Viability, motility, ATP content and fertilizing potential of sperm from Atlantic salmon (Salmo salar L.) in milt stored before cryopreservation

Artificial fertilization is increasingly used in aquaculture, mostly applying short-term cold stored milt. Large scale cryopreservation of milt could be valuable for increased flexibility and acceleration of breeding progress. The aim of this study was to assess viability, motility and ATP content of sperm from Atlantic salmon as a function of storage time, before and after cryopreservation. The objective was also to investigate whether in vitro parameters were associated with sperm fertilizing ability after cryopreservation. Milt from six mature Atlantic salmon males were collected twice, one week apart. The milt was stored undiluted at 5 °C in cell culture flasks for six days. Samples were taken on days 1, 3 and 6 of storage for cryopreservation. In total, 36 batches were diluted to a standardized sperm concentration of 2 × 109 spermatozoa/mL, filled into 0.5 mL French medium straws and cryopreserved. In vitro analyses were assessed on the same sample for the 72 combinations of male, collection week, days of storage and cold stored or frozen-thawed. Fertilization trials with cryopreserved milt were carried out for all 36 batches in triplicate for each combination of male, collection week, storage time and sperm:egg ratios of either 2 or 4 × 106 sperm per egg, respectively, totally 218 experimental units, including two egg controls. There was a significant influence of storage and collection week on sperm quality parameters, both cold stored and cryopreserved, and cryopreservation had a significant effect on all tested sperm quality parameters. High correlations for cold stored vs cryopreserved samples was demonstrated for ATP content (p < 0.00001), motility and velocity parameters (p < 0.001), but not for viability, straightness and linearity. The overall percentage of fertilization achieved was 73.9 ± 1.7%. Sperm collected in week 2 showed significantly lower fertility when cryopreserved after six days of storage than after 1 or 3 days for sperm to egg ratios of 2 × 106 (p < 0.005), while there was no such effect for milt collected in week 1. Several post-thaw sperm parameters were correlated to fertilization rates, while curvilinear velocity best explained variations in fertilization by modelling. Our results suggest that cryopreservation of Atlantic salmon milt should be performed soon after milt collection to maximize the cryopreserved sperm quality. Fertilization results seems not to be compromised by storage for three days before cryopreservation.publishedVersio

Praksisveiledning: Behov, ønsker og forventninger

Hensikt: Ekstern praksis er en viktig del av en bioingeniørutdanning. I praksisstudier møter bioingeniørstudenten erfarne bioingeniører som sine praksisveiledere. Hensikten med denne studien er å beskrive behov og ønsker praksisveiledere innenfor bioingeniørfag har, for å fremme praksisstudier av høy kvalitet. Metode: «Skulle ønske jeg hadde»-seminar etterfulgt av semistrukturerte dybdeintervjuer ble gjennomført med bioingeniører som er praksisveiledere. En kvalitativ innholdsanalyse ble gjennomført hvor mønstre i datamaterialet ble identifisert og kategorisert. Resultat: Våre funn indikerer at praksisveiledere har ulike behov, ønsker og innspill som kan kategoriseres til «Før praksisperioden», «I praksisperioden», «Etter praksisperioden» og generelle rammebetingelser. Praksisveilederne trenger avsatt tid til både å forberede seg og til å gjennomføre veiledning, og behov/ønsker som identifiseres inkluderer (i) kompetanseheving innenfor veiledning, (ii) fora for erfaringsutveksling, (iii) praksisveiledning i team, (iii) mer kontakt, dialog og forventningsavklaringer med utdanningen (og studenten), (iv) harmoniserte praksissteder og (v) tilbakemeldinger om og evaluering av praksisperioden. Konklusjon: Praksisveiledere innenfor bioingeniørfag har behov, ønsker, utfordringer og forventninger som bør kartlegges – med hensikt å fremme kvalitet i utdanningene.Praksisveiledning: Behov, ønsker og forventningerpublishedVersio

Sperm DNA Hypomethylation Proximal to Reproduction Pathway Genes in Maturing Elite Norwegian Red Bulls

Genomic selection in modern farming demands sufficient semen production in young bulls. Factors affecting semen quality and production capacity in young bulls are not well understood; DNA methylation, a complicated phenomenon in sperm cells, is one such factors. In this study, fresh and frozen-thawed semen samples from the same Norwegian Red (NR) bulls at both 14 and 17 months of age were examined for sperm chromatin integrity parameters, ATP content, viability, and motility. Furthermore, reduced representation bisulfite libraries constructed according to two protocols, the Ovation R RRBS Methyl-Seq System (Ovation method) and a previously optimized gel-free method and were sequenced to study the sperm DNA methylome in frozen-thawed semen samples. Sperm quality analyses indicated that sperm concentration, total motility and progressivity in fresh semen from 17 months old NR bulls were significantly higher compared to individuals at 14 months of age. The percentage of DNA fragmented sperm cells significantly decreased in both fresh and frozen-thawed semen samples in bulls with increasing age. Libraries from the Ovation method exhibited a greater percentage of read loss and shorter read size following trimming. Downstream analyses for reads obtained from the gel-free method revealed similar global sperm DNA methylation but differentially methylated regions (DMRs) between 14- and 17 months old NR bulls. The majority of identified DMRs were hypomethylated in 14 months old bulls. Most of the identified DMRs (69%) exhibited a less than 10% methylation difference while only 1.5% of DMRs exceeded a 25% methylation difference. Pathway analysis showed that genes annotated with DMRs having low methylation differences (less than 10%) and DMRs having between 10 and 25% methylation differences, could be associated with important hormonal signaling and sperm function relevant pathways, respectively. The current research shows that RRBS in parallel with routine sperm quality analyses could be informative in reproductive capacity of young NR bulls. Although global sperm DNA methylation levels in 14 and 17 months old NR bulls were similar, regions with low and varying levels of DNA methylation differences can be identified and linked with important sperm function and hormonal pathways.publishedVersio

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups. In both fresh and frozen-thawed semen samples, the proportion of hyperactive spermatozoa, average path velocity, curvilinear velocity and amplitude of lateral head displacement were higher in samples collected at 17 months of age compared to 14 months (PSemen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bullsacceptedVersio

Semen quality parameters including metabolites, sperm production traits and fertility in young Norwegian Red AI bulls

Genomic selection in cattle breeding has gradually allowed younger bulls to be recruited for semen production. In this study, sperm quality parameters, seminal plasma and sperm metabolites, semen production capacity and fertility in young Norwegian Red bulls were analysed. For in vitro analyses of sperm quality and metabolites, ejaculates were collected from the same 25 bulls at both 14 and 17 months of age. Semen production and fertility data were collected for all Norwegian Red bulls in production from December 2017 throughout 2019. Bull fertility was measured as 56 days non-return rate (NR56), for both age groups.acceptedVersionpublishedVersio

Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size

acceptedVersio

RNA sequencing reveals candidate genes and polymorphisms related to sperm DNA integrity in testis tissue from boars

© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Background: Sperm DNA is protected against fragmentation by a high degree of chromatin packaging. It has been demonstrated that proper chromatin packaging is important for boar fertility outcome. However, little is known about the molecular mechanisms underlying differences in sperm DNA fragmentation. Knowledge of sequence variation influencing this sperm parameter could be beneficial in selecting the best artificial insemination (AI) boars for commercial production. The aim of this study was to identify genes differentially expressed in testis tissue of Norwegian Landrace and Duroc boars, with high and low sperm DNA fragmentation index (DFI), using transcriptome sequencing. Results: Altogether, 308 and 374 genes were found to display significant differences in expression level between high and low DFI in Landrace and Duroc boars, respectively. Of these genes, 71 were differentially expressed in both breeds. Gene ontology analysis revealed that significant terms in common for the two breeds included extracellular matrix, extracellular region and calcium ion binding. Moreover, different metabolic processes were enriched in Landrace and Duroc, whereas immune response terms were common in Landrace only. Variant detection identified putative polymorphisms in some of the differentially expressed genes. Validation showed that predicted high impact variants in RAMP2, GIMAP6 and three uncharacterized genes are particularly interesting for sperm DNA fragmentation in boars. Conclusions: We identified differentially expressed genes between groups of boars with high and low sperm DFI, and functional annotation of these genes point towards important biochemical pathways. Moreover, variant detection identified putative polymorphisms in the differentially expressed genes. Our results provide valuable insights into the molecular network underlying DFI in pigs.publishedVersio

Genetiske forskjeller mellom villørret og settefisk i Vorma

Det ble samlet inn vevsprøver fra settefisk i Hunderfossen settefiskanlegg og fra settefisk (fettfinneklippet) gjenfanget i Vorma og fra villfisk fanget i Vorma. Det ble analysert på åtte mikrosatellitter i prøver fra til sammen 93 ørret. All settefisk var fra Hunderfossen, avlet av ville foreldre av Hunder/Lågen stammen. Generelt var den genetiske variasjonen høy med allelantall pr. locus (mikrosatellitt) varierende fra 5,75 til 9,50, og observert heterozygositet (HO) varierte fra 0,772 til 0,825, og observert heterozygositet var høyere enn forventet heterozygositet (HE = 0,694-0,780) i alle grupper. Resultatene tydet på at det er en egen ørretstamme i Vorma, og som sannsynligvis gyter i området nedenfor Svanfossen. Alder og lengde blant villfisken som ble analysert, viste betydelig variasjon i vekst, og det kan være flere subpopulasjoner representert i materialet. Noen har rask «innsjøvekst» som er vanlig i Mjøsa, og som også gjenfanget settefisk viste, mens andre hadde et annet vekstmønster. Det var for eksempel en vill ørret på åtte år som var 325 mm og en som var åtte år og 645 mm lang. Blant fem år gamle og eldre settefisk, var alle ≥ 400 mm. De genetiske analysene viste at villfisken skilte seg noe fra gjenfanget settefisk, og hadde for eksempel hele 15 private alleler. Settefisken fra fiskeanlegget skilte seg fra de andre gruppene, og hadde lavest genetisk variasjon, noe som kunne forventes ettersom den var av bare en årsklasse, basert på et begrenset antall foreldre. De gjenfangede settefiskene representerte til sammen seks årsklasser, som til sammen gav en genetisk variasjon litt mindre enn det som ble funnet for villfisk gruppa som bestod av åtte årsklasser. Beregnet effektiv populasjonsstørrelse var 12,5 til 495 fisk, og den var lavest blant settefisk fra fiskeanlegget. Det er naturlig ettersom fisken det ble tatt prøver av i anlegget var av samme alder/årsklasse, mens de andre gruppene bestod av seks til åtte aldersgrupper/årsklasser. Effektiv populasjonsstørrelse var størst i gruppen gjenfanget settefisk.English: Tissue samples were collected from brown trout in the Hunderfossen hatchery and from stocked fish (finclipped) recaptured in the River Vorma and from wild fish caught in the River Vorma. It was analyzed on eight micro-satellites in samples from a total of 93 trout. All hatchery/stocked fish were from Hunderfossen, bred by wild parents of the Hunder/Lågen strain. Generally, the genetic variation was high with allele numbers per. the locus (microsatellite) ranging from 5.75 to 9.50, and observed heterozygosity (HO) ranged from 0.772 to 0.825, and observed heterozygosity was higher than the expected heterozygosity (HE= 0.694-0.780) in all groups. The results indicated that there is a separate trout population spawning in the River Vorma, and which probably spawns in the area below the dam of Svanfossen. The age and length of the analyzed fish showed considerable variation in growth, and there may be several subpopulations represented in the material. Some have rapid "lake growth" which is common in the Lake Mjøsa, like the recaptured hatchery fish showed, while others had slower. For example, it was a wild trout of eight years measuring 325 mm, whereas one speciemen was eight years old and 645 mm long. Among five-year-old and older hatchery fish, all were ≥ 400 mm. The genetic analyzes showed that the wild fish differed somewhat from the recaptured hatchery, and had, for example, a total of 15 private alleles. The brown trout that were sampled in the hatchery differed from the other groups, and had the lowest genetic variation, which could be expected as comprised only one year-class, and were bred from a limited number of parents. The recaptured hatchery fish represented a total of six year classes, which together gave a genetic variation slightly less than that found for the wild fish group that consisted of eight year classes. Estimated effective population size was 12.5 to 495 fish, and it was lowest for the sample from the hatchery. This was expected since the fish were of only one year class, while the other groups consisted of six to eight age groups/year classes. Effective population size was highhest in the recaptured hatchery group.Vassdragsforbundet for Mjøs

Genetiske forskjeller mellom villørret og settefisk i Vorma

Det ble samlet inn vevsprøver fra settefisk i Hunderfossen settefiskanlegg og fra settefisk (fettfinneklippet) gjenfanget i Vorma og fra villfisk fanget i Vorma. Det ble analysert på åtte mikrosatellitter i prøver fra til sammen 93 ørret. All settefisk var fra Hunderfossen, avlet av ville foreldre av Hunder/Lågen stammen. Generelt var den genetiske variasjonen høy med allelantall pr. locus (mikrosatellitt) varierende fra 5,75 til 9,50, og observert heterozygositet (HO) varierte fra 0,772 til 0,825, og observert heterozygositet var høyere enn forventet heterozygositet (HE = 0,694-0,780) i alle grupper. Resultatene tydet på at det er en egen ørretstamme i Vorma, og som sannsynligvis gyter i området nedenfor Svanfossen. Alder og lengde blant villfisken som ble analysert, viste betydelig variasjon i vekst, og det kan være flere subpopulasjoner representert i materialet. Noen har rask «innsjøvekst» som er vanlig i Mjøsa, og som også gjenfanget settefisk viste, mens andre hadde et annet vekstmønster. Det var for eksempel en vill ørret på åtte år som var 325 mm og en som var åtte år og 645 mm lang. Blant fem år gamle og eldre settefisk, var alle ≥ 400 mm. De genetiske analysene viste at villfisken skilte seg noe fra gjenfanget settefisk, og hadde for eksempel hele 15 private alleler. Settefisken fra fiskeanlegget skilte seg fra de andre gruppene, og hadde lavest genetisk variasjon, noe som kunne forventes ettersom den var av bare en årsklasse, basert på et begrenset antall foreldre. De gjenfangede settefiskene representerte til sammen seks årsklasser, som til sammen gav en genetisk variasjon litt mindre enn det som ble funnet for villfisk gruppa som bestod av åtte årsklasser. Beregnet effektiv populasjonsstørrelse var 12,5 til 495 fisk, og den var lavest blant settefisk fra fiskeanlegget. Det er naturlig ettersom fisken det ble tatt prøver av i anlegget var av samme alder/årsklasse, mens de andre gruppene bestod av seks til åtte aldersgrupper/årsklasser. Effektiv populasjonsstørrelse var størst i gruppen gjenfanget settefisk

Sperm chromatin integrity and DNA methylation in Norwegian Red bulls of contrasting fertility

In this study, the complexity of chromatin integrity was investigated in frozen-thawed semen samples from 37 sires with contrasting fertility, expressed as 56-day non-return rates (NR56). Protamine deficiency, thiols, and disulfide bonds were assessed and compared with previously published data for DNA fragmentation index (DFI) and high DNA stainability (HDS). In addition, in vitro embryo development and sperm DNA methylation were assessed using semen samples from 16 of these bulls. The percentages of DFI and HDS were negatively associated with NR56 and cleavage rate and positively associated with sperm protamine deficiency (p < 0.05). Significant differences in cleavage and blastocyst rates were observed between bulls of high and low NR56. However, once fertilization occurred, further development into blastocysts was not associated with NR56. The differential methylation analysis showed that spermatozoa from bulls of low NR56 were hypermethylated compared to bulls of high NR56. Pathway analysis showed that genes annotated to differentially methylated cytosines could participate in different biological pathways and have important biological roles related to bull fertility. In conclusion, sperm cells from Norwegian Red bulls of inferior fertility have less compact chromatin structure, higher levels of DNA damage, and are hypermethylated compared with bulls of superior fertility