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Effects of different animal baits on the gonad development of YY super-male yellow catfish (Pelteobagrus fulvidruco)

  • Corresponding author: Jie MEI, jmei@mail.hzau.edu.cn
  • Received Date: 2019-03-16
    Accepted Date: 2019-05-29
  • The emergence of novel variety " All-male NO.1” dramatically promoted the development of the yellow catfish industry, of which the cultivation of YY super-male was a critical step. Recently, YY super-male yellow catfish with intersexual gonad emerged largely, which hindered the development of yellow catfish industry. We only found the problem of gonad development in YY super-male breeding with tubificidae by checking the production and breeding records for nearly 10 years. Therefore, four different baits including Artemia salina, Zooplankton, Chironomus plumosus, Tubificidae were used to treat YY super-male for 20 days (11 to 30 days post hatching). The survival rate, body length, and body weight of each group were measured at 60 dph (day post-hatching). Tubificidae treatments significantly increased the body length and weight compared with other baits, and the survival rate of fish fed with Artemia salina was significantly lower than fish fed with the other three baits. In addition, we performed histology analysis on gonadal structure at 60 dph and 1 year old and statistics of fertilization rate at 1 year old. As a result, the fish group fed with Tubificidae displayed 75% intersexual gonad and 25% testis without seminiferous lobule, and the fertilization rate was only 36.70%±4.05%, which was significantly lower than the other groups. In order to study the reasons for the feminization of YY yellow catfish, we measured the estradiol content and found that the estradiol content was low in all four different animal baits. It is speculated that the feminization of YY super-male yellow catfish may be caused by the environmental endocrine disruptors (EDCS) enriched by Tubificidae. Therefore, zooplankton or Chironomus plumosus could be fed in the early stage of large-scale breeding of YY-supermale yellow catfish, whereas Tubificidae should not be fed.
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Effects of different animal baits on the gonad development of YY super-male yellow catfish (Pelteobagrus fulvidruco)

    Corresponding author: Jie MEI, jmei@mail.hzau.edu.cn
  • 1. College of Fisheries, Huazhong Agricultural University, Wuhan    430070, China
  • 2. Wuhan BaiRui Biological Technology Co. Ltd, Wuhan    430070, China

Abstract: The emergence of novel variety " All-male NO.1” dramatically promoted the development of the yellow catfish industry, of which the cultivation of YY super-male was a critical step. Recently, YY super-male yellow catfish with intersexual gonad emerged largely, which hindered the development of yellow catfish industry. We only found the problem of gonad development in YY super-male breeding with tubificidae by checking the production and breeding records for nearly 10 years. Therefore, four different baits including Artemia salina, Zooplankton, Chironomus plumosus, Tubificidae were used to treat YY super-male for 20 days (11 to 30 days post hatching). The survival rate, body length, and body weight of each group were measured at 60 dph (day post-hatching). Tubificidae treatments significantly increased the body length and weight compared with other baits, and the survival rate of fish fed with Artemia salina was significantly lower than fish fed with the other three baits. In addition, we performed histology analysis on gonadal structure at 60 dph and 1 year old and statistics of fertilization rate at 1 year old. As a result, the fish group fed with Tubificidae displayed 75% intersexual gonad and 25% testis without seminiferous lobule, and the fertilization rate was only 36.70%±4.05%, which was significantly lower than the other groups. In order to study the reasons for the feminization of YY yellow catfish, we measured the estradiol content and found that the estradiol content was low in all four different animal baits. It is speculated that the feminization of YY super-male yellow catfish may be caused by the environmental endocrine disruptors (EDCS) enriched by Tubificidae. Therefore, zooplankton or Chironomus plumosus could be fed in the early stage of large-scale breeding of YY-supermale yellow catfish, whereas Tubificidae should not be fed.

  • 黄颡鱼(Pelteobagrus fulvidraco)隶属于鲇形目(Siluriformes),鲿科(Bagridae),黄颡鱼属,是我国重要的淡水经济鱼类之一。在相同的养殖条件下,雄性黄颡鱼比雌性黄颡鱼的生长速度快2~3倍,有更高的经济价值。王达[1]等和丹成等[2]成功分离出黄颡鱼X染色体和Y染色体连锁的分子标记,从而开拓出一条性染色体连锁标记辅助的全雄黄颡鱼培育技术路线。基于此,刘汉勤等[3]通过激素性逆转技术结合黄颡鱼性别连锁分子标记筛选得到XY生理雌鱼,再进行雌核发育获得YY超雄黄颡鱼。黄颡鱼YY父本与XX母本繁殖得到的XY全雄黄颡鱼,在2010年经国家水产原种和良种审定委员会审定,农业部批准为水产新品种黄颡鱼“全雄1号”。利用YY黄颡鱼进行单性育种成为继YY罗非鱼后规模化生产全雄鱼成功的第二例。

    武汉百瑞生物技术有限公司作为全国YY超雄黄颡鱼最大生产基地,从传统的直接下塘培育到现代工厂化循环水培苗大幅度提高了YY超雄黄颡鱼的存活率。使用工厂化循环水培育需对仔鱼进行人工驯化,从动物饵料开口逐渐转食为商业饲料。黄颡鱼孵化后常用的天然开口饵料包括丰年虫和浮游动物,开口一周后投喂水蚯蚓或冰冻红虫,期间投喂少量商业微颗粒饲料进行转食。但在工厂化循环水培苗过程中YY超雄黄颡鱼又出现了大量精卵巢共存的兼性性腺,阻碍了全雄黄颡鱼产业的发展。

    鱼类性腺分化不仅受遗传因素控制,也受溶解氧、光照、水温、PH、外源性激素和污染因子等环境因子的影响[4]。影响鱼类性腺分化因子诸多,而关于饵料对鱼类性腺分化影响的尚未见报道。在鱼类单性育种过程中,常通过性激素拌料投喂来获得性逆转鱼,如大鳞副泥鳅(Paramisgurnus dabryanus)[5]、黄颡鱼[6]、尼罗罗非鱼(Tilapia nilotica)[7]、鲤(Cyprinus carpio)[8]和虹鳟 (Oncorhynchus mykiss)[9]等。此外,环境干扰物可导致鱼类的雌性化效应、雄性化效应和雌雄同体效应[10]。当鱼类长期暴露在双酚A(BPA)下,性腺分化受到干扰,甚至性腺出现两性化(既有精母细胞,又有卵母细胞)[11];雄鱼表现为雌性化,成年雄鱼的精子重量下降,精液稀少[12]。4-NP处理后的青鳉(Oryzias latipes)雄鱼,其性腺也发育成精巢和卵巢共存的兼性性腺[13]

    组织学分析发现,黄颡鱼在13日龄时卵巢开始分化,19日龄形成卵巢腔;而精巢分化时间约为40日龄,精小叶和输精管出现于55日龄[14]。故此推测性腺分化关键时期的雌激素水平升高可能是引起YY黄颡鱼雌性化的主要原因。雌激素摄入的主要途径有水浸泡和饵料摄入。对比分析不同批次的性腺发育正常和异常YY超雄黄颡鱼的生产和养殖记录,发现养殖环境相同,仅投喂水蚯蚓的YY黄颡鱼性腺出现了异常。为了确认是饵料引起YY黄颡鱼性腺退化,实验采用连续20 d(11~30日龄)投喂丰年虫、浮游动物、红虫和水蚯蚓,探讨不同饵料对YY黄颡鱼生长和性腺的影响,以期发现导致YY黄颡鱼性腺发育异常的原因,进而建立一套YY养殖超雄黄颡鱼早期培育技术规范,为全雄黄颡鱼产业的发展提供技术保障。

    • 实验鱼亲本YY超雄黄颡鱼和YY生理雌鱼均来自武汉百瑞生物技术有限公司,并在繁殖前用黄颡鱼性别连锁分子标记进行鉴定。实验鱼苗为亲本YY♂×YY♀人工繁殖所得的超雄黄颡鱼苗。出膜后3 d用浮游动物开口养至10日龄开始分组,每组设置3个平行,每个平行200尾苗。11日龄开始,分别用丰年虫、浮游动物(桡足类和枝角类)、冰冻红虫和水蚯蚓投喂至30日龄。在11~30日龄期间,鱼苗饲养在60 L的循环水水箱中,每天投喂3次。到30日龄后转入4 m3的流水水泥池中,每天用微颗粒饲料投喂2次 (饲料购自湖州亿盛饲料有限公司,主要成分: 粗蛋白≥45.0%,粗脂肪>5.0%,粗纤维≤6.0%, 灰分≤15.0%,水分≤10.0%)。在实验期间,水温保持26~29 °C,溶解氧5.5~6.8 mg/L。每天清理残饵粪便,及时加注新水。

    • 每组平行随机抽取50尾,解剖取性腺,性腺组织在显微镜下经波恩氏液(Bouin’s solution)染色观察性腺并统计拍照,经4%多聚甲醛固定后的性腺材料用酒精梯度脱水、二甲苯透明、常规石蜡包埋后做纵切片,H.E染色,用光学显微镜观察,显微拍照,检査性别并统计性比。

    • 4组中分别挑选规格相同的一年龄YY超雄黄颡鱼,每组各9尾。每条雄鱼取出精巢,称取0.1 g精巢用匀浆器中研磨成白色浆液10 μL,用精子保存液(63 mmol/L NaCl,19 mmol/L KCl, 1.3 mmol/LM CaCl2,4.7 mmol/L MgSO4·7H2O,2.5 mmol/L NaHCO3,pH 7.4)梯度稀释到2 000倍。在显微镜(400×)下用细胞计数板统计每尾黄颡鱼的精子数目并计算每尾黄颡鱼原精液密度。用精子保存液将原精液稀释到8×106 个/μL。YY雌性黄颡鱼进行人工催产后,用毛巾将生殖孔附近水擦干,左手食指和中指由头部向下握住亲鱼左右胸鳍,右手拇指和食指从腹部由上往下轻、缓的挤压,将所有鱼卵挤入同一钵中混合均匀。各组均用100 μL稀释精液(8×106 个/μL)配2.5 g卵(每克约500枚卵),每组3个平行。受精后16 h在解剖镜下统计受精率(受精率=计数受精卵数/计数总卵数×100%)。

    • 4种饵料样本分别称取0.5 g并加入0.5 mL的PBS,用匀浆器充分匀浆后,3 000 r/min离心20 min,收集上清定容至10 mL,用于雌二醇的测量。

      雌二醇的检测采用双抗体夹心酶联免疫法,试剂盒为江苏雨桐生物科技有限公司产品,其灵敏度为0.027 pg/mL。按试剂盒步骤检测。(1)标准品的加样:设置标准品孔和样本孔,标准品孔各加不同浓度的标准品50 μL。(2)加样:分别设置空白孔和待测样本孔。在酶标包被板上待测样品孔中先加样品稀释液40 μL,然后再加待测样品10 μL(稀释5倍)。(3)加酶:每孔加入酶标试剂100 μL,空白孔除外。(4)温育:用封板膜封板后置37 °C温育60 min。(5)洗涤:小心揭掉封板膜,弃去液体,甩干,每孔加满洗涤液,静置30 s后弃去,如此重复5次,拍干。(6)显色:每孔先加入显色剂A 50 μL,再加入显色剂B 50 μL,轻轻震荡混匀,37 °C避光显色15 min。(7)终止:每孔加终止液50 μL,终止反应(此时蓝色立转黄色)。终止液15 min以内进行测定。(8)测定:以空白孔调零,450 nm波长依序测量各孔的吸光度(OD值)。每个样本3个平行空白重复取其均值。(9)用标准品在450 nm波长下的OD450绘制标准曲线。再根据样品的OD值根据标准曲线计算出样本中雌二醇含量。

    • 采用Excel 2010和SPSS 11.0软件统计分析数据,采用单因素方差分析(One-Way ANOVA)检验各组之间的差异显著性。实验数据为平均值±标准差,显著性水平定义为0.05。

    2.   结果
    • 在11~60日龄期间,丰年虫组的存活率为74.82%±5.12%,显著低于其他3组。实验期间我们将该组死鱼放置解剖镜下观察发现有腹水且胃中有大量不能消化的丰年虫壳可能是丰年虫组存活率低的主要原因。60日龄时水蚯蚓组的体长和体质量分别为(6.11±0.30) cm和(3.94±0.34) g,显著高于其他3组(表1)。

      实验组
      experimental group
      存活率/%
      survival
      体长/cm
      total length
      体质量/g
      weight
      水蚯蚓 92.12±0.48a 6.11±0.30a 3.94±0.34a
      红虫 96.47±0.58a 5.76±0.89b 3.31±0.68b
      浮游动物 96.67±0.65a 5.53±0.25bc 3.18±0.44b
      丰年虫 74.82±5.12b 5.30±0.20c 3.16±0.32b
      注:表中同列数据中不同肩注字母表示有显著性差异(P<0.05),相同字母或无字母表示差异性不显著(P≥0.05)。下同
      Note: In the same line, different letter superscripts mean significant differences (P<0.05), and the same letter superscripts or no letter superscripts mean no significant differences (P≥0.05); the same applies below

      Table 1.  Effects of various food on survival rate, total length, and weight at 60 days post-hatching

    • 60日龄时,YY超雄黄颡鱼外表均有雄性生殖突,但从性腺根据形态学和组织学上可划分精小叶完整的精巢、无精小叶的精巢、精巢和卵巢同时存在的兼性性腺。精小叶完整的精巢呈锯齿状(图版-1),切片HE染色观察有大量精原细胞SG (图版-2),而无精小叶的精巢外表细而光滑(图版-3),且精原细胞数目相对较少(图版-4)。兼性性腺中有肉眼可见的卵粒存在(图版-5),切片HE染色观察到精原细胞SG和初级卵母细胞PO同时存在(图版-6)。60日龄的YY超雄黄颡鱼性腺表型统计结果所示:红虫、丰年虫和浮游动物组的YY超雄黄颡鱼的性腺均为精小叶完整的精巢,而水蚯蚓组YY超雄黄颡鱼的性腺有75%为精巢和卵巢同时存在的兼性性腺,25%的为无精小叶的精巢(图2)。一年龄水蚯蚓组YY超雄黄颡鱼同正常雄性黄颡鱼一样存在生殖突,解剖后观察到卵巢和精巢同时存在,且精巢的精小叶不完整蜷缩成一团(图版-7);其他三组YY超雄黄颡鱼的性腺为饱满的乳白色精巢(图版-8)。

      Figure 1.  Statistics of gonad structure at at 60 day post-hatching

      Figure 2.  Standard curve of E2 ELISA

      Figure 图版.  The phenotype and histology of gonad of yellow catfish

    • 水蚯蚓组YY黄颡鱼精液的精子密度为2.97±0.74×107 个/μL,显著低于其他3组(表2)。将4组精液均用精子保存液稀释到8×106 个/μL用于受精实验。水蚯蚓组YY黄颡鱼受精率仅36.70%±4.05%并显著低于其他3组,其他3组受精率均高于85%。以上结果表明:早期投喂水蚯蚓导致黄颡鱼精子密度降低,且受精率变低。

      实验组
      experimental group
      受精率/%
      fertilization efficiency
      精子密度/(107 cell/μL)
      sperm density
      水蚯蚓 36.70±4.05c 2.97±0.74b
      红虫 92.66±0.71a 5.42±0.58a
      浮游动物 88.79±0.29ab 4.57±0.69 a
      丰年虫 85.38±0.56b 4.81±0.72 a

      Table 2.  The sperm density and fertilization rate of one-year-old YY yellow catfish

    • 雌二醇ELISA的标准曲线的相关系数为0.9857(图2),表明相关性较好。相同重量的4种饵料中,水蚯蚓的雌二醇含量最高,(14.67±0.51) pg/mL,但与浮游动物的雌二醇含量[(10.81±0.29) pg/mL]相差不大,而浮游动物组YY超雄黄颡鱼性腺发育正常,水蚯蚓组却出现兼性性腺(图3)。表明水蚓体内的雌二醇含量可能不是引起YY超雄黄颡鱼性腺雌性化的主要原因。

      Figure 3.  E2 concentration in different baits with equal weight

    3.   讨论
    • 水蚯蚓是环节动物门,寡毛纲一些种类的混合群体,由于蛋白质含量高,是水产苗种培育过程中常见的饵料。目前苗种培育过程中喂食的水蚯蚓主要源于人工养殖或天然水域。人工养殖的水蚯蚓主要通过有机污泥和发酵好的牛粪、猪粪或鸡粪等有机粪肥培育。天然水域的水蚯蚓主要生活在腐殖质丰富的水域中,往往含有大量的生活污水和工业废水。畜禽粪便等有机粪肥和腐殖质丰富的水域中通常有环境雌激素污染[15-16]。实验发现投喂有机粪肥培育的水蚯蚓造成大量YY超雄黄颡鱼出现精卵巢共存的兼性性腺,阻碍了全雄黄颡鱼产业的发展。

      杨世杰等[17]研究发现,用水蚯蚓投喂90 d的南方鲇为全雌,但同样喂食水蚯蚓90 d的YY超雄罗非鱼却未能逆转。董然然等[18]进一步用商业化水蚯蚓喂食10 d,即可得到全雌南方鲇。本研究用水蚯蚓喂食YY黄颡鱼20 d(11~30日龄) 有75%精巢和卵巢共存的兼性性腺和25%的为无精小叶的精巢,且受精率和精子密度均显著小于投喂丰年虫、红虫和浮游动物的YY超雄黄颡鱼。鱼类性别分化很容易受到外界因素的影响,尤其是在性腺分化的关键时期。黄颡鱼雌雄性腺分化解剖学上标志出现的时间上分析,在13日龄卵巢便开始分化,19日龄便形成卵巢腔,而精巢分化时间约40日龄,精小叶和输精管出现于55日龄[14]。本研究投喂水蚯蚓的时期(11~30日龄)正好在黄颡鱼卵巢性腺分化的关键时期。为研究水蚯蚓引起YY黄颡鱼雌性化具体原因,实验测量了相同重量的4种饵料中雌二醇含量,发现水蚯蚓的雌二醇含量为(14.67±0.51) pg/mL与浮游动物[(10.81±0.29) pg/mL]相差不大,水蚯蚓组的YY超雄黄颡鱼出现兼性性腺,而浮游动物组的YY超雄黄颡鱼性腺均发育正常。张晖等对多种环境雌激素对淡水鱼联合毒性作用进行了预测和评价[19]。基于张辉等[19]预测,我们发现水蚯蚓的雌二醇含量属于正常值。此外,水蚯蚓雌二醇含量(14.67±0.51 pg/mL)比逆转成YY生理雌鱼所需的雌二醇含量(200 mg/L)[3]低很多。以上分析表明,水蚯蚓体内的雌二醇含量可能不是引起YY超雄黄颡鱼性腺雌性化的主要原因。董然然等[18]利用高效液相色谱法检测3个不同地域捕捞的水蚯蚓体力的环境内分泌干扰物物质(EDCS),表明水蚯蚓体内含有双酚A、己烯雌酚、辛基酚和壬基酚,并用相应EDCS拌料投喂也能将南方鲇(Silurus metidionalis)逆转为全雌鱼。NP能够与生物体内的雌激素受体结合而产生雌激素效应使鱼雌性化。2010年,在武汉、重庆、南京和马鞍山的野生鲤体内,均检测出壬基酚(NP)、辛基酚(OP)[20]。研究表明,2种不同浓度的OP可引起斑马鱼的雌性化[21]。并且大量的研究显示,在鱼类性别分化时期,EDCs可以引起性逆转或雌性间性[22-26]。因此,水蚯蚓体内富集EDCS可能是导致YY黄颡鱼性腺异常的主要原因。

      综上所述,南方鲇和黄颡鱼都隶属于鲇形目,投喂水蚯蚓的南方鲇和YY黄颡鱼的性腺都受到不同程度影响,而YY罗非鱼却正常发育,可能鲇形目对水蚯蚓更为敏感。其他以水蚯蚓喂食的鱼类性腺是否受到影响需进一步研究确认。鲇形目两性生长差异较大如黄颡鱼、斑点叉尾鮰(Ictalurus punctatus)、南方鲇和乌苏里拟鲿(Pseudobagrus ussuriensis),进行单性育种可大幅度提升产量,水蚯蚓则为单性育种提供了新思路。水蚯蚓营养价值高,价格便宜等优点,但在亲本的苗种培育过程中使用水蚯蚓可能会导致鱼苗的性腺发育异常,成鱼的受精率和精子密度变低,需谨慎使用。丰年虫具繁殖速度快,可人工培育等优点,孵化不完全的卵或未滤干净的卵壳被仔鱼使用后,会阻塞消化道,甚至导致死亡。因此,在培育YY超雄黄颡鱼苗种时应该以浮游动物开口,若受其他原因干扰导致捕捞的浮游动物不足时,可用丰年虫代替,使用时需反复去除卵壳,等仔鱼口裂稍大时以商业化红虫培育几天,在此期间适量投喂微颗粒饲料,待转食性成功后再以不同粒径商业颗粒饲料进行工厂化培苗。

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