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Transgene transmission in South American
catfish (Rhamdia quelen) larvae by sperm-
mediated gene transfer

Article in Journal of Biosciences · March 2010
DOI: 10.1007/s12038-010-0006-6 · Source: PubMed

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Seixas F K. omnivorous feeding habit. aquaculture in temperate and subtropical climates. The fertilization rate in the control and DRE treatments groups were higher than in the DR group. time of activity duration http://www. INC and INCSP treatment groups had the lowest rate. PCR. SMGT. E and DR. HR. enhanced green fluorescent protein. 2005). Brazil *Corresponding author (Fax. collares. Campos V F. HEDEN LUIZ M MOREIRA and JOÃO CARLOS DESCHAMPS Biotechnology Center. of Sciences 39 March 2010 . To the best of our knowledge. time of activity duration (TAD). Reidel et al. INCSP. dehydrated/rehydrated/electroporated. J Biosci. microinjection of plasmid DNA into early catfish are found from southern Mexico to central Argentina. DR 44%.ias. activation solution. seminal plasma. Biosci. E 34%. (3) electroporated (E). CEP 96010-900. Introduction consumers (Barcellos et al. E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. 35(1). SM. making the generation of Keywords. PAULO V CAVALCANTI. Dellagostin O A. high transgene. but the E. There were no differences among the DRE and DR. sperm-mediated gene transfer. dehydrated/rehydrated. INC. fertilization and hatching rates. Pelotas. (4) incubated with seminal plasma (INC). However. sperm motility.05). 2001. green fluorescent protein.ac. FABIANA KÖMMLING SEIXAS. GFP. fertilization rate. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%. E. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. University Campus. hatching rate (HR) and sperm morphology were also evaluated. The biological characteristics of semen ejaculates of this The silver catfish (Rhamdia quelen) is a teleost species species are of interest in aquaculture biotechnology (Borges from the Siluridae family and is an important species for et al. Silver In the past. March 2010. Email. incubated in the absence of seminal plasma. Federal University of Pelotas. Cavalcanti P V. polymerase chain reaction. Moreira H L M and Deschamps J C 2010 Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer. EGFP. The hatching rates of the DRE. The sperm of each species has its own peculiarities and biological characteristics. Sperm motility. TAD. The rates of embryo EGFP expression were: DRE 63%. and good acceptance by low transgenesis frequency). human chorionic gonadotrophin. INC 5% and INCSP 25%. VINICIUS FARIAS CAMPOS. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/ rehydrated (DR). which influence the success of mass DNA transfer methods. fertilization rate (FR). hCG. FR. osmotic differential. 35(1). transgene Abbreviations used: AS.t@gmail. late transgene integration at high copy numbers. 2006. SMGT. 35 39–47] DOI 10. ODIR A DELLAGOSTIN. 39–47.in/jbiosci J. hatching rate. DR and control groups were higher than in the INCSP. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups.Academy Biosci. electroporated. DR 40%. electroporation. DRE.1007/s12038-010-0006-6 1. this approach suffers from Brazil. Transgenic South American catfish 39 Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer TIAGO COLLARES*. INC and E treatment groups (P>0. E 25%. 2010). Catfish. © IndianJ.com) The silver catfish (Rhamdia quelen) is an endemic American fish species. this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology. and (5) incubated in the absence of seminal plasma (INCSP). DR. +55 53 32757551. incubated with seminal plasma. [Collares T. Fish farmers are interested in culturing this species significant drawbacks (mosaic distribution of the injected because of its growth rate. embryos represented the state of the art in generating and husbandry of this species is spreading towards southern transgenic fish. RS. (2) dehydrated/rehydrated/electroporated (DRE). INC 8% and INCSP 38%.

treatment 4 – incubation with seminal plasma (INC). 2009).40 Tiago Collares et al. The ejaculates 2. treatment 2 – dehydration. The average corporal weight of the Here. 1971. tubes. we describe the transmission of the enhanced females used in this experiment was 1120 g and the average green fluorescent protein (EGFP) transgene in silver catfish total length was 44 cm. contaminated fresh fish semen are non-motile. it is necessary to develop simple gene transfer and blinding the animals with a humidified towel to avoid methods for use in aquaculture.7 cryopreservation processes (Christensen and Tiersch 1997). semen was collected by immobilizing Thus. few studies have reported on the use the treatments were performed. in the presence or absence of seminal plasma as a means of increasing the rate of transgenic embryos. CA. catfish with an average body weight of 534 g and average The control consisted of fresh semen samples diluted in total length of 38 cm. 2002. UI/kg was used for inducing artificial spermiation. Biosci. March 2010 . which is an advantage for SMGT techniques. After the dehydrated sperm was mixed with an equal volume of J. rehydration and electroporation (DRE). 35(1). aquatic animals produce a huge number of sperm exhibited no sperm motility were considered adequate for cells. stored in properly identified 15 ml Falcon transfer (SMGT) (Brackett et al. 2. Considering that sperm in non- and internalize them into nuclei (Sciamanna et al. Semen was aspirated with graduated have been successfully produced by sperm-mediated gene syringes (5. Dehydrated Aquaculture Station. in 500 l fibre tanks with closed water sperm was obtained by incubation in hyperosmotic (595 circulation. in osmotic differential and/or electroporation and incubation synchrony with the sperm manipulation treatments. hyperosmotic and hyposmotic) were used. 1999. For rehydration. 2006. Spermatozoa of virtually all activation by fluid contamination using an optical phase- species can take up exogenous DNA or RNA molecules contrast microscope (200x). and consequent sperm activation with approaches have been developed. Lu et from each fish was placed on slides and tested for sperm al. other hand. On the the experiments. The plasmid was first obtained in previous trials (305 mOsm/kg). Human chorionic gonadotrophin (hCG) at 400 mOsm/kg) catfish diluents for 1 h at 5ºC.0 ml). During the past few years. 5 µl of fresh semen transgenic fish (Khoo et al. according to Shen et al. Lanes et al. the by sperm cells subjected to SMGT by comparing the eggs were collected in plastic buckets by hand stripping. 2. and treatment 5 The experiments were conducted using six adult male silver – incubation in the absence of seminal plasma (INCSP). treatment 1 – dehydration and rehydration (DR). biology necessitating the development of specific SMGT three female silver catfish were induced with 800 UI of protocols. often used in fish sperm (Mountain View. The linearized vector was The sperm samples were submitted to five treatments: digested with Apa LI restriction enzyme.2 Collection and manipulation of gametes treatment 3 – only electroporation (E). hCG/kg of live weight.5 ml microtubes Plasmid Maxi kit (Eppendorf. A proportion of and incubated at 5ºC in three different catfish diluents: 3:1 of linear:circular DNA was used as exogenous DNA hyperosmotic (595 mOsm/kg). Several transgenic animals NaCl (50 mOsm/kg). Harel-Markowitz et Federal University of Pelotas Biotechnology Center. USA). stress and injuries. 2009). 2009). Lavitrano et al. EGFP has a single excitation peak at 488 nm and mOsm/kg.1 pEGFP-N1 vector preparation collected from the 6 male donors were evaluated for sperm osmolarity using a Wescor 5500 vapour pressure osmometer before sperm dilution. However. EGFP was expressed under the control of the CMV These diluents represent osmolalities of approximately 300 promoter. Germany®). Materials and methods Three diluents with different osmolalities (isosmotic. similar to the average of the donor ejaculates a maximal emission peak at 507 nm. where al. Kang et al. kb. and maintained at 8ºC during transportation to the Shen et al. The urogenital pore was dried with a spermatozoa have been studied to serve as a vector for gene paper towel to reduce the possibility of contamination with transfer in transgenic animal technology and several different water. of sperm as vector of exogenous DNA for generation of Immediately after collection. Animals were maintained at the UFPel isosmotic media without exogenous DNA. 2007. Coward et al. transgenic lines a laborious task (Soroldoni et al. to transfect silver catfish sperm cells at a total of 30 µg/ml and hyposmotic (125 mOsm/kg). 8 h of induction. samples that Naturally. 2006.3 Sperm diluents 2. faeces or urine. scale DNA extraction and purification using the Perfectprep Semen samples were stored in 1. isosmotic (305 mOsm/kg). After 12 h of hormonal induction. each fish species has a particular reproductive To access female gametes and perform in vitro fertilization. 2002. 1992. The isosmotic solution The pEGFP-N1 plasmid was purchased from CLONTECH was based on catfish sperm diluents. 2009). Its whole length was 4. and do not allow amplified in Escherichia coli cells and subjected to large- sperm activation after dilution.

ANOVA was used to evaluate the effect of the treatments on SM. At the station. alkalinity (21%) DRE treatment resulted in motility parameters higher and oxygen saturation (75%). NaCl (50 mOsm/kg) Twenty of the 90-day-old animals from each treatment were activation solution (AS) was used in a semen:AS proportion randomly selected for genomic DNA extraction from muscle of 1:10.1 In vitro reproductive parameters for fertilization with the sperm from each treatment. until use. Differences were considered to be statistically Station for in vitro fertilization to be performed under significant at the 95% confidence level (P<0.05). measuring approximately 0. dehydrated with graded ethanol and dried by the critical point method. Pearson controlled conditions. Ninety animals in a sperm:isosmotic medium DNA complex at 1:1 ratio per treatment were evaluated. TAD. These were weighed and split onto glass plates 3.6). 1 min at 50°C. Biosci. than that with the control treatment (P<0. The pellet was quickly and gently dissolved in isosmotic medium DNA complex. USA). For sperm activation. This was pools for 96 h post-hatching.5%). Three thousand significant effect (P<0. stirred with (table 1). Fisher exact tests and Pearson After treatment. pH (7. the animals. sperm morphology of the followed by 30 cycles of 1 min at 94°C. and TAD analyses after treatment and transport were measured. 35(1). in pools with water 3.6 Evaluation of EGFP expression foreign DNA. and hatching rate (HR). PCR reactions were carried out in an Mastercycler gradient thermal cycler (Eppendorf®.8 Statistical analyses JEOL JOM 7000 scanning electron microscope. After 100 h of embryo conducted with 3. to remove the seminal plasma. CO2 (0. 200 ohms and 2.5 In vitro fertilization followed by the Duncan test for means comparison (SAS Institute Inc. All animals that presented any for 1 h at 5ºC. 2. NC).2 Sperm motility properly controlled for embryo development at standard temperature (23ºC). Cary. After fertilization. INC treatment was done by incubation expression under a fluorescence microscope. Polymerase chain contrast microscope (200x). March 2010 . This was J.05). The last cycle was followed by a final incubation microscopy to evaluate sperm integrity and determine the of 7 min at 72°C. Approximately 9000 eggs were used in each treatment (3000 Statistical analyses demonstrated that the treatments had a eggs per repetition).5% sequencer MegaBace 500 (Amersham Biosciences). reaction (PCR) was performed with pEGFP-N1-specific oligonucleotides (5′. glutaraldehyde in water. After they were post-fixed in 1% osium tetroxide.0 V capacitance. totalling 54 000 eggs. 2. Each PCR product was sequenced in the automatic Semen was dropped onto cover glasses and fixed in 2. a glass stick and then activated with 150 ml of activation solution (NaCl 50 mOsm/kg). FR and HR eggs were mixed with 1 ml of treated semen. yielding a medium that was isosmotic to the original seminal fluid at about 320 mOsm/kg.8 all electroporated samples using a MicroPulser Electroporator cm. they were observed under a 2. both performed in an optical phase.05) on motility.GAAGATGGTGCGCTCCTGGA -3′) to amplify a 500 base pair (bp) fragment.. Purification Kit (Invitrogen®.7 PCR and sequencing analyses There was no sperm activation of the sperm cells during the dilutions. Results of eggs. For the INCSP treatment. Germany) Due to the reduced dimensions of the silver catfish sperm programmed for an initial denaturation step (2 min at 94°C) observed on optical microscopy. sperm motility (SM) correlation coefficients among variables were tested. DR Hatched larvae were maintained in properly controlled sperm samples were submitted to electroporation.5 kV for development. semen samples were transported from the chi-square were used to evaluate EGFP expression and PCR UFPel Biotechnology Center to the UFPel Aquaculture positivity rates. allowing analyses of sperm motility and time of tissue. eggs were maintained in submerged incubators. Transgenic South American catfish 41 hyposmotic (125 mOsm/kg) catfish diluents containing 2. and 1 proposed treatments was visualized by scanning electron min at 72°C. were evaluated for green fluorescent protein (GFP) gene (Bio-rad®. The samples were then stored at –20°C cellular dimensions of the spermatozoa of this species. fertilization rate (FR). USA). Previously induced females were extruded for collection 3. DNA was obtained by the PureLink™ Genomic DNA activity duration (TAD).CGGGACTTTCCAAAATGTCG -3′ 2. TAD. the samples were variable degree of expression during fluorescence analysis washed by centrifugation (500 × g for 10 min at 5ºC) were considered positive for EGFP.4 Sperm morphology and 5′.

6±5. 3. We observed that removal of the proteins of seminal plasma.6±5. PCR. Statistical treatment group did not differ from that of the DRE treatment analyses for this parameter separated the treatments in group although motility rates were significantly different five significantly different groups (table 1).8720* IVF 0. DRE and control treatments.65%) than DRE and control 3.03%. The TAD for (P<0. The FR following DRE treatment (90. INC.16 c 73.77 cd 91.0±0 a 171.65 b 90. dehydrated/rehydrated. hatch rate.01 Parameters: sperm motility. IVF.89 b 38 (34/90) b 25 (5/20) b Treatments: DR. which did not differ among The TAD.7994* 0.3±8.2±5.00 d 65.6±2.6±17. embryo expression of green fluorescent protein (GFP). it Statistical analyses for the FR parameter separated the was possible to observe a significant correlation between treatments into three significantly different groups (table motility x TDA and motility x FR. sperm incubation with seminal plasma.05).0±0 a 0 (0/90) d 0 (0/20) c DR 80. the electroporation that promoted a reorganization of There was no difference in the TAD between the DR and the plasmatic membrane with activation of the sodium DRE treatment groups.05). 35(1).0±0 a 44 (40/90) ab 40 (8/20) ab DRE 90. time of activity duration. a. INCSP. PCR analyses.42 Tiago Collares et al.50 b 78.0±9. However.89 b 8 (7/90) c 5 (1/20) c INC SP 63. as well as other seminal constituents.3 Time of activity duration (TAD) treatments but was significantly higher when compared with the other treatments. TAD.3±7.0±5.09 c 64. E.00 a 89.58 a 88. IVF. but differed when than the DR.3±0. the FR in the control female gametes during the fertilization process. ** P<0. differed from that of the other treatments. March 2010 .05.6±5.58%). dehydrated/rehydrated/electroporated.3±4. DRE.2±5.d. Table 2 shows a significant correlation the length of time that the spermatozoa interact with between FR and motility. A low TAD rate compared with the other groups.5984 ns — HR 0. INC=65.3±2. This demonstrates that other factors besides the not differ from that of the control (89. but not between motility 1). the duration for which the themselves (E=64. it was significantly lower differ from that in the control group.05). The TAD following E treatment and potassium pumps. TAD × FR and TAD × HR (table 2).03 c 73.89 a 63 (57/90) a 60 (12/20) a E 56.77 c 44. did not differ significantly.0±2. DR treatment resulted in a lower FR (78. HR. probably due to a reflex of the osmotic differential plus the groups that underwent the other treatments (P<0. The electroporation group (44.6±5. difference when compared with the incubation treatments It was possible to observe a significant correlation between group in the presence or absence of seminal plasma and TAD × motility. electroporated. hatch rate.3±4. This effect might be due to cellular exhaustion the control group (217. however.6±5.4 In vitro fertilization rate (FR) promoted a significant loss of motility in the INCSP group (P<0.0±9.05) in the same column.9196** 0.6±0. HR.29%) but presence of foreign DNA can influence the HR. GFP. Motility in the DR treatment group did not INCSP treatments. Pearson correlation coefficients among tested variables Motility HR IVF TAD 0. Averages of the treatments for the analysed variables Treatments Motility (%) TAD (s) IVF (%) HR (%) GFP (%) PCR (%) Control 76. there was no the loss of an energy source to maintain sperm motility. in vitro fertilization rate. Biosci.e differ significantly (P<0.77 b 34 (31/90) b 25 (5/20) b INC 53. DR and DRE groups.c.6±5. time of activity duration.51 e 69. * P<0. in other words.3±0. demonstrated significant loss of motility compared with Removal of the constituents of seminal plasma results in the control.58%) did and HR.77 d 65.6±0. is important because it is SP=69. in vitro fertilization rate.5 s) was observed in the INCSP treatment group.0±5.58 c 71.3±2. foreign DNA. stimulating sperm motility and was significantly higher when compared with the INC and cell activity.8507* 0.b.0±0 b 179. Based on the Pearson correlation coefficients.08 b 90.3±5.6202 ns — — ns. TAD. Table 2. Parameters: sperm motility. however.0±5.0 s) was higher than that for resulting from the increase in motility due to the osmotic Table 1.29 a 90.0±2.16% and INC spermatozoa remain motile. J.77 b 217. sperm incubation in the absence of seminal plasma.

However. INC. Pearson correlation Sperm pathology was observed in 20% of all samples coefficients among tested variables demonstrated that the analysed. J. Scanning electron microscopy of silver catfish spermatozoa after treatments: DR. but there was of seminal plasma in isosmotic conditions. E. For this parameter.59) can be a result of the interference in gene regulation of embryonic development caused by the foreign DNA or the effect of manipulation of the gametes. two groups with a significant statistical difference were observed. Figure 1. demonstrates no loss of integrity with all the treatments. tail with diameter of 120 nm and length themselves (table 1). which was acceptable. electroporated. The presence of an acrosome was not observed in osmotic differential was applied to sperm cells. INC and spermatozoa. which was not Incubation of sperm cell/DNA. this did not affect the the success of the HR of eggs.. the same effect for this parameter. Transgenic South American catfish 43 differential plus the electroporation. which again did not differ among and length of 2 µm. dehydrated/ rehydrated/electroporated. of procedures done for sperm manipulation and injury indicating that the duration of the sperm activity can determine caused by the treatments. in the presence or absence observed with the other treatments (figure 1). not previously published. The low correlation between FR in the TAD of the DRE and control groups. sperm incubation in the absence of seminal plasma. 3. Sperm morphology demonstrated agglutination that electroporation is not necessary to increase this rate. which can be observed foreign DNA (table 2). INCSP. and HR (r=0.85). 35(1). Biosci. were measured: head with a diameter of 1 µm INCSP treatment groups. dehydrated/rehydrated. sperm incubation with seminal plasma. DR and DRE treatment groups did not differ characterize the morphology of Rhamdia quelen among themselves but they differed from the E.5 Hatching rate (HR) The embryo HR is the percentage of embryos that emerge 3. March 2010 . showing these cells. This was the result HR had a significant correlation only with the TAD (r=0. regardless of the presence of fertilization process of each treatment. of cells with the E and INCSP treatments. which control. DRE.6 Sperm morphology from fertilized eggs. The Morphology data. Hatching rates increased when an of 20 µm. Scale bars = 2 µm.

05). 34/90) that did not differ from that with E treatment. Japan) (40x). A total of 44% (40/90) and 63% (57/90) of (38%.7 Transient EGFP expression and PCR analyses did not differ from those of E treatment (34%. demonstrating that E did not increase foreign DNA without the need for sophisticated equipment. (A) Negative control (not treated with exogenous DNA (pEGFP-N1 vector). In addition. 31/90). March 2010 . consequently. except for the control group equipment. with 8% Figure 2. INCSP treatment resulted in an expression rate (figure 2). indicating that only osmotic differential can promote the All the treatments were capable of generating animals uptake of foreign DNA and determine the same expression with different degrees of transient GFP expression in rate. 35(1). it is not necessary to use electroporation muscle and/or nervous tissue. 3.44 Tiago Collares et al. the expression rate. Biosci. (E) group incubated in the absence of seminal plasma. (F) negative control (not treated with exogenous DNA). respectively. J. These groups did not differ seminal plasma can lead to an increase in the incorporation of significantly (P>0. (C) electroporated group. Enhanced green fluorescent protein (EGFP) expression in skin tissue of catfish larvae at 100 h post-hatching visualized by fluorescence microscopy (Olympus BX 51. (B) DR-treated group. (D) group incubated with seminal plasma. the results of DR treatment INC treatment resulted in a lower expression rate. transgenic fish from the DR and DRE treatment groups demonstrating that removal of the biological constituents of were observed.

In this study. (25%) fish from the DR. DRE. integration to be affected by the treatments. Gandolfi 1998. as well as between microscopy. Lavitrano et al. showing that this difficulty could be overcome in transgenic fish produced by SMGT in future. One decade later. Shen et by electroporation or not. lane 6. but between the DRE and E groups it was highly all the treatments. The eggs with sperm without seminal plasma submitted to the reaction using genomic DNA of the control group as the template osmotic differential. since a larger number of gametes can pEGFP-N1 vector sequence. 1997. On the other hand. 2009) but this has not been demonstrated under natural was increased in the DRE group when compared with the conditions. 2006. E. corresponding to 500 bp of the mass production. However. and DR and E groups were not significant that the transgene entered the offspring cells following (P>0. of SM by the osmotic differential could favour transfection J. respectively. observed in the E treatment group may have been due to a 2008). INC. 1989. we did not pursue the in vitro fertilization of 5. a similar pattern as demonstrated by high a FR and HR after We did not evaluate transgene integration into the host treatment with DRE. lane 3. SM al. A small cell agglutination into the host cells occurs in a random manner (Wu et al. lane 2. of the concentration. We showed that treatments used in Magnano et al.05). Spadafora 4. producing mosaic animals. 5 of 20 (25%). Fluorescence positivity rate between the DR and DRE. positive control. we report a novel approach the control group (figure 3). was detected in 8 of 20 (40%). be treated at one time just by an osmotic differential. 1 of 20 (5%) and 5 of 20 have demonstrated that electroporation is unnecessary. spermatozoa can be easily transfected in vitro (Lavitrano et al. Our study could not distinguish animal-to-animal (1999) reported a slight reduction in SM after electroporation variation in transgene EGFP expression and sperm potential. (1999) did not pursue the in vitro fertilization by the presence of inhibiting proteins and DNases in the of eggs with non-electroporated sperm rehydrated in the seminal plasma (data not demonstrated). negative control. The presence or absence of seminal plasma proteins strongly interferes with SMGT (Lavitrano et al. Biosci. or through short incubation with al.05). DRE. However. We 12 of 20 (60%). 2006). important statistical differences were observed and RNA molecules present in the medium (Gandolfi 1998. L. except transgenic fish. The difference in the PCR difference favouring DRE was observed. lane 4. with all the treatments. Giordano et al. Transgenic South American catfish 45 (7/90) of the animals expressing GFP. 2009). 1998. it was possible to observe this through the results of the reproductive parameters. Discussion 1998. Hoelker et al. This can be explained Kang et al. of spermatozoa. sperm morphology does not seem embryo. although a numerical of the 20 control fish examined. March 2010 . In SMGT. DR. 2000. Lavitrano et al. evidence suggests that sperm cells are able to acquire DNA However. it is important to consider that the success In this study. We believe that the expression was transient but further studies must be conducted to elucidate this and/or the integration event occurred after the one-cell stage mechanism. Hoelker et al. INC and INCSP treatment since there was no significant difference between the DR groups. 2009).05) (table 1). This agrees with the observations on significant (P<0. Sciamanna et the present study might affect reproductive parameters. INCSP. Foreign DNA was not detected in any and DRE treatment groups (P>0. It Figure 3. lane 7. 2007). PCR detection of the 500 bp fragment of the green has been demonstrated that the presence of seminal plasma fluorescent protein (GFP) gene in purified genomic DNA from protein is important for the maintenance of sperm quality in hatched Rhamdia quelen after different treatments. and the proportion of the transgenic fish. 2006). lane fish. In contrast. E. rates in birds (Harel-Markowitz et al. Khoo et al. Recent studies have demonstrated high integration technical artifact. Lanes et al. FR and HR appear to follow as has been described previously (Alderson et al. PCR products obtained from other animal species and supports the idea that animal the different treatments were confirmed by sequencing. 35(1). All control DNA samples were without the electroporation step. we have shown that Rhamdia quelen of transfection with foreign DNA seems to be a characteristic spermatozoa can be successfully manipulated to generate of the DNA. which is more practical for negative. These results suggest that spermatozoa genome and no animal was found to express EGPF in the of silver catfish can tolerate electroporation after DR. PCR and sequencing analysis results showed E and INCSP. after applying an osmotic differential followed linear and/or circular forms (Sciamanna et al. 2000. Kang et al. In contrast. Indeed. 1 kb DNA plus ladder. 2007. 1992. presence of DNA and therefore they could not ascertain PCR analysis confirmed the presence of the transgene in if an osmotic differential per se could be used to produce DNA extracted from the different treatment groups. It is conceivable that removal of proteins for PCR is the negative control and PCR with pEGFP-N1 as the that block the entrance of foreign DNA and re-establishment template DNA is the positive control. in vitro foreign DNA in the presence or absence of seminal plasma. The PCR product. control and other treatment groups. TAD. lane 1. 2006. entire body.

Baranska W. Dev. manipulation for biotechnical applications. where DNA incubation in the hormone (GH) messenger (m) RNA in a GH-transgenic tilapia absence of seminal plasma produced better results than DNA (Oreochromis niloticus). orbignyanus. Bull. Cell Biol. Kubota H and Parrington J 2007 In vivo gene transfer research areas can significantly reduce the exploitation of into testis and sperm: developments and future application. 2004. Tesfaye D. Res. Forni M. characteristics of jundia Rhamdia quelen (Quoy and Gaimard. Yazawa et al. Gilles M et al. catfish spermatozoa: effect of cryoprotectant. transmission and expression in bovine sperm mediated gene transfer by RT-PCR: effect of transfection This work was supported by the Brazilian Government reagent and DNA architecture. We are grateful to Dr Luis Houdebine L M 2005 Use of transgenic animals to improve Antônio Suíta de Castro. Katz A. J. Bacci M L. Uptake of heterologous genome by mammalian spermatozoa Fertil. Hu et al. Fish Physiol. Muller F. Gurevich M. Hwang G L. Ittzes Lanes C F C. Lanes et al. Jurinitz D F. Homma O. Lim H B and Wong K Y 1992 Sperm cells as acute stressor in jundia (Rhamdia quelen. Lavitrano M. Bracket B G. decrease costs and speed up the research process. Arch. Laboratório de Microscopia human health and animal production. Theriogenology 67 1097–1107 through CNPq and CAPES. Stram Y ecology and biotechnology (Hu et al. Williams D W. Aquaculture Res. 51 1–7 Hwang G L. Caelers et al. it is important to activity in mature spermatozoa of mouse. Acad. 31 45–53 Lavitrano M. Biol Reprod. Di Stefano C. Giovannoni R. 2006). Rahman M A. 14 95–104 Christensen J M and Tiersch T R 1997 Cryopreservation of channel incubation with seminal plasma. DNA binding and transgenic hazards associated with exposure to chemicals in the aquatic animals. Aquaculture 253 317–321 107 1–19 Barcellos L J G. Sampaio L A and Marins L F 2009 Evaluation of I and Krieger M H 2001 Plasma levels of cortisol and glucose DNase activity in seminal plasma and uptake of exogenous in response to capture and tank transference in Rhamdia quelen DNA by spermatozoa of the Brazilian flounder Paralichthys (Quoy & Gaimard). J. stimulating hormone. Proc. Magnano A R. (2009) inferred that DNA uptake by fish is Caelers A.46 Tiago Collares et al. Shore LS. 18 19–23 and its transfer to ova through fertilization. Mar. Farace M G Sci. Strussmann C A and Takashima degradation but is unable to improve the efficiency of bovine F 1999 Effect of an osmotic differential on the efficiency of gene sperm mediated transgenesis. Wang Y P and Zhu Z Y 2006 A perspective on fish gonad technical support. 148 establish new transfer techniques en masse for genes of 1107–1113 interest and those that are important in studies of molecular Harel-Markowitz E. Wassermann G F. 2002. mammals. uptake. Aquaculture Barcellos L J G. Biochem. 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