In vitro embryoproduksjon i norske duroc og landsvin-griser

The use of embryo technology is of interest in pig breeding to increase genetic gain in the breeding program and disseminate genetics of superior animals worldwide with less transportation of live animals and a lower risk of disease transmission. In Norway, breeding programs exists for two breeds with different reproductive characteristics. The Norwegian Duroc sire line has on average 9.7 total number piglets born per litter compared to 14.3 piglets in the Norwegian Landrace dam line. Breed differences within this trait could be related to ovarian characteristics, which may also affect in vitro embryo production. This could require different optimizations to successfully obtain in vitro blastocysts in both breeds. Therefore, the overall aim of this thesis was to establish protocols for porcine in vitro embryo production and staining of oocytes and embryos to acquire knowledge about in vitro maturation, fertilization and embryo culture in Norwegian Duroc and Norwegian Landrace. Firstly, ovarian characteristics and in vitro nuclear and cytoplasmic oocyte maturation were studied for both breeds one day after weaning in paper I. It was observed that Landrace ovaries contained significantly more 3-8 mm surface follicles compared to Duroc, and thus more oocytes can be collected for IVEP. The individual Duroc cumulus-oocyte complexes (COCs) covered a smaller area at time of collection, but a broader cumulus expansion was observed from 0-20 h compared to Landrace. More Duroc oocytes exhibited advanced stages of nuclear maturation at 20 h of maturation, while more Landrace oocytes showed advanced stages of cortical granule (CG) distribution. Nuclear maturation to MII stage at 48 h did not differ between Duroc and Landrace and was consistently high in both breeds. In addition, no significant differences were observed for glutathione content or CG distribution at the end of maturation. It was therefore questioned if subsequent in vitro fertilization and embryo development would be different between the breeds. In paper II, in vitro fertilization was optimized and the effect of two ‘progressively motile sperm to oocyte ratios’ on fertilization and embryo development was assessed. Random sow oocytes of unknown breed in both the luteal and follicular phase of the oestrus cycle were fertilized with frozen-thawed sperm from three Duroc and three Landrace boars at a 250:1 and 500:1 progressively motile sperm to oocyte ratio. Fertilization with the 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared to the 250:1 ratio, while no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates although all IVEP rounds were adjusted for the same number of progressively motile sperm cells present per oocyte. Promising blastocyst yield and high total blastocyst cell numbers were obtained with cryopreserved sperm from both Duroc and Landrace boars. Since differences were observed between the breeds regarding ovarian characteristics, and nuclear and cytoplasmic oocyte maturation parameters at 20 h of maturation, the aim of paper III was to subsequently assess in vitro embryo development. In addition, follicle diameter and follicular fluid (FF) steroid hormones were measured to study possible relation to oocyte developmental competence. In paper I it was observed at the end of the study that parity, and thus age, was higher in Duroc animals compared to Landrace and this may have affected the maturation results. Therefore, only sows with one or two litters were selected in both breeds for the experiments in paper III. On average a larger follicle diameter was observed on Landrace ovaries compared to Duroc. Individual COC area was larger for Landrace at the start of maturation, while cumulus expansion from 0 to 20 h of maturation was greater for Duroc oocytes and this confirmed the results from paper I. After fertilization, cleavage rate was higher for Duroc oocytes and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace boar. No significant differences were observed between the breeds for the individual steroid hormones, but differences in patterns were observed in the steroid pathways. Both the total level of oestrogens, and aromatase products/substrates ratio were higher in Landrace FF than in Duroc, indicating a higher degree of feminization in Landrace. In conclusion, breed differences were found at the early follicular phase regarding in vitro oocyte developmental competence and steroidogenesis. In summary, differences between the breeds were observed during in vitro maturation, fertilization and embryo culture. Results suggest that Duroc oocyte have a better developmental competence during in vitro embryo production. Further studies are recommended to optimize IVEP protocols per breed and to find out how differences between breeds are related to oocyte developmental competence. The work in this thesis has direct practical relevance for Norsvin, as the established in vitro embryo production and staining protocols and acquired knowledge can be further used in follow-up studies related to in vitro embryo production, embryo quality and freezing and storage of embryos. Moreover, the acquired knowledge on embryo handling and quality is valuable for future commercialisation of embryo transfers.Bruk av embryoteknologi er av interesse i svineavl for å øke genetisk framgang i svinepopulasjonene og for å distribuere genetikk fra verdifulle dyr over hele verden. Produksjon og transport av embryo fører til mindre transport av levende dyr og redusert risiko for overføring av smittsomsykdom. I Norge finnes det avlsprogram for to raser med ulike reproduksjonsegenskaper. Den norske farrasen duroc har i gjennomsnitt 9,7 fødte grisunger totalt per kull sammenlignet med 14,3 i norsk landsvin, som er en morrase. Raseforskjellen i antall fødte grisunger kan skyldes forhold knyttet til eggstokkene, noe som også vil kunne påvirke embryoproduksjonen in vitro (IVEP). Dette kan kreve ulike optimaliseringer for å kunne produsere in vitro blastocyster i begge raser. Målet med denne avhandlingen var å etablere protokoller for in vitro embryoproduksjon hos gris samt protokoller for farging av eggceller og embryoer for å skaffe kunnskap om in vitro modning, befruktning og embryo kultur hos duroc og landsvin. I artikkel 1 ble eggstokker fra begge raser samlet én dag etter avvenning. Eggstokkene ble karakterisert og nukleær og cytoplasmatisk modning av oocyttene ble studert. Eggstokkene hos landsvin hadde signifikant flere 3-8 mm follikler sammenlignet med duroc, og dermed flere eggceller tilgjengelige for IVEP. I gjennomsnitt var cumulusoocytt- kompleksene (COCs) hos duroc mindre i størrelse ved starten av kultur (0 timer), men det ble observert en større grad av cumulusekspansjon fra 0-20 timers modning sammenlignet med landsvin. Flere eggceller fra duroc var kommet lenger i nukleær modning etter 20 timers modning, mens flere eggceller fra landsvin viste avanserte stadier av fordeling av de kortikale granula (CG). Prosent fullført nukleær modning til MII-stadiet etter 48 timer var høy og lik i begge raser. Det ble heller ikke observert signifikante forskjeller i innholdet av glutathion eller for CG-fordeling ved slutten av modningen. Det ble derfor interessant å undersøke om påfølgende in vitro befruktning og embryoutvikling ville være forskjellig mellom rasene. I artikkel II ble in vitro fertilisering optimalisert, og effekten av to forskjellige forholdstall for "progressivt bevegelige spermier per eggcelle" ble undersøkt med hensyn på befruktning og embryoutvikling. Til forsøket ble det samlet eggceller fra follikler i både follikel – og lutealfase fra ukjent donor. Til befruktningen ble det benyttet kryokonserverte spermier fra 3 duroc og 3 landsvin råner i et forhold på 250:1 eller 500:1 «progressivt bevegelige spermier per eggcelle». Befruktning med forholdstallet 500:1 resulterte i høyere befruktningsprosent og høyere blastocystutbytte på dag 6 sammenlignet med forholdstallet 250:1, men ingen effekt av forholdstallet ble observert for polyspermi (egg befruktet av 2 eller flere sædceller), celledeling på dag 2 (som angir % befruktning) eller celleantall i blastocystene. Individuelle forskjeller mellom råner ble observert for befruktning, celledeling og blastocystprosent, selv om alle IVEP-runder ble justert for det samme antallet progressivt bevegelige sædceller per eggcelle. Lovende blastocystutbytte og høye totale blastocystcelletall ble oppnådd med kryokonservert sæd fra begge raser. Siden det ble observert forskjeller mellom rasene med hensyn til eggstokkenes karakteristika og parametre for nukleær og cytoplasmatisk modning etter 20 timer, var målet med artikkel III å studere in vitro embryoutvikling i de to rasene. I tillegg ble follikkeldiameter og steroidhormoner i follikkelvæske (FF) målt for å undersøke om disse var relatert til eggcellenes utviklingsevne. I artikkel I ble det observert at kullnummer, og dermed alder, var høyere hos duroc- enn landsvinpurkene, dette kan ha påvirket modningsresultatene. Derfor ble det kun selektert purker som hadde hatt ett eller to kull til forsøkene i artikkel III. I gjennomsnitt ble det observert en større follikkeldiameter på eggstokkene fra landsvin sammenlignet med duroc. Individuelt COC-areal var større for landsvin ved starten av modningen, mens cumulusekspansjonen fra 0 til 20 timers modning var større for duroc og dette bekreftet resultatene fra artikkel I. Etter befruktning var celledeling høyere for duroc, og det høyeste blastocystutbyttet ble oppnådd for duroc-eggceller befruktet med landsvinsæd. Det ble ikke observert signifikante forskjeller mellom rasene for de enkelte steroidhormonene, men forskjeller i mønstre ble observert for steroidveiene. Både det totale nivået av østrogener og forholdet mellom aromataseprodukter og substrater var høyere i landsvin FF enn i duroc, noe som indikerer en høyere grad av feminisering i landsvin. Oppsummert ble det observert forskjeller mellom rasene under in vitro modning, befruktning og embryokultur. Resultatene tyder på at eggceller fra duroc har en bedre utviklingsevne under IVEP. Videre forskning anbefales for å optimalisere IVEP protokoller per rase og for å finne ut hvordan forskjeller mellom rasene er relatert til eggcellenes utviklingsevne. Arbeidet i denne oppgaven har direkte praktisk relevans for Norsvin, da de etablerte protokollene og tilegnet kunnskap kan brukes videre i oppfølgingsstudier knyttet til IVEP, embryokvalitet og embryo frysing og lagring. Dessuten er den tilegnete kunnskapen om embryohåndtering og kvalitet verdifull for fremtidig kommersialisering av embryooverføringer.Het gebruik van embryotechnologie is van belang in de varkensfokkerij om de genetische vooruitgang in het fokprogramma te versnellen en om de genetica van waardevolle dieren wereldwijd te verspreiden. Wanneer dit kan door middel van embryos, is er daarnaast ook minder transport van levende dieren nodig en is er een verlaagd risico op overdracht van ziekten. In Noorwegen zijn er fokprogramma's voor twee rassen met verschillende reproductieve kenmerken. De Noorse Duroc berenlijn heeft gemiddeld 9.7 totaal aantal geboren biggen per worp vergeleken met 14.3 biggen in de Noorse Landras zeugenlijn. Deze rasverschillen kunnen gerelateerd zijn aan kenmerken van de ovaria, die ook van invloed kunnen zijn op in vitro embryo productie. Het is mogelijk dat verschillende aanpassingen in protocollen vereist zijn om succesvol in vitro blastocysten te kunnen produceren in beide rassen. Het hoofddoel van dit proefschrift was om protocollen op te zetten voor in vitro embryo productie (IVEP) in varkens en voor het kleuren van eicellen en embryos om kennis op te doen over in vitro rijping, bevruchting en embryo ontwikkeling in de Noorse Duroc en Landras rassen. In artikel I werden de ovaria en in vitro nucleaire en cytoplasmatische eicelrijping bestudeerd in beide rassen één dag na het spenen. Landras ovaria hadden significant meer 3-8 mm follikels in vergelijking tot Duroc ovaria en hierdoor kunnen dus meer eicellen verzameld worden voor in vitro embryo productie. Gemiddeld waren de Duroc cumulus-eicelcomplexen (COCs) kleiner van grootte op 0 uur, maar een grotere mate van cumulusexpansie werd waargenomen van 0-20 uur rijping in vergelijking met Landras. Meer Duroc eicellen waren na 20 uur rijping in verder gevorderde stadia van nucleaire rijping, terwijl meer Landras eicellen vergevorderde stadia van corticale granule (CG) distributie vertoonden. Het percentage voltooide nucleaire rijping tot MII-stadium na 48 uur was hoog en gelijk in beide rassen. Er werden bovendien geen significante verschillen waargenomen tussen de rassen in het glutathiongehalte of de CG-verdeling aan het einde van de rijping. Het was daarom interessant om te onderzoeken of de daaropvolgende in vitro bevruchting en embryo ontwikkeling verschillend zou zijn in de rassen. In artikel II werd de in vitro bevruchting geoptimaliseerd en werd het effect van twee 'progressief beweeglijke sperma-tot-eicelverhoudingen' op de bevruchting en embryo ontwikkeling bestudeerd. Voor het experiment werden willekeurige eicellen verzamelend in zowel de luteale- als folliculaire fase van de oestruscyclus van onbekende zeugen. Voor de bevruchting werd ingevroren sperma van drie Duroc en drie Landras beren gebruikt in een verhouding van 250:1 en 500:1 progressief beweeglijke sperma per eicel. Bevruchting met de 500:1 verhouding resulteerde in een hoger bevruchtingspercentage en hogere blastocystopbrengst op dag 6 vergeleken met de 250:1 verhouding, maar er werd geen verschil waargenomen voor polyspermie (eicel bevrucht door 2 of meer zaadcellen), celdeling op dag 2 (wat aangeeft hoeveel eicellen bevrucht waren waarna celdeling begon) of het totale blastocyst celaantal. Individuele verschillen tussen de beren werden waargenomen voor bevruchting, celdeling en blastocystopbrengt, hoewel alle IVEP-rondes werden aangepast voor hetzelfde aantal progressief beweeglijke spermacellen die per eicel aanwezig waren. Veelbelovende blastocystopbrengst en hoge blastocyst celaantal werden verkregen met ingevroren sperma van beide rassen. Aangezien er verschillen tussen de rassen werden waargenomen met betrekking tot het aantal follikels op de eierstokken en parameters voor nucleaire en cytoplasmatische rijping na 20 uur, was het doel van artikel III om vervolgens de in vitro embryo opkweek en ontwikkeling te bestuderen. Verder werden de follikeldiameter en steroïde hormonen in folliculaire vloeistof (FF) gemeten om te onderzoeken of deze verband houden met de ontwikkelingscompetentie van de eicel. In artikel I werd aan het einde van het onderzoek waargenomen dat pariteit, en dus leeftijd, hoger was bij Duroc dieren in vergelijking met Landras en dit kan de rijpings resultaten hebben beïnvloed. Daarom werden voor de experimenten in artikel III alleen zeugen met één of twee worpen geselecteerd. Gemiddeld werd een grotere follikel diameter waargenomen op de eierstokken van Landras zeugen in vergelijking met Duroc zeugen. Het individuele COC oppervlak was weer groter voor Landras op 0 uur, terwijl cumulusexpansie van 0 tot 20 uur rijping groter was voor Duroc eicellen en dit bevestigde de resultaten van artikel I. Na de bevruchting was de celdeling op dag 2 hoger voor Duroc eicellen en de hoogste blastocystopbrengst werd verkregen voor Duroc eicellen die waren bevrucht met de Landras beer. Er werden geen verschillen waargenomen tussen de rassen voor de individuele steroïde hormonen, maar er werden verschillen in patronen waargenomen in de steroïde routes. Zowel het totale gehalte aan oestrogenen als de verhouding aromataseproducten/substraten waren hoger in Landras FF dan in Duroc, wat wijst op een hogere mate van feminisering in Landras. Er werden dus verschillen gevonden in de vroege folliculaire fase met betrekking tot ontwikkelingscompetentie van de eicellen en steroïdogenese. Samenvattend, in dit proefschrift werden er verschillen waargenomen tussen de rassen tijdens in vitro rijping, bevruchting en embryo ontwikkelng. De resultaten suggereren dat Duroc eicellen een betere ontwikkelingscompetentie hebben tijdens in vitro embryo productie. Verder onderzoek wordt aanbevolen om de IVEP protocollen te optimaliseren per ras en om uit te zoeken hoe verschillen tussen de rassen verband houden met de ontwikkelingscompetentie van eicellen. Het werk in dit proefschrift heeft direkte praktische relevantie voor Norsvin, aangezien de opgezette in vitro embryo productie en analyse protocollen en verworven kennis verder kunnen worden gebruikt in vervolgonderzoeken met betrekking tot in vitro embryo productie, embryokwaliteit en het invriezen en bewaren van embryos. Bovendien is de verkregen kennis over het hanteren en de kwaliteit van embryo's waardevol voor toekomstige commercialisering van embryo transplantaties.Norsvin Norges forskningsrå

Cumulus Cell and Oocyte Gene Expression in Prepubertal Gilts and Sows Identifies Cumulus Cells as a Prime Informative Parameter of Oocyte Quality

Cumulus cells (CCs) are pivotal during oocyte development. This study aimed to identify novel marker genes for porcine oocyte quality by examining the expression of selected genes in CCs and oocytes, employing the model of oocytes from prepubertal animals being of reduced quality compared to those from adult animals. Total RNA was extracted either directly after follicle aspiration or after in vitro maturation, followed by RT-qPCR. Immature gilt CCs accumulated BBOX1 transcripts, involved in L-carnitine biosynthesis, to a 14.8-fold higher level (p < 0.05) relative to sows, while for CPT2, participating in fatty acid oxidation, the level was 0.48 (p < 0.05). While showing no differences between gilt and sow CCs after maturation, CPT2 and BBOX1 levels in oocytes were higher in gilts at both time points. The apparent delayed lipid metabolism and reduced accumulation of ALDOA and G6PD transcripts in gilt CCs after maturation, implying downregulation of glycolysis and the pentose phosphate pathway, suggest gilt cumulus–oocyte complexes have inadequate ATP stores and oxidative stress balance compared to sows at the end of maturation. Reduced expression of BBOX1 and higher expression of CPT2 in CCs before maturation and higher expression of G6PD and ALDOA after maturation are new potential markers of oocyte quality.publishedVersio

Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

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Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

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Ovarian characteristics and in vitro nuclear and cytoplasmic oocyte maturation in Duroc and Landrace pigs

Differences in total number of piglets born per litter are observed between the Norwegian Duroc (ND) sire and Norwegian Landrace (NL) dam line. The aim of this study was to evaluate ovarian characteristics, and in vitro nuclear and cytoplasmic oocyte maturation in both breeds. One day after weaning, follicular phase ovaries were collected. Ovary length and weight were measured and the number of follicles (< 3 mm and 3–8 mm) was counted. Cumulus-oocyte complexes (COCs) were collected and matured for 48 hr. To assess cumulus expansion, COC area was analysed at 0 and 20 hr. Nuclear maturation and cortical granule (CG) distribution were analysed at 20 and 48 hr, and total glutathione (GSH) was measured at 48 hr to further elucidate cytoplasmic maturation. In first parity sows, a smaller ovary length and fewer 3 to 8 mm follicles were observed in ND compared to NL. For all sows, ND COCs covered a significantly smaller area at 0 hr, but a higher cumulus expansion ratio was observed at 20 hr compared to NL (364 ± 46% versus. 278 ± 27%, p < 0.001). At 20 hr, more ND oocytes exhibited advanced stages of nuclear maturation, while more NL oocytes showed advanced stages of CG distribution. Nuclear maturation to MII stage at 48 hr did not differ between ND and NL oocytes (90.1% and 87.7%, respectively). Moreover, no significant differences were observed for GSH content or CG distribution after maturation. In conclusion, differences with regard to ovarian characteristics as well as to cumulus expansion, and nuclear and cytoplasmic oocyte maturation at 20 hr were observed between the breeds. Further studies are required to determine if this subsequently affects in vitro fertilization and embryo development.publishedVersio

Effect of two 'progressively motile sperm-oocyte' ratios on porcine in vitro fertilization and embryo development

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Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analyzed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P ¼ 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase

Effect of two 'progressively motile sperm-oocyte' ratios on porcine in vitro fertilization and embryo development

Sperm motility and viability of cryopreserved semen vary between boars and straws, which influences the outcomes of in vitro embryo production (IVEP). However, progressive motility is usually not considered during IVEP. The aim of this study was to assess fertilization with a 500:1 and 250:1 ‘progressively motile sperm to oocyte’ ratio on IVEP outcomes using semen from three Duroc and three Landrace boars. Frozen–thawed sperm was centrifuged through a 45/90% Percoll® density gradient and sperm quality parameters were assessed. In vitro matured oocytes were fertilized at the two ratios, a portion was stained 10–12 h after start of fertilization to analyze fertilization and polyspermy, while the remaining zygotes were cultured up to day 7. The 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared with the 250:1 ratio, but no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates, but not for the other IVEP outcomes. In conclusion, a higher fertilization and blastocyst yield was obtained with the 500:1 ratio compared with the 250:1 ratio, while polyspermy level was consistent across ratios. Differences in IVEP outcomes were still observed between the individual boars although adjusted for progressive motility. Promising blastocyst yields and high total blastocyst cell counts were obtained with sperm from both breeds