Channel catfish ovarian fluid differentially enhances blue catfish sperm performance.

For externally fertilizing fishes, interactions between male and female gametes have been shown to have remarkable impacts on sperm performance. Ovarian fluid (OF) and its ability to alter the swimming behavior of fish sperm makes it a determining factor of fertility. With the expansion of channel catfish (Ictalurus punctatus) ♀ × blue catfish (Ictalurus furcatus) ♂ hybrid aquaculture, it is essential to understand the impacts during fertilization and the magnitude such gametic interactions have on sperm performance and subsequent male fertility potential. This study was conducted to address the following: 1) activate blue catfish sperm with/without channel catfish OF to determine impacts on sperm performance and 2) assess if sperm behave differently when activated in the OF from individual females. Sperm (n = 4 males) were activated without OF (control) and with diluted OF from unique females (n = 6), creating 24 experimental crosses. Sperm motility (%), velocity (VCL), and longevity were analyzed using computer assisted sperm analyses software. With OF incorporated in the activation media, sperm velocity was significantly higher than the control at 10, 20, and 30 s post-activation. OF did not have an impact on motility for any females at 10 s and 20 s post-activation but became significantly higher than the control at 30 s. In all cases, OF treatments greatly increased longevity. Male × female interactions were highly significant, such that motility, velocity, and longevity were dependent on specific male-female pairs. This information shows that OF should be incorporated in aquatic media to simulate natural spawning conditions and accurately assess the fluid mechanics of sperm propulsion for each male. Additionally, there are mechanisms that drive gamete interactions that need to be explored further, which may improve selection of male-female pairs for in-vitro fertilization. On a broad scale, our results also help to shed light on the complexities of fertilization and fish reproduction overall, which may have implications for recruitment variability and recovery strategies of threatened and/or endangered freshwater species.

Quantitative trait loci map for growth and morphometric traits using a channel catfish x blue catfish interspecific hybrid system.

Head length, head depth, head width, body depth, body width, caudal depth, and caudal width and total length and BW were measured for 71 backcross full sibs between the interspecific backcross F1 (female channel catfish [Ictalurus punctatus] × male blue catfish [Ictalurus furcatus]) female × blue catfish male. Body measurements were corrected for both size and the relationship between relative body shape and size, which is critical but usually ignored in fish research. Amplified fragment length polymorphism analysis was used for construction of a QTL map with 44 linkage groups. Eleven of 44 linkage groups had at least 1 significant QTL (P ≤ 0.05) and 11 of 44 at P = 0.10. Linkage group 19 was unique as it had multiple QTL for every trait measured, except for caudal width for which no QTL was identified on any linkage group. Approximately half of the markers measured were associated with positive effects (increase in size) on the traits and half had negative effects (decrease in size). Linkage groups 5, 9, 18, 20, 39, and 40 were significant for multiple traits and always had a trait negative effect. Total length is represented on the map by the most linkage groups and the most markers. The linkage relationships found among BW, total length, and the 7 morphometric traits indicated that multiple trait marker-assisted selection to simultaneously increase BW body depth, body width, and caudal depth while decreasing the head traits with the goal to increase body weight and carcass yield would be very difficult. Multiple genetic enhancement approaches would likely be needed to simultaneously improve BW and body conformation.

Differential gene expression in the brain of channel catfish ( Ictalurus punctatus) in response to cold acclimation.

Aquatic ectotherms can adapt to a wide range of temperature changes, but the molecular mechanisms that underlie this adaptability are not well understood. We identified genes that are differentially expressed in the catfish ( Ictalurus punctatus) brain using a cDNA microarray approach to gain an initial understanding of adaptation to low temperature. Among 660 genes analyzed, 61 were differentially expressed when compared at 12 degrees C and 24 degrees C. Gene induction was rapid, occurring within 2 h of the temperature shift. The major categories of differentially expressed genes included (1) genes for chaperones such as Hsp70 and Hsp70/Hsp90 organizing protein; (2) genes for transcription factors and gene products involved in signal transduction pathways such as zinc-finger proteins, calmodulin kinase inhibitor, the nuclear autoantigen SG2NA, interferon regulatory factor 3, and inorganic pyrophosphatase; (3) genes involved in lipid metabolism such as TB2 and acyl CoA binding protein; and (4) genes involved in the translational machinery such as ribosomal proteins. Some genes were induced transiently, whereas others were induced in an enduring fashion. Several genes, primarily ribosomal protein genes, were down regulated, indicating reduced metabolic activities after extended incubation at the low temperature. Thus channel catfish respond to low temperature by adjusting expression of a large number of genes. The rapid induction of proteins involved in signal transductions and chaperones suggests that both de novo synthesis of cold-induced proteins and modification of existing proteins are required for adaptation and tolerance of catfish to low environmental temperature.

Effect of rainbow trout growth hormone complementary DNA on body shape, carcass yield, and carcass composition of F1 and F2 transgenic common carp (Cyprinus carpio).

The effect of rainbow trout growth hormone complementary DNA on body shape, dress-out yield, and body composition were assessed in the F1 and F2 generations of transgenic common carp (Cyprinus carpio). All measurements were compared with those for nontransgenic full-sibling common carp in their respective families, and the fish were communally evaluated in earthen ponds. The body weight and length were highly correlated (P <0.01) in both genotypes in all the families. Head morphometrics were negatively correlated (P <0.05) to weight and length of the fish. Various head, body, and caudal traits grew disproportionately faster in transgenic fish in both generations. The altered body shape of transgenic fish resulted in improved dressing percentage in the F2 generation. The carcass composition of transgenic muscle had a lower percentage of (P <0.01) moisture and lipids and higher (P <0.01) percentage of protein in both generations. Six of the 18 amino acids analyzed in F1 transgenic common carp muscle were higher F1 (P <0.05) than the control genotype; however, amino acid ratios were minimally changed. Also, the fatty acid profiles of both genotypes were minimally altered. Higher histidine and lysine ratios in the diet are recommended for maximum growth and health of transgenic common carp in intensive culture systems on the basis of essential amino acid ratios.

The skeletal muscle alpha-actin gene of channel catfish (Ictalurus punctatus) and its association with piscine specific SINE elements.

The alpha-actin gene of channel catfish (Ictalurus punctatus) was cloned and sequenced. The gene has a similar organization and exhibited a high level of sequence similarity to those from other vertebrate animals. The upstream region of the alpha-actin gene included a TATA box, a CAAT box, three E-boxes, and a CArG box. Nested deletion segments containing these transcriptional motifs were fused to the reporter gene chloramphenicol acetyl transferase (CAT). Transfection of the clones into C2C12 cells indicated that all these motifs are required for transcriptional activities. The channel catfish alpha-actin gene is associated with two distinct short interspersed repetitive elements (SINEs). The first SINE element showed high levels of sequence similarity to the zebrafish Mermaid element, while the second SINE element is not similar to the Mermaid element except for an 8bp sequence CCCCGTGC suggesting their evolutionary linkage. However, the second SINE element appeared to co-exist with the Mermaid element in most cases and therefore was designated as the Merman element. Approximately 9000 copies and 1200 copies of the Mermaid and Merman elements exist per haploid channel catfish genome, respectively. BLAST searches indicated that both the Mermaid and the Merman elements were frequently associated with gene sequences, mostly those of aquatic animals, suggesting their evolutionary origin in association with aquatic organisms and their function in shaping the evolution of genomes in aquatic animals.

Transcribed dinucleotide microsatellites and their associated genes from channel catfish Ictalurus punctatus.

The presence of trinucleotide microsatellites within genes is a well-known cause for a number of genetic diseases. However, the precise distribution of dinucleotide microsatellites within genes is less well documented. Here we report 15 unique cDNAs containing dinucleotide repeats from the channel catfish Ictalurus punctatus. Gene identities of nine of the 15 cDNAs were determined, of which three encode structural genes, and six encode regulatory proteins. Five cDNAs harbored dinucleotide repeats in the 5' untranslated region (5'-NTR), nine in the 3'-NTR, and one in the coding region. The presence of these transcribed dinucleotide repeats and their potential expansion in size within coding regions could lead to disruption of the original protein and/or formation of new genes by frame shift. The low number of dinucleotide repeats within coding regions suggests that they were strongly selected against. All the transcribed microsatellite loci examined were polymorphic making them useful for gene mapping in catfish.

Transcriptional activities in the pituitaries of channel catfish before and after induced ovulation by injection of carp pituitary extract as revealed by expressed sequence tag analysis.

Expressed sequence tag (EST) analysis was adopted to address physiological changes after injection of carp pituitary extract for induction of ovulation. ESTs were analyzed from cDNA libraries constructed from mRNA isolated from channel catfish (Ictalurus punctatus) pituitaries before and after induction of ovulation by injection of carp pituitary extract. One hundred randomly picked clones were analyzed. Of the sequences generated, a large percentage (59%) of ESTs were identified as known genes by identity comparisons. These 59 clones of known gene products represent transcriptional products of 30 genes. The 41 clones of unknown gene products represent 33 genes. Expression of gonadotropin (GtH) alpha-subunit (149%) and prolactin (176%) was slightly enhanced as a result of induced ovulation. Large increases in frequencies of several peptide hormones were observed as a result of induced ovulation: GtH beta-I, 486%; GtH beta-II, 933%; growth hormone, 393%; proopiomelanocortin (POMC), 345%. POMC represented about 21% of all transcriptional activity in the pituitaries after induced ovulation. This is the first study addressing physiological changes after injection of carp pituitary extract, a procedure widely used in catfish hatcheries.

Characterization of an A/T-rich family of sequences from channel catfish (Ictalurus punctatus).

A family of highly repetitive DNA sequences referred to as Xba elements was identified and characterized from the channel catfish (Ictalurus punctatus) genome. The Xba elements represent about 5% to 6% of the total genomic DNA of the channel catfish. The Xba elements are distributed specifically in the channel catfish and blue catfish (I. furcatus), but not in closely related species such as white catfish (Ameiurus catus) and flathead catfish (Pylodictus olivaris). These Xba elements are arranged as head-to-tail tandem repeats. Seven sequences were sequenced. They are A/T-rich (over 65%). Each sequence contains four copies of the ATTA repeat and eight copies of (A)3-6 GT/TG motifs whose function is not known. Each unit of the repeats is 325 bp from the Kansas strain, and 321 pb from the Auburn and Stuttgart strains of channel catfish. The Xba elements are conserved in length within a specific strain, and highly conserved in sequence identity. Sequence identity is more conserved among copies isolated from the same strain than from different strains. The sequence length polymorphism among strains may be useful for identification of strains by polymerase chain reaction analysis. Many features of these elements could make them potentially important for development of homologous recombination expression vectors and position-independent expression vectors.

Inheritance and usefulness of AFLP markers in channel catfish (Ictalurus punctatus), blue catfish (I. furcatus), and their F1, F2, and backcross hybrids.

Eight primer combinations were used to investigate the application of amplified fragment length polymorphism (AFLP) markers in catfish for genetic analysis. Intraspecific polymorphism was low among channel catfish or blue catfish strains. Interspecific AFLP polymorphism was high between the channel catfish and blue catfish. Each primer combination generated from 70 to more than 200 bands, of which 38.6 75.7% were polymorphic between channel catfish and blue catfish. On average, more than 20 polymorphic bands per primer combination were produced as quality markers suitable for genetic analysis. All AFLP markers were transmitted into channel catfish x blue catfish F1 hybrids, except rare markers that were heterozygous in the parents and therefore were segregating in F1 hybrids. The two reciprocal channel catfish x blue catfish F1 hybrids (channel catfish female x blue catfish male; blue catfish female x channel catfish male) produced identical AFLP profiles. The AFLP markers were inherited and segregated in expected Mendelian ratios. At two loci, E8-b9 and E8-b2, markers were found at significantly lower frequencies than expected with F2 and backcross hybrids which had been selected for increased growth rates. The reproducibility of AFLP was excellent. These characteristics of the catfish AFLP markers make them highly useful for genetic analysis of catfish, especially for construction of genetic linkage and quantitative trait loci maps, and for marker-assisted selection.

Gonadotropin alpha-subunit glycoprotein from channel catfish (Ictalurus punctatus) and its expression during hormone-induced ovulation.

Complementary DNA (cDNA) encoding the channel catfish (Ictalurus punctatus) gonadotropin (GTH) alpha-subunit glycoprotein was cloned by polymerase chain reaction (PCR) from a plasmid library made from pituitary RNA. Complete cDNA cloning was achieved by carrying out two PCR reactions: one with an upstream sense primer plus the universal sequencing primer, located downstream of the poly(A) sequence of the cDNA in the plasmid vector, to amplify the downstream portion of the cDNA; the other with a downstream antisense primer plus the reverse-sequencing primer, located upstream of the very 5' end of the cDNA sense strand in the plasmid vector, to amplify the upstream portion of the cDNA. The two amplified fragments overlapping about 70 bp. Nucleotide sequence analysis revealed that the catfish GTH alpha-subunit was 658 bp encoding 116 amino acids and harboring a 5' nontranslated region (NTR) of 42 bp and a 3' NTR of 265 bp. The deduced amino acid sequence of the catfish GTH alpha-subunit is highly conserved with those from other cloned teleost GTH alpha-subunits. The GTH alpha-subunit was highly expressed even before induction for ovulation in females during spawning season. Administration of carp pituitary extract (a spawning-inducing reagent) induced only 1.4-fold higher expression of the GTH alpha-subunit RNA, but included very rapid egg maturation and ovulation. This unexpected result indicated that the GTH alpha-subunit may not be the limiting factor for ovulation and spawning, which may be regulated by the change of proportional coupling of the GTH alpha-subunit with specific beta-subunit during hormone-induced ovulation.

Effects of sperm concentration and egg number on fertilization efficiency with channel catfish (Ictalurus punctatus) eggs and blue catfish (I. furcatus) spermatozoa.

The mean sperm concentration of 10 blue catfish (Ictalurus furcatus ) was 1.03 x 10(10) per gram of testis. Testis weighed 3.9 and 17.2 g, with a mean of 6.6 g per fish. Fertilization rate of channel catfish (Ictalurus punctatus ) eggs fertilized with 5.00 x 10(4) to 1.20 x 10(7) blue catfish spermatozoa per egg was 17 to 87%, with an overall mean of 65%. Sperm concentrations of 5.0 x 10(4)/egg exhibited a lower, 16.6% (P < 0.05) fertilization rate than higher sperm concentrations (1.25 x 10(5) to 1.20 x 10(8)/egg). Batches of 450, 2,000, 5,000, 8,000 and 11,000 eggs were similarly fertilized with various sperm concentrations. Mean fertility rate ranged from 25 to 67%, with an overall mean of 53%. The largest egg mass produced the lowest (P < 0.05) fertilization rate. A combination of 450 eggs per batch and 5.0 x 10(5) to 1.20 x (8) sperm per egg produced the highest rate of fertilization (67 to 87%).

Transgenic fish and its application in basic and applied research.

Since 1985, transgenic fish have been successfully produced by microinjecting or electroporating desired foreign DNA into unfertilized or newly fertilized eggs using many different fish species. More recently, transgenic fish have also been produced by infecting newly fertilized eggs with pantropic, defective retroviral vectors carrying desired foreign DNA. These transgenic fish can serve as excellent experimental models for basic scientific investigations as well as in biotechnological applications. In this paper, we will review the current status of the transgenic fish research and its potential application in basic and applied research.

Structure of the channel catfish (Ictalurus punctatus) growth hormone gene and its evolutionary implications.

A DNA fragment of 1.6 kilo base pairs (kb), encoding part of the channel catfish (Ictalurus punctatus) growth hormone (GH) gene, was generated by the polymerase chain reaction (PCR) using 2 degenerate synthetic oligonucleotides (30 and 33 mer) derived from the N- and C-terminal amino acid sequences of the catfish GH polypeptide as amplification primers and with catfish genomic DNA as a template. This DNA fragment was used as a probe for the isolation of a catfish GH gene from a genomic library constructed in a lambda phage cloning vector, lambda Dash II. Three positive clones were isolated, and their complete nucleotide sequences were determined. Nucleotide sequences from clones 1 and 3 were identical, whereas clone 2 had 2 base substitutions. The gene spans approximately 3 kb and is comprised of 5 exons and 4 introns. The initiation codon, the termination codon, and the canonical polyadenylation sequence were identified. The amino acid sequence deduced from the predicted coding region of the gene is in agreement with that of the native GH polypeptide sequence. A sequence (TATAAAA) matching the TATA box consensus sequence was located at nucleotide positions -30 to -23. Furthermore, 2 sequences corresponding to the mammalian Pit-1/GHF-1 binding sites (consensus sequence TT[AA]TATNCAT) were identified in the 5' flanking region starting at positions -113 and -134. Another sequence (GTACCAGTGA) conserved among the GH genes of the channel catfish and other known animal species was also identified at position -220. The biological functions of this sequence remain to be determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression and inheritance of RSVLTR-rtGH1 complementary DNA in the transgenic common carp, Cyprinus carpio.

Transgenic common carp, Cyprinus carpio, possessing the long terminal repeat (LTR) sequence of avian Rous sarcoma virus (RSV) fused to the rainbow trout (rt) growth hormone (GH1) complementary DNA (cDNA) were produced by microinjection. Initial studies showed that the transgenic common carp transmitted the foreign DNA to a significant fraction of their progeny in three of four crosses of transgenic males with control females. These progeny grew 20 to 40% faster than their nontransgenic full siblings. In this study, additional experiments were conducted to evaluate inheritance and expression of the foreign GH gene in transgenic common carp, and the growth performance of these transgenic fish. Four P1 (parental generation produced by microinjection) x nontransgenic controls, four P1 x P1, and one P1 x F1 matings of transgenic carp containing RSVLTR-rtGH1 cDNA were made. The percentages of transgenic progeny resulting from these matings were: 0, 32, 42, 100 (4 progeny only), 21, 21, 31, 30, and 23%, respectively. All crosses except 1 siblot (control x P1) exhibited progeny ratios below the expected 50 or 75% transgenic. These results indicate that most of these transgenic P1 had the foreign gene in their germ line but were mosaics, and at least one transgenic individual did not have the RSVLTR-rtGH1 cDNA in the gonadal tissue. Both P1 and F1 transgenic fish produce trout growth hormone mRNA and polypeptide as determined by reverse transcription polymerase chain reaction amplification, RNA dot-blot hybridization, and radio-immunobinding assay. Growth response by families of F1 transgenic fish to the addition of rtGH1 cDNA varied widely.(ABSTRACT TRUNCATED AT 250 WORDS)

Transfer, expression, and inheritance of salmonid growth hormone genes in channel catfish, Ictalurus punctatus, and effects on performance traits.

We examined expression and inheritance of salmonid growth hormone genes RSVLTR-rtGH1 cDNA and RSVLTR-csGH cDNA, transferred to channel catfish (Ictalurus punctatus) by microinjection. One to 9 copies of the foreign DNA were inserted in either head-to-tail tandem array at single insertion sites or single copies at multiple insertion sites. All P1 transgenic catfish evaluated produced salmonid growth hormone regardless of the construct. Five P1 x P1 matings were accomplished. The spawning rate and fertility of these P1 transgenics in artificial spawning conditions were comparable to those of normal channel catfish. In two of three years, 100% spawning and 100% hatch were obtained. Percent transgenic progeny observed in the five matings were 20, 52, 7, 47, and 0%, which was lower (P < 0.001, chi 2) than the 75% inheritance expected assuming the P1 brood stock had at least one copy of the foreign gene integrated and were not mosaics in the germ line. At least 7 of 10 P1 were mosaics, and a minimum of 2 of 10 P1 did not possess the salmonid growth hormone genes in their germ line. P1 transgenics grew at the same rate as their nontransgenic full siblings, which is not surprising because the P1 were mosaics. F1 transgenic progeny in two families possessing RSVLTR-csGH cDNA grew 26% faster, to 40 to 50 gm, than their nontransgenic full siblings when evaluated communally. One F1 progeny group produced by RSVLTR-rtGH1 cDNA x RSVLTR-csGH cDNA mating and one F1 progeny group (parents either RSVLTR-rtGH1 cDNA or RSVLTR-csGH cDNA) grew at the same rate as normal full siblings when grown communally to 25 gm and 60 mg, respectively. In families where F1 progeny grew faster than controls, the range in body weight and coefficient of variation for the transgenic full siblings were less than those for controls. In families where F1 progeny grew at the same rate as controls, range in body weight and coefficient of variation were similar for transgenic and normal individuals. The percent deformities observed in P1 transgenics (13.6%) was higher (P < 0.05) than in microinjected P1 nontransgenics (5.1%). Percent deformities in transgenics and control F1 channel catfish was not different (p > 0.05; 0.5 and 2.8%, respectively).

Gene transfer, expression and inheritance of pRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus).

A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.

Effects of inbreeding on economic traits of channel catfish.

Inbred channel catfish (Ictalurus punctatus) were produced from two generations of full-sib matings to study the effect of inbreeding on reproduction, growth and survival. A randomly mated control line was propagated from the same base population to be used for the evaluation of the inbred fish. First generation inbred (I1) and control (C1) lines comprised five full-sib families each. Second generation inbred (I2) and control (C2) lines were produced by mating each male catfish from the I1 or C1 line to two females in sequence, one from the I1 and one from the C1 line. The design also produced two reciprocal outcross lines to be compared to their contemporary inbred and control lines. The coefficient of inbreeding for the inbred line increased from 0.25 in generation 1 to 0.375 in generation 2. The inbreeding coefficient was zero for all other lines. The resulting fish were performance tested in two locations, Tifton, Georgia and Auburn, Alabama and no genotype-environment interactions occurred. Results indicated that one generation of inbreeding increased number of days required for eggs to hatch by 21%, but did not significantly influence spawn weight or hatchability score. However, inbred females produced more eggs/kg body weight than control females. Two generations of full-sib mating in Georgia did not depress weight when expressed as a deviation to random controls but was depressed 13-16% when expressed as a deviation to half-sib out-crosses. Second generation inbreds produced in Alabama exhibited a 19% depression for growth rate when compared to either random or half-sib outcross controls. Survival rates at various age intervals was not decreased by inbreeding. The amount of inbreeding depression varied among families and between sexes.