ovarian histology and reproductive health of short mackerel...

Original Article

Ovarian histology and reproductive health of short mackerelRastrelliger brachysoma (Bleeker 1851) as threatened marine fish in Thailand

Sinlapachai Senarat1 Wannee Jiraungkoorskul2 and Jes Kettratad1

1 Department of Marine Science Faculty of ScienceChulalongkorn University Pathum Wan Bangkok 10330 Thailand

2 Department of Pathobiology Faculty of ScienceMahidol University Ratchathewi Bangkok 10400 Thailand

Received 29 September 2015 Revised 8 April 2016 Accepted 16 May 2016

Abstract

The ovarian structure and reproductive health of short mackerel Rastrelliger brachysoma in the upper Gulf ofThailand from non-breeding (October to November 2013) and breeding seasons (December 2013 to February 2014) wereexamined by histological and histochemical techniques The results revealed that the ovary in this species was enclosed bythe tunica albuginea It was considered as an asynchronous type which could be classified into seven stages based on itshistological characteristics These seven stages were oogonium chromatin nucleolar perinucleolar oil droplet and corticalalveolar early late and post vitellogenic stages During the oogenic processes the change and characterization of inclusionsincluding lipids cortical alveoli and yolk granules were detected by histolochemical techniques The ovarian histopathologyin R brachysoma was detected This includes ovarian degeneration ovarian atrophy and atresia in different stages (oogoniaprevitellogenic stages and vitellogenic stages) as well as melano-magcrophage centers Atretic follicles and ovarian atrophywere also observed and could negatively affect the reproductive health of the species which could lead to populationdeclines in R brachysoma as well as threaten other marine fishes connected through the food web in Thailand

Keywords ovary histopathology Rastrelliger brachysoma reproductive failure

Songklanakarin J Sci Technol39 (2) 225-235 Mar - Apr 2017

1 Introduction

The short mackerel Rastrelliger brachysoma (Bleeker1851) is the principal resource supporting a large fisheryindustry in Thailand Currently hypothesized reproductivescheme of the R brachysoma in the Gulf of Thailand includedthree periods The first period extends from January to Marchand the second period is from June to August Both periodsare in the vicinity of the spawning ground of the PrachuapKhiri Khan and Surat Thani Provinces The third reproduc-tive season extends from October to November The spawn-

ing ground in this period is in the vicinity of Samut Sakhonand Samut Songkram Provinces (Menaveta 1980) Thishypothesized reproductive scheme was based on the gonado-somatic index (GSI) the gross morphology and coloration ofthe ovarian tissue (Sritakon et al 2006 Sutthakorn 1998)Nonetheless histological aspects of the ovarian tissue inR brachysoma have not yet been identified Using suchtechniques will give more accurate results and would increasethe baseline knowledge of their reproductive developmentand reproductive cycle

To date this species is listed as a threatened marinefish in Thailand (Senarat et al 2015) due to the occasionaldeclines in the natural population The declines were likelycaused by over fishing and the deterioration of their naturalhabitat Previous studies suggested that the Upper Gulf of

Corresponding authorEmail address jeskchulaacth kettratadjesgmailcom

httpwwwsjstpsuacth

S Senarat et al Songklanakarin J Sci Technol 39 (2) 225-235 2017226

Thailand which is the prime habitat of R brachysoma hasenvironmental stressors and high pollutants which includeheavy metals and petroleum hydrocarbon (Wattayakorn2012) Previous studies on health assessment of Rbrachysoma showed severe sign of histopathology such asvarious lesions and parasites in the liver brain and testis(Senarat et al 2015 Senarat et al unpublished data) How-ever comprehensive study on the reproductive health offemale R brachysoma is still lacking

In this article we primarily attempted to identify theovarian structure and reproductive mode of R brachysomaduring its annual reproductive season using histological andhistochemical analyses In addition to reproductive healthwe assessed the R brachysoma ovarian tissue using histo-pathological biomarker This information is vital for theprovision of scientific advice to aquaculture developmentFor example the study of the oocyte quality and the func-tionseffects of neurotransmitter and other neurohormoneswhich are important in the maturation of the ovary canincrease the success rate in aquaculture of this species whichwould ultimately help increase the natural population of thisspecies

2 Materials and Methods

Adult short mackerel Rastrelliger brachysoma witha standard body length about 15-19 cm (average 165plusmn049cm) and total weight about (6868plusmn067 g) were consideredas late ndash development stage of the ovarian histology Theywere obtained by bamboo strake trap during non-breeding(October to November 2013 n = 30) and breeding seasons(January to February 2014 n = 30) from the area of SamutSongkram Province in the Upper Gulf of Thailand (13deg16rsquo184 N 100deg02rsquo134 E) Identification of all fish was madeaccording to the taxonomic key of FAO (Food and AgricultureOrganization [FAO] 2010) Fish were then euthanized by therapid cooling shock method (Wilson et al 2009)

The light microscopic observation the ovarian tissueof fish was removed from the body cavity and fixed inDavidsonrsquos fixative (48 hrs) Then they were processed usingstandard histological techniques (Bancroft amp Gamble 2002)The gonadal paraffin block was cut at 5 microm thickness andstained with Harrisrsquos hematoxylin and eosin (HampE) andMassonrsquos trichrome (MT) periodic acid-schiff (PAS) Grocottmethenamine silver (GMS) alcian blue pH 25 (AB) and reti-culin (Rt) methods (Puchtler and Waldrop 1978 Vidal 1988Bancroft and Gamble 2002) Additionally frozen tissues werecut at 10 microm thicknesses and stained with oil red O (ORO)(Culling 1963) The ovarian structure oogenesis with sizemeasurement was assessed to detail according to the guide-lines of Dietrich and Krieger (2009) and Brown-Peterson et al(2011) under light microscopy The numbers of atretic follicleswere quantified (50-100 oocytesfish per section) in middlearea of three ovarian sections and followed the guidelinesunder light microscopy (10x and 40x) of Blazer (2002)

For ultrastructural observation small pieces ofovarian atrophy were cut and rapidly pre-fixed in 25glutaraldehyde in 01 M phosphate buffer pH 74 at 4degC andwashed in the buffered fixative solution Thereafter theywere post-fixed with 1 osmium tetraoxide (OsO4) and thenprocessed using standard ultrastructural techniques (Rowdenamp Lewis 1974) The thin sections were cut by ultramicrotomeabout 90 nm thicknesses stained with uranyl acetate andlead citrate Lastly the ultrathin sections were observed andphotographed using a JEM-2100 at 200kV

Histopathological analysis the histopathologicalalterations of ovarian tissue were observed and recorded asa mean prevalence for each period

3 Results

Based on anatomical analysis the asymmetrical elon-gation of R brachysoma ovaries were paired organs locatedin the abdominal cavity Cross sections of the immature ovaryin non-breeding season were histologically surrounded bytunica albuginea including a thick layer of connective tissueand numerous blood vessels (Figures 1a 1b 1c 2r) Theywere suspended by mesovarium from the peritoneal dorsal

Figure 1 Light photomicrograph (a b c e f) and schematic sum-mary of ovarian structure (d g) adipose tissue (ad) bloodvessel (Bv) chromatin nucleolar stage (Ch) epitheliumcells (Ec) early vitellogenic stage (Ev) germinal compart-ment (GC) lipid and cortical alveoli stage (Lc) latevitellogenic stage (Lv) ovigerous fold (Of) oognia (Og)perinucleolar stage (P) post vitellogenic stage (Pv)stromal compartment (SC) tunica albuginea (Ta) Scalebar a c f = 100 microm e = 200 microm

227S Senarat et al Songklanakarin J Sci Technol 39 (2) 225-235 2017

wall Overall histological sections in both immature andmature ovaries revealed that the ovigerous fold protrudedinto a central lumen where it could be distinguished into twocompartments (i) a germinal compartment which was coveredwith the ovigerous fold The ovigerous fold were composedof epithelial cells and oogonia (ii) A stromal compartmentwhich contained the different of oogenic stages (Figure 1aand schematic diagram 1d) The ovary in this species was

described as an asynchronous developmental type whichwas distinctly classified into seven stages based on the cellsize histological features of each developing oocyte andstaining properties are given as follows (Figures 1a-1g and2a-2v)

Oogonia the smallest among oogenic cells werelocated and scattered within the somatic epithelial cells in thegerminal epithelium The oogonium was an oval-rounded cell

Figure 2 Schematic summary of oogenesis (a-g) and light photomicrograph (h-v) h-m o-q based on HampE and n r-v based on MTBalbianirsquos bodies (B) circumnuclear ring (Bc) cortical alveoli (Ca) chromatin nucleolar stage (Ch) early vitellogenic stage (Ev)follicular complex (Flc) granulosa cells (G) yolk granules (Gn) inner zona pellucida (IZ) lipid and cortical alveoli stage (Lc)lipid droplets (Ld) late vitellogenic stage (Lv) micropyle (Mp) nucleus (N) nucleolus (no) ovigerous fold (Of) oognia (Og)ovigerous lamellae (OI) outer zona pellucida (OZ) perinucleolar stage (P) post vitellogenic stage (Pv) theca cell (T) tunicaalbuginea (Ta) zona pellucida (Z) Scale bar i-v = 100 microm h = 20 microm


about 25-30 microm in diameter This cell contained a sphericalnucleus (18-20 microm in diameter) with a prominent nucleolusabout 3-4 microm in diameter Its cytoplasm was stained slightblue by HampE It was noted that the oogonium was attachedwithin clusters or oogonial cysts inside the ovigerous foldEach oogonium was surrounded by a few squamous-shapedprefollicular cells (Figures 2a 2h)

Chromatin nucleolar stage oocyte significantlyincreased in size (30-40 microm) The nucleus of oocytes (15-20microm) had a weakly basophilic nucleoplasm showing a sphericalform and enlarged size Single and large spherical nucleoliwere present The ooplasm had a thin layer of progressivelybasophilic cytoplasm Their follicle cells formed a squamouscell layer which surrounded the oocyte (Figures 2b 2h)

Perinucleolar stage oocyte was 80-100 microm in diameterThe nucleus about 50 microm in diameter was in the central areaMultiple nucleoli of 5-8 microm in diameter had spherical shapeand were arranged around the nuclear membrane Balbianirsquosbodies were first present around the periphery There was aslight decrease in basophilic property of the ooplasm Thenthe circumnuclear ring was seen during the oocyte underlight microscope The follicle cells were elongated and flataround the oocyte (Figures 2c 2i)

Oil droplets and cortical alveolar stage oocyte werelarge They attained a size of 150-180 microm in diameter Multiplenucleoli at the periphery of the nuclear membrane progres-sively decreased in size (about 3-4 microm) At first appearancespherical oil droplets were seen primarily encircling thenucleus Their sizes were 10 microm in diameter Later oval corticalalveoli appeared with clear content as vesicles At the end oildroplets and cortical alveoli increased in both numbers andsize throughout the ooplasm at the basophilic area Duringthis stage the zona pellucida as an acidophilic acellular layerwas first detected The layer of simple granulosa cells andtheca cells was similar to that seen in the previous stage(Figures 2d 2j)

Early vitellogenesis stage oocyte was characterized byan increase in cell size to about 250-320 microm in diameter and adecrease of cell basophilia The beginning of ooplasm of theoocyte was filled with numerous small yolk granules The oildroplets increased in size and began to migrate throughoutof the ooplasm Numerous cortical alveoli were still detectedand seemed to increase in number The follicular complex ofthis oocyte consisted of three well-developed layers zonapellucida granulosa cell and theca cell The zona pellucidawas 20 microm thick It was thicker than the previous stage It wasdistinctly striated and strongly positive with eosin In thisstage the layer of granulosa cell underwent morphologicalchange in shape from squamous to low columnar epitheliumBasement membrane and theca cells were seen (Figures 2e2k 2o)

Late vitellogenesis stage oocyte was 280-350 microm indiameter It displayed reddish-stain with acidophilia accord-ing to the most accumulation of yolk deposition (10-15 microm)The periphery of the ooplasm was filled by lipid globules anda few cortical alveoli (around 15-18 microm in diameter) The

irregular and eccentric nucleus appeared to move near theanimal pole Later the micropyle in an oocyte was well-developed and occurred in the zona pellucida (Figure 2n)The zona pellucida was also present as a thick and striatedlayer (Figure 2g) In addition the zona pellucida could beseparated into two layers a thick internal layer and a thinexternal layer The layers of granulosa and theca cells weresimilar to those described in the previous stage (Figures 2f2l 2p)

The post vitellogenesis oocyte stage or maturatingoocyte was the largest oocyte (320-370 microm diameter) amongoogenic stage because the yolk granules of the oocyte werenot completely fused as a homogenous material Enlargedyolk granules were also present at this stage The nucleuswas not visible at this stage A few lipid droplets and corticalalveoli were rarely observed at the periphery of the ooplasmThe follicular complex was slightly flattened under histologi-cal changes but the zona pellicida could be distinctly classi-fied into two layers the outer layer was located near thegranulosa cells whereas the inner layer was exhibited nearto the oocyte surface (Figures 2g 2m 2q)

Histochemistry of the ovarian tissue revealed thatnumerous cortical alveoli were positively stained with MT asbluish and with PAS as pinkish This indicated the presenceof a glycoprotein Also it slightly stained with AB as bluishindicating the presence of carbohydrate or acid mucopoly-saccharide (Figure 2) Another inclusion yolk granules in thevitellogeneic stage also strongly stained with MT as reddishand slightly stained with PAS reactions (Figures 2n 2p 3b3e) The zona pellucida also strongly showed slight PASreaction during lipid droplet and cortical alveolar stagethroughout the post vitellogenic stage indicating thepresence of the protein (Figures 3b and 3e) Surprisingly theinner zona pellucida of the post vitellogenic stage was exclu-sively seen than the outer zona pellucida (Figure 3e)

Among lipids lipid droplet and cortical alveolar stagethe most intense ORO reaction appeared in the lipid dropletsIt first appeared near nuclear membrane and then it distri-buted along the mid and peripheral regions This distributionpattern could be found throughout the post vitellogenicstage (Figures 2f 2g 2h 2i) Furthermore MT PAS and ABnegative staining indicated empty vesicles (Figures 2 and 3)

MT staining was positive at the connective tissuewith the covering epithelial cells that separated the two com-partments of the ovary (Figure 2k) Additionally they werepositively reticulin stained which located at the basementmembrane of the active germinal epithelium This germinalepithelium included the separation between the germinalepithelium and the ovigerous fold from the stromal compart-ment (Figure 4a) and the separation between the granulosacells and the thecal cell layers (Figure 4e 4f) Moreoveroogonia within the cell nest were also separated from theovigerous fold by the same basement membrane (Figure 4b4c) Based on the Massonrsquos trichrome (MT) and reticulin (Rt)stain the reticulated fiber was found in the connective tissuethat formed compartmentalization


Figure 3 Light photomicrograph of oogenesis a-d based on PAS e-h based on ORO and i-l based on AB cortical alveoli (Ca) earlyvitellogenic stage (Ev) yolk granules (Gn) inner zona pellucida (IZ) lipid and cortical alveoli stage (Lc) lipid droplets (Ld)late vitellogenic stage (Lv) nucleus (N) ovary (O) ovigerous fold (Of) oviferous lamellae (Ol) outer zona pellucida (OZ)perinucleolar stage (P) post vitellogenic stage (Pv) tunica albuginea (Ta) Scale bar a-d f-h j-l = 40 microm e i = 100 microm

Histopathological alterations and prevalence of lesionsin the ovarian parenchyma were found in both seasons (Fig-ures 7-9 and Table 1) In contrast to the breeding seasonwhen the ovarian atrophy was not found the non-breeding

season had 133 prevalence of fish with ovarian atrophyAll fish that showed ovarian atrophy and had asymmetricalovaries The smaller ovary exhibited histopathologicaldamage This included atrophy in some ovigerous folds


Figure 4 Light photomicrograph of ovarian structure (O) andoogenesis a-g based on RT basement membrane (Bm)Elastic fiber (head arrow) blood vessel (Bv) earlyvitellogenic stage (Ev) germinal compartment (GC) latevitellogenic stage (Lv) mesovarium (Mo) ovigerous fold(Of) oognia (Og) oogonia cyste (Ogc) oviferous lamellae(Ol) perinucleolar stage (P) post vitellogenic stage (Pv)stromal compartment (SC) tunica albuginea (Ta) Scalebar a c g = 100 microm f = 200 microm

Figure 5 Gross anatomy (A) and light photomicrograph of histo-pathological alterations of the ovarian structures (B-J)oogonia degeneration () previtellogenic degeneration() mitochondrial degeneration (Dm) exocytotic vesicle(Ev) fibrosis (Fs) granulosa cell (G) ovarian atrophy(Oa) ovigerous atrophy (Ova) normal ovary (No) nucleo-lus (Nu) zona pellucida (Z) Scale bar a b = 02 cm f =200 microm c d g = 50 microm e = 20 micromwhich contained degenerate oocytes in both oogonia and

previtellogenic stages (chromatin nucleolar perinucleolar oildroplet and cortical alveolar stages) At high magnificationovarian fibrosis was observed among oogenic stages Thiswas indicated by bluish when stained with MT methodUltrastructural analysis showed that their previtellogenicstages exhibited the degeneration and swelling of mitochon-dria in the ooplasm At the same time the largest exocytoticvesicles with both irregular and oval shapes were detectednear periphery of oocyte and nucleoplasm Other importantfindings at the light microscopic level included the highestprevalence of atretic follicles which was the ovarian histo-pathology symptom that exhibited the degeneration and dis-organization in different stages of oocytes The atresia inoogonia and the previtellogenic stages was shown in non-breeding season with 100 prevalence whereas the breedingseason fish showed atresia in many stages including oogonia(at 833 prevalence) previtellogenic stage (at 933 pre-

valence) and especially vitellogenic stage (early late and postvitellogenic stages) (100 prevalence) Moreover the atreticvitellogenic stage showed irregular disorganized and dis-integrated follicular cells The atretic vitellogenic stage wasfound with 933 prevalence in breeding season Theamount of atretic vitellogenic stage in the breeding seasonwas approximately 802 cells of atretic follicles per 100 oocytes(Table 2) Some ovarian parenchyma showed both blooddilation and congestion together with accumulating themelanomacrophage centers (as yellowish brown with GMSmethod) These lesions were slightly shown at 233 pre-valence in the non-breeding season and at 333 prevalencein breeding season Moreover an unidentified parasite amongthe ovarian tissue based on GMS stain was detected in onlynon-breeding season at 10 prevalence


Figure 6 Light photomicrograph of histopathological alterations of the ovarian structures (a-h) atresia of previtellogenic stage (Ap)atresia in vitellogenic stage (Av) blood congestion (Bv) degeneration of follicular cell (Df) ovarian degeneration (Od) Scale bara d e f = 100 microm b c g h = 50 microm

Figure 7 Light photomicrograph of histopathological alterations of the ovarian structures (a-j) atresia of previtellogenic stage (Ap)malanomacrophage center (MMC) ovarian degeneration (Od) white blood cell infiltration (WBI) unidentified parasites (Ud)Scale bar a c e g i j = 50 microm b d = 20 microm


4 Discussion

Based on the histological structure the ovarian tissueof R brasochyma was the asynchronous oocyte develop-ment type which is similar to all other scombrids (Chellappaet al 2010) This indicated a protracted spawning period withmultiple spawning At the initiation phase of oogenic stagethe oogonium was observed and they were the smallestamong female germ cells They was proliferated within cellnests They were characterized as being enclosed by pre-follicular cells in the germinal compartment This feature wascommonly found in most teleost (Grier 2000 Selman et al1993) Then it progressed to the primary growth stage In thisstage the primary growth stage is characterized by basophiliccell due to a period of intense RNA synthesis together withribosome production to support the oocyte development(Wallace and Selman 1990) Generally this stage can bedivided into two phases based on nuclear morphologicalcharacteristics (i) the chromatin nucleolus and (ii) the peri-nucleolar stages as in this species The characterization ofthe chromatin nucleolus stages of this fish was seen butrarely observed as shown in Thunnus orientalis (Chen etal 2006) and other teleosts oocyte (Sarasquete et al 2002)This probably was caused by the rapid transformationprocess of oogonia turning into the chromatin nucleolus stage

and then it turning into the perinucleolar stage (Sarasqueteet al 2002 and Mandich et al 2002) The perinucleolar stagewas first observed in the Balbianirsquos body as a non-homo-geneous structure with oval shape and basophilic staininglocated near the nucleus membrane A similar characteristicwas also found in Oryzias latipes (Wallace amp Selmann 1981)The functional structure of Balbianirsquos body was uncertainbut it was believed to be a center for the formation of variousorganelles It was region rich in nucleic acid (Hamaguchi1993) RNA and proteins (Selman amp Wallace 1989)

Afterwards two types of inclusion occurred lipiddroplet and cortical alveoli Lipid droplet inclusion occurredprior to the cortical alveoli inclusion in the cortical alveolarstages The sequence of these inclusions were used in theegg type postulation in most teleost fishes (Brown-Petersonet al 1988 Selman amp Wallace 1989) The ovary pattern of Rbrachysoma agreed with the some marine fishes includingDicentrachus labrax (Mayer et al 1988) and C undecimalis(Neidig et al 2000) Our inclusion sequence confirmed thatthe egg in R brachyoma is a pelagic type Although thestructural role of lipids is still unclear it is concerned withenergy sources for embryo development (Wiegand 1996)whereas cortical alveoli were negatively stained with HampEand ORO but were strongly positive to MT as greenish colorindicating the presence of the polysaccharide The staining

Table 1 Alteration in prevalence () of dominant histopathological observations in the ovariantissue of Rastrelliger brachysoma caught during non-breeding (October to November2013 n = 30) and breeding seasons (January to February 2014 n = 30) from SamutSongkram Province the Upper Gulf of Thailand

Alteration prevalence () Ovarian lesions

Non-breeding Breeding

Ovarian degeneration 1000 (n = 30) 1000 (n = 30)Ovarian atrophy 133 (n = 4) -Ovarian fibrosis 100 (n = 3) 100 (n = 3)Atresia in oogonia 1000 (n = 30) 833 (n = 25)Atresia in previtellogenic stage 1000 (n = 30) 933 (n = 28)Atresia in vitellogenic stage - 1000 (n = 30)Irregular and disorganized with breakdown of follicular cells - 933 (n = 28)Accumulation of melanomacrophage centers 233 (n = 7) 333 (n = 10)Unidentified parasites 100 (n = 3) -

- data not found

Table 2 Oocyte quality in different stages of the atretic follicles in Rastrelligerbrachysoma caught from the Upper Gulf of Thailand

Seasons Atresia


Oogonia 2390plusmn303 (50 oocytes) 1960plusmn442 (50 oocytes)Pre-vitellogenic stage 6350plusmn383 (100 oocytes) 5740plusmn483 (100 oocytes)Vitellogenic stage - 8020 plusmn 345 (100 oocytes)


pattern in R brachysoma was similar to that of other fishspecies (Selman et al 1988 Wallace and Selman 1990)The role and function of this inclusion was concerned withphysiological roles especially the prevention of polyspermyafter ovulation (Nagahama 1983)

The early vitellogenic oocyte started either synthesisor accumulation of yolk granules Hence it increased in sizeWallace and Selman (1990) reported that these yolk granulesin the ooplasm were the material stored during the secondarygrowth stage Their structural roles were usually related to thenutrition and metabolic activities in the embryonic develop-ment as described in most fish species (Chen et al 2006)Then entering into late vitellogenic stage the oocyte showedseveral processes in both cytoplasm and nucleus In thecytoplasm the characterization of yolk granule in this speciesis similar to Serra Spanish (Chellappa et al 2010) The yolkwas not fused until it entered into the mature stage Thisfeature was suggested to be typical of pelagic egg (Wallaceand Selman 1981) which is different from demersal eggsin which the beginning of yolk fusion was observed duringthe early vitellogenesis stage This was also found inGasterosteus aculeatus and Apeltes quadracus (Selman andWallace 1989) Then the nucleus of the late vitellogenesisstage first migrates to the animal pole and the micropyle wasobserved This pattern was also observed in other teleostfishes including Fundulus heteroclitus (Kuchnow amp Scott1977) and Torientalis (Chen et al 2006) The micropylefunction is concerned with the entering location of the sper-matozoa to the oocyte surface without acrosomal reaction(Bartsch and Britz 1997) The final stage of oocyte in thisspecies is the post-ovulatory stage or mature oocyte withprocesses involving the nucleus region No nucleus withgerminal vesicle breakdown was observed under the firstmeiotic cell division This breakdown is widely used as anindicator of the final maturation in the teleost fish (West1990)

Our observation found that atretic follicles mainlyappeared in vitellogenic stages It is possible that the endo-crinological activities in both decreasing of gonadotropin(GTH) and estrogen may be responsible for such atreticfollicles Similarly Wood and Van Der Kraak (2002) found thatatretic follicles were observed in the female Oncorynchusmykiss after exposed to low level of 17-estradiol (E2)Although it is weakly evidenced in this case the atreticfollicles seemed to be the results of the reduced oocytenumber which is due to impairment of the oogenic process aswell as reproductive failure The reduced ovarian size whichis caused by the ovarian atrophy was observed to be rela-tively low frequency in the non-breeding season Unfortu-nately the effects of estrogen mimics on ovarian atrophy wasonly known from laboratory work None have been reportedfrom filed experiment For instance Sridevi et al (2015) whoreported that the ovarian atrophy together with oocytesdegeneration of the Clarias gariepinus was observed afterthe exposure in both estrogens 17-ethynylestradiol (EE2)and diethylstilbesterol (DES) Both of these were classified as

endocrine disruptive chemicals (EDCs) These two pollutantswere also reported as chemicals that induce significant inci-dence of ovarian atrophy in Dicentrarchus labrax during earlydevelopment (Blazquez et al 1998) It strongly suggestedthat the reduction and the impairment of female reproductivetissue may be directly caused by these reagents and thendisrupted via the endocrine system through the hypothala-mus-pituitary-gonadal pathway (Sridevi et al 2015) There-fore we hypothesized that the Upper Gulf of Thailand mayaccumulate various pollutants particularly EDCs This shouldurgently be investigated in further research However basedon this result it should be expected that the ovarian impair-ment would have a significant impact on fecundity andfertility of R brachysoma population Also it is should benote that the occurrence of the ovarian atrophy referring toovarian morphological abnormalities is a case-effective anduseful sensitive indicator of the exposure of the model estro-genic compounds

The ovarian tissue was noticeably seen as melano-macrophage centers with evidence of chronic inflammation aswell as an inflammatory response in some areas It could besuggested the occurrence of melanomacrophages is theresult of the long-term injury of this organ This is similar towhat was reported in Danio rerio which were exposed to 17-ethinylestradiol (93 ngL 177 days) (Schatildefers et al 2007)We also observed unidentified parasites in ovarian tissue ofthe R brachysoma Even though the identity of the parasitecould not be confirmed it was suggested that the presenceof parasite in the system could potentially be detrimental tothe host (Barber et al 2000 Hecker amp Karbe 2005)

An important conclusion from in this study was thatthe histological and histochemical characteristics of theovarian tissue in R brachysoma should be applied to usein aquacultural development It is also suggested that Rbrachysoma is a threatened marine fish in the Upper Gulf ofThailand and there is still much research required to providefurther information on the effects of the EDCs and some heavymetals on ovarian tissue in R brachysoma We emphasizeagain the importance of understating the effect of overallhistopathological alterations especially atretic follicles andovarian atrophy on the reproductive health of R brachysomato the improvement of aquaculture of this species whichwould ultimately help in ensuring the suitability and safetyof food consumption by humans

Acknowledgements

This research was supported by The 100th Anniver-sary Chulalongkorn University Fund for Doctoral ScholarshipWe also specially thank Dr David V Furman for critically read-ing the manuscript The experimental protocol was approvedby the Animal Care and Use Committee of Faculty of Sciencein accordance with the guide for the care and use of labora-tory animal prepared by Chulalongkorn University (ProtocolReview No 1423003)


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Selman K Wallace R A amp Barr V (1988) Oogenesis inFundulus heteroclitus V The relationship of yolkvesicles and cortical alveoli Journal of ExperimentalZoology 246 42-56

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Selman K Wallace R A Sarka A amp Qi X (1993) Stagesof oocyte development in the zebrafish Brachydaniorerio Journal of Morphology 218 203-224


Senarat S Kettratad J Poolprasert P Boonyoung PKangwanrangsan N amp Jiraungkoorskul W (2015)Hepatic histopathology in Rastrelliger brachysoma(Bleeker 1851) from The Upper Gulf of ThailandAsia-Pacific Conference on Engineering and AppliedScience (pp 369-374) Osaka Japan

Sridevi P Chaitanya RK Prathibha Y Balakrishna SLDutta-Gupta A amp Senthilkumaran B (2015) Earlyexposure of 17aacute-ethynylestradiol and diethylstilbestrolinduces morphological changes and alters ovariansteroidogenic pathway enzyme gene expression incatfish Clarias gariepinus Environtal Toxicology30 439-451

Sritakon T Songkaew N Chotithammo S amp Vechprasit S(2006) Reproductive biology of short mackerelRastrelliger brachysoma (Bleeker 1851) and Indianmackerel R kanagurta (Cuvier 1817) in the South-ern Gulf of Thailand Bangkok Thailand Departmentof Fisheries

Sutthakorn P (1998) Biological aspects of short mackerelRastrelliger brachysoma (Bleeker 1851) of theAndaman Sea Coast of Thailand Phuket ThailandAndaman Sea Fisheries Department Center Depart-ment of Fisheries

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Wallace R A amp Selman K (1981) Cellular and dynamicaspects of oocyte growth in teleosts AmericanZoologist 21 325-343

Wallace R A amp Selman K (1990) Ultrastructural aspects ofoogenesis and oocyte growth in fish and amphibiansJournal of Electron Microscopy Technique 16 175-201

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Wilson J M Bunte R M amp Carty A J (2009) Evaluation ofrapid cooling and tricaine methanesulfonate (MS222)as methods of euthanasia in zebrafish (Danio rerio)Journal of the American Association for LaboratoryAnimal Science 48(6) 785-789

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Thailand which is the prime habitat of R brachysoma hasenvironmental stressors and high pollutants which includeheavy metals and petroleum hydrocarbon (Wattayakorn2012) Previous studies on health assessment of Rbrachysoma showed severe sign of histopathology such asvarious lesions and parasites in the liver brain and testis(Senarat et al 2015 Senarat et al unpublished data) How-ever comprehensive study on the reproductive health offemale R brachysoma is still lacking

In this article we primarily attempted to identify theovarian structure and reproductive mode of R brachysomaduring its annual reproductive season using histological andhistochemical analyses In addition to reproductive healthwe assessed the R brachysoma ovarian tissue using histo-pathological biomarker This information is vital for theprovision of scientific advice to aquaculture developmentFor example the study of the oocyte quality and the func-tionseffects of neurotransmitter and other neurohormoneswhich are important in the maturation of the ovary canincrease the success rate in aquaculture of this species whichwould ultimately help increase the natural population of thisspecies

2 Materials and Methods

Adult short mackerel Rastrelliger brachysoma witha standard body length about 15-19 cm (average 165plusmn049cm) and total weight about (6868plusmn067 g) were consideredas late ndash development stage of the ovarian histology Theywere obtained by bamboo strake trap during non-breeding(October to November 2013 n = 30) and breeding seasons(January to February 2014 n = 30) from the area of SamutSongkram Province in the Upper Gulf of Thailand (13deg16rsquo184 N 100deg02rsquo134 E) Identification of all fish was madeaccording to the taxonomic key of FAO (Food and AgricultureOrganization [FAO] 2010) Fish were then euthanized by therapid cooling shock method (Wilson et al 2009)

The light microscopic observation the ovarian tissueof fish was removed from the body cavity and fixed inDavidsonrsquos fixative (48 hrs) Then they were processed usingstandard histological techniques (Bancroft amp Gamble 2002)The gonadal paraffin block was cut at 5 microm thickness andstained with Harrisrsquos hematoxylin and eosin (HampE) andMassonrsquos trichrome (MT) periodic acid-schiff (PAS) Grocottmethenamine silver (GMS) alcian blue pH 25 (AB) and reti-culin (Rt) methods (Puchtler and Waldrop 1978 Vidal 1988Bancroft and Gamble 2002) Additionally frozen tissues werecut at 10 microm thicknesses and stained with oil red O (ORO)(Culling 1963) The ovarian structure oogenesis with sizemeasurement was assessed to detail according to the guide-lines of Dietrich and Krieger (2009) and Brown-Peterson et al(2011) under light microscopy The numbers of atretic follicleswere quantified (50-100 oocytesfish per section) in middlearea of three ovarian sections and followed the guidelinesunder light microscopy (10x and 40x) of Blazer (2002)

For ultrastructural observation small pieces ofovarian atrophy were cut and rapidly pre-fixed in 25glutaraldehyde in 01 M phosphate buffer pH 74 at 4degC andwashed in the buffered fixative solution Thereafter theywere post-fixed with 1 osmium tetraoxide (OsO4) and thenprocessed using standard ultrastructural techniques (Rowdenamp Lewis 1974) The thin sections were cut by ultramicrotomeabout 90 nm thicknesses stained with uranyl acetate andlead citrate Lastly the ultrathin sections were observed andphotographed using a JEM-2100 at 200kV

Histopathological analysis the histopathologicalalterations of ovarian tissue were observed and recorded asa mean prevalence for each period

3 Results

Based on anatomical analysis the asymmetrical elon-gation of R brachysoma ovaries were paired organs locatedin the abdominal cavity Cross sections of the immature ovaryin non-breeding season were histologically surrounded bytunica albuginea including a thick layer of connective tissueand numerous blood vessels (Figures 1a 1b 1c 2r) Theywere suspended by mesovarium from the peritoneal dorsal

Figure 1 Light photomicrograph (a b c e f) and schematic sum-mary of ovarian structure (d g) adipose tissue (ad) bloodvessel (Bv) chromatin nucleolar stage (Ch) epitheliumcells (Ec) early vitellogenic stage (Ev) germinal compart-ment (GC) lipid and cortical alveoli stage (Lc) latevitellogenic stage (Lv) ovigerous fold (Of) oognia (Og)perinucleolar stage (P) post vitellogenic stage (Pv)stromal compartment (SC) tunica albuginea (Ta) Scalebar a c f = 100 microm e = 200 microm































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- data not found


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4 Discussion








- data not found


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ovarian histology and reproductive health of short mackerel...

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