Effect on growth and reproduction of hormone immersed and masculinized fighting fish Betta splendens

To produce all-male progenies in the fighting fish, Betta splendens, six groups of fry were subjected to discrete immersion treatment at different 17α-methyltestosterone (MT) doses (viz. 100, 200, 500, 700, 900, and 1,000 μg/l) for a constant duration (3 hr/day) and frequency (second, fifth, and eighth day after hatching). The treatment at 900 μg/l led to 98% masculinization and 71% survival at sexual maturity. Treated groups, which showed significant deviation from the 1:1 sex ratio, were classified into two different series: S1 and S2. The groups that showed nearly cent-percent masculinization were classified as S1, and the other groups were classified as S2. The S1 males showed remarkably slower growth and attained 3.5 cm total length compared to 6.0 cm attained by a normal male. The S2 males attained 5.4 cm total length. Apart from these morphological defects, both S1 and S2 males suffered functional (decreased sperm count and sperm motility) and behavioral defects (incomplete embracing during mating) in their reproductive ability, leading to approximately 50% and 30% reduction in fecundity per mating, respectively. The cumulative fecundity loss suffered by the S1 male during its active reproductive phase is discussed. When normal and sex-reversed males were presented, a female preferred the former. Progeny testing of the sex-reversed males showed the occurrence of 12.75% males, indicating the possible role of autosomal genes in the sex determination mechanism of this species. Discrete immersion treatment at optimal/super-optimal doses ensured not only a higher percentage of masculinization, but also a higher frequency of homogametic males (XX)

Production and progeny testing of androgenetic rosy barb Puntius conchonius

Protocol for androgenetic cloning of the rosy barb, Puntius conchonius, with contrasting gray and golden strains is described. At the intensity of 4.2 W/m<SUP>2</SUP>, UV irradiation for 3.0 min inactivates the maternal genome in eggs of the gray barb. Following activation by the golden barb sperm, 24-min old eggs are shocked at 41&#176;C for 2 min to restore diploidy. Maternal genomic inactivation is confirmed by the (i) golden body color, (ii) karyotyping, and (iii) progeny testing of F<SUB>1</SUB>-F<SUB>3</SUB> progenies. Estimates of stage-specific mortality of haploid and diploid androgenotes indicate no change in the time scale or developmental sequence, when sperm of related strain is used for activation, and when haploid genome regulates the development. Survival of androgenetic clones remains constant for the F<SUB>1</SUB>, F<SUB>2</SUB>, and F<SUB>3</SUB> progenies and is about 15% and 7% at hatching and sexual maturity, respectively. Homozygosity of the androgenotes is shown to inflict greater mortality. Between F<SUB>1</SUB> and F<SUB>3</SUB> generations, the heterozygosity of the androgenetic clone is decreased, as evidenced by reduction in size hierarchy. Though the reproductive performance of the F<SUB>1</SUB>, F<SUB>2</SUB>, and F<SUB>3</SUB> supermales is superior to the normal ones, the realized fecundity remains equal around 80 progenies per brood. The 92 crosses involving 16 supermales and 10 normal dams yield 75-100% male progenies, confirming the possible operation of XX&#x2640;:XY&#x2642; sex determination system. The frequency of unexpected occurrence of female progenies is about 8%, the causes for which are discussed

GFP reporter gene confirms paternity in the androgenote Buenos Aires tetra, Hemigrammus caudovittatus

A protocol for successful induction of androgenetic cloning of the Buenos Aires tetra (BT), Hemigrammus caudovittatus, with contrasting gray and golden strains is described. At the intensity of 4.2W/m<SUP>2</SUP>, UV irradiation for 2.75 min totally inactivated the maternal genome in eggs of gray BT. Following activation by sperm of golden BT, the 25-min-old embryos were shocked at 41&#176;C for 2 min to restore diploidy. Interestingly, the hatching success of the haploid fry was always higher than that of the diploid fry, indicating that the enhanced homozygosity (Y<SUP>2</SUP>Y<SUP>2</SUP>) is more deleterious than haploidy. Maternal genomic inactivation was confirmed by (i) expression of green fluorescent protein (GFP) gene in the 6-16 hr old live haploid and aneuploid embryos, (ii) golden body color in the diploid fry and adult and (iii) progeny testing. Survival of androgenotes was 10% at hatching and 6% at sexual maturity. Reproductive performance of F<SUB>0</SUB> and F<SUB>1</SUB> males (Y<SUP>2</SUP>Y<SUP>2</SUP>) was superior to that of normal ones (X<SUP>1</SUP>Y<SUP>2</SUP>), but that of the F<SUB>0</SUB> and F<SUB>1</SUB> females (X<SUP>2</SUP>X<SUP>2</SUP>) was inferior to the control (X<SUP>1</SUP>X<SUP>2</SUP>). Of 21 crosses involving homozygous androgenetic (Y<SUP>2</SUP>Y<SUP>2</SUP>) males and heterozygous (X<SUP>1</SUP>X<SUP>2</SUP>) females, 7 of them (33%) produced 3-9% unexpected female progenies. But only a single cross (14%) generated 3-4% unexpected female progenies, when 7 pairs of homozygous androgenetic (Y<SUP>2</SUP>Y<SUP>2</SUP>) males and (X<SUP>2</SUP>X<SUP>2</SUP>) females were crossed. Hence, the paternal autosomes, inherited by the homozygous androgenetic female (X<SUP>2</SUP>X<SUP>2</SUP>), produced female progenies in significantly less number of crosses, also at lower frequencies than the crosses with heterozygous females (X<SUP>1</SUP>X<SUP>2</SUP>), which carried an equal number of paternal and maternal autosomes. However, progenies resulting from the cross between gray female (X<SUP>1</SUP>X<SUP>2</SUP>) and golden male (Y<SUP>2</SUP>Y<SUP>2</SUP>), after undergoing androgenesis, were males, with paternal chromosomes alone, indicating that the presence of Y<SUP>2</SUP>Y<SUP>2</SUP> appears to override the modifying effect of autosomes, but the paternal or maternal autosomes seemed to override the single Y<SUP>2</SUP> present with X<SUP>1</SUP> or X<SUP>2</SUP>, and induced the production of unexpected female progenies. Using Double sex Mab3 related transcription factor (DMRT 1)-specific primers, PCR analyses of the genomic DNA of the normal (X<SUP>1</SUP>Y<SUP>2</SUP>) and androgenetic males (X<SUP>1</SUP>Y<SUP>2</SUP>) produced two amplicons of 237 and 300 bp length. However, they were not detectable in the female (X<SUP>1</SUP>X<SUP>2</SUP>) genomic DNA, which amplified only one amplicon of 100 bp. Genomic DNA extracted from the 18 unexpected female progenies expressed the (X<SUP>1</SUP>Y<SUP>2</SUP>) genotype-specific banding pattern with two amplicons of 237 and 300 bp length and thereby confirmed that they were genotypic males. A partial sequencing of the male-specific sequence indicated that DMRT 1-specific primer was bound to the fragment of the genomic DNA of the male tetra, although the male-specific sequence of DMRT 1 was not completely detectable

Growth enhancement and food conversion efficiency of transgenic fish Labeo rohita

Three family lines of fast growing transgenic rohu Labeo rohita (rohu) were generated by electroporated-sperm-mediated transfer of the vectors harboring CMV promoter or grass carp β-actin promoter fused to endogenous rohu GH (rGH) cDNA. The gene transfer efficiency was 25%. The transgenic rohu (family line 1) with CMV promoter showed a growth enhancement of four times normal size, whereas those (family lines 2 and 3) generated with β-actin promoter grew 4.5 and 5.8 times faster than their respective control siblings. Southern analysis confirmed the transgene extrachromosomal (Te) persistence until the 60th week in family 1. The individuals of family lines 2 and 3, however, showed integration (Ti), as well as persistence as extarchromosomal copies (Te) until the age of 30 weeks. Mosaicism of the transgene was shown at the levels of its presence and expression. The ectopic expression of rGH mRNA was confirmed by RT-PCR. Feeding experiments revealed that the transgenic rohu ate food at a lower rate but grew more efficiently than their control siblings