| The loach M. anguillicaudatus (Cobitidae; Cobitinae; Misgurnus), and large scale loach P. dabryanus (Cobitidae; Cobitinae; Paramisgurnus) had been cited as one of the aquaculture species with a high commercial value. The diploid (2n=50), tetraploid (2n=100) loach and large scale loach (2n=48) are common in wild populations of China. So, these species are very useful in producing triploid by crossing between diploid and tetraploid, which is very important for aquaculture. The purposes of this study as follows, diploid (D) and tetraploid (T) loach (M.anguillicaudatus), large scale loach (P. dabryanus) (P) and their reciprocal hybrids were produced by artificial fertilization. Nine genetic crosses were made. These were (♀×♂):D×D, D×T, D×P, T×D, T×T, T×P, P×D, P×T and P×P. Then, the growth performance, reproduction and cytology of the progenies were studied. The main objectives of this study include:1. The growth performance and reproduction of the progenies(1) Fertilization and relative hatching rates:The highest fertilization rate was 93.10% (P×P), and significantly higher than other groups (P<0.05). The group (D×T) had a lowest fertilization rate (66.62%), significantly lower than other groups (P<0.05). And the fertilization rates of other groups in order were P×D>P×T>D×P>T×P>D×D> T×D>T×T. The highest relative hatching rate was 78.42%(T×T), and significantly higher than other groups (P<0.05). The group (PXT) had a lowest relative hatching rate (55.91%), significantly lower than other groups (P<0.05). The result showed that the fertilization capacity of sperm from tetraploid loach was lower than those of diploid loach and large scale loach, and the fertilization capacity of eggs form large scale loach was higher than others. But all of the crosses had lower relative hatching rates, with the large scale loach as the female parent.(2) Survival rate:The highest survival rate was 52.77%(D×D), followed by 50.31% (P×P). The lowest survival rate were P×D (19.05%), D×P (17.89%) and P×T (15.44%). It was found that all of the intergeneric crosses had lower survival rate, and the survival rate of reciprocal crosses between diploid and tetraploid loach had a medium level. In conclusion, D×D and P×P had the advantage of survival rate.(3) Growth performance:Under the similar rearing conditions, P×P showed significant growth advantage, total length and body weight were significantly higher than others. The average total length and body weight was 142.34 mm and 9.74 g at 46weeks, respectively. All of the crosses had lower growth rate, with the diploid loach as the female parent. It revealed that the possible existence of maternal effects on growth performance, because of the diploid loach generally have a smaller size, but the large scale loach generally grows to a larger size, has a faster growth rate. In conclusion, P×P had the advantage of growth rate.(4) Gonadal development:Comparison of gonadal development among different cytotypes of the progenies on 46 weeks. The results revealed that the gonad of TXT and P×P were the most mature, followed by D×D, D×T and T×D. The intergeneric crosses between M. anguillicaudatu and P. dabryanus showed the tardy gonadal development. Although all the progenies had different gonad maturation, they could produce mature gametes from the histological section.2. The cytology studies of the progenies(1) Erythrocyte and its nucleus size:Blood smears of the progenies were prepared, stained with Wright-Giemsa fluid. The major axis and minor axis of both the erythrocyte and its nucleus were recorded under oil magnification. Area and volume of both the erythrocyte and nucleus were calculated by following the formula for oblate spheroids or ellipsoids. Through statistical analysis, the results showed that the size will not increased in proportion, when the level of ploidy was increased. Among them, the erythrocyte volume of the triploid T×D and T×P was 525.91μm3 and 581.83μm3, respectively. However, the erythrocyte volume of the tetraploid T×T was 523.36μm3. It was found that the erythrocyte volume of triploid is larger than tetraploid. Therefore, the erythrocyte size can not be used as a basis to determine the ploidy of loach. However, the nucleus area and volume can reflect the changes in ploidy. The ratio of nucleus area and volume among diploid, triploid and tetraploid was about 2.00:2.50:2.98 and 2.00:2.61:3.34, respectively. Although there was no present theoretical 2:3:4, all of the progenies of nuclear size were increased with the increasing of ploidy. Therefore, it is concluded that the nuclear size measurement can be used as a good Methods for identification of ploidy status in M. anguillicaudatus(2) DNA content:The DNA content and ploidy status of the progenies were determined by flow cytometry. The CRBC (chicken red blood cell) and diploid M. anguillicaudatus red blood cell were used as an internal standard, respectively. We acquired the same result by the two ways:D×D, D×P, P×D and P×P were diploid, D×T, T×D, P×T and T×P were triploid, T×T was tetraploid. The ratio of relative DNA contents of the three types was approximately 2:3:4. The DNA contents of the progenies were calculated by using chicken erythrocytes DNA contents (2.50 pg/N) as standard, and the values were 3.16 pg/N,5.08 pg/N,3.11 pg/N,4.87 pg/N,6.73 pg/N,4.98 pg/N,2.86 pg/N, 4.98 pg/N and 3.03 pg/N, respectively. In conclusion, flow cytometry is a sensitive and rapid method for examining the DNA content and ploidy status of fish.(3) Chromosome counts and karyotype:The chromosome specimens of the progenies were obtained by the PHA colchicine hypotonic-air drying technique. Then, the karyotypes were analyzed and compared, the results showed that, D×D has 2n=50 chromosomes, consisting of 8 metacentrics (m),6 submetacentrics (sm) and 36 telocentrics (t) chromosomes. PXP has 2n=48 chromosomes, consisting of 12 m,4 sm and 32 t. Both of the two species have a fundamental arm number (NF) of 64. T×T has 4n=100 chromosomes, consisting of 16m,12sm,72t, NF=128. D×P and P×D are 2n=49, 10m+5sm+34t, NF=64; D×T and T×D are 2n=75,12m+9sm+54t, NF=96; T×P and P×T are 2n=74,14m+8sm+52t, NF=96. It was found that the karyotypes of diploid and triploid hybrids are intermediate between the parental species, consisting of a haploid maternal set and a haploid paternal set. |
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