Spotted wolffish (Anarhichas minor) sperm cryopreservation in 5-mL cryovials.

In spotted wolffish Anarhichas minor aquaculture, cryopreservation is used to secure sperm availability throughout the entire spawning season. Under current protocols, sperm is cryopreserved in 0.5-mL straws. This implies thawing a considerable number of straws for insemination with cryopreserved sperm. In this work, we scale up the spotted wolffish sperm cryopreservation procedure through the development of a protocol for sperm cryopreservation in 5-mL cryovials. Different freezing (distances from the liquid nitrogen surface) and thawing rates were tested. The best results were obtained with cryovials frozen at a distance of 1.5 cm from the liquid nitrogen surface and thawed either at 15 or 10 °C for 4 and 6 min, respectively. Under these conditions, similar percentage of motile cells, sperm velocity and percentage of viable cells were obtained in comparison with the sperm cryopreserved in the traditional 0.5-mL straws. This protocol will facilitate the process of insemination with cryopreserved sperm in the spotted wolffish hatcheries.

Sex-biased miRNA expression in Atlantic halibut (Hippoglossus hippoglossus) brain and gonads.

The role of miRNA in fish sexual development is not elucidated yet. We profiled miRNAs in gonads and brains of Atlantic halibut using SOLiD sequencing technology. We found tissue- and sexually dimorphic expression of several miRNAs, including miR-29a, miR-34, miR-143, miR-145, miR-202-3p, miR-451, and miR-2188. miR-9 and miR-202 were abundant in brain and gonads, respectively. In the next step, we selected some miRNAs showing differential expression patterns between sexes and performed RT-qPCR on 3 age groups: juveniles, 3-year-, and 5-year-olds. In brains, miR-451 was significantly down-regulated in juveniles compared to adults. let-7a, miR-143, and miR-202-3p were up-regulated in gonads of mature males compared to immature females at the same age. We investigated the effect of suppressing aromatase cytochrome P450 enzyme on miRNA expression at the onset of sex differentiation through masculinization with Fadrozole or 17-α-methyltestosterone. We found significant differences in miRNA expression between masculinized individuals and untreated controls. miR-202-3p was significantly down-regulated in female juveniles compared to male juveniles. The expression levels of let-7a and miR-451 were restored after termination of the masculinization treatment. Our data give a first insight into miRNA involvement in sexual development in teleosts.

Profiling of key apoptotic, stress, and immune-related transcripts during embryonic and postembryonic development of Atlantic cod (Gadus morhua L.).

We investigated the profiles of 25 genes involved in apoptosis (bcl-x2, casp3, casp8, ccar1, mcl1, and tpt1), immunity (bty, cathl, ifng, il1b, il6, il8, il10, lyzg, and tfa), oxidative stress (cat, gpx4, gsh-px, hsp70, hsp90a, and sod1), and stress axis (crh, pomc, grl1, and mlr) during Atlantic cod development and compared the mRNA transcript levels between samples from farmed (FB) and wild broodstock (WB) using real-time polymerase chain reaction. The suitability of nine endogenous housekeeping genes and an external standard (luciferase) as reference genes was also evaluated. The cycle threshold values of all housekeeping genes differed significantly throughout Atlantic cod development. Fertilization and hatching rates were significantly higher in WB group (95 ± 1.8% and 89 ± 2.8%, respectively) compared with FB (75 ± 3.4% and 66 ± 3.2%, respectively). Eleven target genes, namely, ccar1, casp3, bcl-x2, mcl-1, cat, gsh-px, hsp70, sod1, lyzg, il8, and grl were expressed in both groups at fertilization stage, indicating their maternal transfer. Among them, transcripts of gsh-px were more abundant in WB eggs, while the expression of hsp70 was significantly higher in FB eggs. In FB larvae, expression of cat, hsp70, hsp90a, pomc, mlr, grl1, bclx2, and il6 was significantly higher at hatching and the expression of cat, gpx4, casp3 and ccar1 was significantly higher at first feeding stages, than in WB group. These findings give an insight into the expressional changes in certain category of genes involved in the embryonic development of Atlantic cod, which may eventually determine the ultimate quality of the larvae.

Expression of vasa and nanos3 during primordial germ cell formation and migration in Atlantic cod (Gadus morhua L.).

Primordial germ cells (PGCs), progenitors of gametes, are specified very early in embryonic development and undergo an active migration to the site where the future gonads will form. While the developmental pattern of PGCs during embryogenesis has been documented in few model teleost fishes, there is currently no information available for any representative of Superorder Paracanthopterygii. This includes Atlantic cod (Gadus morhua), which is a historically important food fish in both fisheries and aquaculture industries. In the present study, we cloned and characterized vasa and nanos3 and used them as germ cell markers in Atlantic cod. Sequencing results showed prospective vasa and nanos3 mRNA contained the domains used to describe their respective protein family. Furthermore, phylogenetic analysis using the amino acid sequence placed Atlantic cod Vasa distinct from representatives of three other taxonomic Superorders. Atlantic cod Nanos3 was placed with other homologues from the Nanos3 subfamily. Expression of both genes was detected from the first cleavage division; both were specifically expressed in Atlantic cod PGCs from the 32-cell stage. While nanos3 expression ceased during early somitogenesis, vasa was strongly expressed throughout embryonic development. Using vasa as a marker, we described the Atlantic cod PGC migration pattern. We demonstrated that Atlantic cod PGCs migrate ventral to the trunk mesoderm. With the exception of Pacific herring (Clupea pallasii), PGCs in other described teleost fishes migrate lateral to the trunk. The results from this study are the first step toward understanding germ line formation in Atlantic cod.

Karyotyping and cytogenetic mapping of Atlantic cod (Gadus morhua Linnaeus, 1758).

The Atlantic cod (Gadus morhua) is an important natural resource for northern societies and is now also considered to be a promising candidate for aquaculture. In recent years, much effort has been directed towards the development of genomic tools, and genome initiatives for Atlantic cod have been established. Despite the growing attention devoted to the Atlantic cod genomics, basic aspects of its genome structure and organization remain unknown. Thus, the present work aims to study cytogenetic features of the Atlantic cod as a contribution to the knowledge of this species' genome. The Atlantic cod displays a diploid number of 46 chromosomes, with a karyotypic formula 16 m/sm + 30 st/t. Conventional karyotyping was improved by chromosomal mapping of two classes of repetitive sequences. 18S rDNA clusters were assigned to pairs 2 and 4; small amounts of 18S rDNA clusters were occasionally detected on pair 5. These findings could not be related to the geographical origin of the specimens, but were consistent with the variability of these repeated genes in fish in general. 5S ribosomal gene clusters, apparently corresponding to a single 5S rDNA class, were detected on twelve chromosomes (pairs 11, 12, 14, 17, 20 and 21). The present update of the existing but meagre information on the karyotype of Atlantic cod, plus the first physical mapping of repetitive genes in this species herein, opens the way for an integrated approach that combines genetic and physical mapping with the assembly of the genome of this commercially important species.

Semen characteristics and their ability to predict sperm cryopreservation potential of Atlantic cod, Gadus morhua L.

There is a lack of biomarkers or indices that can be used to predict the quality of fish semen samples following the freezing and thawing cycle. In the present study, a series of semen indices were tested to assess if they could accurately forecast the cryopreservation potential of Atlantic cod (Gadus morhua) semen. Fresh and frozen-thawed sperm activity variables were compared, and relationships between frozen-thawed sperm activity and fertilization success were examined. In comparison with fresh sperm, activity variables of frozen-thawed spermatozoa were reduced. Of the 18 males examined, mean (± SEM) spermatocrit of fresh sperm was 40.72 ± 4.23%, osmolality of the seminal plasma 366.32 ± 4.95 mOsmol/kg, pH 8.32 ± 0.04, protein concentration 1.05 ± 0.08 mg/mL, anti-trypsin activity 153.83 ± 19.25 U/L, and total antioxidant capacity 0.15 ± 0.03 µmol Trolox equivalents/mL. Frozen-thawed fertilization success was highly variable among males with values ranging from 18.5 to 90.2%. Regressions yielded significant positive relationships between frozen-thawed motility, velocity, track crossing frequency, and subsequent fertilization success. Sequential multiple regressions explained up to 95% of the variation in frozen-thawed sperm activity. Spermatocrit and pH of fresh semen were negatively related, whereas osmolality and antioxidant capacity were positively related to frozen-thawed motility and velocity. Each of these indices can be measured within minutes of collecting a fresh sample of semen and are thus early indicators of the capacity of semen samples to withstand cryopreservation. These results have many benefits for conservation of wild stocks, aquaculture production, and for understanding semen biology and cryobiology of fishes.

Variation in size and location of the Ag-NOR in the Atlantic halibut (Hippoglossus hippoglossus).

The distribution of differentially stained chromatin was studied in the Atlantic halibut (Hippoglossus hippoglossus) chromosomes (2n=48). Four pairs of homologous chromosomes were identified using a combination of traditional cytogenetic staining techniques (Giemsa/DAPI/CMA3/Ag-NO3). Chromosome 1 showed a length polymorphism (1(S)-short, 1(L)-long isoforms of the chromosome 1) which was related to the variation of the size of the Ag-NORs. In one specimen the Ag-NOR was translocated from chromosome 1 into the telomeric region on the q-arm of the chromosome 2 forming a derivative chromosome der(2)t(1(S);2)(q?;q?). Four Ag-NOR genotypes have been shown: 1(S)1(S), 1(S)1(L), 1(L)1(L) and 1(S) der(2)t(1(S);2)(q?;q?). The chromosome rearrangements did not leave any interstitially located telomeric sequences and the telomeres were confined to the ends of the chromosomes. A single chromosomal location of 5S rDNA clusters was found using the PRINS technique. In the extended metaphase spreads two adjacent clusters of 5S rDNA could be seen on one chromosome while condensed chromatin gave a single hybridization signal. Double 5S rDNA signals on the same chromosome arm suggested paracentric inversion of the minor rDNA site. 5S rDNA clusters were not co-localized with Ag-NORs. Although female and male karyotypes were compared no sex related cytogenetic markers were found.

Parental effects on fertilization and hatching success and development of Atlantic halibut (Hippoglossus hippoglossus L.) embryos and larvae.

The parental effects on fertilization and early life history traits were studied in Atlantic halibut, Hippoglossus hippoglossus L. Sperm from 12 different males were used to fertilize eggs of two females in separate crosses. The fertilization success were generally high, above 80% of developing embryos at 16-cell stage in 20 of 24 crosses with an average of 85.9+/-17.6% and 87.2+/-16.5% for female A and female B, respectively. Corresponding hatching success was 74.8+/-17.7% and 41.6+/-20.1%, respectively. The relationship between fertilization success and hatching success was positive. The parental influence on hatching was dominated by a strong and significant (p<0.001) maternal effect; however, the paternal effect was also significant (p<0.001). Furthermore, there was no relationship between fertilization success, hatching success and larvae viability as a high number of larvae developed locked jaws (i.e., were not functional). There was a significant (p<0.01) difference in yield of functional larvae of female A (43%) and female B (56%), and also between crosses sired by different males. The standard length of offspring of female A (12.4+/-0.5 mm) and B (12.6+/-0.6 mm) were significantly (p<0.001) different, and also significantly influenced by both the female (p<0.001) and the male (p<0.001). The present paper provides strong indications that not only the female, but also the male parent influences quantitative features of early development of their offspring.

The stability of telomereless chromosome fragments in adult androgenetic rainbow trout.

The study provides new data on the stability of gamma radiation-induced chromosome fragments of a putative maternal nuclear genome in an androgenetic vertebrate, rainbow trout (Oncorhynchus mykiss Walbaum). The fragments were found in five of 16 examined individuals and they were mostly centromeric parts of metacentric or subtelocentric chromosomes. Chromosome fragments were identical in all cells of a given androgenetic individual, indicating that segregation of chromosome fragments is active from the early cell divisions. Most of the fragments were telomereless, i.e. they had no telomeric sequences on their ends. This shows that telomeres are not necessary for stability of chromosomal structures in a vertebrate genome. In one individual, the interstitial telomeric sites were found in chromosomes, which could be the effect of joining chromosome fragments.

Are sex steroids involved in the sexual growth dimorphism in Eurasian perch juveniles?

Individual growth and food intake were monitored in Eurasian perch (Perca fluviatilis L.) juveniles (13.5+/-3.4 g initial body weight) to determine whether androgens and estrogens may mediate sex-related growth differences. Fish were individually tagged with chips and implanted with cocoa butter containing 20 microg of either 17alpha-methyltestosterone (MT) or 17beta-estradiol (E(2)) per gram of fish body; controls were implanted with cocoa butter without hormones. All fish were bled at the end of the experiment for measurement of E(2) in females and testosterone (T) in males (MT was not measured) and triiodothyronine (T3) in both genders. Survival, gonadosomatic index and hepatosomatic index were not affected by steroid treatments. Relative food intake (RFI), feed efficiency (FE) and specific growth rate (SGR) were higher in females than in males in all treatments. MT treatment significantly lowered RIF, FE and SGR in both sexes, while E(2) treatment showed no significant effect on growth and feeding parameters. In contrast to E(2) and T concentrations, T3 levels were significantly and positively correlated with SGR and RFI. The results provide evidence that MT may affect sexually related growth dimorphism by decreasing food intake and FE in Eurasian perch.

Androgenesis in rainbow trout using cryopreserved spermatozoa: the effect of processing and biological factors.

In addition to producing homozygous lines for biomedical and genomic research and monosex stocks for commercial purposes, androgenesis is the biotechnology considered most promising and reliable for recovering complete nuclear genome information from cryopreserved fish cells. That is because procedures of cryopreserving spermatozoa, contrary to procedures for oocytes or entire eggs, are being well developed. Application of androgenesis in genome banking programs addresses the needs of both the aquaculture industry (safeguard for valuable strains and lines) and natural resource conservation (in vitro protection of endangered species or populations). The present study was focused on successful production of an androgenetic rainbow trout stock using cryopreserved spermatozoa. Our report constitutes the first time a full factorial analysis of processing and biological factors affecting androgenesis efficacy has been presented. Also for the first time, the survival of androgenetic individuals during 2 years of life was recorded. Remarkably high survival rates were observed in one of the two experiments-up to 42.5 +/- 2.8% of hatched larvae, 22.5 +/- 0.1% of swim-up larvae and 10.5% of androgenetic alevins 0.4 g. Mortality rates in androgenetic groups were high especially during the first 6 months. In all, 114 androgenetic individuals (0.9%) survived 2 years. Cryopreservation of spermatozoa generally did not affect androgenesis efficiency significantly, however, this effect was significantly dependent on the method of ploidization shock and on the duration of treatment. Significant interactions were revealed between the irradiation dose and the magnitude of pressure applied, and between the treatment of sperm and duration of pressure shock. Individual variability of spermatozoa donors significantly affected androgenesis efficiency regardless of their genetic (outbred or inbred) origin. Genetic source of the oocytes, contrary to spermatozoa, proved to be an important factor. Following findings of other researchers that androgenesis using cryopreserved spermatozoa is possible, we demonstrated that viable stock could be successfully established from cryopreserved nuclear genome information. Complex statistical analysis of previously developed procedures resulted in information-rich data regarding factorial interactions helpful for developing protocols in genome-restoration programs.

Effect of extender composition and equilibration time on fertilization ability and enzymatic activity of rainbow trout cryopreserved spermatozoa.

The effects of extender composition and equilibration time on fertilizing ability of cryopreserved spermatozoa from rainbow trout, Oncorhynchus mykiss, were investigated. In addition, enzyme activity in supernatants from thawed sperm was assessed. The use of the two extenders: Erdahl & Graham's + 10% DMA (dimethyl acetamide) + 10% egg yolk and 0.3 M glucose + 10% DMA yielded the highest post-thaw fertilization rates. We observed interactions between extender constituents and the equilibration of diluted semen. This indicates a multifactorial effect of the extender constituents on spermatozoal resistance against injuries. The 10-min equilibration of spermatozoa in extender before freezing generally lowered the fertilization ability of spermatozoa, except for DMA-based extenders. The addition of egg yolk to the extender was generally beneficial, especially in DMA- and DMSO-based extenders. The use of low-density lipoprotein fraction showed no advantage to full-yolk or free-of-yolk extenders. Aspartate aminotransferase and lactate dehydrogenase leakage from damaged spermatozoa correlated negatively with the ability of cryopreserved spermatozoa to fertilize eggs. Each factor tested, when analyzed separately, did not give general information about its effect on the fertilization ability of cryopreserved sperm. The multifactorial analysis of the important factors in cryopreservation of trout spermatozoa showed their cumulative effect. This is the most likely reason for divergent information reported elsewhere on the effect of various factors in the cryopreservation of rainbow trout spermatozoa.

Principles for the management of deep infections following total hip replacement.

Current methods are presented for the management of deep infection of total hip replacements. The goal of treatment is to maintain the improvement of function gained by hip replacement. In cases without loosening, precise surgical debridement without removal of implants is indicated. Management in septic loosening depends on the general medical condition of the patient, the local bone stock, and the spread of infection. Satisfactory bone stock allows for reimplantation of a new prosthesis, prefably using antibiotic-loaded cement, after scrupulous removal of all potentially infected tissues and particles. The reimplantationcan be done as a one- or two-stage operation. In the two0stage procedure, antibiotic-loaded cement spacer can be used, but both two- and one-stage reimplantation have similar results as regards infection control. Definitive removal of the prosthesis is indicated in patients whose general medical condition is poor, who have no usable bone stock, or who present with a persistent Gram-negative bacterial infection. This procedure is not fully effective, however (infection brought under control in 83% of patients), although it does not cause significant negative effect for the functional status of this group of patients.

Principles for the diagnosis of infections following total hip arthroplasty.

Based on authorial material consisting of 19 patients operated in the period 1993-2000 for infections following total hip replacements, and on information from the literature, the authors discuss the suitability of various diagnostic methods. It is particularly important to diagnose infections with a slow course, without the classic clinical signs. The diagnostic criteria accepted by the Disease Control Center in Atlanta are presented, along with the radiological symptoms of latent infections and scintigraphic methods of varying sensitivity and specificity to regards to hip endoprosthesis infections. The article presents the typical changes in the value of erythrocyte precipitation and CRP concentration during the first year following a non-complicated hip arthroplasty, which has a significant impact on the interpretation of results when there is a suspicion of early infection. The decisive test for the diagnosis of a slow infection in a joint with loosened endoprosthesis with obvious clinical signs of infection is peri-operative examination of frozen scraps of the joint capsule for the presence of infiltrations of neutrophil leukocytes.

Results of therapy of infection complicating total hip replacement.

An aim of the management of infection complicating total hip arthroplasty is to control infection and maintain satisfactory hip function. In the period 1993-2000 effectives of therapy of deep injection after total hip replacement in 91 patients was assessed. Infection without loosening of endoprosthesis was diagnosed in 33 hips, septic loosening in 58. In 33 cases without loosening surgical debridement without removal of prosthesis was done with good control of infection in 25 hips. Permanent removal of prosthesis done in 42 cases resulted in recurrence of infection in 6 hips. One stage reimplantation done in 12 patients succeded in 9 hips. Two-stage reimplantation in 3 hips resulted in recurrence of infection in 2 cases. Antibiotic-embedded cement spacer (gentamycin with vancomycin) was used in I case with unsatisfactory result. Recurrent infection was observed in 13 cases of 48 hips treated without implant removal or by reimplantation of new prosthesis.

The effect of egg yolk, low density lipoproteins, methylxanthines and fertilization diluent on cryopreservation efficiency of northern pike (Esox lucius) spermatozoa.

The effect of egg yolk, low density lipoproteins (LDL) as well as methylxanthines (caffeine and theophylline) and fertilization diluent on cryopreservation efficiency of northern pike, Esox lucius, spermatozoa was tested. Milt was cryopreserved in pellets on dry ice then stored in liquid nitrogen. The extender consisted of 0.6 M sucrose + 15% DMSO supplemented with egg yolk or LDL fractions. The most effective results (77.3% hatched larvae vs 74.1% in the control group) were obtained from extender that contained only 0.6 M sucrose + 15% DMSO and was used for freezing, while the fertilization diluent was used for thawing. Addition of egg yolk or LDL to the extender did not improve the results. The presence of caffeine in the thawing solution significantly lowered fertilization rate of cryopreserved spermatozoa, whereas theophylline did not significantly affect the results. The addition of fertilization diluent to the eggs prior to insemination was superior to the other treatments. The proposed procedure constitutes a complete method for the efficient cryopreservation of northern pike semen.

Activity of aspartate aminotransferase and acid phosphatase in cryopreserved trout sperm.

Milt of brown, rainbow and brook trout was cryopreserved. Activity of aspartate aminotransferase (AspAT) and acid phosphatase was assayed both in supernatants and in spermatozoa obtained from thawed sperm samples; additionally, post-thaw motility was evaluated. Enzyme activities differed according to fish species and were strongly affected by the type of cryoprotectant used. The activity in supernatants was usually higher than that in spermatozoa because of protein leakage from injured cells. AspAT activity in cryopreserved spermatozoa correlated positively with fertilization success in all three species. There was a negative correlation between activity of extracellular (supernatant) AspAT and fertilization rates in variants with dimethyl sulfoxide and dimethylacetamide-based extenders. The motility of thawed sperm, determined microscopically, provided some information on the cryopreservation efficiency of trout milt.

[Evaluation of lower extremity function following removal of the endoprosthesis]. / Ocena funkcji konczyny dolnej po usunieciu endoprotezy stawu biodrowego.

The authors made clinical evaluation of 35 patients after the removal of the hip endoprosthesis due to infection. The age of the patients varied from 18-82 years (average 56 years). Majority of patients (26) estimated the result of treatment as good; only 3 among them wanted a revision hip arthroplasty. Observation revealed good general performance and painless gait in 11 subjects; poor hip stability in 21 cases, and significant functional impairement in 3 patients. Removal of the prosthesis resulted in marked relief of pain, and enhanced the healing of infection.