3.70 to 4.30 and DHA contents ranging from 10.5 to 13.2 mol% (Chatzifotis et al., 2004).Knowing the essential fatty acid content of the broodfish gonads can help establish their nutritional status, which is of utmost importance since it can markedly affect ovarian maturation, reproductive performance, and offspring quality (Izquierdo et al., 2001).Furthermore, information on the chemical composition and fatty acid profile of the gonads can be used as indicators of dietary lipid and essential fatty acid requirements of broodfish, which may contribute to the formulation of a balanced diet for the culture of this ornamental species.On the other hand, knowledge obtained on the stages of gonadal development of local king angelfish during the period of this study could help establish basic reproductive aspects, like the size or age at which the organisms reach sexual maturity in this area, and the length of their reproduction season, which is relevant for the development of a protection program.
We concluded that, during the months of July and August, close to 80% of wild Holacanthus passer captured from El Datil in the Kino Bay area, had spent gonads, and in about 20% of them the gonads were ripe, which indicates that fish were reproducing earlier than populations in southern areas, most likely in the spring.The quantitative composition of fatty acids of the gonad confirmed that they are a very rich source of HUFA of both, the n-3 and n-6 family.DHA was quantitatively the most important fatty acid of all, and its concentration in the gonad changed in relation to its developmental stage, therefore, it might serve as an additional maturation index.A seasonal study is warranted to confirm these observations, and to describe the remaining stages of gonad development throughout the year in this location for this species.

Table 1 .
Means and standard error of the mean (S.E.) of biological parameters, gonadosomatic index (GSI), and condition factor (K) of wild female and male Holacanthus passer captured at El Datil during June and July 2014.

Table 2 .
Means and standard error of the mean (S.E.) of the proximate composition of gonads from wild female and male Holacanthus passer captured at El Datil during June and July 2014.Mean values of females or males of different developmental stages (Ripe ♀ : 5; Spent ♀: 25; Ripe ♂: 3; Spent ♂: 12) with different superscripts within the same column, are significantly different (P < 0.05).

Table 3 .
Means and standard error of the mean (S.E.) of fatty acids (mg of fatty acid g -1 of gonad wet weight) from ripe and spent gonads of wild H. passer females captured at El Datil during June and July 2014.

Table 4 .
. Means and standard error of the mean (S.E.) of fatty acids (mg of fatty acid g -1 of gonad wet weight) from ripe and spent gonads of wild H. passer males captured at El Datil during June and July 2014.