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1.
本研究分离培养尼罗罗非鱼(Oreochromis niloticus)精巢支持细胞(Sertoli cells, SCs),建立并优化尼罗罗非鱼精巢支持细胞分离培养体系。无菌条件下,取发育至第Ⅲ期的尼罗罗非鱼精巢,PBS清洗后,剪碎精巢组织,0.25%胰蛋白酶消化,用含10%犊牛血清(Newborn bovine serum, NBS)的L-15培养液终止消化,过滤、离心,获得细胞。差速贴壁法获得SCs后,在26℃、无CO2饱和湿度恒温培养箱中培养。分别采用含10% NBS的L-15、M199、F12培养液,含5%、10%、15% NBS的L-15培养液,含1%罗非鱼血清的L-15+10% NBS培养液培养尼罗罗非鱼SCs。各组每2 d取细胞计数,绘制SCs生长曲线;甲基绿染色,倒置显微镜下观察SCs的形态。尼罗罗非鱼SCs培养1~2 d,细胞贴壁;培养3~5 d,细胞完全贴壁并迅速增殖。单个SCs呈不规则多边形,其核位于胞质中央,呈卵圆形,胞质中可见吞噬颗粒和空泡,空泡聚集于支持细胞胞质的两极或散布于核的四周。SCs在L-15培养液中贴壁生长,在F12、M199培养基中较难贴壁。与F12、M199培养液相比,L-15培养液更有助于尼罗罗非鱼SCs生长增殖(P<0.01)。与添加5%和15% NBS相比,在L-15培养基中添加10% NBS更有助于尼罗罗非鱼SCs生长增殖(P<0.05)。与未添加尼罗罗非鱼血清相比,在10% NBS+L-15培养中添加1%尼罗罗非鱼血清,能显著促进SCs生长增殖(P<0.05)。研究表明,采用胰酶消化差速贴壁法获得尼罗罗非鱼精巢支持细胞,10% NBS+1%罗非鱼血清+L-15培养液培养,能显著促进尼罗罗非鱼精巢支持细胞生长增殖。  相似文献   

2.
钙敏感受体(calcium-sensing recceptor, CaSR)在 Ca2+ 刺激下可参与调控细胞凋亡等生理过程, 在机体适应逆境胁迫中发挥重要作用。为研究吉富罗非鱼(Genetically Improved Farmed Tilapia, GIFT)CaSR 基因的特点及其在缺氧胁迫下参与细胞凋亡的调控机制。本研究利用 RT-PCR 技术克隆了吉富罗非鱼 CaSR cDNA 全长序列, 利用 qRT-PCR 技术分析了该基因在不同组织中的表达模式, 并进一步检测了缺氧胁迫下(0.55 mg/L)肝脏中该基因和细胞凋亡相关基因 mRNA 的表达变化, 同时利用 ELISA 法检测了肝脏中抗氧化酶活性的变化, 以及通过 HE 和 TUNEL染色法分别观察了肝细胞的形态变化和凋亡情况。结果显示, 吉富罗非鱼 CaSR cDNA序列全长 3265 bp, 包括 21 bp 5′非编码区、2823 bp 开放阅读框和 421 bp 3′非编码区, 编码 940 个氨基酸。CaSR 基因 mRNA 在不同组织中均有表达, 其中肌肉中表达量最高, 肾脏次之; 组织切片观察发现缺氧可导致肝脏组织结构损伤, 促进肝细胞凋亡; 与对照组(5.0 mg/L)相比, 缺氧可增强 SOD、CAT 和 GSH-Px 抗氧化酶活性, 上调 CaSR mRNA 的表达, 并引起 Bcl-2、Caspase-3 和 P53 凋亡基因 mRNA 的表达变化。研究结果表明, CaSR 可能通过介导 Ca2+调控细胞凋亡, 从而参与吉富罗非鱼的缺氧应对机制。  相似文献   

3.
将雄性幼鲻(Mugil cephalus)分为4组,分别投喂含17β-雌二醇饲料组、含三苯氧胺(Tamoxifen)饲料组、含17β-雌二醇和三苯氧胺(Tamoxifen)混合饲料,以及对照组,旨在进一步研究17β-雌二醇对幼鲻精子发生的作用机制.同时,采用组织学和免疫组织化学方法对17β-雌二醇影响鲻精巢精子发生及其作用机制进行分析,结果表明,雌激素明显促进鲻精原细胞有丝分裂及增殖和精子发生;Tamoxifen组与混合组于实验后30 d取材观察,两组精巢切片均显示,雄性鲻精巢发育停滞,精原细胞坏死、凋亡,Sertoli's细胞形态结构发生改变、数量下降.这进一步证实雌二醇激发精子发生必须经其受体的介导.雌激素受体(ER)在精巢的免疫组织化学定位揭示,ER在雌激素组免疫活性最高,对照组次之,混合投喂组免疫活性非常弱,而Tamoxifen组则显免疫阴性反应,这为雌激素对鲻精巢的精子发生起着关键作用的论点提供可靠确凿的新证据,同时表明在鲻早期精子发生中,一旦雌激素不足或缺失,精巢的发育质量和生精能力就会受到严重的影响.  相似文献   

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在盐度为0、10、20、30时测定了萨罗罗非鱼(Sarotherodon melanotheron)、尼罗罗非鱼(Oreochromis niloticus)和以色列红罗非鱼(Oreochromis sp.)鳃和肾中Na+-K+-ATPase活性。结果表明:(1)不同盐度对不同组织的Na+-K+-ATPase活性有显著影响,鱼的品种及其他交互作用对Na+-K+-ATPase活性均无显著影响;(2)在实验盐度范围内,无论是鳃和肾,萨罗罗非鱼、尼罗罗非鱼和以色列红罗非鱼的Na+-K+-ATPase活性均随盐度的升高而增高;3种罗非鱼中,萨罗罗非鱼的Na+-K+-ATPase活性随盐度的升高增大最为剧烈,以色列红罗非鱼次之,尼罗罗非鱼最小;低盐度时尼罗罗非鱼的Na+-K+-ATPase活性相对较高,高盐度时萨罗罗非鱼的Na+-K+-ATPase活性相对较高,Na+-K+-ATPase的活性与罗非鱼的耐盐能力有着一定的联系;(3)盐度大于7.19和11.94时,萨罗罗非鱼鳃中的Na+-K+-ATPase活性分别开始高于以色列红罗非鱼和尼罗罗非鱼;盐度大于18.42时,萨罗罗非鱼肾中的Na+-K+-ATPase活性开始高于尼罗罗非鱼;(4)除了在盐度20和30中的尼罗罗非鱼及盐度30的以色列红罗非鱼,是肾中的Na+-K+-ATPase活性高于鳃外,其他均为鳃中的Na+-K+-ATPase活性高于肾,肾中Na+-K+-ATPase活性在不同盐度之间的变化较鳃剧烈;罗非鱼的鳃比肾在渗透压调节上起的作用更大。  相似文献   

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用兔抗血清对抗促黄体素生成素受体(LHR)或称绒毛膜促性腺激素受体(CGR)和雄激素受体(AR)进行LHR和AR免疫组织化学定位,以揭示外源性促性腺激素(鲤脑垂体激素和hCG)诱发日本鳗鲡精子发生及其内分泌机制。结果表明,经过注射激素处理后的实验组与注射前的对照组相比较,其精巢发育和精子发生出现十分显著的变化。组织学切片观察显示,激素处理前鳗鲡精巢处于精原细胞增殖期,而两种激素混合注射后第10天,实验组可见精小叶中精原细胞的有丝分裂和初级与次级精母细胞的数量显著的增加。注射后第35天,靠近生殖上皮除有少量精原细胞外,精小叶中有大量初级精母细胞和次级精母细胞和少数精子细胞以及管腔中存在少量精子。在注射后第83天,日本鳗鲡完成了精子发生和精巢发育成熟以及释精。免疫组织化学染色结果进一步揭示,激素处理前,LH受体免疫活性分布在生殖上皮,显示强的免疫阳性反应;激素处理后,LH受体定位在Sertoli细胞和间质细胞以及精原细胞和初级与次级精母细胞的胞膜上,均显示强的免疫阳性反应。激素处理前,雄激素受体定位在生殖上皮和早期生精细胞的胞膜上;激素处理后,AR则定位在这些生精细胞的核或胞质,而精子细胞和精子显示免疫阴性反应。这些结果首次证明了这两种激素诱导鳗鲡精子发生和成熟的作用机制是通过LH受体和雄激素受体的介导。  相似文献   

6.
魏静  于林田  周林燕  张小萍  王德寿 《水产学报》2015,39(12):1763-1772
为探讨低等脊椎动物Foxp1与免疫细胞活化及机体雌激素水平的相关性,本研究首次分离克隆了尼罗罗非鱼 (Oreochromis niloticus) Foxp1的开放阅读框序列,并通过real-time PCR对其mRNA表达水平进行检测。结果显示,尼罗罗非鱼具有两种不同基因编码的Foxp1分子,分别命名为OnFoxp1a与OnFoxp1b;OnFoxp1a为1710 bp,由15个外显子编码569个氨基酸,OnFoxp1b为2040 bp,由16个外显子编码679个氨基酸;OnFoxp1a/b均含有Foxp亚家族的特征性结构,即锌指结构、亮氨酸拉链样结构和叉状螺旋结构;与OnFoxp1a相比,OnFoxp1b与高等脊椎动物Foxp1具有更近的亲缘关系。Real-time PCR检测结果显示,OnFoxp1a/b几乎在所有组织中均有表达,OnFoxp1a在精巢中有高水平表达, OnFoxp1b在心脏中有高水平表达,同时OnFoxp1b在免疫相关组织如鳃、脾脏、肾脏、肠等均有中等水平表达;淋巴细胞多克隆刺激剂PHA、PMA、LPS刺激尼罗罗非鱼外周血单个核细胞(PBMC),结果显示,50 μg/mL PHA和50 ng/mL PMA分别刺激6、12、24 h均显著增强OnFoxp1b的表达 (p<0.05),20 μg/mL LPS刺激后,OnFoxp1b的表达出现先降低后升高的趋势 (p<0.05);而OnFoxp1a的表达除50 μg/mL PHA 刺激24 h后有所升高,其余均无显著变化;雌激素处理6月龄雄性尼罗罗非鱼48 h,OnFoxp1a/b在肠、肾脏中的表达显著上调 (p<0.05),而脾脏中无显著变化 (p>0.05)。综上所述,低等脊椎动物硬骨鱼类两种不同基因编码的Foxp1a/b均为哺乳动物Foxp1的同源分子,但其序列、结构特征、表达模式迥异,提示其功能发生歧化;同时两者的表达与淋巴细胞活化及机体雌激素水平密切相关。  相似文献   

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本研究以腹腔注射无乳链球菌(Streptococcus agalactiae, WC1535菌株)后的尼罗罗非鱼(Oreochromis niloticus,GIFT strain)为研究对象,研究不同水温下无乳链球菌在尼罗罗非鱼体内的动态分布及消除规律。首先,分析25℃、29℃和33℃3个实验组罗非鱼的感染累积死亡率;其次,对3个实验组罗非鱼进行无乳链球菌的体外分离、培养和计数;然后,分析感染后不同实验组罗非鱼脑、肝脏、脾脏和肾脏组织中无乳链球菌的浓度。结果显示,随着水温的升高罗非鱼的累积死亡率也随之升高,25℃、29℃和33℃组的累积死亡率分别为6.67%、25.56%和78.90%。菌落统计结果显示,随着水温的升高,无乳链球菌在罗非鱼体内的增殖速度加快,同时,单位质量组织(脑、肝脏、脾脏和肾脏)中无乳链球菌的最大载菌量也随之升高。本研究还发现,无乳链球菌在罗非鱼脾脏中的浓度最高,在肾脏中的存活时间最长。综上可知,尼罗罗非鱼感染无乳链球菌后,体内各组织中链球菌的增殖、消除速度均与水温密切相关。本研究为研究无乳链球菌的致病机制奠定基础,也为通过合理调控水温及施药等措施防治尼罗罗非鱼链球菌病的暴发提供科学依据。  相似文献   

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为获得具有单一克隆特性的能稳定传代培养的罗非鱼巨噬细胞系,本研究从尼罗罗非鱼腹腔中分离纯化巨噬细胞,采用EB病毒(Epstein-Barr virus,EBV)感染,筛选单克隆细胞的方法建立了尼罗罗非鱼巨噬细胞系,并对其进行了EBV感染鉴定、电镜观察、端粒酶活性检测、致癌性评估、核型分析以及分子生物学鉴定。研究表明,EBV已整合到尼罗罗非鱼巨噬细胞中且稳定表达,经30代稳定传代,该细胞系仍维持较好的增殖状态;该细胞系表面不平滑,有明显的钝圆形突起和细长的伪足,表现为典型的巨噬细胞形态;端粒酶活性显著高于未经感染的巨噬细胞,而与He La细胞差异不显著,且该细胞系不具有致癌性,说明永生化细胞系构建成功。核型分析结果发现,该细胞系具有44条染色体,其核型公式为2 n=2 x=44=4 sm+17 st+1 t。PCR检测发现,该细胞系存在CD33和CD205的转录本,这些都是单核巨噬细胞的标志物,经18S r RNA检测证明该细胞系来自尼罗罗非鱼巨噬细胞。永生化尼罗罗非鱼巨噬细胞系已被成功建立,该细胞系为研究罗非鱼链球菌HSP70-肽疫苗的高保护率,以及罗非鱼的免疫防御机制提供了工具。  相似文献   

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采用同源克隆技术获得了佛罗里达红罗非鱼(Oreochromis sp.)前原黑色素浓集激素1(prepro-melanin concentrating hormone 1,pmch1)基因c DNA全长序列,将其命名为flpmch1;并通过实时荧光定量PCR(q RTPCR)分析天生黑斑佛罗里达红罗非鱼组织表达谱和低温胁迫后其各组织表达差异。结果表明,flpmch1 c DNA序列全长629 bp,开放阅读框411 bp,编码136个氨基酸,存在12个潜在磷酸化位点和1个长度为199 bp的Cp G岛。Flpmch1与24个物种Pmch1氨基酸序列比对结果表明,Flpmch1与尼罗罗非鱼(O.niloticus)、奥利亚罗非鱼(O.aureus)、莫桑比克罗非鱼(O.mossambicus)、布氏新亮丽鲷(Neolamprologus brichardi)的Pmch1相似性最高。系统进化树分析显示硬骨鱼纲Pmch聚成一个大支,Flpmch1与尼罗罗非鱼Pmch1进化地位最接近。组织表达谱分析显示,flpmch1 m RNA在多个组织中均有表达,其中在脑中表达量最高,且粉白色皮肤中的表达量显著高于黑色皮肤(P0.01)。低温胁迫后flpmch1 m RNA各组织表达量较对照组均呈下调表达。推断flpmch1可能参与了佛罗里达红罗非鱼天生黑斑的形成和过冬黑化过程。  相似文献   

10.
为了探究补体C9在尼罗罗非鱼(Oreochromis niloticus)免疫中发挥的作用,本研究克隆并分析了尼罗罗非鱼补体C9基因(OnC9)。结果显示:OnC9的cDNA序列全长2 502 bp,包含1 761 bp的开放阅读框(ORF),编码586个氨基酸。氨基酸序列同源性分析表明,OnC9与牙鲆(Paralichthys olivaceus)补体C9氨基酸相似性最高,达73.0%,与其他鱼类补体C9的相似性介于49.3%~71.4%之间。实时荧光定量PCR检测结果表明,OnC9基因在所检测的各个组织或器官中表达水平有明显差异,在肝脏中表达量最高,其次是肠道、后肾、皮肤、肌肉、鳃,在脑、脾脏、心脏、头肾、胸腺和血液中表达量最低。在无乳链球菌(Streptococcus agalactiae)感染鱼体后,肝脏、后肾等组织中OnC9表达量表现为在感染12 h、48 h(或36 h)、120 h先后出现三个峰值的波动表达的规律。说明OnC9在无乳链球菌感染尼罗罗非鱼后的免疫应答中发挥作用。  相似文献   

11.
Initial appearance and development of Leydig cells (LCs) during testicular differentiation in tilapia,Oreochromis niloticus, were investigated histologically. In addition, changes of testosterone levels in gonadal tissue and serum were examined by radioimmunoassay. In the gonads of fry at 23–26 days after hatching, initial testicular differentiation was confirmed by the observation of the differentiation of connective tissues into tissues which are characteristic of the adult testis. LCs, which were identified by the ultrastructural features (a moderate number of mitochondria with tubular cristae, well developed smooth endoplasmic reticulum and many free ribosomes) appeared initially at the time of testicular differentiation. LCs increased in number rapidly in the testes of fish at 70 days after hatching. Concomitant with this increase, spermatogonia increased in number. Testosterone was detectable in the fish at 40–50 days after hatching, but levels in tissue and serum were low. Testosterone levels increased gradually in the fish beginning at 70 days after hatching and increased still more at 100–150 days accompanying active spermatogenesis.  相似文献   

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In fish, sex steroids initiate and/or accelerate the maturation of the brain-pituitary-gonad axis. In order to obtain information on the steroid milieu during the pubertal development of male African catfish, we have monitored the conversion of [3H]-pregnenolone and [3H]-androstenedione by testis and [3H]-pregnenolone by interrenal tissue fragmentsin vitro. Pubertal development occurs between two and six months of age. Testicular development proceeds through four main stages that are characterised histologically by the presence of spermatogonia (stage I), spermatogonia and spermatocytes (stage II), spermatogonia, spermatocytes and spermatids (stage III), and all germ cells including spermatozoa (stage IV). 11β-Hydroxyandrostenedione and cortisol were the main products of testes and interrenal tissue, respectively, in all stages of the pubertal development; a change in the steroidogenic pattern was not observed during this period. The interrenal tissue displayed no significant conversion of [3H]-pregnenolone to 11-oxygenated androgens. Blood plasma was analyzed for the presence of five androgens; testosterone, 11β-hydroxytestosterone, 11β-hydroxyandrostenedione, androstenetrione, and 11-ketotestosterone. 11-Ketotestosterone was the quantitatively dominating androgen in the circulation at all stages of development, which was more pronounced in stages III and IV. The obvious differences between thein vitro andin vivo results, namely 11β-hydroxyandrostenedione being the main testicular productvs. 11-ketotestosterone dominating in the blood, may indicate that 11β-hydroxyandrostenedione is converted to 11-ketotestosterone at extratesticular sites.  相似文献   

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The initial appearance and the development of Leydig cells (LCs), the sites of steroid hormone production in the testis, were investigated ultrastructurally during testicular differentiation in the Japanese eel, Anguilla japonica. In addition, the effects of a single injection of human chorionic gonadotropin (HCG; 5 IU g body weight-1) on histological changes of the testes and serum 11-ketotestosterone (11-KT) were examined at various stages (15–18, 20–23, 26–29, 32–35, 38–41 and 46–50 cm body length (BL)) of testicular differentiation. Testicular differentiation was morphologically characterized by the development of loose connective tissue on the medial side in animals 18–29 cm in BL. Ultrastructurally, LCs were first identified in the loose connective tissue of the testis of the 23 cm fish. In the testes of fish over 32 cm, clusters of LCs were distributed throughout the interstitial region accompanying the increase in number of spermatogonia. In fish larger than 32 cm, spermatogenesis was induced by administration of HCG; serum 11-KT levels were also raised. On the other hand, there was no effect on spermatogenesis or serum 11-KT levels in fish less than 29 cm, or in the controls. These result suggests that morphological differentiation of LCs occurs in testis of the 23 cm eel, and subsequently, the testes of eels of BL more than 32 cm acquire the capability to produce steroid hormones.  相似文献   

17.
薛永霞  卫赛超  张菊  陈舜胜 《水产学报》2019,43(7):1661-1677
为探究上海熏鱼制作过程中不同加工阶段风味物质的变化,本研究以尼罗罗非鱼肉为对象,通过高效液相色谱法、氨基酸自动分析法和固相微萃取—气相色谱—质谱联用技术对上海熏鱼4个加工阶段(生鲜罗非鱼、一次浸渍、一次浸渍后油爆和上海熏鱼成品)的风味物质进行分析和鉴定。结果显示,各阶段IMP含量呈逐渐上升趋势,是主要的鲜味核苷酸。游离氨基酸总量和呈味氨基酸含量也逐渐升高,并呈显著性差异,其中天冬氨酸和谷氨酸对上海熏鱼风味影响最大。生鲜罗非鱼、一次浸渍、一次浸渍后油爆和上海熏鱼成品中挥发性物质依次为50、84、78和82种,主要由醛类、酮类、醇类和烃类构成。因此,浸渍和油爆是上海熏鱼风味形成的主要原因,使得罗非鱼鱼体的腥味得以有效改善。  相似文献   

18.
Recently, we developed an intraspecies spermatogonial transplantation technique in a pelagic egg spawning marine teleost, nibe croaker Nibea mitsukurii. Nibe croaker is an ideal candidate recipient for spermatogonial transplantation since it has a short generation time and small body size. In the present study, yellowtail Seriola quinqueradiata spermatogonia were transplanted into nibe croaker larvae, and the behavior of transplanted spermatogonia in recipient gonads was observed. Three weeks post-transplantation, yellowtail spermatogonia were incorporated into the gonads of 72 out of 88 recipients. An antiproliferating cell nuclear antigen was detected in incorporated yellowtail spermatogonia, suggesting that the xenogenic germ cells were proliferating in recipient gonads. Yellowtail vasa-positive spermatogonia survived for 11 months after transplantation in the gonads of recipient fish. Thus, we showed that the microenvironment in nibe croaker gonads can support the colonization, proliferation, and survival of germ cells derived from a different taxonomic family.  相似文献   

19.
Glutathione and its Related Enzymes in the Nile Fish   总被引:2,自引:0,他引:2  
Glutathione (GSH) and related enzymes, glutathione transferase (GST), glutathione peroxidase (GPx) and glutathione reductase (GR) form an important phase 2 biotransformation enzymes system. The objective of this study was to compare this enzymes system in three fish species from the river Nile, Oreochromis niloticus, Claris lazera and Cyprinus carpio in order to establish the main differences and to purify and characterize GST from the liver of O. niloticus.The level of GSH and the activity of GST, GPx and GR in the liver, kidney and gills of the three fish species were examined. A simple reproducible procedure for the purification of GST from the liver of O. niloticus to homogeneity, which includes chromatography on DEAE- cellulose followed by affinity chromatography on GSH-sepharose was established. The molecular mass was found to be 25,460 Da by SDS-PAGE. The Michaelis-Meneten constants (Km) of the enzyme for GSH and CDNB were 0.35 mM and 0.42 mM, respectively. The affinity purified enzyme exhibited maximum pH at pH 8.0 and increasing pH above 8.0 did not affect the observed maximum. The purified enzyme acts readily on CDNB, less readily on some standard transferase substrates (1,2-dichloro-4-nitrobenzene and p-nitrophenethyl bromide) and not at all on others (bromosulphophthalein and p-nitrobenzyl chloride). Bromosulfophthalein, cibacron blue and hematin inhibited CDNB-conjugating activity of the purified enzyme with IC50 0.079, 3.98 and 0.126 μM, respectively.  相似文献   

20.

In the present study, the tilapia Oreochromis mossambicus were exposed to food deprivation for a period of 6 or 12 days and changes in the luteinizing hormone (LH) immunoreactivity in the proximal pars distalis (PPD) of the pituitary gland and the testicular activity were examined. Intensely immunoreactive LH content was noticed in the PPD of the pituitary gland in the initial controls, controls on days 6 and 12, and fasting fish on day 6, whereas the LH immunoreactivity was moderate or weak in fasting fish on day 12. In addition, although the mean gonadosomatic and hepatosomatic indices among different experimental groups did not show any statistically significant difference, the mean numbers of spermatogonia, spermatocytes, and spermatids were significantly lower in food-deprived fish on days 6 or 12 compared to those of controls. The inhibition of the spermatogenesis was accompanied by the presence of abundant spermatozoa in the lumen of seminiferous tubules of the testis in food-deprived fish, whereas the occurrence of spermatozoa was relatively infrequent in initial controls and controls. Furthermore, refeeding to food-deprived fish on day 6 onwards resulted in occurrence of few intensely stained LH secreting cells and significantly higher numbers of spermatocytes and spermatids concomitant with sparse spermatozoa in majority of tubules compared to those of food-deprived fish. These results suggest that prolonged exposure to food-deprivation causes suppression of the LH secretory activity in the pituitary gland and disruption in the spermatogenesis in O. mossambicus.

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