Efecto del tratamiento de superovulación sobre la calidad embrionaria en conejo

MASTER INTERUNIVERSITARIO EN MEJORA GENÉTICA ANIMAL Y BIOTECNOLOGÍA DE LA REPRODUCCIÓN E ec o o a ian s imula ion ea men s on he emb yo quali y in abbi Tesis de Mas e Valencia, Julio 2016 José Isaí Cedano Cas o Di ec o : D . José Sal ado Vicen e An ón I ABSTRACT Supe o ula ion in animals is used o p oduce a maximum numbe o ans e able emb yos pe dono , in o de o suppo bo h gene ic imp o emen p og ams and ex si u conse a ion o o op imize o he bio echnologies. O e ime, he use o his bio echnology has shown a iable ou comes as a consequence o se e al ac o s, such as he o igin o exogenous ho mone, i s adminis a ion mode, he dono and he en i onmen . Nowadays he use o gonado opins such as FSH, LH, hCG and eCG has enabled us o achie e he supe o ula ion in di e en species success ully. Howe e , he posology and he e ec o bo h gonado opins when used simul aneously (e.g. FSH and LH) i is no ye clea ly de ined. The supe o ula ion in abbi does and in o he species, has had a g ea ad ance in ecen yea s, changing he eCG use by he ecombinan gonado opins and jumping he mo phological classi ica ion o he molecula analysis o emb yos. The objec i e o his s udy was o e alua e he e ec o long-ac ing FSH-CTP and hFSH alone o supplemen ed wi h hLH on o a ian s imula ion in abbi does and de e mine he impac o i s s imula ion on in i o and in i o de elopmen o esh and c yop ese ed emb yos. Ou ou comes showed ha bo h he o ula ion a e and he mean o eco e ed ans e able emb yos pe dono we e simila among he di e en o a ian s imula ion g oups (FSH-CTP, FSH-CTP+ hLH, hFSH, hFSH+ hLH), bu much highe han he con ol g oup. In i o iabili y o supe o ula ed emb yos om FSH-CTP g oup was simila o he emb yos om he con ol g oup un il blas ocys s s age a 48 hou s, bu i was highe a expanded blas ocys s age o FSH- CTP g oup in ela ion o he con ol g oup a 48 hou s. The emb yo de elopmen a any s age was a ec ed by he i i ica ion p ocess. Howe e , i seems ha o a ian s imula ion did no a ec he SOX2 and OCT4 gene exp ession. NANOG gene o e exp ession was obse ed in emb yos om FSH-CTP, FSH-CTP+ hLH and hFSH in conco dance wi h he highe in i o emb yo de elopmen o hese g oups. In i o iabili y o emb yos om bo h FSH-CTP and FSH-CTP+ hLH expe imen al g oups was simila o he emb yos iabili y om he con ol g oup. The in i o emb yo iabili y a implan a ion s age was a ec ed by he i i ica ion p ocess, bu oe al losses and bi h a e was no a ec ed. Ne e heless, i seems ha he i i ica ion did no a ec he in i o iabili y o emb yos om FSH-CTP and hFSH g oups in ela ion o he con ol g oup. II The e was no di e ence in he li e size and weigh a bi h and weaning among he expe imen al g oups. The i i ica ion did no a ec he li e size a bi h and weaning bu he pup weigh who came om he i i ied emb yos was i e pe cen highe han he one in he esh g oups. By con as , weigh a weaning was lowe han he esh g oup. In conclusion, he esul s o his s udy sugges ha he use o FSH-CTP (3 µg/doe) is enough o supe o ula e abbi does wi hou comp omising he quan i y and quali y o emb yos, ei he esh o i i ied. In addi ion, he use o his ho mone educes he equency o animal handling, imp o ing he animal wel a e oo. Keywo ds: supe o ula ion, long ac ing FSH-CTP, hFSH, hLH, gene exp ession, emb yo iabili y, i i ica ion. III RESUMEN La supe o ulación en animales se emplea pa a p oduci el máximo núme o de emb iones ans e ibles po donan e, bien con el in de apoya a los p og amas de mejo a gené ica y la conse ación ex si u o bien pa a la op imización de o as bio ecnologías. A lo la go del iempo, el uso de es a bio ecnología ha mos ado esul ados muy a iables como consecuencia de a ios ac o es ales como el o igen de las ho monas exógenas, el modo de aplicación de las mismas, las hemb as donan es y el ambien e. Hoy en día el uso de gonado opinas como la FSH, LH, hCG y eCG pe mi e consegui sa is ac o iamen e la supe o ulación de hemb as de di e en es especies, sin emba go, aún no es á cla amen e de inida la posología y la consecuencia de usa dos gonado opinas conjun amen e (po ejemplo, FSH y LH). Recien emen e la supe o ulación en conejas ha enido g andes a ances, cambiando el uso de la eCG po gonado opinas ecombinan es y e aluando la calidad emb iona ia a a és del análisis molecula de los emb iones, al igual que en o as especies. El obje i o de es e es udio ue e alua el e ec o de la FSH-CTP de la ga acción y la hFSH solas o suplemen adas con hLH sob e la es imulación o á ica en conejas y de e mina el impac o de dicha es imulación sob e el desa ollo in i o e in i o de emb iones escos y c iop ese ados. Nues os esul ados mos a on que la asa de o ulación ue simila en e los g upos de es imulación o á ica (FSH-CTP, FSH-CTP+ hLH, hFSH, hFSH+ hLH), al igual que la media de emb iones ans e ibles ecupe ados po donan e. La iabilidad in i o de los emb iones p oceden es del a amien o con FSH-CTP ue simila al g upo con ol has a el es adio de blas ocis o a las 48 ho as, sin emba go, se obse ó un mayo núme o de blas ocis os expandidos en el g upo FSH-CTP en e al con ol. Sin emba go, pa ece que la es imulación o á ica no a ec ó la exp esión de los genes SOX2 y OCT4, mien as que hubo una sob eexp esión del gen NANOG, en conco dancia con el mayo desa ollo in i o de los emb iones de los g upos FSH-CTP, FSH- CTP+ hLH y hFSH. El p oceso de i i icación a ec ó el desa ollo in i o e inc emen ó la exp esión génica de SOX2, OCT4 y NANOG en los emb iones. Sin emba go, pa ece que la i i icación no a ec ó la iabilidad in i o de los emb iones de los g upos FSH-CTP y hFSH. La iabilidad in i o de los emb iones de los g upos expe imen ales FSH-CTP y FSH-CTP+ hLH ue simila al g upo con ol. IV El p oceso de i i icación a ec ó a la iabilidad de los emb iones has a la implan ación, pe o las pé didas e ales y la asa de pa os no se ie on a ec adas. Sin emba go, la i i icación no a ec ó a la iabilidad in i o de los emb iones p o enien es de los a amien os FSH-CTP y hFSH en elación al g upo con ol. El amaño y peso de la camada ue simila en e los dis in os g upos de supe o ulación es udiados. El p oceso de supe o ulación no a ec ó al amaño de camada al nacimien o y des e e. Sin emba go, mien as que el peso al nacimien o de los gazapos p o enien es de emb iones i i icados ue un cinco po cien o mayo que los p o enien es de emb iones escos, en el momen o del des e e su peso ue meno . En conclusión, los esul ados de es e es udio sugie en que el uso de FSH-CTP (3 µg/coneja) es su icien e pa a supe o ula conejas sin comp ome e la can idad y la calidad de los emb iones, ya sean escos o i i icados. Además, el uso de es a ho mona educe el manejo de los animales, mejo ando el bienes a animal. Palab as cla e: supe o ulación, FSH-CTP de acción p olongada, hFSH, hLH, exp esión génica, iabilidad emb iona ia, i i icación. V INDEX ABSTRACT ----------------------------------------------------------------------------------------------------------------------------- i RESUMEN ------------------------------------------------------------------------------------------------------------- iii 1. INTRODUCTION ------------------------------------------------------------------------------------------------ 1 1.1. Gonado opin biochemis y --------------------------------------------------------------------------------------- 1 1.1.1. Equine cho ionic gonado opin (eCG) --------------------------------------------------------------------------------- 2 1.1.2. Human cho ionic gonado opin (hCG) -------------------------------------------------------------------------------- 2 1.1.3. Follicle-s imula ing ho mone (FSH) ------------------------------------------------------------------------------------ 3 1.1.4. Lu einizing ho mone (LH) ------------------------------------------------------------------------------------------------- 5 1.2. Rabbi ep oduc i e physiology ---------------------------------------------------------------------------------- 6 1.3. Manipula ion o abbi o a ian unc ion and consequences--------------------------------------------- 8 2. OBJECTIVES ---------------------------------------------------------------------------------------------------- 11 3. MATERIALS AND METHODS ------------------------------------------------------------------------------- 12 3.1. Animals an e hical s a emen ----------------------------------------------------------------------------------- 12 3.2. Expe imen al design: he ollowing scheme shows he expe imen al wo k. ---------------------- 13 3.3. Ho monal ea men ---------------------------------------------------------------------------------------------- 14 3.4. Emb yo eco e y --------------------------------------------------------------------------------------------------- 14 3.5. Emb yo i i ica ion p ocedu e --------------------------------------------------------------------------------- 16 3.6. Expe imen 1: In i o de elopmen and quali y o emb yos ------------------------------------------ 17 3.6.1. In i o cul u e ------------------------------------------------------------------------------------------------------------- 17 3.6.2. RNA ex ac ion and quan i a i e PCR analysis ------------------------------------------------------------------- 18 3.7. Expe imen 2: In i o su i al ---------------------------------------------------------------------------------- 19 3.7.1. Emb yo ans e ----------------------------------------------------------------------------------------------------------- 19 3.7.2. Emb yo su i al a e and e al losses ------------------------------------------------------------------------------- 20 3.8. S a is ical analysis-------------------------------------------------------------------------------------------------- 21 4. RESULTS -------------------------------------------------------------------------------------------------------- 22 4.1. E alua ion o o a ian s imula ion ea men s on he o a ian esponse and eco e y a iables ------------------------------------------------------------------------------------------------------------------------ 22 4.2. Expe imen 1. In i o de elopmen and quali y o eco e ed emb yos --------------------------- 22 4.2.1. E alua ion o in i o emb yo de elopmen a 24 and 48 hou s -------------------------------------------- 22 4.2.2. E alua ion o ela i e gene exp ession o blas ocys a e in i o cul u e ------------------------------ 26 4.3. Expe imen 2: In i o su i al o eco e ed emb yos ---------------------------------------------------- 27 4.3.1. E alua ion o emb yo iabili y a e ans e --------------------------------------------------------------------- 27 4.3.2. Li e size a bi h and weaning in expe imen al g oups ------------------------------------------------------ 30 4.3.3. Pup weigh a bi h and weaning ------------------------------------------------------------------------------------- 32 5. DISCUSSION --------------------------------------------------------------------------------------------------- 34 6. CONCLUSION -------------------------------------------------------------------------------------------------- 40 7. REFERENCES --------------------------------------------------------------------------------------------------- 41 VI INDEX OF TABLES Table 1. Resul s o supe s imula ion ea men induced by eCG in abbi s. ---------------------------------------- 9 Table 2. Resul s o supe s imula ion ea men induced by FSH in abbi s. -------------------------------------- 10 Table 3. Lis o p ime s used o quan i a i e eal- ime polyme ase chain eac ion. --------------------------- 19 Table 4. E ec o o a ian s imula ion ea men s on eco e y a iables. ----------------------------------------- 23 Table 5. E ec o Emb yo ype on in i o de elopmen . -------------------------------------------------------------- 25 Table 6. E ec o Emb yo ype on genes exp ession in blas ocys . ------------------------------------------------- 27 Table 7. E ec o Emb yo ype on implan a ion and bi h a e. ----------------------------------------------------- 29 Table 8. E ec o Emb yo ype on li e size a bi h and weaning. ------------------------------------------------- 31 Table 9. E ec o Emb yo ype on pup weigh a bi h and weaning. ---------------------------------------------- 33 Table 10. The es ima ed means o co a iables and hei coe icien . ---------------------------------------------- 33 INDEX OF FIGURES Figu e 1. human Cho ionic Gonado opin molecule (Leao and Es e es 2014). ------------------------------------ 3 Figu e 2. Follicle-s imula ing ho mone (Leao and Es e es 2014). ----------------------------------------------------- 4 Figu e 3. Lu einizing ho mone (Leao and Es e es 2014). --------------------------------------------------------------- 5 Figu e 4. Emb yo and oe al de elopmen in abbi . --------------------------------------------------------------------- 8 Figu e 5. Line A abbi . --------------------------------------------------------------------------------------------------------- 12 Figu e 6. Expe imen al scheme. ---------------------------------------------------------------------------------------------- 13 Figu e 7. Emb yo eco e y. ---------------------------------------------------------------------------------------------------- 15 Figu e 8. No mal ( ans e able) emb yos ---------------------------------------------------------------------------------- 15 Figu e 9. O a ian ollicles wi h sca s ---------------------------------------------------------------------------------------- 16 Figu e 10. Emb yo de elopmen . -------------------------------------------------------------------------------------------- 18 Figu e 11. T ans e s eps on he ecipien abbi doe. ----------------------------------------------------------------- 20 Figu e 12. T ans e able emb yos eco e ed by dono doe. ----------------------------------------------------------- 24 Figu e 13. In i o de elopmen emb yos a 24 hou s. ---------------------------------------------------------------- 24 Figu e 14. In i o de elopmen emb yos a 48 hou s. ----------------------------------------------------------------- 25 Figu e 15. Blas ocys a e a 48 hou s o di e en o a ian ea men by ype o emb yos. ------------------ 26 Figu e 16. Rela i e gene exp ession by s imula ion ea men . ------------------------------------------------------ 27 Figu e 17. Su i al a e o emb yos a implan a ion and bi h. ------------------------------------------------------ 28 VII Figu e 18. Implan a ion a e o di e en o a ian ea men by ype o emb yos. ------------------------------ 29 Figu e 19. Bi h a e o di e en o a ian ea men by ype o emb yos. ----------------------------------------- 30 Figu e 20. li e size pe ecipien doe a bi h and weaning. --------------------------------------------------------- 31 Figu e 21. Weigh a bi h. ---------------------------------------------------------------------------------------------------- 32 Figu e 22. Weigh a weaning. ------------------------------------------------------------------------------------------------ 33 I 1. INTRODUCTION Supe o ula ion is he a i icially induced elease o a la ge - han-no mal numbe o oocy es om he o a y p omo ed by exogenous ho mones adminis a ion (Gibbons e al. 1981, and Pa k e al. 2015). In animals i is common o use his ep oduc i e echnology o p oduce he maximum numbe o ans e able emb yos pe dono (Taylo e al. 2007) in o de o suppo bo h gene ic imp o emen p og ams and ex si u conse a ion, o o op imize o he bio echnologies such as cul u e, cloning, c yop ese a ion o ansgenesis (Sal e i e al. 2007, and Viudes-de-Cas o e al. 2009). Despi e ex ensi e esea ch e o s in he pas 60 yea s, a iabili y in he supe o ula ion esponse among indi iduals is he majo p oblem in all species. Di e se ac o s in luence he success o supe o ula ion such as he gonado opin p epa a ion, i s adminis a ion mode, he o a ian physiological s a us, he dono and he en i onmen (Taylo e al. 2007). Al hough conside able p og ess has been made in he s udy o olliculogenesis, manipula ion o o a ian unc ion, gonado opin biochemis y and ac o s inhe en o he dono animal, he applica ion o supe o ula ion emains a challenge. 1.1. Gonado opin biochemis y Follicle-s imula ing ho mone (FSH), lu einizing ho mone (LH), human cho ionic gonado opin (hCG) and equine cho ionic gonado opin (eCG) a e membe s o he gonado opin amily. They a e glycop o ein ho mones which a e cha ac e ized by a he e odime ic s uc u e composed o α-subuni no-co alen ly linked o a speci ic β-subuni . The α-subuni is common o all gonado opin amily membe s and con ains 92 amino acid esidues, wi h i e disulphide bonds con ibu ing o i s e ia y s uc u e. The β-subuni is speci ic o each ho mone al hough hey all show some deg ee o homology because hey bind o a common α -subuni . LH and hCG sha e common biological p ope ies and hei β-subuni s possess a high deg ee o sequence homology (Comba nous 1988, and Ryan e al. 1988). The FSH and LH a e sec e ed by he pi ui a y gland, while hCG and eCG a e sec e ed by he placen a in p egnan women and ma es, espec i ely. The e a e pha maceu ical companies ha p oduce and dis ibu e eCG, hCG, FSH om po cine, o ine, equine o bo ine pi ui a y and ecombinan FSH and LH (Leao and Es e es 2014). 8 placen a (Ho man e al. 1998). The bi h akes place 30-32 days pos -coi um, depending on he numbe o e uses ges a ed and he gene ic lineage (Figu e 4). Figu e 4. Emb yo and oe al de elopmen in abbi . 1.3. Manipula ion o abbi o a ian unc ion and consequences In abbi s, in o de o ensu e he maximum numbe o no mal emb yos eco e ed pe dono , bo h eCG and pi ui a y de i ed FSH ha e been commonly used o induce supe o ula ion (Kanayama e al. 1994, Kau man e al. 1998, Hashimo o e al. 2004, Mehaisen e al. 2005, Mehaisen e al. 2006, and Sal e i e al. 2007). Mo e ecen ly, ecombinan human LH and FSH ha e been used (Viudes-de- Cas o e al. 2009 and 2015, and Co ell e al. 2010). The eCG p olongs plasma hal -li e and can nega i ely a ec emb yonic de elopmen (Taneja e al. 1990). The use o FSH has ad an ages o e eCG, bu is a mo e ime-consuming p o ocol due o i s sho e hal -li e. FSH equi es o be adminis e ed wice daily o e 3–4 days o s imula e he same amoun o ollicula g ow h ha would esul om a single injec ion o eCG (Rena d e al 1982; Joly, 1997, and Kau man e al. 1998). 9 As shown in he ables 1 and 2, esul s o supe o ula ion ea men s a y, and one o he easons o his may be he a iable LH : FSH a io when p epa a ions isola ed om he pi ui a y gland a e used. In abbi s, he e ec o LH on supe o ula ion has been s udied using pu i ied po cine FSH ob aining highly a iable esul s (Hashimo o e al. 2004, and Sal e i e al. 2007). Mo e ecen ly, Viudes-de- Cas o e al. (2015) using ecombinan LH and FSH sugges ed ha he window o LH in abbi s is FSH dose dependen . I seems ha he endogenous LH concen a ion is enough o duplica e he o ula ion a e o does ea ed wi h low FSH doses (Viudes-de-Cas o e al. 2009), bu i is insu icien o inc ease ollicula ec ui men when highe doses o FSH a e used (Viudes-de-Cas o e al. 2015). On he o he hand, se e al s udies in di e en species ha e demons a ed an inc ease in an i- gonado opin an ibodies in animals epea edly ea ed wi h gonado opins (Swanson e al. 1996, F ancois e al. 1999, Van and Da is 2001, combelles and Albe ini 2003, Mehaisen e al. 2006, and Viudes-de-Cas o e al. 2009). Table 1. Resul s o supe s imula ion ea men induced by eCG in abbi s. OI: O ula ion induc ion, RR: Response a e, OR: O ula ion a e, RE: Reco e ed emb yos, RO: Reco e ed oocy es. IV: in a enously TREATMENT AUTHORS DOSES OI RR OR RE RO eCG López- Beja and López- Ga ius 2000 80 UI 30 UI hCG (IV) - - 17.8 - Besen elde e al. 2002 120 UI 180 UI hCG (IV) - 36.3 24.3 3.2 Tsiligianni e al. 2004 20 UI/Kg 120 UI hCG (IV) - 28.7 14.2 4.31 Mehaisen e al. 2005 50 UI - - 15.2 10.1 - 200 UI - - 19.1 8.8 - Mehaisen e al. 2006 20 IU/kg 75 UI hCG (IV) 80.6 19.2 14.1 - Badawy e al. 2016 50 UI - - 20.54 - - 10 Table 2. Resul s o supe s imula ion ea men induced by FSH in abbi s. OI: O ula ion induc ion, RR: Response a e, OR: O ula ion a e, RE: Reco e ed emb yos, RO: Reco e ed oocy es. FSHo: o ine FSH, FSHp: po cine FSH, hFSH: ecombinan human FSH, hLH: ecombinan human LH. IM: in amuscula ly. IV: in a enously. SC: subcu aneously The usual pa ame e s o e alua e emb yo quali y a e mo phologic c i e ia acco ding o In e na ional Emb yo T ans e Socie y classi ica ion. The e o e, he e alua ion o he exp ession p o ile o genes ela ed o emb yo de elopmen (Viudes-de-Cas o e al. 2015) o some ac o s ela ed o hei me abolism (Co ell e al. 2015) may help o assess he quali y o he emb yos p oduced by supe s imula ion p o ocols. Se e al au ho s ha e obse ed ha supe o ula ion ea men s wi h high doses o eCG o FSH may gene a e a highe numbe o abno mal and imma u e ollicles by inc easing he cys ic and o TREATMENT AUTHORS DOSES OI RR OR RE RO FSHo Kau man e al. 1998 6 x 0.5 mg/12h (SC) 150 UI hCG (IV) - 25.6 19.4 2.25 8 x 0.5 mg/12h (SC) - 23.5 23.5 0.75 FSHp Cheng e al. 1999 6 x 0.3 mg/12h 25 UI hCG/kg - 28.3 15.5 2.1 FSHo Mehaisen e al. 2006 3 x 0.6 mg/24h 75 IU hCG (IV) 87.5 25.2 17.7 - FSHp Sal e i e al. 2007 5 x 9 µg/12h (IM) 1.6 µg buse elin (IM) 77.8 26.7 21.2 - FSHp +LHp(20%) 81.3 27.1 20.3 - hFSH Viudes-de- Cas o e al. 2009 3 x 25 UI/24h (IM) 2 µg buse elin (IM) 84.94 20.24 15.59 1.62 hFSH+ hLH(5%) 88.42 18.46 15.93 0.96 hFSH+ hLH(10%) 77.17 18.41 13.72 0.59 hFSH Viudes-de- Cas o e al. 2015 18.75 UI/12h (IM) 1 µg buse elin (IM) 74.1 20.4 15.9 - hFSH+ hLH(10%) 73.5 20.6 12.2 - hFSH 37.50 UI /12h (IM) 79.0 28.2 25.0 - hFSH+ hLH(10%) 80.7 41.5 34.0 - 11 hemo hagic ollicles (Ga cía Ximénez and Vicen e 1990, Schmid e al. 1992, Kim e al. 1988, Cheng e al. 1999, Hashimo o e al. 2004). Addi ionally, hese ea men s may dec ease he emb yo eco e y a es (Kennelly and Foo e 1965, Ha ez 1969, and Schmid e al. 1992), p oduce cy ogene ic de ec s and ch omosomal al e a ions in emb yos (Fechheime and Bea y 1974, and Pau le e al. 1975), making hem mo e sensi i e o c yop ese a ion p o ocols and dec easing hei po en ial abili y o p oduce li e pups (Mehaisen e al. 2006) and a ec ing he oocy e me abolism and consequen ly he u u e emb yo de elopmen (Co ell e al. 2015). O he au ho s ha e shown no di e ences be ween supe o ula ed and non- ea ed emales on emb yo quali y, de elopmen al capaci y and he exp ession p o ile o OCT4, NANOG, and SOX2 genes o bo h esh and ozen- hawed emb yo (Sal e i e al. 2007, Viudes-de-Cas o e al. 2009 and 2015). 2. OBJECTIVES The cu en s udy was pe o med o e alua e he e ec o long-ac ing FSH-CTP and h FSH alone o supplemen ed wi h hLH on o a ian s imula ion in abbi does, and o de e mine he impac o his s imula ion on in i o and in i o de elopmen o esh and c yop ese ed emb yos. The speci ic objec i es we e:  Compa ison o he ecombinan human gonado opins ( hFSH, hLH and FSH-CTP, Co i olli opin al a) on he o a ian esponse, ecunda ion and emb yo eco e y a e.  E alua ion o in i o emb yo de elopmen and he exp ession o de elopmen plu ipo ency genes: oc ame ‐binding ansc ip ion ac o (OCT4), NANOG homeobox (NANOG) and sex de e mining egion Y-box 2 (SOX2).  Assessmen o he in i o iabili y o emb yos de i ed om he di e en supe o ula ion ea men s. 12 3. MATERIALS AND METHODS 3.1. Animals an e hical s a emen The esea ch was ca ied ou a he expe imen al a m o he Ins i u e o Science and Animal Technology (ICTA), Poly echnic Uni e si y o Valencia. All animals we e handled in acco dance wi h he p inciples o animal ca e published by Spanish Royal Dec ee 53/2013 (BOE 2013). The expe imen s we e app o ed by he Commi ee o E hics and animal Wel a e o he Poly echnic Uni e si y o Valencia (p ocedu e 2015/VSC/PEA/00061). One hund ed six y- wo nullipa ous does o 18 – 20 weeks old we e used (Figu e 5). Does belonged o a New Zealand Whi e line selec ed o li e size a weaning (Line A) (Es any e al. 1992). Animals we e housed in la -deck cages, ed wi h a s anda d pelle die ad libi um and had ee access o wa e . An al e na ing cycle o 16 h ligh s and 8 h o da k was used. Figu e 5. Line A abbi . 13 3.2. Expe imen al design: he ollowing scheme shows he expe imen al wo k. Figu e 6. Expe imen al scheme. 14 3.3. Ho monal ea men O a ian s imula ion was induced using Co i ol opin al a (FSH-CTP; Elon a, 150 mic og amos/0.5 ml; Me ck Sha p & Dohme S.A.; Spain) alone o in combina ion wi h ecombinan human LH ( hLH; Lu e is 75; Se ono Eu ope L d., London, Uni ed Kingdom); and ecombinan human FSH ( hFSH; Gonal-F 75; Se ono Eu ope L d., London, Uni ed Kingdom) alone o in combina ion wi h hLH. Rabbi does, weighing 3.9 o 4.2 kg, we e assigned andomly o i e expe imen al g oups: 1. G oup FCT-CTP: 20 abbi does we e subcu aneously ea ed once wi h 3 µg o FSH-CTP. 2. G oup FSH-CTP + hLH: 20 abbi does we e subcu aneously ea ed once wi h 3 µg o FSH-CTP and in amuscula ly ea ed wi h a 10% o hLH dis ibu ed in i e equal doses a 12-hou s in e al. 3. G oup hFSH: 20 abbi does we e in amuscula ly ea ed wi h 3 µg o hFSH dis ibu ed in i e equal doses a 12-hou s in e als. 4. G oup hFSH + hLH: 20 abbi does we e in amuscula ly ea ed wi h 3 µg o hFSH in combina ion wi h a 10% o hLH dis ibu ed in i e equal doses a 12-hou s in e als. 5. Con ol g oup: 26 emales we e ea ed in amuscula ly wi h saline solu ion (0.2 ml) a he same ime as he o he g oups. Does we e insemina ed wi h 1 ml o pooled spe m om e ile males o he same line (Line A) 60 h a e he i s gonado opin injec ion, and o ula ion was induced wi h 1 µg buse elin ace a e (Sup e ac ; Hoechs Ma ion Roussel, S.A., Mad id, Spain) gi en in amuscula ly. 3.4. Emb yo eco e y Emb yo eco e y was ca ied ou in 12 ba ches (Figu e 7). Females we e eu hanized 72 h a e a i icial insemina ion wi h an in a enous injec ion o 0.6 g pen oba bi al sodium (Dole hal; Ve oquinol, Mad id, Spain), and he ep oduc i e ac was immedia ely emo ed. Emb yos we e eco e ed by pe usion o each u e ine ho n wi h 10 mL Dulbecco´s phospha e bu e ed saline (HyClone™ DPBS liquid Wi hou Calcium, Magnesium, Phenol Red; HyClone Labo a o ies, Logan, U ah, USA) con aining 0.2% bo ine se um albumin (AMRESCO® Albumin, Boi ine, (BSA); Solon, USA), 0.133 g/L CaCl2, 0.100 g/L MgCl2, an ibio ics (penicillin G sodium 300.000 IU/L, penicillin G p ocaine 700.000 UI, and dihyd os ep omycin sul a e 1250 mg/L; Peni e 1; Di asa Fa ma ic, Ba elona, Spain) 10 ml/L. 15 Figu e 7. Emb yo eco e y. The eco e ed luid was collec ed in o s e ile Pe i dishes (P60) o examina ion unde a s e eomic oscope. Emb yos we e sco ed by mo phologic c i e ia acco ding o In e na ional Emb yo T ans e Socie y classi ica ion (IETS). B ie ly, only emb yos in mo ula o ea ly blas ocys s ages wi h homogenous cellula mass, and sphe ical mucin coa and zona pellucida we e ca alogued as no mal ( ans e able) emb yos (Figu e 8). Figu e 8. No mal ( ans e able) emb yos. 16 The o ula ion a e, which was es ima ed coun ing he o a ian ollicles wi h sca unde he mic oscope s e eoscope (Figu e 9), he numbe o oocy es and he no mal and abno mal emb yos we e eco ded. Figu e 9. O a ian ollicles wi h sca s. The eco e y a e was calcula ed as he pe cen age o ela ion be ween numbe o emb yos plus oocy es and he o ula ion a e. The e iliza ion a e was calcula ed as he pe cen age o ela ion be ween he numbe o no mal emb yos and he numbe o emb yos and oocy es eco e ed. 𝐫𝐞𝐜𝐨𝐯𝐞𝐫𝐲 𝐫𝐚𝐭𝐞 = ((𝑡𝑜𝑡𝑎𝑙 𝑒𝑚𝑏𝑟𝑦𝑜𝑠+ 𝑜𝑜𝑐𝑦𝑡𝑒𝑠) 𝑜𝑣𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 ⁄) 𝐟𝐞𝐫𝐭𝐢𝐥𝐢𝐳𝐚𝐭𝐢𝐨𝐧 = (𝑛𝑜𝑟𝑚𝑎𝑙 𝑒𝑚𝑏𝑟𝑦𝑜𝑠 (𝑡𝑜𝑡𝑎𝑙 𝑒𝑚𝑏𝑟𝑦𝑜𝑠 + 𝑜𝑜𝑐𝑦𝑡𝑒𝑠) ⁄) 3.5. Emb yo i i ica ion p ocedu e Vi i ica ion was ca ied ou in 12 ba ches. A o al o 810 emb yos we e i i ied and de- i i ied using he me hodology desc ibed by Vicen e e al. (1999). The i i ica ion p ocedu e was ca ied ou in wo s eps a 20 °C. In he i s s ep, emb yos we e placed o 2 min in a i i ica ion solu ion consis ing o 10% ( / ) dime hyl-sulphoxide (1.75M DMSO, Sigma) and 10% ( / ) e hylene glycol (2.23 M EG, Sigma) in DPBS supplemen ed wi h 0.2% (w/ ) o BSA. In he second s ep, emb yos we e suspended 17 o 1 min in a solu ion o 20% ( / ) DMSO and 20% EG in DPBS supplemen ed wi h 0.2% (w/ ) o BSA. Then, emb yos suspended in he i i ica ion medium we e loaded in o 0.25 ml plas ic s aws (IMV, L’Aigle, F ance) be ween wo d ops o DPBS sepa a ed by ai bubbles. Finally, he s aws we e sealed and di ec ly plunged in o liquid ni ogen. De- i i ica ion p ocedu e was pe o med by placing he F ench minis aws o 10 cm om apou ni ogen un il i i ied ac ion begin o ice o ma ion (milking aspec 20-30 sec) and hawed by subme ging he s aws in o a wa e ba h a 20 °C o 10 sec. The i i ica ion medium was emo ed in wo s eps. In he i s s ep, he emb yos we e expelled wi h he medium in o a solu ion o DPBS wi h 0.33M suc ose o 5 min, and in he second s ep he emb yos we e washed in a solu ion o DBPS o ano he 5 min. De i i ied emb yos we e sco ed and only undamaged emb yos we e ca alogued as ans e able. 3.6. Expe imen 1: In i o de elopmen and quali y o emb yos 3.6.1. In i o cul u e In i o cul u e was pe o med in 12 ba ches. A o al o 530 esh and 450 de- i i ied emb yos we e cul u ed o 48 h in Tissue Cul u e Medium 199 (TCM199) + 10% Fe al Bo ine Se um (FBS, Sigma-Ald ich Quimica S.A., Spain) and an ibio ic (penicillin G sodium 300.000 IU/L, penicillin G p ocaine 700.000 UI, and dihyd os ep omycin sul a e 1250 mg/L; Peni e 1; Di asa Fa ma ic, Ba elona, Spain) a 38.5 °C, 5% CO2 and sa u a ed humidi y. Bo h de i i ied and esh emb yos we e assessed acco ding o hei de elopmen al s age a 24 and 48 hou s (non-de eloped, blas ocys , expanding blas ocys and expanded blas ocys ) (Figu e 10). 24 Nº ans e able emb yos 0 10 20 30 40 50 60 a a a a b FSH - CTP FSH - CTP + hLH hFSH hFSH + hLH Con ol Figu e 12. T ans e able emb yos eco e ed by dono doe. a,bValues on he ba s wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). (leas squa e mean ± s anda d e o ). a Blas ocys Expanded blas ocys De elopmen a e FSH-CTP FSH-CTP+ hLH hFSH hFSH+ hLH Con ol aa b a ab aab bb 1 3 9 1 3 9 1 3 6 1 5 8 1 4 9 0.2 0.6 0.0 0.4 0.8 1.0 Figu e 13. In i o de elopmen emb yos a 24 hou s. a,bValues a he same emb yo s age wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). numbe in o he column: numbe o blas ocys a 24 h. (leas squa e mean ± s anda d e o ) 25 ab Blas ocys Expanded blas ocys De elopmen a e FSH-CTP FSH-CTP+ hLH hFSH hFSH+ hLH Con ol ba c ab a ab a bc c 1 7 3 1 6 7 2 0 3 1 8 2 1 6 8 0.2 0.6 1.2 0.0 0.4 0.8 1.0 Figu e 14. In i o de elopmen emb yos a 48 hou s. a,b,cValues a he same emb yo s age wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). numbe in o he column: numbe o blas ocys a 48 h. (leas squa e mean ± s anda d e o ) Table 5. E ec o Emb yo ype on in i o de elopmen . (leas squa e mean ± s anda d e o ) a,bValues in he same column wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). Emb yo ype cul u ed emb yos 24 Hou s 48 Hou s Blas ocys Expanded blas ocys Blas ocys Expanded blas ocys Vi i ied 450 0.56 ± 0.024b 0.12 ± 0.016b 0.87 ± 0.016b 0.50 ± 0.024b F esh 530 0.90 ± 0.014a 0.42 ± 0.022a 0.96 ± 0.009a 0.65 ± 0.021a 26 FSH - CTP i i ied Blas ocys a e blas os blas os: 0,8902 blas os: 0,9804 blas os: 0,8981 blas os: 0,9907 blas os: 0,7586 blas os: 0,8923 blas os: 0,8718 blas os: 0,9804 FSH - CTP + hLH i i ied hFSH + hLH esh hFSH esh FSH - CTP esh FSH - CTP + hLH esh Con ol esh hFSH i i ied Con ol i i ied hFSH + hLH i i ied 0.0 0.2 0.4 0.6 0.8 1.0 bab ba ba bbc c Figu e 15. Blas ocys a e a 48 hou s o di e en o a ian ea men by ype o emb yos. a,b,cValues on he ba s wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). numbe in o he column: numbe o blas ocys a 48 h. (leas squa e mean ± s anda d e o ) 4.2.2. E alua ion o ela i e gene exp ession o blas ocys a e in i o cul u e Resul s o o a ian s imula ion ea men on SOX2, OCT4 and NANOG gene exp ession o emb yos a e shown in Figu e 16. No signi ican di e ence among ela i e alues o SOX2 and OCT4 gene exp ession om di e en expe imen al g oups was de ec ed. While o a ian s imula ion g oups showed simila ela i e alue o NANOG gene exp ession, FSH-CTP, FSH-CTP+ hLH and hFSH g oups showed signi ican ly highe NANOG gene exp ession han he con ol g oup (Figu e 16). Exp ession o genes main aining he plu ipo en emb yonic s em cell pheno ype we e a ec ed by emb yo ype, being he ela i e abundance o SOX2, OCT4 and NANOG highe o i i ied g oup (Table 6). In addi ion, in e ac ion be ween o a ian s imula ion ea men and emb yo ype was signi ican o SOX2 gene exp ession. Vi i ied and esh emb yos om hFSH g oups had simila gene exp ession (0.67 ± 0.121 and 0.68 ± 0.121, da a no shown in ables). 27 SOX2 OCT4 NANOG Rela i e abundance FSH-CTP FSH-CTP+ hLH hFSH hFSH+ hLH Con ol aa aab b 0.2 0.6 1.2 0.0 0.4 0.8 1.0 Figu e 16. Rela i e gene exp ession by s imula ion ea men . a,bValues a he same block o columns wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). (leas squa e mean ± s anda d e o ) Table 6. E ec o Emb yo ype on genes exp ession in blas ocys . (leas squa e mean ± s anda d e o ) a,bValues in he same column wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). 4.3. Expe imen 2: In i o su i al o eco e ed emb yos 4.3.1. E alua ion o emb yo iabili y a e ans e Emb yos om o a ian s imula ed does wi h hFSH+ hLH showed he lowes emb yo su i al a es o implan a ion and bi h (0.53 ± 0.042 and 0.33 ± 0.039, espec i ely) (Figu e 17). Foe al losses in his ea men g oup was also he highes (0.34 ± 0056), while FSH-CTP and FSH-CTP + hLH o a ian s imula ion ea men did no inc ease s a is ically he oe al losses wi h espec o he Con ol g oup (0.22 ± 0.042, 0.20 ± 0.044 and 0.13 ± 0.037, o FSH-CTP, FSH-CTP+ hLH and Con ol g oup, Emb yo ype Numbe o samples SOX2 OCT4 NANOG Vi i ied 20 0.70 ± 0.056a 0.80 ± 0.051a 0.51 ± 0.036a F esh 20 0.42 ± 0.053b 0.66 ± 0.048b 0.38 ± 0.034b 28 espec i ely; da a no shown in ables). Mo eo e , he emb yos om does ea ed only wi h hFSH showed lowe iabili y a bi h ela ed o con ol g oup. F om a o al o 56 ecipien s does, 86.2% (25/29) o i i ied g oup and 85.2% (23/27) o esh g oup ga e bi h (Table 7). In spi e o he numbe o ecipien gi en bi h was simila be ween esh and i i ied emb yos, he implan a ion a e and he bi h a e we e a ec ed by he i i ica ion p ocess. No di e ence in oe al losses was obse ed be ween emb yo ype g oups (Table 7). a Implan a ion Bi h Su i al a e FSH-CTP FSH-CTP+ hLH hFSH hFSH+ hLH Con ol a ab ab b c a b a a 0.2 0.6 0.0 0.4 0.8 1.0 9 99 79 27 58 37 57 66 14 67 3 Figu e 17. Su i al a e o emb yos a implan a ion and bi h. a,b,cValues a he same block o columns wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). Numbe in o he “implan a ion” column: N° implan ed emb yos a 11 days o p egnancy. Numbe in o he “bi h” column: N° bo n pup (leas squa e mean ± s anda d e o ) 29 Table 7. E ec o Emb yo ype on implan a ion and bi h a e. (leas squa e mean ± s anda d e o ) a,bValues in he same column wi h di e en supe sc ip s a e s a is ically di e en (P<0.05) In addi ion, in e ac ion be ween o a ian s imula ion ea men and emb yo ype ( i i ied o esh) was ound. FSH-CTP, FSH-CTP+ hLH and hFSH g oups had simila implan a ion a e o esh and i i ied emb yos, in he same way, FSH-CTP, hFSH and hFSH+ hLH g oups showed simila bi h a e o esh and i i ied emb yos (Figu e 18 and 19). FSH - CTP i i ied Implan a ion a e Con ol i i ied hFSH esh Con ol esh hFSH + hLH i i ied hFSH + hLH esh hFSH i i ied FSH - CTP + hLH esh FSH - CTP + hLH i i ied FSH - CTP esh bc e a ab bc ed bc cd a bc 5 54 43 6 5 6 3 16 63 73 83 74 6 0.2 0.6 0.0 0.4 0.8 1.0 Figu e 18. Implan a ion a e o di e en o a ian ea men by ype o emb yos. a-eValues on he ba s wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). Numbe in o he columns: N° implan ed emb yos a 11 days o p egnancy. (leas squa e mean ± s anda d e o ) Emb yo ype N° ecipien s N° p egnan abbi s (%) Implan ed emb yos a e Deli e y a e (%) Foe al losses a e Bi h a e Vi i ied 29 27 (93.1) 0.55 ± 0.026b 25 (86.2) 0.23 ± 0.030 0.42 ± 0.025 F esh 27 27 (100) 0.72 ± 0.026a 23 (85.2) 0.22 ± 0.029 0.55 ± 0.029 30 Bi h a e FSH - CTP i i ied Con ol i i ied hFSH esh Con ol esh hFSH + hLH i i ied hFSH + hLH esh hFSH i i ied FSH - CTP + hLH esh FSH - CTP + hLH i i ied FSH - CTP esh dc e ab bd de ece de a bd 3 83 72 65 03 62 52 0 2 63 04 3 0.2 0.6 0.0 0.4 0.8 1.0 Figu e 19. Bi h a e o di e en o a ian ea men by ype o emb yos. a-eValues on he ba s wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). Numbe in o he columns: N° bo n pup (leas squa e mean ± s anda d e o ) 4.3.2. Li e size a bi h and weaning in expe imen al g oups The li e size a bi h and weaning a e shown in he Figu e 20. The o a ian s imula ion ea men did no signi ican ly a ec he li e size a bi h and weaning (Figu e 20 and Table 8). T ans e able emb yos as co a iable did no show s a is ical di e ence (12.9 and 0.4 ± 0.31 o es ima ed mean and i s coe icien o ans e able emb yos, espec i ely). 31 Li e size Li e size a weaned Numbe o pup 0 2 4 6 8 10 FSH-CTP FSH-CTP+ hLH hFSH hFSH+ hLH Con ol Figu e 20. li e size pe ecipien doe a bi h and weaning. (leas squa e mean ± s anda d e o ) Table 8. E ec o Emb yo ype on li e size a bi h and weaning. (leas squa e mean ± s anda d e o ) Emb yo ype Numbe o ecipien T ans e ed emb yos Li e size a bi h Li e size a weaning Vi i ied 29 397 5.4 ± 0.63 4.4 ± 0.46 F esh 27 324 6.9 ± 0.66 4.9 ± 0.46 32 4.3.3. Pup weigh a bi h and weaning The weigh a bi h and weaning we e no signi ican ly a ec ed by he o a ian s imula ion ea men (Figu e 21 and 22). The i i ica ion p ocess a ec ed signi ican ly he pup weigh a bi h and weaning. The highe weigh obse ed o he i i ied emb yos g oup a bi h (3.1 g. mo e han esh emb yos g oup) was compensa ed by he esh emb yos du ing he lac a ion, showing highe weigh a weaning (Table 9). The use o co a iables such as he li e size a bi h and weaning and he age a weaning did no a ec he analysis esul s. The es ima ed means o co a iables and hei coe icien a e shown in Table 10. Weigh (g) 0 10 20 30 40 50 60 70 7 57 67 3 6 14 6 FSH - CTP FSH - CTP + hLH hFSH hFSH + hLH Con ol Figu e 21. Weigh a bi h. Numbe in o he columns: N° bo n pup (leas squa e mean ± s anda d e o ) 33 FSH - CTP Weig h (g) 0 200 400 600 800 FSH - CTP + hLH hFSH hFSH + hLH Con ol 5 4 2 9 4 2 5 9 6 3 Figu e 22. Weigh a weaning. Numbe in o he columns: N° li e young abbi s a weaning (leas squa e mean ± s anda d e o ) Table 9. E ec o Emb yo ype on pup weigh a bi h and weaning. (leas squa e mean ± s anda d e o ) Emb yo ype Numbe o bo n pup Numbe o weaned pup Bi h weigh Pups weigh a weaning Vi i ied 156 113 59.2 ± 1.04a 544,9 ± 17,27b F esh 175 134 56.1 ± 0.92b 649,8 ± 15,98a a,bValues in he same column wi h di e en supe sc ip s a e s a is ically di e en (P<0.05). Table 10. The es ima ed means o co a iables and hei coe icien . Cha ac e Es ima ed mean Co a iable coe icien Li e size a bi h 7.8 -2.2 ± 0.37 Days a weaning 31.6 11.2 ± 4.07 Li e size a bi h 7.6 -38.1 ± 8.08 Li e size a weaning 6.3 -28.4 ± 7.51 40 6. CONCLUSION  Does ea men wi h FSH-CTP and hFSH alone o supplemen ed wi h hLH achie ed o a ian supe s imula ion. Supe o ula ion ea men s wi h hFSH o FSH-CTP alone did no a ec emb yos in i o de elopmen , while hFH adminis a ion supplemen ed wi h hLH comp omised he in i o iabili y a 48 hou s. On he o he hand, he exp ession o plu ipo en genes (SOX2 and OCT4) was no a ec ed and an o e exp ession o NANOG gene was obse ed, in conco dance wi h he highe in i o de elopmen o supe s imula ed emb yos.  The li e size and he weigh a bi h and weaning we e non-a ec ed by supe s imula ion ea men s.  Vi i ica ion caused a dec ease in he in i o de elopmen a 48 hou s in all g oups, excep in FSH-CTP supplemen ed wi h hLH. Ne e heless, i i ica ion had no in luence on implan a ion a e o hFSH and FSH-CTP g oups.  Vi i ica ion p ocess did no a ec he li e size, bu had an impo an e ec on he weigh a bi h, being highe han he esh g oup. By con as , weaning weigh was lowe han in he con ol g oup.  The esul s o his s udy sugges ha he use o FSH-CTP (3 µg/doe) is enough o supe o ula e abbi does wi hou comp omising he quan i y and quali y o emb yos, ei he esh o i i ied. In addi ion, he use o his ho mone educes he equency o animal handling, imp o ing he animal wel a e. 41 7. REFERENCES Adams CE. 1965. P eimplan a ion s ages o p egnancy. In: Wols en- holme GEW, O’Conno CM, edi o s. Ciba. Found. Symp.p. 345. Badawy, A.Y. e al., 2016. E ec o Inc eased O ula ion Ra e on Emb yo and Foe al Su i al as a Model o Selec ion by O ula ion Ra e in Rabbi s. Wo ld Rabbi Science, 24(No embe 2015), pp.1–8. Bakke , J. and Baum, M.J., 2000. Neu oendoc ine egula ion o GnRH elease in induced o ula o s. F on ie s in neu oendoc inology, 21(3), pp.220–262. Balen, A.H. e al., 2004. Pha macodynamics o a single low dose o long-ac ing ecombinan ollicle- s imula ing ho mone (FSH-ca boxy e minal pep ide, co i olli opin al a) in women wi h Wo ld Heal h O ganiza ion g oup II ano ula o y in e ili y. Jou nal o Clinical Endoc inology and Me abolism, 89(12), pp.6297–6304. Bank, H. and Mau e , R.R., 1974. Su i al o ozen abbi emb yos. Expe imen al Cell Resea ch, 89(1), pp.188–196. A ailable a : h p://www.sciencedi ec .com/science/a icle/pii/0014482774902018 [Accessed June 6, 2016]. Ba uselli, P.S. e al., 2011. Timed emb yo ans e p og ams o managemen o dono and ecipien ca le. The iogenology, 76(9), pp.1583–1593. Be gendah, M. and Veldhuis, J.D., 2001. Is he e a physiological ole o gonado ophin oligosaccha ide he e ogenei y in humans? Human Rep oduc ion, 16(6), pp.1058–1064. Besen elde , U., Haas, C. and B em, G., 2000. Rep oduc ion echnology and gene ans e in abbi s. In: P oceedings o 7 hWo ld Rabbi Cong ess,Valencia Spain. ol A pp.37–59. Bó, G.A. and Maple o , R.J., 2014. His o ical pe spec i es and ecen esea ch on supe o ula ion in ca le. The iogenology, 81(1), pp.38–48. A ailable a : h p://linkinghub.else ie .com/ e ie e/pii/S0093691X13003853. Boi i, C. e al., 2006. Rep oduc i e physiopa hology o he abbi doe. Recen ad ances in abbi sciences, pp 3 -19. Boye , L. e al., 2005. Co e ansc ip ional egula o y ci cui y in human emb yonic s em cells. Cell, 122(6), pp.947–956. A ailable a : ci eulike-a icle-id:350303 h p://dx.doi.o g/10.1016/j.cell.2005.08.020. 42 B owning, J., Reyes, L. and Wol , R., 1980. Compa ison E idence o Se um P oges e one , in P egnan Rabbi s : Recogni ion o P egnancyi and Es adiol-1713 and Pseudop egnan o Pos implan a ion a e. Biology o ep oduc ion, 23, pp.1014–1019. Campbell, R.K., 2005. Molecula pha macology o gonado opins. Endoc ine, 26(3), pp.291–6. A ailable a : h p://www.scopus.com/inwa d/ eco d.u l?eid=2-s2.0- 24644472873&pa ne ID= ZO x3y1. Ca ney, E.W. and Foo e, R.H., 1990. E ec s o supe o ula ion, emb yo eco e y, cul u e sys em and emb yo ans e on de elopmen o abbi emb yos in i o and in i o. Jou nal o ep oduc ion and e ili y, 89(2), pp.543–51. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/2401982. Ca alho, P.D. e al., 2014. Use o a single injec ion o long-ac ing ecombinan bo ine FSH o supe o ula e Hols ein hei e s: A p elimina y s udy. The iogenology, 82(3), pp.481–489. A ailable a : h p://linkinghub.else ie .com/ e ie e/pii/S0093691X14002337. Cen eno, M.L. e al., 2004. Exp ession o α4 and α7 nico inic ecep o s in he b ains em o emale abbi s a e coi us. B ain Resea ch, 1012(1-2), pp.1–12. Ci e, J. e al., 1999. E ec o emb yo c yop ese a ion on ep oduc i e and g ow h ai s in abbi s. Ann Zo ech 48(1), pp.15-24. Cheng, H. e al., 1999. De elopmen o abbi emb yos du ing a 96-h pe iod o in i o cul u e a e supe o ula o y ea men unde condi ions o ele a ed ambien empe a u e. Anim Rep od Sci, 56(3- 4), pp.279–290. A ailable a : h p://www.ncbi.nlm.nih.go /en ez/que y. cgi?cmd=Re ie e&db=PubMed&dop =Ci a ion&lis _ui ds=10497922. Chu, T., Du o , I. and Si a d, M.A., 2012. E ec o o a ian s imula ion on oocy e gene exp ession in ca le. The iogenology, 77(9), pp.1928–1938. A ailable a : h p://dx.doi.o g/10.1016/j. he iogenology.2012.01.015. Coce o, M.J. e al., 2011. The e iciency o in i o o ine emb yo p oduc ion using an unde ined o a de ined ma u a ion medium is de e mined by he sou ce o he oocy e. Rep oduc ion in domes ic animals, 46(3), pp.463–70. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/20887399 [Accessed June 5, 2016]. Cole, L.A., 2010. Deg ada ion p oduc s o hCG, hype glycosyla ed hCG, and ee b-subuni . In Human 43 Cho ionic Gonado opin (hCG). , pp.109–119. Eds LA Cole & SA Bu le . Else ie , Inc. Comba nous, Y., 1988. ela ionships gonado opins. Rep oduc ion, nu i ion, de elopmen , 28, pp.211–228. Combelles, C.M.H. and Albe ini, D.F., 2003. Assessmen o Oocy e Quali y Following Repea ed Gonado opin S imula ion in he Mouse. Biology o Rep oduc ion, 68(3), pp.812–821. A ailable a : h p://www.biol ep od.o g/cgi/doi/10.1095/biol ep od.102.008656. Co ell, C. e al., 2015. E ec o di e en supe o ula ion s imula ion p o ocols on adenosine iphospha e concen a ion in abbi oocy es. Zygo e, 23(4), pp.507–13. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/24735658 [Accessed Feb ua y 4, 2016]. Cupps, P.T., 1991. Rep oduc ion in Domes ic Animals Fou h. P. T. Cupps, ed., A ailable a : h ps://books.google.com/books?hl=es&l =&id=bbb-ow0N7K4C&pgis=1 [Accessed Feb ua y 3, 2016]. Denke , H.W., 2000. S uc u al dynamics and unc ion o ea ly emb yonic coa s. Cells, issues, o gans, 166(2), pp.180–207. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/10729727 [Accessed June 5, 2016]. De oey, P. e al., 2009. A double-blind, non-in e io i y RCT compa ing co i olli opin al a and ecombinan FSH du ing he i s se en days o o a ian s imula ion using a GnRH an agonis p o ocol. Human Rep oduc ion, 24(12), pp.3063–3072. Dias, F.C.F. e al., 2013. Leng h o he ollicula g owing phase and oocy e compe ence in bee hei e s. The iogenology, 79(8), pp.1177–1183. A ailable a : h p://dx.doi.o g/10.1016/j. he iogenology.2013.02.016. Doody, K. e al., 2010. LH concen a ions do no co ela e wi h p egnancy in FSH/GnRH an agonis cycles. Rep oduc i e BioMedicine Online, 20(4), pp.565–567. A ailable a : h p://dx.doi.o g/10.1016/j. bmo.2009.12.019. Duijke s, I.J.M. e al., 2002. Single dose pha macokine ics and e ec s on ollicula g ow h and se um ho mones o a long-ac ing ecombinan FSH p epa a ion (FSH-CTP) in heal hy pi ui a y-supp essed emales. Human ep oduc ion, 17(8), pp.1987–93. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/12151425. 44 Du ne in, C.I. e al., 2008. E ec s o ecombinan LH ea men on olliculogenesis and esponsi eness o FSH s imula ion. Human Rep oduc ion, 23(2), pp.421–426. Es any, J. e al., 1992. Selec ion esponse o g ow h a e in abbi s o mea p oduc ion. Gene ics Selec ion E olu ion, 24, pp.527–537. A ailable a : h p://www.gse- jou nal.o g/a icles/gse/pd /1992/06/GSE_0999-193X_1992_24_6_ART0004.pd . Ezcu a, D. and Humaidan, P., 2014. A e iew o lu einising ho mone and human cho ionic gonado opin when used in assis ed ep oduc i e echnology. Rep oduc i e Biology and Endoc inology, 12(1), pp.95. A ailable a : h p://www. bej.com/con en /12/1/95. Fause , B.C.J.M. e al., 2009. Ad ances in ecombinan DNA echnology: Co i olli opin al a, a hyb id molecule wi h sus ained ollicle-s imula ing ac i i y and educed injec ion equency. Human Rep oduc ion Upda e, 15(3), pp.309–321. Fechheime , N.S. and Bea y, R.A., 1974. Ch omosomal abno mali ies and sex a io in abbi blas ocys s. Jou nal o Rep oduc ion and Fe ili y, 37, pp.331–341. Fische , R., 2007. Unde s anding he ole o LH: my hs and ac s. Rep oduc i e biomedicine online, 15(4), pp.468–77. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/17908413 [Accessed Feb ua y 3, 2016]. Fou nie , T., Guibou denche, J. and E ain-B ion, D., 2015. Re iew: hCGs: Di e en sou ces o p oduc ion, di e en glyco o ms and unc ions. T ophoblas esea ch. 29, pp.60-65. A ailable a : h p://ac.els-cdn.com/S0143400415002696/1-s2.0-S0143400415002696-main.pd ?_ id=2bd5ee52- 935c-11e5-99 e-00000aab0 02&acdna =1448446158_54132eb63972d587d 569c287c8e5696 [Accessed No embe 25, 2015]. F anco, J.G. e al., 2009. E ec s o ecombinan LH supplemen a ion o ecombinan FSH du ing induced o a ian s imula ion in he GnRH-agonis p o ocol: a ma ched case-con ol s udy. Rep oduc i e biology and endoc inology : RB&E, 7, pp.58. A ailable a : h p://www.pubmedcen al.nih.go /a icle ende . cgi?a id=2701434& ool=pmcen ez& ende ype =abs ac . Ga cía M.L., 2000a. Recons i ución de una línea de conejos a pa i de emb iones c ioconse ados. A ch Zoo ec, 49, pp. 81-86. 45 Ga cía M.L., 2000b. Vi i ied emb yos ans e o wo selec ed spanish abbi lines o U uguay. 7 h Wo ld Rabbi Cong ess, Uni e sidad Poli écnica de Valencia, Spain, Vol. A, pp. 139-142. Ga cía-Ximénez, F. and Vicen e, J.S., 1992. E ec o o a ian cys ic o haemo hagic ollicles on emb yo eco e y and su i al a e ans e in hCG-o ula ed abbi s. Rep oduc ion, nu i ion, de elopmen , 32(2), pp.143–9. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/1388691 [Accessed June 9, 2016]. Gibbons, H. e al., 1981. Impac s o applied gene ics : mic o-o ganisms, plan s, and animals., DIANE Publishing. A ailable a : h ps://books.google.com/books?id=eyu3Al1OjQ8C&pgis=1 [Accessed Ap il 17, 2016]. Gin he , O.J. e al., 2009. T ea men wi h human cho ionic gonado opin (hCG) o o ula ion induc ion is associa ed wi h an immedia e 177 ß-es adiol dec ease and a mo e apid LH inc ease in ma es. Animal Rep oduc ion Science, 114(1-3), pp.311–317. Gosselin, N. e al., 2000. Dec eased LH pulsa ili y du ing ini ia ion o gonado opin supe o ula ion ea men in he cow: E idence o nega i e eedback o he han es adiol and p oges e one. The iogenology, 54(4), pp.507–521. Gu h ie, H.D., Pu sel, V.G. and Wall, R.J., 1997. Po cine ollicle-s imula ing ho mone ea men o gil s du ing an al enoges -synch onized ollicula phase: E ec s on ollicle g ow h, ho mone sec e ion, o ula ion, and e iliza ion. Jou nal o Animal Science, 75(12), pp.3246–3254. Ha ez E.S.E., 1969. Fe al su i al in unde c owded and o e c owded unila e ally p egnan u e i in he abbi . In P oc.: 6 h In e na ional Cong ess Rep oduc ion Animal, 22-26 July, 1969. Pa is, F ance. 1, pp.575 Ha ez, E.S.E. and Ha ez, B., 2000. Rep oduc ion in Fa m Animals Se en h Edi ion. A ailable a : h p:// eebookbank.ne /book-lis ing/ ep oduc ion-in- a m-animals-7 h-edi ion/. Ha cou -B own, F., 2002. Tex book o Rabbi Medicine. Else ie Heal h Sciences, 3(3), pp.356-360. A ailable a : h p://www.ques ia.com/PM.qs ?a=o&docId=26347764. Ha kness, J.E. e al., 2010. Biology and Medicine o Rabbi s and Roden s, 5 h Edi ion. , pp.472. A ailable a : h p://eu.wiley.com/WileyCDA/WileyTi le/p oduc Cd-0813815312.h ml [Accessed Feb ua y 3, 2016]. 46 Hashimo o, S., Ku amochi, T. and Aoyagi, K., 2004. Re ined Po cine Follicle S imula ing Ho mone P omo es he Responsi eness o Rabbi s o Mul iple-O ula ion T ea men . , 53(Decembe 2003), pp.395–397. He le , A. e al., 1991. Supe o ula ion o dai y cows wi h pu i ied FSH supplemen ed wi h de ined amoun s o LH. The iogenology, 35(3), pp.633–643. A ailable a : h p://www.sciencedi ec .com/science/a icle/pii/0093691X9190459Q [Accessed May 29, 2016]. Hesse , M., Mo is, J. and Gibbons, J., 2011. Ad ances in Recombinan Gonado opin P oduc ion o Use in Bo ine Supe o ula ion. Rep oduc ion in Domes ic Animals, 46(5), pp.933–942. A ailable a : h p://doi.wiley.com/10.1111/j.1439-0531.2011.01767.x. Hillie , S.G., 1994. Cu en concep s o he oles o ollicle s imula ing ho mone and lu einizing ho mone in olliculogenesis. Human ep oduc ion (Ox o d, England), 9(2), pp.188–91. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/8027271 [Accessed June 5, 2016]. Hillie , S.G., 2001. Gonado opic con ol o o a ian ollicula g ow h and de elopmen . Molecula and Cellula Endoc inology, 179(1-2), pp.39–46. Ho man, L.H. e al., 1998. P oges e one and Implan ing Blas ocys s Regula e Muc1 Exp ession in Rabbi U e ine Epi helium. Endoc inology, 139(1), pp.266–271. A ailable a : h p://p ess.endoc ine.o g/doi/pd /10.1210/endo.139.1.5750. Holmes, P. V, Schillinge , E. and Janson, P.O., 1985. E ec s o high and low p eo ula o y concen a ions o p oges e one on o ula ion om he isola ed pe used abbi o a y. Jou nal o Rep oduc ion and Fe ili y, pp.393–399. Ja e, R.B., Lee, P.A. and A. Rees Midgley, J., 1969. Se um Gonado opins Be o e, a he Incep ion o , and Following Human P egnancy. The Jou nal o Clinical Endoc inology & Me abolism, 29(9), pp.1281– 1283. Joly, T.1996. C yop ese a ion o gene ic esou ces in abbi species: p ac ical applica ion. 6 h Wo ld Rabbi Cong ess, Toulouse 2, pp. 293-298. Joly, T. 1997. E ablissemen d'une c yobanque de semences ou d'emb yons pou la conse a ion ex si u de la di e si e gene ique chez les mammi e es domes iques: l'exemple du lapin (O yc olagus cuniculus). Thèse nou eau doc o a . Ins i u na ional des sciences appliquées de Lyon, pp. 143. 47 Kahkesh, F.B. e al., 2010. E ec o o ap im, o a ide, HCG, LHRH-A2, LHRHA2+CPE and ca p pi ui a y in benni (Ba bus sha peyi) a i icial b eeding. Global Ve e ina ia, 5(4), pp.209–214. Kanayama, K. e al., 1994. Simpli ica ion o supe o ula ion induc ion by using poly inylpy olidone as a sol en o FSH in abbi s. The Jou nal o e e ina y medical science / he Japanese Socie y o Ve e ina y Science, 56(3), pp.599–600. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/7948403 [Accessed June 5, 2016]. Kau man, R.D. e al., 1998. Supe o ula ion o abbi s wi h FSH al e s in i o de elopmen o i i ied mo ulae. The iogenology 50:1081-1092,, 50(1998), pp.1081 – 1092. Kennelly, J.J. and Foo e, R.H., 1965. Supe o ula o y esponse o p e- and pos -pube al abbi s o comme cially a ailable gonado ophins. Jou nal o Rep oduc ion and Fe ili y, 9, pp.177–188. Kim, J.K. e al., 1988. S udies on simpli ied p ocedu es o eezing and hawing o bo ine emb yos. Pa I. E ec s o PMSG and hCG injec ion on he induc ion o supe o ula ion in abbi . Ko ean J. Anim. Sci. 30, pp. 519–524. Koma su, K. and Fujimo i, T., 2015. Mul iple phases in egula ion o Nanog exp ession du ing p e- implan a ion de elopmen . De elopmen , g ow h & di e en ia ion, 57(9), pp.648–56. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/26660234 [Accessed June 5, 2016]. Kuma , P. and Sha ma, A., 2014. Gonado opin- eleasing ho mone analogs: Unde s anding ad an ages and limi a ions. Jou nal o Human Rep oduc i e Sciences, 7(3), pp.170. A ailable a : h p://www.pubmedcen al.nih.go /a icle ende . cgi?a id=4229791& ool=pmcen ez& ende ype =abs ac [Accessed Oc obe 12, 2015]. La a a, R., Baselga, M. and Vicen e, J.S., 2011. Does s o age ime in LN2 in luence su i al and p egnancy ou come o i i ied abbi emb yos? The iogenology, 76(4), pp.652–657. A ailable a : h p://dx.doi.o g/10.1016/j. he iogenology.2011.03.018. La a a, R. e al., 2015. Emb yo i i ica ion in abbi s: Consequences o p ogeny g ow h. The iogenology, 84(5), pp.674–680. A ailable a : h p://linkinghub.else ie .com/ e ie e/pii/S0093691X15002265. Leao, R. and Es e es, S., 2014. Gonado opin he apy in assis ed ep oduc ion: an e olu iona y pe spec i e om biologics o bio ech. Clinics, 69(4), pp.279–293. A ailable a : h p://www.scopus.com/inwa d/ eco d.u l?eid=2-s2.0-84898744189&pa ne ID= ZO x3y1. 48 Lee, K.Y. and DeMayo, F.J., 2004. Animal models o implan a ion. Rep oduc ion, 128(6), pp.679–695. Leoni, G. e al., 2001. Sheep emb yos de i ed om FSH/eCG ea men ha e a lowe in i o iabili y a e i i ica ion han hose de i ed om FSH ea men . Rep oduc ion, nu i ion, de elopmen , 41(3), pp.239–246. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/11592721 [Accessed June 9, 2016]. Lé y, D.P. e al., 2000. The ole o LH in o a ian s imula ion: exogenous LH: le ’s design he u u e. Human ep oduc ion (Ox o d, England), 15(11), pp.2258–2265. Lin, W.W. and Rami ez, V.D., 1991. E ec o ma ing beha io on lu einizing ho mone- eleasing ho mone elease in emale abbi s as moni o ed wi h push-pull cannulae. Neu oendoc inology, 53(3), pp.229–35. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/2041585 [Accessed Ap il 16, 2016]. López, M., 1995. E aluación de la iabilidad de emb iones de conejo de di e sos es adios p eimplan aciones c ioconse ados medan e p ocedimien os de congelación ápida. Ph. D. Uni e si a Au ònoma Ba celona, Ba celona. López-Béja , M. and López-Ga ius, F., 2000. In i o and in i o su i al o i i ied abbi emb yos. The iogenology, 53, pp.259. Loumaye, E. e al., 2003. Clinical e idence o an LH ‘ceiling’ e ec induced by adminis a ion o ecombinan human LH du ing he la e ollicula phase o s imula ed cycles in Wo ld Heal h O ganiza ion ype I and ype II ano ula ion. Human Rep oduc ion, 18(2), pp.314–322. Ly on, F.D.C. and Poyse , N.L., 1982. P os aglandin p oduc ion by he abbi and placen a in i o u e us. J. Rep od Fe .66, pp.591-599. Mamo, S. e al., 2008. Exp ession p o iles o he plu ipo ency ma ke gene POU5F1 and alida ion o e e ence genes in abbi oocy es and p eimplan a ion s age emb yos. BMC molecula biology, 9, pp.67. A ailable a : h p://www.pubmedcen al.nih.go /a icle ende . cgi?a id=2507718& ool=pmcen ez& ende ype =abs ac . Mannae s, B. e al., 1991. Compa a i e in i o and in i o s udies on he biological cha ac e is ics o ecombinan human ollicle-s imula ing ho mone. Endoc inology, 129(5), pp.2623–30. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/1935792 [Accessed June 8, 2016]. 49 Ma co-Jiménez, F. e al., 2013. In i o de elopmen o i i ied abbi emb yos: E ec s o i i ica ion de ice, ecipien geno ype, and asynch ony. The iogenology, 79(7), pp.1124–1129. Mau e , R.R., Hun , W.L. and Foo e, R.H., 1968. Repea ed supe o ula ion ollowing adminis a ion o exogenous gonado ophins in Du ch-bel ed abbi s. Jou nal o Rep oduc ion and Fe ili y, 15, pp.93– 102. McIn osh, J.E.A., Moo , R.M. and Allen, W.R., 1975. P egnan ma e se um gonado ophin: a e o clea ence om he ci cula ion o sheep. Jou nal o ep oduc ion and e ili y, 44, pp.95–100. De Medei os, S.F. and No man, R.J., 2009. Human cho iogonado ophin p o ein co e and suga b anches he e ogenei y: Basic and clinical insigh s. Human Rep oduc ion Upda e, 15(1), pp.69–95. Mehaisen, G.M.K. e al., 2005. E ec o eCG dose and o ula ion induc ion ea men s on emb yo eco e y and in i o de elopmen pos - i i ica ion in wo selec ed lines o abbi does. Animal ep oduc ion science, 90(1-2), pp.175–184. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/16257606. Mehaisen, G.M.K. e al., 2006. In i o and in i o iabili y o i i ied and non- i i ied emb yos de i ed om eCG and FSH ea men in abbi does. The iogenology, 65(7), pp.1279–1291. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/16171855. Milligan, S., 1982. Induced o ula ion in mammals. , pp.1–46. Mocé, M.L., Blasco, A. and San ac eu, M.A., 2010. In i o de elopmen o i i ied abbi emb yos: E ec s on p ena al su i al and placen al de elopmen . The iogenology, 73(5), pp.704–710. Moo , R.M., K uip, T.A.M. and G een, D., 1984. In ao a ian con ol o olliculogenesis: Limi s o supe o ula ion? The iogenology, 21(1), pp.103–116. Mundim, T.C.D. e al., 2009. Changes in gene exp ession p o iles o bo ine emb yos p oduced in i o, by na u al o ula ion, o ho monal supe s imula ion. Gene ics and Molecula Resea ch, 8(4), pp.1398– 1407. Mu phy, B.D. and Ma inuk, S.D., 1991. Equine cho ionic gonado opin. Endoc ine e iews, 12(1), pp.27–44. A ailable a : h p://www.ncbi.nlm.nih.go /pubmed/2026120 [Accessed Feb ua y 4, 2016]. Pa k, S. e al., 2015. Repea ed Supe o ula ion ia PMSG / hCG Adminis a ion Induces 2-Cys Pe oxi edoxins Exp ession and O e oxida ion in he Rep oduc i e T ac s o Female Mice. , 38(9),