UNIVERSITATEA DE TIINE AGRICOLE I MEDICIN VETERINAR CLUJ-NAPOCA COALA DOCTORAL

FACULTATEA DE ZOOTEHNIE I BIOTEHNOLOGII

Ing. Hettig Andrea Klara

VITRIFICAREA OVOCITELOR PROVENITE DE LA SUINE: EFECTUL CONCENTRAIEI DE CRIOPROTECTOR I A TIMPULUI DE EXPUNERE

REZUMAT AL TEZEI DE DOCTORAT

Conductor tiinific Prof. dr. ing. Miclea Vasile

Cluj-Napoca 2011

CUPRINS INTRODUCERE ............................................................................................................... 4 PARTEA I .......................................................................................................................... 4 CAPITOLUL 1. BAZELE CRIOBIOLOGIEI .............................................................. 4 1.1. ELEMENTELE CRIOBIOLOGIEI.......................................................................... 4 1.2. STRUCTURA OVOCITEI I EFECTELE NEGATIVE ALE CRIOCONSERVRII..................................................................................................... 4 CAPITOLUL 2 .................................................................................................................. 5 AGENII CRIOROTECTORI UTILIZAI N PROCESUL DE............................... 5 VITRIFICARE .................................................................................................................. 5 2.1. CRIOPROTECTORI PERMEABILI SAU INTERNI ............................................. 5 2.2. CRIOPROTECTORI IMPERMEABILI SAU EXTERNI ....................................... 6 CAPITOLUL 3 .................................................................................................................. 6 EFECTELE CRIOPROTECTORILOR......................................................................... 6 3.1. TOXICITATEA I DAUNELE PRODUSE DE CRIOPROTECTORI ................... 6 3.1.1. Influena timpului de expunere, a concentraiei i a temperaturii asupra toxicitii substanelor crioprotectoare....................................................................... 6 3.1.2. Evitarea daunelor i a toxicitii....................................................................... 7 CAPITOLUL 4 .................................................................................................................. 7 CRIOCONSERVAREA PRIN VITRIFICARE METODE UZUALE ..................... 7 PARTEA II ........................................................................................................................ 7 CERCETRI PROPRII ................................................................................................... 7 SCOPUL I OBIECTIVELE CERCETRII ................................................................ 7 SCHEMA EXPERIMENTAL GENERAL ............................................................... 8 CAPITOLUL 5 .................................................................................................................. 8 METODE DE VITRIFICARE UTILIZATE ................................................................. 8 5.1 VITRIFICAREA PRIN METODA SOPS A OVOCITELOR SUINE IMATURE ... 8 5.1.1. Material biologic............................................................................................... 8 5.1.2. Medii utilizate.................................................................................................... 9 5.1.3. Metoda de lucru................................................................................................. 9 5.1.3.1. Recoltarea ovarelor i a ovocitelor............................................................. 9 5.1.3.2. Vitrificarea ovocitelor prin metoda SOPS ................................................. 9 5.1.3.3. Decongelarea ovocitelor........................................................................... 10 5.1.3.4. Splarea ovocitelor i evaluarea morfologic .......................................... 10 5.1.3.5. Denudarea ovocitelor vitrificate i evaluarea viabilitii prin colorarea cu diacetat de fluorescein i iodur de propidiu...................................................... 10 5.2 VITRIFICAREA PE SUBSTRAT SOLID (SSV) A OVOCITELOR SUINE IMATURE..................................................................................................................... 10 2

5.2.1.Crioconservarea ovocitelor prin metoda vitrificrii pe substrat solid (SSV) .. 10 5.2.2. Decongelarea i evaluarea ovocitelor ............................................................ 11 5.3. VITRIFICAREA PRIN METODA SOPS A OVOCITELOR SUINE MATURATE IN VITRO................................................................................................ 11 5.3.1. Materialul biologic.......................................................................................... 11 5.3.2. Medii utilizate.................................................................................................. 11 5.3.3. Metoda de lucru............................................................................................... 12 5.3.3.1. Recoltarea i maturarea ovocitelor........................................................... 12 5.3.3.2. Crioconservarea ovocitelor maturate in vitro prin metoda SOPS............ 12 5.3.3.3. Evaluarea morfologic post-vitrificare a ovocitelor maturate in vitro..... 12 5.3.3.4. Evaluarea viabilitii post-vitrificare a ovocitelor maturate in vitro........ 12 CAPITOLUL 6 ................................................................................................................ 13 REZULTATE I DISCUII .......................................................................................... 13 6.1. INFLUENA CONCENTRAIEI DE CRIOPROTECTORI I A TIMPULUI DE EXPUNERE ASUPRA VITRIFICRII PRIN METODA SOPS A OVOCITELOR SUINE IMATURE.............................................................................. 13 6.2. INFLUENA CONCENTRAIEI DE CRIOPROTECTORI I A TIMPULUI DE EXPUNERE ASUPRA VITRIFICRII PRIN METODA SSV A OVOCITELOR SUINE IMATURE ........................................................................................................ 15 6.3. ANALIZA COMPARATIV A REZULTATELOR VITRIFICRII PRIN TEHNICILE SOPS I SSV A OVOCITELOR SUINE IMATURE .............................. 16 6.4. VITRIFICAREA PRIN METODA SOPS A OVOCITELOR SUINE MATURATE IN VITRO................................................................................................ 17 CAPITOLUL 7. ............................................................................................................... 18 CONCLUZII GENERALE I RECOMANDRI ....................................................... 18 BIBLIOGRAFIE SELECTIV ..................................................................................... 21

INTRODUCERE Dei istoria vitrificrii dateaz de mai mult de trei sferturi de secol, domeniul este probabil, nc n faza incipient pentru a fi utilizat la nivelul ntregului su potenial. Ca ntotdeauna, natura ajut cercettorii n descoperirea abordri noi n vitrificare, dar n cea mai mare parte, procesele din natur rmn ascunse i dificile pentru a fi imitate n mod direct. Vitrificare este o abordare viabil pentru crioconservarea unei game largi de sisteme vii. Principiile sale fizice i biologice continu s fie mai bine nelese, iar acest lucru conduce la aplicaii din ce n ce mai numeroase i de succes. Cu toate acestea, specialitii n criobiologia reproduciei, posed att mijloace ct i motivaia de a rspunde la una din cele mai interesante provocri ale criobiologiei: aducerea celulelor ntr-o stare de animaie suspendat i repornirea metabolismului acestora, astfel nct s devin posibil nceperea unei noi viei. PARTEA I CAPITOLUL 1. BAZELE CRIOBIOLOGIEI 1.1. ELEMENTELE CRIOBIOLOGIEI Criobiologia reprezint unul dintre domeniile biologice care a cunoscut o dezvoltare spectaculoas n ultimele decenii. Domeniul are ca i obiect de studiu totalitatea modificrilor pe care le sufer celulele, esuturile, organele i organismele integre (bios = via), atunci cnd sunt supuse aciunii temperaturilor sczute n general (grecescul crios = frig) i a celor negative, n special. Partea aplicativ a acestui domeniu poart denumirea de crioconservare i reprezint totalitatea metodelor, mijloacelor i tehnicilor prin care, cu ajutorul frigului, se urmrete conservarea de lung durat a vieii indiferent de forma sub care aceasta se prezint (celule, esuturi, organe, organisme integre). Crioconservarea se bazeaz pe principiul c temperaturile sczute determin, la nivel intracelular, o reducere a micrii moleculare i, implicit, o scdere semnificativ a metabolismului pn la nivele extreme, rezultnd aa numita anabioz (Ladoi, 1997). 1.2. STRUCTURA OVOCITEI I EFECTELE NEGATIVE ALE CRIOCONSERVRII Ovocitele sunt mai dificile de crioconservat dect embrionii, datorit faptului c acestea sunt celule sferice, mari, cu o suprafa mic (raportat la volum) i conductivitate hidraulic sczut (Leibo, 1980). Ovocita este cea mai mare celul din corpul majoritii speciilor de mamifere (Wassarman, 1988). Aproximativ 80% din volumul acestora este ap. Atunci cnd sunt rcite la temperaturi sub 0C, se poate forma ghea intracelular, probabilitate care este dependent de rata scderii temperaturii (Leibo et al.,1978). Deteriorrile pot fi att la nivel intracelular (nucleu, citoplasma cu organitele celulare: mitocondrii, actine/microfilamente, fus de diviziune, microtubuli) ct i extracelular (zona pellucida).

CAPITOLUL 2 AGENII CRIOROTECTORI UTILIZAI N PROCESUL DE VITRIFICARE Vitrificarea este o solidificare a soluiilor fr formarea cristalelor de ghea prin creterea vscozitii n timpul scderii temperaturii. Lipsa total a cristalelor de ghea din timpul vitrificrii este datorat concentraiei mari de crioprotectori. Cristalele mici de ghea formate la decongelarea lichidelor vitroase (devitrificarea) nu sunt considerate periculoase atta timp ct nu are loc recristalizarea mediului (unirea cristalelor mici de ghea i formarea cristalelor mari) (Ada Cean, 2009). Crioprotectorii folosii la crioconservarea entitilor biologice (ovocite, spermatozoizi, embrioni) sunt clasificate n dou grupe mari: crioprotectori permeabili sau interni crioprotectori impermeabili sau externi 2.1. CRIOPROTECTORI PERMEABILI SAU INTERNI Crioprotectori permeabili sau interni, au o greutate molecular mic ce le face posibil trecerea prin membrana celular, oferind protecie intern ovocitelor, embrionilor mpotriva frigului; crioprotectorii interni formeaz legturi de hidrogen cu moleculele de ap, prevenind astfel formarea cristalelor de ghea i a efectului de soluie. Aceti compui chimici se pot difuza prin membrana celular, permeabiliznd celula i echilibrndu-se cu citoplasma. nlocuiesc marea majoritate a cantitii de ap intracelular fr s deshidrateze total celula. Crioprotectorii interni solidific la temperaturi mai joase dect apa, reducnd astfel substanial cantitatea de ghea intracelular format la o temperatur dat. Aceast descretere a cantitii de ghea intracelular diminueaz deteriorrile cauzate de perturbarea fizic a organitelor i a membranei celulare. n plus, crioprotectori sunt i o soluie tampon ce ofer protecie mpotriva stresului srurilor. Acionnd ca solvent, reduce concentraia soluiei n fraciunea de ap rmas n interiorul celulei pn cnd sistemul este rcit la o temperatur suficient de sczut (Swain i Smith, 2010). Muli ageni crioprotectori au fost utilizai cu succes n conservarea spermatozoizilor, embrionilor i a ovocitelor. Succesul utilizrii fiecrui tip de crioprotector n parte, depinde de viteza cu care acetia traverseaz membrana celular, rat dictat de diferii factori, cum ar fi spre exemplu vscozitatea. Totui permeabilitatea nu depinde numai de crioprotectorul n sine ci i de proprietile membranei celulare, ce, variaz n funcie de tipul i stadiul celular (Pedro et al., 2005). Din categoria crioprotectorilor permeabili cei mai importani sunt: etilen glicolul (EG), dimetil sulfoxidul (DMSO), propilen glicolul (1,2 propandiol, propilen diol, PROH) i glicerolul. Din punct de vedere a toxicitii, etilen glicolul este cel mai puin toxic dintre crioprotectorii permeabili i prezint o permeabilitate mai mare n ceea ce privete traversarea membranar, motiv pentru care, a fost integrat n protocolul de vitrifcare din prezentul studiu.

2.2. CRIOPROTECTORI IMPERMEABILI SAU EXTERNI Crioprotectori externi, au o greutate molecular mare neputnd traversa membrana celular, dar ofer o protecie extern celulelor. Crioprotectorii permeabili faciliteaz vitrificarea, stabilizeaz membrana n timpul decongelrii, reduce ocul osmotic n timpul ndeprtrii crioprotectorului intern din celule (Tucker i Liebermann, 2007). Aceti compui cresc osmolaritatea spaiului extracelular, ce duce la deshidratarea celulei i deci la reducerea riscului formrii cristalelor de ghea intracelulare. De asemenea a fost postulat c, unii crioprotectori impermeabili se absorb pe suprafaa membranar, inhibnd formarea cristalelor de ghea din imediata vecintate a celulei prin meninerea gheii n stare amorf. Astfel, ageni crioprotectori externi, sunt des incluse n compoziia mediilor de decongelare a celulelor pentru a preveni ocul osmotic. ocul osmotic este descris ca un fenomen, ce apare atunci cnd presiunea osmotic a compartimentrii intracelulare este mai mare dect cel extracelular, i se datoreaz deshidratrii celulare. n timpul dezgherii, datorit influxului de ap pot avea loc lezri i traumatizri ale celulelor. Din aceast categorie de crioprotectori fac parte: macromoleculele (ficolul, polivinil pirolidona, polivinil alcoolul, polietilen glicolul), zaharurile (trehaloza, sucraza, glucoza, latoza, lactoza). n prezentul studiu s-a utilizat trehaloza ca i crioprotector extern.

CAPITOLUL 3 EFECTELE CRIOPROTECTORILOR 3.1. TOXICITATEA I DAUNELE PRODUSE DE CRIOPROTECTORI n ceea ce privete criobiologia gameilor, de obicei, sunt efectuate msuri de evaluare a adecvrii sau toxicitii a unui agent crioprotector. La spermatozoizi, aceste msuri includ urmrirea efectului asupra mobilitii, capacitrii i a capacitii fertilizante. La ovocite, se evalueaz deteriorrile produse de crioprotectori asupra fusului de diviziune, citoscheletul actinelor, aranjarea cromozomilor i asupra capacitrii fertilizante. Cu privire la embrioni, studiile de toxicitate sunt ndreptate spre evaluarea dezvoltrii embrionare ulterioare i asupra efectelor pe diferite ci moleculare i biochimice de semnalizare. Din pcate, mecanismele exacte prin care se manifest toxicitatea agenilor crioprotectori sunt necunoscute. Exemple de leziuni induse de expunerea n mod abuziv la diferii crioprotectori ar fi nlturarea i fuziunea membranelor celulare i activitate enzimatic precar. Ele duc la modificri ale structurilor i funciilor organitelor i la perturbarea interaciunilor proteice (Fahy et al., 1990; Anchordouguy et al., 1987). 3.1.1. Influena timpului de expunere, a concentraiei i a temperaturii asupra toxicitii substanelor crioprotectoare Condiiile n care are loc expunerea la crioprotector pot influena toxicitatea acestora asupra gameilor. Factori, cum ar fi timpul de expunere, concentraia de crioprotector, precum i temperatura de expunere au repercusiuni asupra efectului agentului crioprotector respectiv. Agenii crioprotectori nu sunt toxici n cazul n care celulele sunt expuse la aciunea lor un timp scurt, ns o expunere mai ndelungat, sau o concentraie ridicat, poate duce la metabolizarea agentului crioprotector, ceea ce se concretizeaz n afectarea funciei i a viabilitii celulare. 6

3.1.2. Evitarea daunelor i a toxicitii Alegerea unui crioprotector adecvat, optimizarea concentraia acestuia, precum i monitorizarea timpului i a temperaturii de expunere, influeneaz poteniale daune pe care un crioprotector le poate provoca ntr-o celul. Prin urmare, o abordare simpl pentru a reduce toxicitatea presupune combinarea diferiilor crioprotectori, att permeabili ct i impermeabili, reducndu-se astfel concentraia individual i atenuarea daunelor provocate, meninnd n acelai timp efectele generale de protecie. Modul de introducere i de evacuare a crioprotectorilor este un alt mijloc de a reduce toxicitatea sau daunele rezultate. Adugarea progresiv a agenilor crioprotectori, sau eliminarea treptat a acestor compui n timpul decongelrii, ajut la reducerea stresului osmotic. CAPITOLUL 4 CRIOCONSERVAREA PRIN VITRIFICARE METODE UZUALE Odat cu dezvoltarea cunotinelor n domeniul criobiologiei i n special al crioconservrii rapide, au aprut numeroase tehnici de vitrificare a ovocitelor i embrionilor tuturor speciilor de animale, precum i a gameilor i embrionilor umani. Cele mai folosite metode sunt: vitrificarea n paiete deschise (OPS), paiete super fine (SOPS), cryoloop, pe substrat solid (SSV), cryotip, cryotop, n paiete hemy-straw, n paiete nchise. n prezentul studiu, ovocitele suine imature au fost vitrificate cu paiete (metoda SOPS i pe substrat solid (metoda SSV). PARTEA II CERCETRI PROPRII SCOPUL I OBIECTIVELE CERCETRII Cercetrile urmresc mbuntirea rezultatelor vitrificrii ovocitelor provenite de la suine utiliznd metodele SOPS i SSV. Pentru realizarea scopului menionat s-au fixat urmtoarele obiective: 1. Determinarea concentraiei optime de etilen glicol pentru vitrificarea ovocitelor suine imature prin metoda SOPS. 2. Stabilirea timpului optim de echilibrare a ovocitelor suine imature crioconservate prin metoda SOPS n funcie de concentraia etilen glicolului n mediul de vitrificare. 3. Determinarea concentraiei optime de etilen glicol pentru vitrificarea ovocitelor suine imature prin metoda SSV. 4. Stabilirea timpului optim de echilibrare a ovocitelor suine imature crioconservate prin metoda SSV n funcie de concentraia etilen glicolului n mediul de vitrificare.

5. Determinarea efectului concentraiei de crioprotector i a timpului de echilibrare n funcie de metoda de vitrificare utilizat. 6. Crioconservarea ovocitelor suine maturate in vitro aplicnd cea mai bun tehnic de vitrificare, concentraie de crioprotector i timpul de echilibrare optim. SCHEMA EXPERIMENTAL GENERAL

VITRIFICAREA OVOCITELOR SUINE

IMATURE

Vitrificarea ovocitelor prin metoda SOPS

Vitrificarea ovocitelor prin metoda SSV

Determinarea influenei concentraiei de EG

Determinarea influenei timpului de echilibrare

Determinarea influenei concentraiei de EG

Determinarea influenei timpului de echilibrare

MATURATE IN VITRO

Ovocite nedenudate

Ovocite denudate

Fig. 1. Schema experimental general CAPITOLUL 5 METODE DE VITRIFICARE UTILIZATE 5.1 VITRIFICAREA PRIN METODA SOPS A OVOCITELOR SUINE IMATURE 5.1.1. Material biologic Materialul biologic utilizat este reprezentat de ovocite suine imature, recoltate din ovare obinute de la abatorul SC. Turism Vlcele SRL judeul Cluj. Pentru prelevarea ovarelor s-au selectat scrofie prepubere aparinnd hibrizilor comerciali, cu greutate maxim de pn la 110 kg. Pentru vitrificarea ovocitelor suine imature prin metoda SOPS s-au folosit un numr de 5.337 de gamei.

5.1.2. Medii utilizate Mediul de transport Pentru transportul ovarelor s-a folosit o soluie salin de 0,9% NaCl n ap pur, suplimentat cu antibiotice (penicilin, streptomicin i gentamicin). Mediul de recoltare a ovocitelor Mediu de recoltare a ovocitelor este mediul stoc M 199 1x coninnd Earles salts, aprovizionat de la Sigma. Medii de congelare Mediul de baz (MB), este folosit la toate variantele experimentale i conine: tampon fosfat salin (PBS) 10x, ap deionizat i ser fetal bovin (FSB). Mediul de previtrificare: este compus din mediu de baz (MB, crioprotectorul ce ofer ovocitelor protecie intern, reprezentat n cazul nostru de etilen glicol (EG) i un crioprotector de protecie extern, trehaloza Mediul de vitrificare: conine aceleai substane pe care le conine i mediul de previtrificare, diferenele fiind date de concentraia acestora, n funcie de variantele experimentale. Mediile de decongelare Mediile de decongelare conin tampon fosfat salin (PBS) i trehaloz. Se utilizeaz 3 medii de decongelare avnd concentraii descresctoare de trehaloz. Mediul de splare a ovocitelor Mediul de splare are n componena sa tampon fosfat salin (PBS) suplimentat cu 0,4 % albumin seric bovin (BSA). Mediu de colorare a ovocitelor Mediul de colorare este alctuit din PBS i doi colorani fluoresceni, respectiv diacetat de fluorescein (FDA) i iodur de propidiu (PI). 5.1.3. Metoda de lucru 5.1.3.1. Recoltarea ovarelor i a ovocitelor Dup sacrificarea scroafelor, ovarele sunt colectate ntr-un recipient termoizolant ce conine o soluie salin de NaCl 0,9% la temperatura de 37C. Acestea sunt transportate n laborator ntr-un interval de 4-6 ore. n laborator se detaeaz de restul de oviduct i burs ovarian dup care sunt splate i puse ntr-un recipient ce conine soluie salin suplimentat cu antibiotice. Ovocitele au fost recoltate prin metoda punciei, doar din foliculi nonatretici ce au diametru cuprins ntre 2-5 mm. Cu ajutorul unei seringi de 10 ml prevzut cu un ac de 18G, se ptrunde n corticala ovarian i ptrunderea n ct mai muli foliculi. Dup recoltarea ovocitelor din foliculi, acestea sunt evaluate la stereomicroscop i transferate ntr-o alt plac cu mediu de recoltare n scopul splrii acestora de resturile foliculare. 5.1.3.2. Vitrificarea ovocitelor prin metoda SOPS Ovocitele splate de dou ori n mediu TCM sunt supuse echilibrrii cu agenii crioprotectori n doi pai: previtrificare i vitrificare. Previtrificarea: grupuri de cte 10 ovocite sunt luate cu o pipet Pasteur i puse n picturi de 30 l de mediu de previtrificare. Ovocitele sunt meninute n mediile de previtrificare timp de 4 minute. Vitrificarea: ovocitele sunt transferate n micropicturile de mediu de vitrificare ce conine concentraia final de etilen glicol i trehaloz. Picturile au dimensiuni de 2 l. 9

Dup expirarea timpului de expunere la concentraiile finale de EG i trehaloz (specific fiecrei variante experimentale), ovocitele sunt ncrcare n paietele SOPS, prin simpla atingere a micropicturii cu vrfului pipetei. Mediul mpreun cu ovocitele sunt urcate n paiete prin capilaritate. Tubul cu paietele este apoi transferat n container i stocat pn la evaluare. 5.1.3.3. Decongelarea ovocitelor Paietele se scot pe rnd din tubul ce conine azot, se ine n aer timp de 5 secunde, dup care ovocitele sunt trecute succesiv prin mediile de decongelare (Vajta, 2000). Evacuarea ovocitelor se face prin atingerea mediului cu vrfului paietei SOPS. Printr-un fenomen invers capilaritii ovocitele sunt evacuate n picturi i sunt lsate timp de 5 minute (Somfai, 2007) n fiecare mediu n parte. Dup trecerea ovocitelor i prin ultimul mediu de decongelare, ovocitele sunt luate cu o pipet n mediul de splare. 5.1.3.4. Splarea ovocitelor i evaluarea morfologic Ovocitele sunt splate de 3 ori n picturi de 60 l de PBS suplimentat cu BSA 0,4%. pentru a scpa de urmele de crioprotectori. Dup splare, ovocitele sunt examinate n vederea evalurii morfologice. Examinarea s-a realizat la un microscop cu inversie Olympus. Au fost cuantificate ovocitele care au prezentat straturile cumulusului aderente, bine ataate de ovocit, zon pellucida intact, membran bine delimitat, ovoplasma uniform granulat i compact 5.1.3.5. Denudarea ovocitelor vitrificate i evaluarea viabilitii prin colorarea cu diacetat de fluorescein i iodur de propidiu Denudarea ovocitei s-a realizat astfel: 15-20 de ovocitele au fost plasate n 30 l PBS, dup care s-a luat nc 200 l PBS ntr-o micropipet i s-a pipetat peste ovocite. Prin micri repetate de pipetare majoritatea celulelor cumulus s-au desprins de ovocit. Colorarea ovocitelor a fost realizat dup denudare. ntr-o prim etap pe o plac Petri sunt puse picturi de 30 l n care sunt plasate grupuri de 10-15 ovocite. Placa cu picturile sunt transferate la termostat la 37C pentru un timp de 10 minute, dup care sunt evaluate la microscop prevzut cu lamp UV. Cele vii, sunt verzi fluoresceni datorita activitii enzimatice (colorate cu FDA) i necolorate cu PI datorit integritii membranare. Cele moarte nu se coloreaz cu FDA, dar se coloreaz cu PI. 5.2 VITRIFICAREA PE SUBSTRAT SOLID (SSV) A OVOCITELOR SUINE IMATURE Materialul biologic, mediile utilizate precum i marea parte a metodei de lucru sunt aceleai ca la metoda SOPS, motiv pentru care vor fi descrise doar procedurile ce difer. Pentru vitrificarea ovocitelor suine imature prin metoda SSV s-au folosit un numr de 5.164 de gamei. 5.2.1.Crioconservarea ovocitelor prin metoda vitrificrii pe substrat solid (SSV) Previtrificare: grupuri de cte 10 ovocite sunt luate cu o pipet Pasteur i puse n picturi de 30 l de mediu de previtrificare. Ovocitele sunt meninute n mediile de previtrificare timp de 4 minute, n cazul tuturor variantelor experimentale 10

Vitrificare: dup expirarea timpului de previtrificare, ovocitele sunt transferate n micropicturile de vitrificare de 2l, ce sunt plasate pe folii de aluminiu, reprezentnd substratul solid. Dup expirarea timpului de echilibrare (specific fiecrei variante experimentale) a ovocitelor n mediul de vitrificare, folia este luat de un col cu ajutorul unei pense lungi i este plasat ntr-un tub, ce este imersat n azot lichid, urmnd ca apoi tubul prevzut cu guri, s fie nchis i transferat i stocat pn la decongelare i evaluare, n containerul cu azot lichid. 5.2.2. Decongelarea i evaluarea ovocitelor La decongelare foliile de aluminiu au fost luate una cte una din tubul cu azot lichid i au fost puse pe placa de nclzire, ce a fost nclzit n prealabil la temperatura de 37C. Astfel, picturile cu ovocite s-au dezgheat pe folia de aluminiu i cu ajutorul unei pipete automate au fost luate i plasate n mediile de decongelare. Dup meninerea ovocitelor n fiecare mediu de decongelare timp de 5 minute, acestea au fost splate n PBS, colorate cu coloranii fluoresceni i evaluate att din punct de vedere morfologic ct i din punct de vedere al viabilitii. Procedeul este acelai ca la ovocitele vitrificate prin metoda SOPS, motiv pentru care nu vor fi descrise i n acest capitol. 5.3. VITRIFICAREA PRIN METODA SOPS A OVOCITELOR SUINE MATURATE IN VITRO 5.3.1. Materialul biologic Materialul biologic este reprezentat de ovocitele suine imature recoltate din ovarele de scroafe abatorizate. 5.3.2. Medii utilizate Mediul de transport al ovarelor i mediile de recoltare Sunt identice cu cele descrise in subcapitolul 5.2.2, motiv pentru care acestea nu vor fi redate i n capitolul acesta. n cele ce urmeaz se vor descrie subpunctele specifice acestui capitol. Mediul de maturare ovocitar Mediul de maturare a fost reprezentat de M 199 suplimentat cu L-glutamin (0,1 g/l), piruvat de sodiu (0,1 mg/ml), Folligon (10 UI/ml) ca nlocuitor al hormonului hipofizar FSH, Chorulon (10 UI/ml) ca nlocuitor al LH din vivo (Intervet), ser fetal de viel 10%, penicilin (100 UI/ml), streptomicin (100 g/ml). Verificarea i corectarea pH-ului s-a realizat la valoarea de 7,4. Picturi de 30 l de mediul de maturare a fost distribuit n placi Petri. Peste picturi a fost pus ulei mineral pentru a evita evaporarea acestor cantiti mici de mediu. Mediul de crioconservare: Este reprezentat de mediul de baz la care se adaug crioprotectorii. Mediul de previtrificare conine MB, 15% EG i 0,25M trehaloz. Mediul de vitrificare conine MB, 45% EG i 0,5M trehaloz Mediul de decongelare Este acelai ca i n cazul vitrificrii ovocitelor imature. Mediul de colorare

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Mediul de colorare difer fa ceea utilizat n cazul ovocitelor imature prin faptul c acesta conine un al treilea colorant: Hoechst 33258. 5.3.3. Metoda de lucru 5.3.3.1. Recoltarea i maturarea ovocitelor Metoda de recoltare a ovocitelor este aceeai ca i ceea descris n subcapitolul 5.1.3. Ovocitele au fost alese pentru maturare pe baza evalurii morfologice. nainte de plasarea ovocitelor n mediul de maturare, plcile Petri au fost lsate n incubator timp de 3 ore. Grupuri de cte 10 ovocite au fost plasate n picturile de medii de maturare. Plcile Petri cu ovocite au fost lsate n incubator la temperatura de 37C, cu 5% CO2 n aer, timp de 44 ore. 5.3.3.2. Crioconservarea ovocitelor maturate in vitro prin metoda SOPS Deoarece cele mai bune rezultate n cazul vitrificrii ovocitelor imature s-a obinut prin metoda SOPS, cu 45% etilen glicol n mediul de vitrificare, cu un timp de echilibrare de 40 secunde, am recurs la aceti parametri i n cazul vitrificrii ovocitelor maturate in vitro. Aadar, dup 44 ore de maturare, ovocitele au fost transferate din picturile de maturare n picturi de 60 l de PBS suplimentat cu BSA (0,4%). Ovocitele au fost splate n acest mediu de 2 ori dup care au fost supuse procesului de vitrificare. Procedura este aceeai ca i n cazul vitrificrii ovocitelor imature prin metoda SOPS. 5.3.3.3. Evaluarea morfologic post-vitrificare a ovocitelor maturate in vitro Complexele ovocit-cumulus au fost observate la microscop. n vederea evalurii morfologiei ovocitelor s-au utilizat urmtorii indicatori: creterea diametrului complexului ovocit-cumulus i a spaiului perivitelin, mucifierea i expandarea celulelor cumulus ooforus. Dup evaluarea morfologic, ovocitele sunt supuse denudrii, ce se realizeaz mecanic, prin pipetri repetate. Ovocitele astfel pregtite, sunt supuse apoi evalurii viabilitii. 5.3.3.4. Evaluarea viabilitii post-vitrificare a ovocitelor maturate in vitro Pentru a se obine i un alt punct de vedere asupra rezultatelor vitrificrii ovocitelor suine maturate in vitro, s-a realizat n paralel colorarea fluorescent a acestora, utilizndu-se cei trei colorani fluoresceni. Fiecare dintre acetia indic un alt aspect referitor la viabilitatea celular. Consecutiv denudrii ovocitele au fost incubate n mediul care coninea coloranii fluoresceni (FDA, PI i Hoechst 33258) timp de 10 minute i apoi observate n lumin ultraviolet la microscopul inversat iar imaginile preluate cu ajutorul programului Cell F* (Olympus). S-a nregistrat numrul de celule care prezentau fluorescen verde sau roie precum i cele n al cror spaiu perivitelin s-a observat primul globul polar ce s-a colorat albastru.

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CAPITOLUL 6 REZULTATE I DISCUII 6.1. INFLUENA CONCENTRAIEI DE CRIOPROTECTORI I A TIMPULUI DE EXPUNERE ASUPRA VITRIFICRII PRIN METODA SOPS A OVOCITELOR SUINE IMATURE Rezultatele evalurii morfologice ale ovocitelor suine imature vitrificate prin metoda SOPS n figura 2 sunt prezentate rezultatele obinute n urma evalurii morfologice a ovocitelor vitrificate cu 35%, 40%, 45% i 50% etilen glicol n mediul de vitrificare i timp de echilibrare n mediu de 30, 40, 50 i 60 secunde. Aa cum reiese i din figura 2, cele mai bune rezultate cu privire la integritatea morfologic post-congelare s-au obinut n cazul vitrificrii ovocitelor cu 45% etilen glicol n mediul de vitrificare i timp de echilibrare de 40 secunde. Valoarea procentual medie efectuat pe cinci repetiii este de 77,66% 0,75. Aadar, mai mult de 70% din totalul ovocitelor supuse vitrificrii au prezentat dup decongelare o granulaie uniform a ovoplasmei, celulele cumulus ooforus bine ataate celulei i zona pellucida integr.

% 80 70 60 50 40 30 20 35% 40 s e c 40% 45% 50% 30 s e c 60 s e c 50 s e c

Fig. 2. Rezultatele evalurii morfologice ale ovocitelor vitrificate prin metoda SOPS cu diferite concentraii de EG i timpi de echilibrare La aceast concentraie de crioprotector i timp de expunere majoritatea ovocitelor realizeaz schimbul transmembranar ap i crioprotectorul permeabil (EG), realizndu-se astfel gelificarea ovoplasmei i intrarea acesteia n stare solid, vitroas n momentul scderii temperaturii. Uor sub aceast valoare (76,58 2,10) este cea obinut n cazul utilizrii mediului de vitrificare cu 40% EG i timp de echilibrare de 50 secunde. Demn de menionat sunt i 13

procentele de 75,54% 1,29, obinute n urma vitrificrii ovocitelor cu 40% EG n mediul i timp de expunere de 60 secunde. Toate aceste valori menionate, reprezint vrfurile din figura 2. De asemenea, se poate observa foarte bine inferioritatea rezultatelor obinute la concentraiile de 35% i 50% de EG, indiferent de timpul de echilibrare. Rezultatele evalurii viabilitii ovocitelor suine imature vitrificate prin metoda SOPS Rezultatele cu privire la viabilitatea post-congelare a ovocitelor vitrificate cu 35%, 40%, 45% i 50% etilen glicol n mediul de vitrificare i timp de echilibrare n mediu de 30, 40, 50 i 60 secunde, sunt prezentate n figura 3.

% 50 40 30 20 10 0 35% 40 sec 40% 45% 30 sec 60 sec 50 sec

50%

Fig. 3. Rezultatele evalurii viabilitii ovocitelor vitrificate prin metoda SOPS cu diferite concentraii de EG i timpi de echilibrare Ca i n cazul morfologiei, cele mai multe ovocite vii s-au nregistrat n cazul vitrificrii acestora cu 45% EG n mediu i 40 secunde timp de echilibrare. Valoarea procentual medie pe cinci repetiii este de 46,80% 0,32, evideniindu-se foarte bine punctul cel mai nalt n figura 3. Ovocitele prezentau o activitate enzimatic intens, esterificnd diacetatul de flouarescein i colorndu-se verde la lumina UV a microscopului. Cu 1,01% sub valoarea mai sus menionat este varianta experimental cu 45% EG n mediul de vitrificare i timp de echilibrare 50 secunde. Aceast valoare reprezint al doilea vrf n figura 3. Prin crioconservarea n mediul de vitrificare cu 40% EG, valorile procentuale medii ale ovocitelor vii dup decongelare sunt reduse cu 10%-17%. n cazul concentraiei de 35% se reduc cu 15%- 19% fa de valorile celor cu 45%. Aceste concentraii de crioprotector nu sunt suficiente pentru a aduce celulele n stare vitroas n momentul imersrii n azot lichid. indiferent de timpul de expunere. Se pot observa forte bine valorile cele mai mici obinute n cazul vitrificrii ovocitelor suine imature cu 50% etilen glicol, indiferent de timpii de echilibrare. Acest fenomen se explic prin faptul c la aceast concentraie ridicat, crete toxicitatea crioprotectorului, ne mai avnd efecte benefice asupra vitrificrii. 14

6.2. INFLUENA CONCENTRAIEI DE CRIOPROTECTORI I A TIMPULUI DE EXPUNERE ASUPRA VITRIFICRII PRIN METODA SSV A OVOCITELOR SUINE IMATURE Rezultatele evalurii morfologice ale ovocitelor suine imature vitrificate prin metoda SSV n figura 4 sunt prezentate sugestiv, rezultatele obinute n urma evalurii morfologice a ovocitelor vitrificate cu 35%, 40%, 45% i 50% etilen glicol n mediul i timp de echilibrare n mediu de 30, 40, 50 i 60 secunde.

% 80 70 60 50 40 30 20 10 0 35% 40% 45% 50% 30sec 60sec 50sec 40sec

Fig. 4. Rezultatele evalurii morfologice ale ovocitelor vitrificate prin metoda SSV cu diferite concentraii de EG i timpi de echilibrare Vitrificarea ovocitelor suine imature cu concentraia etilen glicolului de 45% la timp de echilibrare de 40 secunde a dat cele mai bune rezultate cu privire la integritatea morfologic a acestora. Procentul mediu la aceti parametri este de 74,81% 1,26. Urmat de aceast valoare este ceea obinut n cazul vitrificrii cu mediu ce conine 40% etilen glicol i timpul de expunere este de 40 secunde (72,83% 1,23). Rezultatele evalurii viabilitii ovocitelor suine imature vitrificate prin metoda SSV n figura 5 sunt prezentate rezultatele cu privire la viabilitatea post-congelare a ovocitelor vitrificate cu 35%, 40%, 45% i 50% etilen glicol n mediul de vitrificare i timp de echilibrare n mediu de 30, 40, 50 i 60 secunde.

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% 50 40 30 20 10 0 35% 40s e c 40% 45% 30s e c 60se c 50se c

50%

Fig. 5. Rezultatele evalurii viabilitii ovocitelor vitrificate prin metoda SSV cu diferite concentraii de EG i timpi de echilibrare Valoarea maxim cu privire la viabilitatea post-congelare este de 42,54% 0,26, reprezentnd punctul cel mai nalt din figura 5. Aceast valoare s-a obinut n cazul vitrificrii ovocitelor n mediu cu 45% etilen glicol i timp de expunere de 40 secunde. 6.3. ANALIZA COMPARATIV A REZULTATELOR VITRIFICRII PRIN TEHNICILE SOPS I SSV A OVOCITELOR SUINE IMATURE n cazul ambelor metode vitrifcare (SOPS i SSV) cele mai bune rezultate s-au obinut n urma vitrificrii ovocitelor suine imature, n mediu cu 45% etilen glicol i timp de echilibrare de 40 secunde. Rezultatele vitrificrii cu 35%, 40% i 50% etilen glicol, sunt inferioare celor obinute la 45%, motiv pentru care n figura 6 sunt trecute comparaiile dintre metode, doar cu concentraia de 45% cu cei patru timpi de echilibrare. (30. 40, 50 i 60 secunde).

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90 80 70 60 50 40 30 20 10 0

%
77.66 71.29 70.06 74.81 72.28 73.67 69.3 60.39

46.80 42.54 30.03 30.41

45.79

40.96

39.36

40.17

45 % 30

45 %

Morfologie

Fig. 6. Analiza comparativ a rezultatelor vitrificrii cu 45% EG prin metoda SOPS i SSV Rezultatele cele mai bune att din punct de vedere a morfologiei ct i din punct de vedere a viabilitii post-congelare s-au obinut prin vitrificarea ovocitelor prin metoda SOPS, cu timp de echilibrare de 40 secunde. 6.4. VITRIFICAREA PRIN METODA SOPS A OVOCITELOR SUINE MATURATE IN VITRO O influen major n reuita vitrificrii, o are stadiul de maturare al ovocitei. Prerile privind vitrificarea ovocitelor imature sau maturate n prealabil sunt mprite. Unii autori susin c vitrificarea ovocitelor suine este mai reuit cnd acestea adic sunt imature, iar ali autori susin contrariul. Ovocitele maturate au fost crioconservate n paiete (SOPS), prin vitrificare n mediu cu 45% etilen glicol i 0,5M trehaloz i 40 secunde timp de expunere la aciunea crioprotectorilor. Aceast metod i variant experimental a dat rezultatele cele mai bune n cazul vitrificrii ovocitelor imature, motiv pentru care, a fost desemnat pentru crioconservarea ovocitelor suine maturate in vitro. O parte din ovocitele maturate au avut ataate la zona pellucida celulele cumulus ooforus (complex ovocit- cumulus- COC integru) i o alt parte au fost denudate nainte de vitrificare. n figura 7 sunt prezentate rezultatele evalurii (morfologice, a viabilitii i a prezenei globului polar n spaiul perivitelin), obinute n urma vitrificrii ovocitelor suine mature, n prezena celulelor cumulus ooforus i denudate.

45 % 50 se cS O PS 45 % 50 se cS SV 45 % 60 se cS O PS 45 % 60 se cS SV
Viabilitate

SO PS

SO PS

SS V

45 % 30 se c

40 se c

45 %

40 se c

se c

SS V

17

% 100 80 60 40 20 0

92.34

94.72 79.17

49.77 41.99 38.21

CO C
Morfol ogi e Vi abi l i tate GP

Denudate

Fig. 7. Rezultatele evalurilor morfologice, a viabilitii i a prezenei GP la COC i a ovocitelor denudate i vitrificate n urma evalurii morfologice, a rezultat un procent mediu de 92,34%, COC cu celulele cumulus ooforus expandat, granulaie uniform a citoplasmei, zona pellucida intact, spaiu perivitelin uor mrit, caracteristic stadiului MII. Din punct de vedere statistic, aceste valori, sunt semnificativ mai mari (p< 0,001) n comparaie cu cele obinute n urma vitrificrii ovocitelor denudate (41,99%) i a celor imature. n ceea ce privete viabilitatea din totalul de 212 COC, vitrificate prin tehnica SOPS, 94,72%, au fost numrate vii n urma evalurii prin tripla coloraie Procentul mediu privind viabilitatea post-congelare al ovocitelor denudate i vitrificate (38,21%), este cu mult sub cele obinute la vitrificarea COC.

CAPITOLUL 7. CONCLUZII GENERALE I RECOMANDRI n urma vitrificrii a 10.849 ovocite provenite de la suine prin dou metode diferite de crioconservare, respectiv n paiete (SOPS) i pe substrat solid (SSV) cu patru concentraii de etilen glicol (35%, 40%, 45%, 50%) i patru timpi de expunere (30, 40, 50 i 60 secunde) sau desprins urmtoarele concluzii: 7.1. Concluzii privind determinarea influenei concentraiei de crioprotectori i a timpului de expunere asupra vitrificrii prin metoda SOPS a ovocitelor suine imature Concentraia de 45% EG n mediul de vitrificare la timp de expunere de 40 secunde, are cea mai favorabil aciune asupra capacitii ovocitelor crioconservate de a-i pstra structura morfologic integr i viabilitatea dup decongelare. Concentraiile de 35% i 50% EG n mediul de vitrificare indiferent de timpul de expunere, nu reuesc s pstreze structura morfologic i viabilitatea ovocitelor crioconservate. Ovocitele improprii pentru a fi utilizate n lucrrile de fecundaie in vitro cresc brusc atingnd procente ridicate. Influena timpului de echilibrare asupra reuitei crioconservrii ovocitelor suine imature este dependent de concentraia de etilen glicol n mediul de vitrificare.

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 Timpii optimi de echilibrare din cadrul fiecrei concentraii a EG n mediul de vitrificare determin diferene superioare i asigurate statistic, comparativ celorlalte loturi. 7.2. Concluzii privind influena concentraiei de crioprotectori i a timpului de expunere asupra vitrificrii prin metoda SSV a ovocitelor suine imature Echilibrarea ovocitelor n medii cu 45% EG, independent de perioada de timp experimentat, d cele mai bune rezultate. Concentraia de 35% EG n mediul de vitrificare, indiferent de timpul de echilibrare, nu asigur condiiile necesare deshidratrii suficient a ovocitelor, iar ca urmare structura morfologic se deterioreaz determinnd pierderea calitii acestora. Utilizarea mediilor de vitrificare cu 50% EG, la oricare dintre timpii de echilibrare, are efect toxic, calitatea morfologic i viabilitatea ovocitelor reducndu-se la diferene asigurate statistic fa de celelalte variante experimentale. n general, creterea concentraiei de crioprotector reduce timpul de echilibrare a ovocitelor atingnd un optim dup care calitatea ovocitelor vitrificate scade puternic. 7.3. Concluzii privind analiza comparativ a rezultatelor vitrificrii ovocitelor suine imature prin metodele SOPS i SSV Metoda SOPS este superioar metodei SSV deoarece determin pstrarea integritii morfologice i a viabilitii ovocitelor suine imature vitrificate la majoritatea variantelor experimentale. Metoda SSV prezint valori superioare metodei SOPS n privina pstrrii integritii morfologice a ovocitelor vitrificate n cazul concentraiei de 50% EG independent de timpul de echilibrare. Diferenele dintre metode privind pstrarea integritii morfologice i a viabilitii ovocitelor sunt datorate specificului tehnicii aplicate. n cazul SSV timpul n care se ajunge la temperatura de - 196C este mai lung comparativ vitrificrii prin SOPS ceea ce explic superioritatea ultimei metode. 7.4. Concluzii privind vitrificarea prin metoda SOPS a ovocitelor suine maturate in vitro Vitrificarea ovocitelor suine maturate in vitro, duce la pstrarea structurii morfologice, precum i o sporire a numrului de ovocite vii post-vitrificare. Crioconservarea ovocitelor suine maturate i nedenudate d rezultate superioare celor obinute prin vitrificarea ovocitelor maturate i denudate. Prezena celulelor cumulus ooforus favorizeaz schimburile dintre mediul de vitrificare i ovocite determinnd sporirea gradului de reuit a vitrificrii. Recomandri: Utilizarea timpilor de echilibrare i a mediilor de vitrificare care au concentraii optime de crioprotectori, specifice fiecreia dintre metodele experimentale. Vitrificarea ovocitelor suine imature prin metoda SOPS folosind concentraii de 45% EG i timpi de echilibrare de 40 secunde. Crioconservarea ovocitelor suine maturate in vitro prin metoda SOPS n mediu de vitrificare cu 45% EG i timp de echilibrare 40 secunde.

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 Vitrificarea ovocitelor suine maturate in vitro care au cumulus ooforus integru (nedenudate). n cazul scderii concentraiei de EG n mediul de vitrificare este necesar sporirea duratei de echilibrare pentru a optimiza rezultatele crioconservrii ovocitelor suine. Pentru optimizarea tehnicii de lucru, este necesar ca numrul de ovocite din micropicturile de mediu de vitrificare cu volum de 2,5l s fie optim, respectiv reducerea acestora s se coreleze cu cheltuielile efectuate. Elemente de originalitate: mbuntirea tehnicii de vitrificare prin reducerea numrului de ovocite suine pe micropicturile de mediu i depistarea situaiilor de pierdere a caliti acestuia pe baza culorii, n momentul scderii temperaturii. Determinarea concentraiei de 45% etilen glicol i a timpului de echilibrare de 40 secunde ca i parametrii optimi pentru reuita vitrificrii ovocitelor suine imature prin metoda SOPS i SSV. Stabilirea procentului de pstrare a integritii morfologice i a viabilitii ovocitelor suine imature vitrificate prin metodele SOPS i SSV, la concentraii de 35%, 40%, 45%, 50% etilen glicol i timpi de echilibrare de 30, 40,50 i 60 secunde. Precizarea influenei timpului de echilibrare a ovocitelor suine imature n medii de vitrificare care au concentraii specifice de crioprotector, asupra morfologiei i viabilitii acestora. Utilizarea celei mai bune concentraii de etilen glicol i a timpului optim de vitrificare a ovocitelor suine imature este o soluie de urmat pentru crioconservarea ovocitelor suine maturate in vitro.

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BIBLIOGRAFIE SELECTIV

1. ANCHORDOGUY T.J., A.S. RUDOLPH, F.F. CARPENTER, J.H. CROWE, 1987, Modes of interaction of cryoprotectants with membrane phospholipids during freezing, Cryobiology, 24: 324 331. 2. CEAN ADA (cs. TELEA), 2009, Tez de doctorat, Cercetri privind congelarea rapid a embrionilor de mamifere, USAMVB Timioara. 3. LEIBO S.P., 1980, Water permeability and its activation energy of fertilized and unfertilized mouse ova, Journal Membrane Biology, 53:179-188. 4. FAHY G.M., T.H. LILLEY, H. LINSDELL, M.S. DOUGLAS, H.T. MERYMAN, 1990, Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms, Cryobiology, 27: 247 268. 5. LEIBO S.P., J.J. MCGRATH AND E.G. CRAVALHO, 1978, Microscopic observation of intracellular ice formation in unfertilized mouse ova as a function of cooling rate, Cryobiology, 15:257-271. 6. PEDRO P.B., E. YOKOYAMA, S.E. ZHU, 2005, Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants, Journal of Reproduction and Development, 51: 235 246. 7. SOMFAI, T., M. OZAWA, J. NOGUCHI, H. KANEKO, N.W.K. KARJA, M. FARHUDIN, A. DINNYES,T. NAGAI, K. KIKUCHI, 2007, Developmental competence of in vitro-fertilized porcine oocytes after in vitro maturation and solid surface vitrification: effect of cryopreservation on oocyte antioxidative system and cell cycle stage, Cryobiology 55: 115126. 8. TUCKER M., N.J. LIEBERMAN, 2007, Vitrification in assisted reproduction, Ed. Informa Healthcare. 9. VAJTA G., 2000, Vitrification of the oocytes and embryos of domestic animals, Animal Reproduction Science, 60-61:357-364. 10. WASSARMAN P., 1988, The mammalian ovum, Page 69 in The Physiology of Reproduction. E. Knobil and J. Neill, eds. Raven Press, New York.

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UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE DOCTORAL SCHOOL

FACULTY OF ANIMAL HUSBANDRY AND BIOTECHNOLOGIES

Eng. Hettig Andrea Klara

PhD THESIS

VITRIFICATION OF SWINE OOCYTES: EFFECT OF CRYOPROTECTANT CONCENTRATION AND EXPOSURE TIME

Scientific coordinator, Prof. dr. ing. Miclea Vasile

Cluj-Napoca 2011

TABLE OF CONTENT INTRODUCTION..............................................................................................................4 PART I ................................................................................................................................4 CHAPTER 1 CRYOBIOLOGY BASIS ..........................................................................4 1.1. CRYOBIOLOGY ELEMENTS................................................................................4 1.2. STRUCTURA OVOCITEI I EFECTELE NEGATIVE ALE CRIOCONSERVRII .....................................................................................................4 CHAPTER 2 .......................................................................................................................5 CRYOPROTECTIV AGENTS USED IN VITRIFICATION.......................................5 2.1. PENETRATING OR PERMEABLE CRYOPROTECTANTS................................5 2.2. NON-PENETRATING OR PERMEABLE CRYOPROTECTANTS......................5 CHAPTER 3. THE CRIOPROTECTANTS EFFECTS................................................6 3.1. CRYOPROTECTANT TOXICITY AND DAMAGE..............................................6 3.1.1. The effects of exposure time, concentration, and temperature on toxicity ........6 3.1.2. Avoiding damage and toxicity............................................................................6 CAPITOLUL 4...................................................................................................................7 PART II...............................................................................................................................7 OWN RESEARCHES .......................................................................................................7 GOAL AND OBJECTIVES..............................................................................................7 EXPERIMENTAL DESIGN ............................................................................................8 CHAPITER 5. VITRIFICATION METHODES USED................................................8 5.1 IMMATURE SWINE OOCYTES VITRIFICATION BY THE SOPS TECHNIQUE ..........................................................................................................................................8 5.1.1. Biological material ............................................................................................8 5.1.2. Used media.........................................................................................................8 5.1.3. Working methods ...............................................................................................9 5.1.3.1. Ovaries and oocytes collection ...................................................................9 5.1.3.2. Oocyte vitrification by the SOPS method ..................................................9 5.1.3.3. Oocytes warming ........................................................................................9 5.1.3.4. Oocyte washing and morphological evaluation..........................................9 5.1.3.5. Oocyte denuding and viability evaluation through FDA and PI staining.10 5.2. IMMATURE SWINE OOCYTES VITRIFICATION BY THE SSV TECHNIQUE ........................................................................................................................................10 5.2.3.1.Crioconservarea ovocitelor prin tehnica vitrificrii pe substrat solid (SSV) ................................................................................................................................10 5.2.3.2. Oocyte warming and evaluation ...............................................................10 5.3. SOPS VITRIFICATION OF IN VITRO MATURATED OOCYTES....................10 5.3.1. Biological material ..........................................................................................10 5.3.2. Used media.......................................................................................................11 5.3.3. Working methods .............................................................................................11 5.3.3.Oocyte collection and maturation .................................................................11 2

5.3.3.2. Vitrification by the SOPS method ............................................................11 5.3.3.3. Morphological assessments of in vitro maturated oocytes after vitrification.............................................................................................................11 5.3.3.4 Viability assessments of in vitro maturated oocytes after vitrification .....11 CHAPTER 6. RESULTS AND DISCUSSION .............................................................12 6.1. INFLUENCE OF CRIOPROTECTANT CONCENTRATION AND EQUILIBRATION TIME ON SOPS VITRIFIED IMMATURE SWINE OOCYTES .12 6.2. THE INFLUENCE OF CRIOPROTECTANT CONCENTRATION AND EQUILIBRATION TIME ON SSV VITRIFIED IMMATURE SWINE OOCYTES ....13 6.3. COMPARATIVE ANALISYS OF IMMATURE SWINE OOCYTES VITRIFICATION RESULTS BY SOPS AND SSV METHODS ..................................15 6.4. SOPS VITRIFICATION OF IN VITRO MATURATED SWINE OOCYTES .......15 CHAPTER 7. GENERAL CONCLUSIOSN AND RECOMMENDATIONS ...........16

INTRODUCTION Although the history of the concept goes back more than three quarters of a century, the field is probably still in the infancy of its full potential. As always, nature may have preceded biologists in discovering viable approaches to vitrification, but for the most part natures examples remain both recondite and difficult to emulate directly. Vitrification is a viable approach to cryopreservation of a wide range of living systems. Its physical and biological principles are continuing to become better understood, and this is leading to more numerous and more successful applications. Nevertheless, reproductive cryobiologists have ample means and ample incentive to follow natures lead and develop their own approaches to answering one of biologys most interesting challenges, the goal of arresting life in a state of suspended animation and restarting it at the right time to enable new lives to begin (Tucker, 2007). PART I CHAPTER 1 CRYOBIOLOGY BASIS 1.1. CRYOBIOLOGY ELEMENTS Cryobiology is one of the biological fields that have grown spectacularly in recent decades. The objectives of this domain are all the changes which suffers cells, tissues, organs and organisms (bios= life), when they are exposed to low temperatures in generally and negative (crios = cold), especially. Cryopreservation is based on the principle that low temperatures determine the intracellular level, a reduction in molecular motion and thus a significant decrease in metabolism to extreme levels, resulting the so-called anabiosis (Ladoi, 1997). 1.2. STRUCTURA OVOCITEI I EFECTELE NEGATIVE ALE CRIOCONSERVRII Oocytes are more difficult to cryopreserv than embryos, because they are spherical cells, large, with a small area (reported to the volume) and low hydraulic conductivity (Leibo, 1980). The oocyte is the largest cell in the body of most species of mammals (Wassarman, 1988). Approximately 80% of their volume is water. When cooled to temperatures below 0C, intracellular ice can form, which is likely dependent on the temperature-decreasing rate (Leibo et al., 1978). The damages produced by cryopreservation can be both intracellular (regarding to nucleus, cytoplasm with cell organelles: mitochondria, actin/microfilaments, microtubules/spindle) and extracellular (zona pellucida).

CHAPTER 2 CRYOPROTECTIV AGENTS USED IN VITRIFICATION Vitrification is the solidification of the solutions without ice crystal formation by increasing the viscosity during the decreasing temperature. The total absence of ice crystals during vitrification is due to the high concentrations of cryoprotectant. Small crystals of ice are not considered dangerous as long as there is no recrystallization environment (union of small crystals of ice crystals forming large) (Ada Cean, 2009). The cryoprotectors used for biological entities preservation are divided in two major groups: penetrating or permeable cryoprotectants non-penetrating cryoprotectants 2.1. PENETRATING OR PERMEABLE CRYOPROTECTANTS Penetrating cryoprotectants are generally small, nonionic compounds with a high solubility in water at low temperatures. Given time, these chemical compounds can diff use through cellular membranes, permeating the cell and equilibrating within the cytoplasm, replacing the bulk of intracellular water without over-dehydrating the cell. Permeating cryoprotectants solidify at lower temperatures than water and thus subsequently reduce the amount of intracellular ice formation at a given temperature. This decreased amount of intra cellular ice-crystal formation mitigates damage resulting from physical perturbations to cellular organelles and membranes. Additionally, penetrating cryoprotectants provide buffering against salt-induced stress by acting as solvent, reducing the solute concentration in the remaining water fraction inside the cell until the system it is cooled to a low enough temperature. Success using each type of permeating cryoprotectant depends in part on the speed at which they can cross cellular membranes, and this rate is dictated by various factors, such as viscosity. However, permeation is dependent not only upon the cryoprotectant itself but also upon the membrane properties of the cell, which vary between cell types as well as cell stages. (Pedro et al., 2005). The most important permeable cryoprotectants are: ethylene glycol (EG), dimethyl sulfoxide (DMSO), propylene glycol (PROH) and glycerol. Ethylene glycol is the less toxic compared to the other permeable cryoprotectants, reason why it id used in our vitrification protocol. 2.2. NON-PENETRATING OR PERMEABLE CRYOPROTECTANTS Non-penetrating cryoprotectants are generally long chain polymers too large to diff use into cells. These compounds act to increase osmolarity of the extracellular space, which results in cellular dehydration and thus reduces the chance of intracellular ice crystal formation. It is also postulated that some non-permeating cryoprotectants may absorb on membrane surfaces, inhibiting ice crystal formation in the immediate vicinity of the cell by keeping ice in the amorphous state. Additionally, non-penetrating cryoprotectants are oft en included in media used for warming/thawing of cells to help to avoid osmotic 5

shock. Osmotic shock describes the phenomenon that occurs when the osmotic pressure of the intracellular compartment is greater than the extracellular compartment, which occurs because of cellular dehydration. Upon thawing, traumatic cell expansion and lysis can occur following the accompanying water influx. The most important impermeable cryoprotectants are macromolecules (Ficoll, polyvinyl pirrolydone, polyvinyl alcohol, polyethylene glycol), sugars (trehalose, sucrose, glucose, lactose, lactose). In the present study, trehalose was used in the vitrification protocol. CHAPTER 3. THE CRIOPROTECTANTS EFFECTS 3.1. CRYOPROTECTANT TOXICITY AND DAMAGE Regarding reproductive biology and assessing suitability/toxicity of a cryoprotective agent, certain endpoint measures are commonly assessed. In sperm, these endpoints often include effects on motility, capacitation, and ability to fertilize. In oocytes, assessment of damage from cryoprotectants oft en focuses on the meiotic spindle, actin cytoskeleton, chromosomal arrangement, and ability to fertilize. Regarding embryos, toxicity studies tend to examine ability to continue embryonic development, or effects on various molecular and biochemical signaling pathways. Unfortunately, the exact mechanisms by which the toxicity of cryoprotective agents arises are unknown. Examples of damage induced by improper exposure to various cryoprotectants range from removal or fusing of cellular membranes to impair enzyme activity, altered organelle structure/function, and perturbed protein interactions (Fahy, 1986; Fahy et al., 1990; Anchordouguy et al., 1987). 3.1.1. The effects of exposure time, concentration, and temperature on toxicity The conditions under which cryoprotectant exposure occurs can influence their toxicity for reproductive cells. Factors such as time of exposure, concentration of cryoprotectant, as well as temperature of exposure all influence the effect of the particular protecting agent. While cryoprotectants themselves are generally nontoxic when exposed to cells for short periods, extended exposure or exposure at elevated concentrations can result in metabolism of the cryoprotectant agents, which can subsequently disrupt cellular function and viability. 3.1.2. Avoiding damage and toxicity Selection of a suitable cryoprotectant and optimizing its concentration, as well as monitoring the time and temperature of exposure, all influence the potential damage a cryoprotectant may cause to a cell. Therefore, a common approach to reduce toxicity has emerged that entails combining various cryoprotectants, both permeating and nonpermeating, thereby reducing individual concentrations and mitigating damage while maintaining the overall protective effects. Methods of introducing and removing cryoprotectants offer another means to reduce toxicity or resulting damage. Stepwise addition of cryoprotective agents, or gradually increasing concentrations, has been useful. Additionally, stepwise removal of these 6

compounds upon warming/thawing helps to minimize osmotic stress. Furthermore, altering the ionic composition of the carrier solution in which the cryoprotectant was dissolved offers another means of mitigating damage. Specifically, replacing sodium with choline attenuates damage from the solute effect and may benefit from increasing solution viscosity. CAPITOLUL 4 CRIOCONSERVAREA PRIN VITRIFICARE METODE UZUALE With the development of cryobiology and especially the fast cryopreservation, there are numerous techniques for vitrification of oocytes and embryos of all species of animals and human gametes and embryos The most used technique are: vitrification in open pull straw (OPS), super fine open pull straw (SOPS), cryoloop, on solid surface (SSV), cryotip cryotop, the hemy-straw system and closed pull straws. Super fine open pull straw (SOPS) and solid surface vitrification (SSV) techniques are used in our research, for immature swine oocytes vitrification.

PART II
OWN RESEARCHES GOAL AND OBJECTIVES The researches aimed at improving the method of cryopreservation of swine oocytes by the SOPS and SSV technique To achieve that goal the following objectives were set: 1. Define the optimal ethylene glycol concentration on immature swine oocytes vitrification by the SOPS technique. 2. Establish the optimal exposure time on immature swine oocytes vitrification by the SOPS technique considering the concentration of the ethylene glycol in the vitrification medium. 3. Define the optimal ethylene glycol concentration on immature swine oocytes vitrification by the SSV technique. 4. Establish the optimal exposure time on immature swine oocytes vitrification by the SSV technique considering the concentration of the ethylene glycol in the vitrification medium. 5. Determine the effect of cryoprotectant concentration and exposure time depending on the vitrification technique used. 6. Vitrification of in vitro maturated swine oocytes, applying the best technique, cryoprotectant concentration and optimal equilibration time.

EXPERIMENTAL DESIGN
SWINE OOCYTE VITRIFICATIO

IMMATURE SOPS technique SSV technique

Defining the influence of EG concentration

Defining the optimal exposure time

Defining the influence of EG concentration

Defining the optimal exposure time

IN VITRO MATURATED

With cumulus ooforus cells

Denuded

CHAPITER 5. VITRIFICATION METHODES USED 5.1 IMMATURE SWINE OOCYTES VITRIFICATION BY THE SOPS TECHNIQUE 5.1.1. Biological material The biological material used was represented by immature swine oocytes harvested from prepuberal saws ovaries obtained from SC. Turism Vlcele SRL slaughterhouse from Cluj County. For ovaries were collected from prepuberal gilts belonging to commercial hybrids, with maximum weight up to 110 kg. 5.1.2. Used media Transportation medium For transport of the ovaries 0,9% NaCl saline solution in pure water, supplemented with antibiotics (penicillin, gentamycine and streptomycin) was used. Oocytes collecting medium For oocytes collection, M 199 1X containing Earles salts, supplied by Sigma was used. Cryopreservation media Holding medium (MB), containing phosphate buffered solution (PBS) 10x, pure water and bovine fetal (FSB). Previtrification medium: is composed of MB, ethylene glycol 15% (EG) for internal protection and trehalose (0,25M) for extracelullar protection. Vitrification medium: contains the same substance that contains previtrificare medium, the differences being given by their concentration, depending on the experimental groups. Warming solution: consist of PBS and trehalose with three decreasing concentration (0,5M, 0,25M and 0,125M). 8

Washing meium: composed of PBS supplemented with 0,4 % bovine serum albumine (BSA). Stainig solution: is composed of PBS and two complementary dyes namely fluorescein diacetate (FDA) and propium iodide (PI). 5.1.3. Working methods 5.1.3.1. Ovaries and oocytes collection Ovaries were collected in a thermo-isolated container containing a 0,9% NaCl saline solution at 37C. They were transported to the laboratory within 4-6 hours. In the laboratory, the oviductal tissue is detached from the ovaries washed and placed in a recipient with salt solution suplimented with antibiotics. Oocytes were collected by the follicular aspiratory method from the follicles with a 2-5 mm diameter. After collection oocytes assessment was performed. 5.1.3.2. Oocyte vitrification by the SOPS method Oocytes washed two times and equilibrated in cryopreservation media in a stepwise manner. Previtrification: groups of 10 oocytes were taken with a Pasteur pipette, and placed into 30 l droplets of previtrification solution. Oocytes were maintained in the solution for 4 minutes. Vitrification: oocytes were transferred into the vitrification micro-droplets (2l) media containing the final concentration of ethylene glycol and trehalose. After the exposure time expires (specific to each experimental variants), oocytes were loaded onto SOPS, with a touch of the micro-droplet with pipette tip. The medium with the oocytes were loaded by capillary action in the pipette. The pipettes are places in a tub with liquid nitrogen and then transferred and stored until evaluation in a container. 5.1.3.3. Oocytes warming The straw were taken from the tube containing nitrogen, and kept in air for 5 seconds, after which the oocytes are passed sequentially through thawing media with decreased concentration of trehalose (Vajta, 2005) and kept for 5 minutes (Somfai, 2007) in each medium. Oocytes evacuation was performed by the simply touch of the pipette tip to the media through a reverse capillary phenomena. 5.1.3.4. Oocyte washing and morphological evaluation Oocytes were washed three times in drops of 60 ml of PBS supplemented with 0.4% BSA, to evacuate the remaining the cryoprotectant. After washing, oocytes were morphologically assessed by Olympus microscope with inversion. Oocytes that showed adherent cumulus layers, firmly attached to oocytes, zona pellucida intact, well-defined membrane, evenly granulated and compact ooplasm were considered morphologically normal cells and there were counted.

5.1.3.5. Oocyte denuding and viability evaluation through FDA and PI staining Oocyte denuding was achieved as follows: 15-20 oocytes were placed in 30 ml PBS, after which 200 ml PBS that previously was taken into a micropipette and was pipetted over the oocytes. By repeated pipetting, most of the cumulus cells were separated from oocytes. Oocytes staining were performed after denudation. In a first step on a Petri dish with 30 l, droplets were placed groups of 10-15 oocytes. The Petri dish was then placed in the thermostat to 37C for 10 minutes, after which they are evaluated under a microscope equipped with UV lamp. The viable cells were green fluorescent due to enzymatic activity (stained with FDA) and uncolored with PI due to membrane integrity. The death cells were not stained with FDA but stained with PI. 5.2. IMMATURE SWINE OOCYTES VITRIFICATION BY THE SSV TECHNIQUE Biological material, media used and most of the working method are the same as described at SOPS method, so that will be described only procedures that differs. For immature swine oocytes vitrification by the SSV technique were used 5164 gametes. 5.2.3.1.Crioconservarea ovocitelor prin tehnica vitrificrii pe substrat solid (SSV) Previtrification: it is the same as described at the SOPS vitrification method. Vitrificarea: after previtrification, oocytes were transferred into the 2l vitrification micro-droplets that were placed on aluminum foils, representing the solid substrate. After equilibration in the vitrification media (time specific or each experimental group) the aluminum foils were taken from a corner with a long forceps and placed in a tube that is immersed in liquid nitrogen, then the tube was closed and transferred and stored in a liquid nitrogen container until evaluation. 5.2.3.2. Oocyte warming and evaluation At thawing, aluminum foils were taken one by one from the tube with liquid nitrogen and were placed on heating plate, which was previously heated at 37 C. Thus, the droplets with oocytes were thawed on aluminum foil, with an automatic pipette were taken and placed in the thawing media. After maintaining the oocytes in each of the thawing media for 5 minutes, they were washed in PBS, stained with fluorescent dyes and evaluated (the morphology and viability). The procedure is the same like in the case of SOPS vitrified oocytes, so that will not be described in this chapter. 5.3. SOPS VITRIFICATION OF IN VITRO MATURATED OOCYTES 5.3.1. Biological material The biological material was represented by immature oocytes collected from ovaries of slaughtered sows.

10

5.3.2. Used media Transportation medium of the ovaries and collection medium are identical to those described in subsection 5.2.2, which is why they will not be mentioned in this chapter. It will be described the specific points of this chapter. Maturation medium For oocyte maturation, M 199 was supplemented with L-glutamine (3.4 g/l), Chorulon (10 IU/ml), Folligon (10 IU/ml), fetal bovine serum 10%, penicillin (100 g /ml) and streptomycin (100 IU/ml). The pH was checked an corrected to 7,4. Cryopreservation medium: consist of holding medium (HM) with cryoprotectants. The prevetrification medium was composed of HM, 15% EG and 0,25M trehalose. The composition of vitrification solution was HM, 45% EG and 0,5M trehalose. Warming solution is the same we used for immature oocytes Staining solution: differs from that used for immature oocytes in that it contains a third dye: Hoechst 33258. 5.3.3. Working methods 5.3.3.Oocyte collection and maturation Method of harvesting oocytes is the same as described in section 5.1.3.Oocytes with a uniform ooplasm and compact cumulus cell mass were washed with harvest medium. Then were placed in 30 l droplets of maturation medium, covered in paraffin oil and incubated for 44 hours at 37C in an atmosphere with 5% CO2. 5.3.3.2. Vitrification by the SOPS method Because the best results for immature oocytes was obtained by vitrification with the SOPS technique, with 45% ethylene glycol in the vitrification solution, with 40 seconds exposure time, we chose this technique and parameters for in vitro maturated oocytes preservation. Therefore, after 44 hours of maturation, oocytes were transferred from maturation drops in 60 l of PBS supplemented with BSA (0.4%). Oocytes were washed two times and there were undergone to the vitrification process. The procedure is the same as in immature oocytes vitrification by the SOPS method. 5.3.3.3. Morphological assessments of in vitro maturated oocytes after vitrification Cumulus-oocyte complexes were observed under microscope. To assess the morphology of oocytes were used the following indicators: cumulus oocyte complex diameter extension, perivitelin space growth, cumulus ooforus cells expansion (according to the criteria developed by Downs, 1989). After morphological evaluation, oocytes were denuded mechanically by repeated pipetting. Thus prepared were undergone to viability assessment. 5.3.3.4 Viability assessments of in vitro maturated oocytes after vitrification The oocytes were transferred to PBS containing 1 g/ml 3, 6 fluorescein diacetate (FDA), 50 g/ml propidium iodide (PI), and 20 g/ml Hoechst 33258, incubated for 15 minutes and viewed under the UV light of the inverted microscope. Live oocytes appeared green (FDA stained), and their chromosomes were labelled with the Hoechst 11

33342 being blue under UV light. The cytoplasm of dead oocytes ware not coloured with FDA but they were stained with PI, thus appearing red. CHAPTER 6. RESULTS AND DISCUSSION 6.1. INFLUENCE OF CRIOPROTECTANT CONCENTRATION AND EQUILIBRATION TIME ON SOPS VITRIFIED IMMATURE SWINE OOCYTES Results of morphological evaluation of SOPS vitrified swine immature oocytes In figure 2 are presented the results of morphological evaluation of oocytes vitrified by 35%, 40%, 45% and 50% ethylene glycol in the vitrification medium and 30, 40, 50 and 60 seconds exposure time. As shown in Figure 2, the best results after the morphological integrity evaluation post-thawing were obtained when oocytes were vitrified with 45% ethylene glycol in the medium while exposure time is 40 seconds. The averaged value over five repetitions percentage is 77.66% 0.75. Therefore, more than 70% of oocytes after thawing showed a uniform ooplasm, well-attached cumulus cells attached and zona pellucida without rupture.

% 80 70 60 50 40 30 20 35% 40% 45% 50% 30 s e c 40 se c 60 se c 50 s e c

Fig. 2. Morphological assessment of oocytes vitrified with different concentrations of EG and equilibration times At this concentration of cryoprotectant and exposure for the majority of oocytes achieved adequate membrane exchange between water and permeable crioprotectorul (EG), thus achieving ooplasm gelling and it becomes solid, vitreous at low temperatures. Slightly below this value (76.58 2.10) is obtained oocytes were vitrified with 40% EG in the medium and exposed for 50 seconds. Noteworthy are the percentages of 75.54% 1.29, obtained from oocytes vitrified with 40% in the medium and exposure time 60 seconds. All the above values represent peaks in figure 2, also it is seen well the inferiority results in extreme concentrations of ethylene glycol (35% and 50%) regardless equilibrating time. Results of viability assessments of SOPS vitrified swine immature oocytes 12

The results on post-freezing viability of vitrified oocytes by 35%, 40%, 45% and 50% ethylene glycol in the medium and 30, 40, 50 and 60 seconds exposure time, are presented in Figure 3. As morphology, most oocytes were recorded live when they were exposed to vitrification media containing 45% EG for 40 seconds. Average percentage value of five repetitions is 46.80% 0.32, showed very well the highest point in Figure 3. Oocytes showed an intense enzyme activity, colored green at the UV light of the microscope.

% 50 40 30 20 60 sec 10 0 35% 40% 45% 50% 30 sec 40 sec 50 sec

Fig.3. Viability assessment of SOPS vitrified oocytes with different concentrations of EG and equilibration times With 1.01% below the experimental version mentioned above are the results with 45% EG in the vitrification medium, while exposure time is 50 seconds. This value is the second peak highest in figure 3.By cryopreservation with 40%, EG the average percentage values of live oocytes after thawing are reduced by 10% -17%. If the concentration of 35% is reduced by 15% - 19% compared to values of 45%. These concentrations of cryoprotectants are not enough, regardless of exposure time, to bring cells in a vitreous solid state when immersed in liquid nitrogen. It can be seen very well the lowest points obtained for immature swine oocytes vitrified with 50% ethylene glycol in the media, regardless of time of equilibration. This phenomenon is explained by the fact that at this high concentration increases the toxicity of cryoprotectant, and it has no longer beneficial effect. 6.2. THE INFLUENCE OF CRIOPROTECTANT CONCENTRATION AND EQUILIBRATION TIME ON SSV VITRIFIED IMMATURE SWINE OOCYTES Results of morphological evaluation of SSV vitrified swine immature oocytes The results of morphological evaluation of oocytes vitrified by 35%, 40%, 45% and 50% ethylene glycol in the vitrification medium and 30, 40, 50 and 60 seconds exposure time are presented in figure 4.

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% 80 70 60 50 40 30 20 10 0 35% 40% 45% 50% 30sec 60sec 50sec 40sec

Fig. 4. Morphological assessment of SSV vitrified oocytes with different concentrations of EG and equilibration times Immature swine oocytes vitrification with ethylene glycol concentration of 45% and 40 seconds exposure time, gave the best results on morphological integrity assessment. The average percentage of morphological normal oocytes after thawing is 74.81% 1.26. Followed by this value are the average obtained when ethylene glycol concentration was 40% with the same exposure time (72.83% 1.23). Results of viability assessments of SSV vitrified swine immature oocytes In figure five, there are presented the results on post-freezing viability of oocytes vitrified by 35%, 40%, 45% and 50% ethylene glycol in the media and 30, 40, 50 and 60 seconds exposure time.

% 50

40

30 20 10 0 35% 40% 45% 50% 30s e c 40s e c 60s e c 50s e c

Fig.5. Viability assessment of SSV vitrified oocytes with different concentrations of EG and equilibration times The maximum value on post-freezing viability 42.54% 0.26 is representing the highest point in Figure 5. This value was obtained when oocytes were exposed 40 seconds to vitrification medium with 45% ethylene glycol.

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6.3. COMPARATIVE ANALISYS OF IMMATURE VITRIFICATION RESULTS BY SOPS AND SSV METHODS

SWINE

OOCYTES

The best results for swine immature oocytes vitrification has been obtained, in case of both methods (SOPS vs. SSV), by using a concentration of 45% ethylene glycol in the media and 40 seconds equilibration time. The vitrification results using 35%, 40% and 50% ethylene glycol in the media, are lower than those obtained with 45% EG, which is why in figure 6 are presented the analysis between the two method with 45% EG and the four exposure time.
90 80 70 60 50 40 30 20 10 0
PS PS PS PS V V SS V SS 50 se cS O SS SO SO SO SS 45 % 60 se c V

%
77.66 71.29 70.06 74.81 72.28 73.67 69.3 60.39

46.80 42.54 30.03 30.41

45.79

40.96

39.36

40.17

45 % 30 se c

45 % 50 se c

se c

45 %

45 %

Morfologie

Viabilitate

Fig. 6. Morphology and viability of oocytes vitrified with 45% EG by SOPS and SSV methods As shown in figure 6, the best results both in terms of morphology and postthawing viability of the oocytes were obtained by SOPS vitrification with 45% EG and 40 seconds exposure time. 6.4. SOPS VITRIFICATION OF IN VITRO MATURATED SWINE OOCYTES A major influence on a successful vitrification has the oocyte stage. Opinions on immature or mature oocytes vitrification in advance are divided. Some authors claim that swine oocytes vitrification is successful if they are immature, and other authors argue the opposite. Mature oocytes were vitrified by the SOPS method with 45% ethylene glycol in the medium plus 0.5 M trehalose. The were maintained 40 seconds in the vitrification media. This method and experimental design gave the best results when immature oocytes were vitrified, therefore, has been designated for cryopreservation of in vitro maturated swine oocytes. Some of the oocytes were vitrified with the cumulus ooforus cells enclosed (COC- cumulus oocyte complex) and some of them were denuded before preservation. In the figure below are presented the results regarding morphology and viability assessments and the presence of the first polar body (PB) in the perivitelin space.

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45 % 60 se c

40 se c

se c

30

45 %

40

45 %

% 100 80 60

92.34

94.72 79.17

49.77 41.99 38.21

40 20 0 COC
Morfologie Viabilitate GP

Denudate

Fig. 7. Morphology, viability and PB presence assessments after vitrification of COCs and denuded oocytes The results of morphological assessment, shows that an average of 92.34% COCs has cumulus ooforus cells expanded, uniform grained cytoplasm, intact zona pellucida, slightly increased perivitelin space, characteristic to the MII stage oocytes. Statistically, these values are significantly higher (p <0.001) compared with those obtained from denude oocytes (41.99%). Regarding viability, from 212 COCs vitrified by the SOPS technique, an average of 94.72% were counted viable by triple staining assessment. The average percentage of post-thawing viability of denuded oocytes is well below (38.21%), those achieved in vitrification COC. CHAPTER 7. GENERAL CONCLUSIOSN AND RECOMMENDATIONS An amount of 10.849 swine oocytes were vitrified by two different cryopreservation methods (SOPS and SSV) with four concentration of ethylene glycol (35%, 40%, 45%, 50%) and four exposure times (30, 40, 50 and 60 seconds). The following conclusions results: 7.1. Conclusions regarding cryoprotectant concentration and exposure time influence on SOPS vitrified immature swine oocytes The 45% ethylene glycol concentration in the vitrification medium and 40 seconds exposure time had the most favorable action on the ability of cryopreserved oocytes to keep their morphological structure integrity and viability after thawing, regardless of exposure time used. Concentrations of 35% and 50% ethylene glycol in vitrification medium regardless of exposure time, fail to preserve the morphological structure and viability of the vitrified thawed oocytes. Oocyte unsuitable for in vitro fertilization reaches suddenly high percentages. The influence of exposure time on the success of immature swine oocytes preservation is dependent on the concentration of ethylene glycol in the vitrification media. Optimal exposure times of each vitrification media determine statistically superior differences compared to other groups. 7.2. Conclusions regarding cryoprotectant concentration and exposure time influence on SSV vitrified immature swine oocytes

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 Exposing oocytes to 45% EG in vitrification medium, gives the best results. regardless the period, Concentration of 35% EG in vitrification medium, at any equilibration period does not provide sufficient conditions for oocytes dehydration and their morphological structure deteriorates and causing quality loss. Vitrification media with 50% EG regardless exposure time has a toxic effect, on the viability and morphological quality of oocytes with statistically differences compared to other experimental groups. In general, increasing the concentration of cryoprotectant reduces equilibration period, reaching an optimum quality of vitrified oocytes, then decreases strongly. 7.3. Conclusions regarding comparative analisys of immature swine oocytes vitrification results by SOPS and SSV methods SOPS method is superior compared to the SSV method because maintains in most experimental variants the morphological integrity and viability of the vitrified swine immature oocytes SSV method shows higher values on SOPS method of preserving the morphological integrity of vitrified oocytes when 50% ethylene glycol was used, regardless time. Differences between methods of preserving morphological integrity and viability of oocytes are due to the specific technique used. SSV, reaches - 196 C later than the SOPS, which explains the superiority of the last method. 7.4. Conclusions regarding sops vitrification of in vitro maturated swine oocytes In vitro maturated swine oocytes vitrification leads to an increasedand viability after. Cryopreservation of matured swine oocytes with cumulus ooforus cells attached gives superior results compared to denuded oocytes The presence of these cells promote trade between vitrification media and the oocyte increasing the success of vitrification. Recommendations Use time balancing and vitrification media have with optimal cryoprotectant concentrations, specific to each experimental method. Immature swine oocytes vitrification by the SOPS method using 45% ethylene glycol and 40 seconds equilibration times. Cryopreservation of in vitro maturated swine oocytes by SOPS method with 45% ethylene glycol and 40 seconds exposure time. Vitrification of in vitro maturated swine oocytes with cumulus ooforus cells attached. If the concentration of EG decrease in the vitrification medium is necessary to increase the exposure time to optimize the results. To optimize the technique, it is necessary that the number of oocytes from a 2.5 l volume of vitrification media to be optimal, and correlated with spending cuts.

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Originality elements Improved vitrification technique by reducing the number of swine oocytes on droplets and quality detection based on the aspect of the droplets when temperatures drop. Defining the 45% ethylene glycol concentration and 40 seconds equilibration time as the optimal parameters for immature swine oocytes successfully vitrification by the SOPS and SSV methods. Establish the optimal of concentrations of 35%, 40%, 45%, 50% ethylene glycol and equilibration times of 30, 40.50 and 60 seconds to maintain the morphological integrity and viability of SOPS and SSV vitrified immature swine oocytes. Specification of exposure time influence, for each concentration of ethylene glycol in the vitrification media. Vitrification of the maturated swine oocytes with the best concentration and exposure time defined for immature oocytes vitrification.

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