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
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