Breeding of XX Females （XX♀）， YY Physiological Females （YY♀）， YY Supermales （YY♂） of Pureline Yellow Catfish （Pelteobagrus fulvidraco Richardson）

Qiankai TAN Zhifa TANG Zhixue TANG Xiuli CHEN Xiaolong WU Tonghui LU

AbstractMature wildtype yellow catfish （Pelteobagrus fulvidraco Richardson） individuals with excellent traits have been screened from the Yangtze River as broodstock to establish the germplasm bank of pureline yellow catfish by artificial gynogenesis technique and hormonal sex reversal method. Based on pure lines of yellow catfish， supermales and physiological females of yellow catfish were selected by GMT technique， hormonal sex reversal method and testcrossing to establish the germplasm bank of YY physiological females （YY♀）. The propagation system of male yellow catfish （XY♂） was established based on the combination of supermales （YY♂） and pureline female （XX♀） for largescale production of pureline yellow catfish males， which effectively overcome the disadvantages in the production of common fingerlings and male fingerlings using lowgrade fish with slow growth and small size at the bottom of the fish grader as broodfish， such as progeny germplasm degeneration， loss of growth vigor and reduction of breeding benefits. The technical route of breeding pureline YY physiological females （YY♀） and YY supermales （YY♂） laid the foundation for largescale production of environmentally friendly yellow catfish males with pure germplasm and strong stress resistance， and provided an efficient， stable， healthy， environmentally friendly， energysaving and incomeincreasing approach for sustainable development of yellow catfish breeding industry.

Key wordsYellow catfish （Pelteobagrus fulvidraco Richardson）； Pure line selection； XX females （XX♀）； YY physiological females （YY♀）； YY supermales （YY♂）

Received： September 20， 2018Accepted： October 28， 2018

Supported by Implementation Plan of Subsidy Project for the Reform and Construction of Grassroots Agricultural Technology Extension System.

Qiankai TAN （1974- ）， male， P. R. China， senior agronomist， devoted to research about aquaculture.

*Corresponding author. Email： eptqk@126.com.

It has been 20 years since yellow catfish （Pelteobagrus fulvidraco Richardson） has transformed from purely uncultivated to completely artificial cultivated. At present， some yellow catfish breeding farms sell large rapidgrowing adult individuals without germplasm screening， while preserving lowgrade fish with slow growth and small size at the bottom of the fish grader for one year of breeding as broodfish to produce common fingerlings or allmale fingerlings. Combined with inbreeding， the germplasm quality declined year by year. Therefore， it is necessary to establish the germplasm bank of pureline yellow catfish. In addition， the ratio of common yellow catfish females to males is 50%. The growth speed of yellow catfish males is 2-3 times that of females. Although allmale breeding has been rising in recent years， YY supermales （YY♂） for allmale production are in short supply and become expensive with uneven quality. Moreover， due to lack of detection techniques， it is difficult for ordinary nurseries and the majority of the households to detect and identify the parent YY♂ and male fingerlings （XY♂）. These factors become the bottleneck for the healthy development of yellow catfish breeding industry. Therefore， it is necessary to purify and rejuvenate pureline yellow catfish germplasms to produce yellow catfish females and supermales as male broodstock for largescale breeding of commercial fish.

Liu et al. produced YY supermales of yellow catfish from XY females via gynogenesis and obtained XY physiological females by hormonal sex reversal method. Subsequently， YY supermales were produced from XY physiological females via gynogenesis. Yellow catfish males were obtained by crossing between YY supermales and common XX females. There is certain controversy about the viability and fertility of YY supermales and the physiological females. So far， whether YY supermales and the physiological females are survivable and fertile has been concerned by researchers[1-10]. By hormonal sex reversal method and breeding technique， Yamamoto first confirmed the presence of VY supermales of Oryzias latipes. Through hormonal sex reversal， gynogenesis， androgenesis and breeding technique， YY supermales were obtained. It has been confirmed that YY supermales are survivable and fertile. The feminization of YY supermales is a key step for largescale breeding of YY supermales and a critical factor that restricts the production of yellow catfish males. However， realizing the feminization of YY supermales of yellow catfish has become a key factor restricting largescale production of YY supermales and males.

Materials and Methods

Establishment of germplasm bank

Mature wildtype yellow catfish broodstock with excellent traits were screened from the Yangtze River. After seven years of breeding， pureline YY supermales （YY♂） and YY physiological females （YY♀） of yellow catfish were successfully bred through bioengineering， cell engineering， genetic engineering techniques and genetic methods. At present， pureline yellow catfish supermales have been sold to different hatcheries with billions of subgenerations， increasing the income of thousands of farmers.

Experimental treatments and design

Overview of the test site

Enping City of Guangdong Province is located at 112.19 E， 22.12 N， belonging to the typical southern subtropical climate， with an average annual temperature of 23.1 ℃. The highest and lowest temperatures are 36.4 ℃ and 4.9 ℃， respectively. The annual rainfall is 2 559.9 mm. The maximum daily rainfall is 185.5 mm.

The experiment was carried out in Haifa Fish Farm of Shangnan Management District， Datian Town， Enping City， Jiangmen City， Guangdong Province. It covers an area of 100 000 m2， including a cement pool of 13 300 m2 and a hatching greenhouse of 1 500 m2. There are 8 universal circular hatching channels， 50 universal hatching incubators， 80 breeding pots， 8 parental storage ponds， and 20 selfflowing irrigation ponds.

Experimental design

The specific procedures are as follows：

（1） Collection of spawns： Wildtype yellow catfish females and males with large size and excellent traits were selected as broodstock from the Yangtze River for intensive cultivation. Wildtype yellow catfish females were injected with oxytocic hormone. At the effective time， spawns were collected artificially.

（2） Sperm inactivation： Mature allogeneic sperms （such as sperms of Silurus soldatovi meridionalis Chen， Leiocassis longirostris） were diluted in sperm preservation solution， irradiated using an ultraviolet light with a wavelength of 2 537 angstroms for 25 minutes to inactivate the genetic material of sperms， and preserved before use.

（3） Artificial insemination： Inactivated sperms and spawns were mixed evenly and added with 4 ‰ saline to activate sperms， thus stimulating fertilization and development of spawns.

（4） Cold shock treatment： Within 1-2 minutes after fertilization， fertilized eggs were treated by cold shock in 4 ℃ water for 15-20 minutes， to block the meiosis of eggs and discharge of secondary polar body， thus restoring gynogenetic diploids.

（5） Artificial hatching： After cold shock treatment， spawns were placed in 25-30 ℃ water and artificially hatched according to the normal method to obtain pureline female progenies XX♀.

（6） Production of physiological males： At five days after initial feeding of females， some individuals were separated and cultivated independently， which were transferred to physiological males XX♂ after 40 days of feeding with 17αmethyltestosterone bait （insect）. The physiological males XX♂ and yellow catfish females were cultivated independently to sexual maturity.

（7） Group expansion by crossing： Pureline yellow catfish females XX♀ were crossed with physiological males XX♂ to expand the group for largescale production of pureline females. After cultivation， all the females were preserved in the germplasm bank of pureline yellow catfish females.

Data processing

SPSS.17.0 software was used for analysis of variance， Ftest， and Duncans multiple range test. After test cross between candidate YY females and normal XY males， the sex ratio of the progenies was analyzed. Theoretically， the ratio of females to males of the progenies was 0∶N （N indicates the total number of test cross progenies）， suggesting that the probability of femalemale ratio （0∶N） was 95%-100%， and the candidate broodstock could be confirmed as YY females. Similarly， after test cross between candidate YY supermales and normal XX females， the sex ratio of the progenies was analyzed. Theoretically， the ratio of females to males of the progenies was 0∶N （N indicates the total number of test cross progenies）， suggesting that the probability of femalemale ratio （0∶N） was 95%-100%， and the candidate broodstock could be confirmed as YY males.

Results and Analysis

Establishment of the germplasm bank of pureline XX females （XX♀）

In view of the problems existing in the production of common fingerlings and male fingerlings of yellow catfish over the years， yellow catfish germplasms were purified and rejuvenated， to establish the pure lines by traditional methods （selecting breeding and cross breeding）， which requires 8-10 generations of mating. However， the gene locus can be completely homozygous by inhibiting the first cleavage via artificially inducing gynogenesis. The gender determination mechanism of yellow catfish is female homogamety. Therefore， the progenies produced are all females. Wildtype yellow catfish broodstock with large size and excellent traits were screened from the Yangtze River. Pureline female progenies XX♀ were produced through artificial gynogenesis. At four days after initial feeding， the progenies were cultivated independently and transferred to physiological males XX♂ by feeding with 17αmethyltestosterone bait. The physiological males XX♂ and yellow catfish females XX♀ were cultivated independently to sexual maturity. XX♀ and XX♂ were mated to expand the pureline female group （XX♀）.

Breeding of YY supermales （YY♂） and YY physiological females （YY♀） by GMT technique and test cross technique

Pureline XX females （XX♀） were crossed with wildtype males （XY♂） to obtain the hybrid progenies （XY♂∶XX♀=1∶1）. At five days after initial feeding of the hybrid progenies， 500 individuals were cultivated independently to sexual maturity. Another 500 individuals were transferred to physiological females （XY♀） by EE2 estradiol bait （insect） and cultivated independently to sexual maturity.

The hybrid progenies XY were separately mated with 10-20 physiological females to cultivate the progenies independently. If the female parent was a physiological female， among the progenies， supermales， males and females accounted for 25%， 50% and 25%， respectively. If the female parent was a normal female， the ratio of female progenies to male progenies was 1∶1. At five days after initial feeding， 500 individuals in each group were cultivated independently to sexual maturity. Another 500 individuals were transferred to physiological females by EE2 estradiol and cultivated independently to sexual maturity.

After test cross， YY supermales （YY♂） and YY physiological females （YY♀） were selected.

In the present study， based on the breeding of pureline females， highquality yellow catfish supermales and physiological females were bred by GMT technique， hormonal sex reversal method and test cross technique. The specific procedures are as follows：

Breeding of highquality yellow catfish males

Pureline XX females （XX♀） were crossed with wildtype yellow catfish males to obtain highquality XY♂ and XX♀ progenies.

Preparation of highquality physiological females （XY♀）

Five hundred highquality hybrid progenies were fed with fairy shrimp. After five days， 250 individuals were cultivated independently and fed with EE2 estradiol bait： The fairy shrimp were soaked in 150 μg/L EE2 solution for one hour. Live insects were filtered and fed three times a day for 45 consecutive days. Thus， the progenies were transferred to physiological females （XY♀） and fed with normal yellow catfish feed to sexual maturity. The remaining 250 individuals were cultivated independently to sexual maturity. When the males and females were distinguishable， the females were eliminated and males （XY♂） were preserved.

Breeding of YY males （YY♂） and YY physiological females （YY♀）

The seminal vesicles were collected from two welldeveloped highquality yellow catfish males， cut into pieces， diluted with sperm preservation solution， and stored in a 5-7 ℃ refrigerator.

About 10-20 welldeveloped pureline physiological females were selected， and injected with oxytocic hormone. Spawns were collected artificially and stored separately. Each individual was inseminated artificially with the above （XY♂） sperms. The fertilized eggs were hatched and cultivated independently. At five days after initial feeding， 500 progenies of each female were preserved， 50% of which were cultivated independently after artificial feminization. The remaining 250 progenies were cultivated independently according to the conventional method.

The above fingerlings were cultivated independently in small cages until the males and females could be distinguished. The proportion of males and females in each nontransferred group was investigated. If the males and females both accounted for 50%， the female parent was XX♀， and the female parent and progenies （including feminized progenies） were all eliminated. If the ratio of females to males was 1∶3， the female parent was XY♀， and there were 25% YY supermales among the progenies； among the progenies of the female， nontransferred individuals and transferred females were cultivated independently to maturity.

Screening of pureline YY supermales （YY♂） and YY physiological females （YY♀） by test cross

The candidate males （YY♂） were mated with common females （XX♀） （1∶1）. The parents and progenies were cultivated independently until the males and females could be distinguished. If the progenies were all males， the male individual was YY supermale （YY♂）， otherwise it was eliminated. The candidate physiological females （YY♀） were mated with common males （XY♂）. The progenies were cultivated independently. If progenies were all males， the female individual was YY physiological female （YY♀）， otherwise it was eliminated.

Establishment of the germplasm bank of pureline YY supermales （YY♂）

Physiological females （YY♀） were crossed with YY supermales （YY♂）， and the progenies were 100% supermales. At five days after initial feeding， some progenies were fed with EE2 estradiol bait for 45 days， which were transferred to YY physiological females （YY♀）. The physiological females and nontransferred YY supermales （YY♂） were cultivated to sexual maturity， which were crossed for largescale breeding of supermales. The supermales were stored in the germplasm bank.

The screened YY supermales （YY♂） and YY physiological females （YY♀） were mated artificially. The progenies were all supermales. In accordance with the above method， some YY supermales （YY♂） were artificially transferred to YY physiological females （YY♀） and cultivated to maturity for group expansion， which were preserved in the germplasm bank of yellow catfish supermales.

Qiankai TAN et al. Breeding of XX Females （XX♀）， YY Physiological Females （YY♀）， YY Supermales （YY♂） of Pureline Yellow Catfish （Pelteobagrus fulvidraco Richardson）

Establishment of the propagation system of pureline yellow catfish males

Yellow catfish supermales （YY♂） and females （XX♀） were crossed for largescale artificial propagation of pureline yellow catfish males （XY♂）. When there were enough yellow catfish supermales （YY♂） and mature female broodstock （XX♀） in the germplasm bank， male yellow catfish fingerlings could be propagated massively and supplied to the households. Generally， one supermale （YY♂） could be inseminated with 1.0-3.5 million spawns of pureline female yellow catfish. If the broodstock was well cultivated， both the general fertilization rate and hatching rate could be above 95%.

Discussions

Germplasm bank of pureline XX females （XX♀）

Establishment of pureline yellow catfish germplasm bank

Mature wildtype yellow catfish broodstock with large size and excellent traits were screened from the Yangtze River. Pureline female progenies （XX♀） were produced through artificial gynogenesis. By hormonal sex reversal， physiological males （XX♂） were obtained and cultivated to sexual maturity for largescale propagation. Pureline yellow catfish females （XX♀） were propagated massively as the female parent and cultivated to sexual maturity.

Establishment of pureline supermale yellow catfish germplasm bank and genetic testing laboratory

On the basis of successfully established pureline yellow catfish germplasm bank， pureline YY supermales （YY♂） and YY physiological females （YY♀） were bred using pure lines by GMT technique， test cross technique and hormonal sex reversal method. Through crossing and group expansion， YY♂ broodstock were obtained for largescale propagation and production of pureline male yellow catfish fingerlings.

In order to prevent gene mutation of very few progenies during cell division， common males （XY♂） might be mixed into supermales， which affected the quality of supermale group. All the supermales （YY♂） used for production of male yellow catfish were genetically detected to ensure that all the males used in production were supermales.

Fig. 1Propagation system of pureline yellow catfish males

Establishment of the propagation system of pureline yellow catfish males （male yellow catfish breeding group）

When enough pureline supermales and females were stored， pureline males could be produced according to sales prospects， so as to meet the demand for fingerlings.

Advantages of the established breeding system

Compared with existing techniques， "breeding pureline yellow catfish females and supermales for largescale production of male yellow catfish" had various advantages as follows：

（1） All the parents used for the production of yellow catfish progenies were pure lines purified and rejuvenated by cell engineering and genetic breeding techniques. The genetic loci were completely homozygous， so the propagated progenies were pure lines.

（2） Pureline females were used as female parents to produce male yellow catfish， which exhibited various advantages such as pure germplasm， obvious growth vigor， strong resistance to diseases and stresses， low feed coefficient， environment friendly and high food safety factor compared with lowgrade fish， thereby greatly improving the economic and social benefits of commercial fish farming.

（3） This study provided a practical technical route for sustainable development of yellow catfish breeding industry and production of sufficient pureline YY supermales， and provided the material basis for producing male yellow catfish according to sales prospects.

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