Neuroanatomical and gene expression features of the rabbit accessory olfactory system. Implications of pheromone communication in reproductive behaviour and animal physiology

INTERNATIONAL DOCTORAL
SCHOOL OF THE USC
Paula
Rod íguez Villamayo
PhD Thesis
Neu oana omical and gene
exp ession ea u es o he abbi
accesso y ol ac o y sys em.
Implica ions o phe omone
communica ion in ep oduc i e
beha iou and animal physiology.
Doc o al P og amme in Basic and Applied Resea ch in Ve e ina y Sciences
Lugo, 2023
TESE DE DOUTORAMENTO
NEUROANATOMICAL AND GENE EXPRESSION
FEATURES OF THE RABBIT ACCESSORY
OLFACTORY SYSTEM. IMPLICATIONS OF
PHEROMONE COMMUNICATION IN
REPRODUCTIVE BEHAVIOUR AND ANIMAL
PHYSIOLOGY
Paula Rod íguez Villamayo
ESCOLA DE DOUTORAMENTO INTERNACIONAL DA
UNIVERSIDADE DE SANTIAGO DE COMPOSTELA
PROGRAMA DE DOUTORAMENTO EN INVESTIGACIÓN
BÁSICA E APLICADA ÁS CIENCIAS CLÍNICAS VETERINARIAS
LUGO
ANO 2023
DECLARACIÓN DO AUTOR/A DA TESE
D./Dna.
Paula Rod íguez Villamayo
Tí ulo da
ese:
Neu oana omical and gene exp ession ea u es o he
abbi accesso y ol ac o y sys em. Implica ions o
phe omone communica ion in ep oduc i e
beha iou and animal physiology
P esen o a miña ese, seguindo o p ocedemen o axei ado ao
Regulamen o, e decla o que:
1) A ese aba ca os esul ados da elabo ación do meu aballo.
2) De se o caso, na ese aise e e encia ás colabo acións que
i o es e aballo.
3) Con i mo que a ese non inco e en ningún ipo de plaxio
dou os au o es nin de aballos p esen ados po min pa a a
ob ención dou os í ulos.
4) A ese é a e sión de ini i a p esen ada pa a a súa de ensa e a
e sión imp esa coincide coa p esen ada en o ma o
elec ónico
E comp omé ome a p esen a o Comp omiso Documen al de
Supe isión no caso de que o o ixinal non es ea na Escola.
En Lugo, de no emb o de 2022
Fdo: Paula Rod íguez

AUTORIZACIÓN
DO
DIRECTOR
/
TITOR DA TESE
Neu oana omical and gene exp ession ea u es o he
abbi accesso y ol ac o y sys em. Implica ions o
phe omone communica ion in ep oduc i e beha iou
and animal physiology
D. Paulino Ma ínez Po ela (di ec o )
D. Pablo Sánchez Quin ei o (di ec o )
D. José Manuel Ci uen es Ma ínez ( i o )
INFORMAN:
Que a p esen e ese, co espóndese co aballo ealizado po Dna Paula Rod íguez
Villamayo , baixo a nosa di ección/ i o ización, e a
u o izamos
a súa
p esen ación,
conside ando
que eúne os
equisi os
esixidos no R
egulamen o
de Es udos de
Dou o amen o da USC,
e
que
como di ec o es/ i o des a
non inco en nas
causas de
abs ención
es ablecidas
na Lei
40/2015.
De aco do co indicado no Regulamen o de Es udos de Dou o amen o, decla amos
amén que a p esen e ese de dou o amen o é idónea pa a se de endida en base á
modalidade de COMPENDIO DE PUBLICACIÓNS,
nos que a pa icipación da
dou o anda oi decisi a pa a a súa elabo ación e as publicacións se axus an
ao Plan de In es igación.
En Lugo, de no emb o de 2022
Fdo: Paulino Ma ínez Fdo: Pablo Sánchez Fdo: José Manuel Ci uen es
Eu Paula Rod íguez Villamayo , decla o que es a ese de
dou o amen o non p esen a con lic os de in e és.
En Lugo, de no emb o de 2022
Fdo: Paula Rod íguez
Published in: Animals (2022) 12:308. doi:
h ps://doi.o g/10.3390/ani12030308. Q1 in he ca ego y Ag icul u e,
dai y & Animal science. Impac ac o : 3.231.
Con ibu ion: Me hodological and expe imen al design. Pa icipa ion
in he ield s udy. Pe o ming da a analysis. W i ing he o iginal d a .
W i ing, e iewing and edi ing. Funding acquisi ion.
Con ic o in e es : The au ho s decla e ha he e is no con lic o
in e es .
Copy igh © 2022 Villamayo PR, Gullón J, Yáñez U, Sánchez M,
Quin ei o P, Sánchez- Ma ínez P, Quin ela L. This is an open access
a icle dis ibu ed unde he C ea i e Commons A ibu ion licence
(CC-BY). No special pe mission is equi ed o euse all o pa o
a icle published by MDPI, including igu es and ables. Fo a icles
published unde an open access C ea i e Common CC BY license, any
pa o he a icle may be eused wi hou pe mission p o ided ha he
o iginal a icle is clea ly ci ed (see Annex V).

PhD
chap e
Jou nal
IF
5-yea
IF
Qua ile
Rank
ISS
N
I
Jou nal o
Ana omy
2.479
(2017)
2.789
Ana omy &
Mo phology
(Q1)
4/21
0021
-
8782
II
B ain
S uc u e and
Func ion
3.622
(2018)
4.019
Ana omy &
Mo phology
(Q1, D1)
1/21
1863
-
2653
III
Genomics
6.205
(2019)
4.149
Gene ics &
he edi y (Q1)
18/178
0888
-
7543
IV
F on ie s in
Molecula
Neu oscience
6.261
(2021)
6.187
Neu oscience
(Q1)
54/274
1662
-
5099
V
Animals
3.231
(2021)
3.312
Ve e ina y
Sciences
(Q1)
16/144
2076
-
2615
ABBREVIATIONS
Abb e ia ions
.
2MB2: 2-me hyl-bu -2-enal.
Also e e ed as MP in chap e
II
Aas: Amino acids
AEMPS: Spanish agency o
medicines and medical de ices
AI: A i icial insemina ion
Ano: Anoc amin
AOB: Acceso y ol ac o y bulb
AOS: Accesso y ol ac o y
sys em
APHR: Aph odisin
AQPs: Aquapo ins
A g: A ginase
Bn: bouin liquid
Bpi b3: BPI Fold Con aining
Family B Membe 3
BSI-B4: Bandei aea
simplici olia
CB: Calbindin
CCK: Cholecys okinin
CF: Combined emale
CM: Combined male
CNS: Cen al ne ous sys em
CR: Cal e inin
DAG: Diacylglyce ol
DE: Di e en ially exp essed
DEGs: Di e en ially exp essed
genes
Dlk1: Del a like non-canonical
No ch ligand 1
Dll4: Del a-like 4 signaling
gene
ECL: Ex e nal cellula laye
EPL: Ex e nal plexi o m laye
ESP1: Exoc ine gland-
sec e ing pep ide 1
ESPs: Exoc ine sec e o y
pep ides
ESR: Es ogen
Es : Es ogen ecep o
EU: Eu opean Union
FC: Fold change
FDR: False disco e y a e
FEL1A: Majo alle gen I

polypep ide chain 1
F-F: Female- emale; emia-
emia
FISH: Fluo escence in si u
hyb idiza ion
FLP: Female-speci ic lac imal
gland p o ein
FPRs: Fo myl pep ide
ecep o s
F : bu e ed o malin
Fsh : Follicle-s imula ing
ho mone ecep o
GAP-43: G ow h-associa ed
p o ein 43
GFAP: Glial ib illa y acidic
p o ein
GG: G ünebe g ganglion
GHRHR: G ow h ho mone-
eleasing ho mone ecep o
GlL: Glome ula laye
GLS: Glu aminase
GnRH: Gonado opin eleasing
ho mone
GO: Gene on ology
GPA1: Alpha subuni o he
he e o ime ic guanine
nucleo ide-binding G p o ein
G L: G anula laye
Gαi2: G p o ein, subuni αi2
Gαo: G p o ein, subuni αo
H2-M : A mul igene amily o
non-classical class I majo
his ocompa ibili y complex
(MHC) genes
ICL: In e nal cellula laye
IN: In e neu ons
IP3: Inosi ol 1,4,5-
iphospha e
IPL: In e nal plexi o m laye
KEGG: Kyo o Encyclopedia o
Genes and Genomes
KO: Knockou
LCN: Lipocalins
LCN1_B: Lipocalin 1B
LCN2: Lipocalin 2
LEA: Lycope sicon esculen um
Lhcg : Lu einizing
ho mone/cho iogonado opin
ecep o
LOT: La e al ol ac o y ac
M&M: Ma e ial and Me hods
sec ion
MAP-2: Mic o ubule
associa ed p o ein 2
MCL: Mi al cell laye
MeA: Medial amygdala
MHC: Majo
his ocompa ibili y complex
MOB: Main ol ac o y bulb
MOE: Main ol ac o y
epi helium
MOS: Main ol ac o y sys em
MP: Mamma y phe omone
MSP: Male-speci ic
submandibula sali a y gland
p o eins
Mup4: Majo u ina y p o ein 4
MUPs: Majo u ina y p o eins
MYA: Millions o yea s ago
NC: Nasal ca i y
NCBI: Na ional Cen e o
Bio echnology In o ma ion
OB: Ol ac o y bulb
OC: O al ca i y
OMP: Ol ac o y ma ke p o ein
ORs: Ol ac o y ecep o s
Ox : Oxy ocin ecep o
PAS: Pe iodic acid-Schi
PB: Phospha e bu e
PBS: Phospha e bu e ed saline
PCA: P incipal componen
analyses
PE: Pai ed-end
Pg : P oges e one ecep o
Pg mc: P oges e one ecep o s
memb ane componen
PLC: Phospholipase C
Pp: pala ine p ocess o he
incisi e bone
PRG: P oges e one
RD: Real Dec e o
RIN: RNA in eg i y numbe
RNAseq: RNA sequencing
RT-PCR: Re e se ansc ip ion
polyme ase chain eac ion
SBT: 2-sec-bu yl-4,5-
dihyd o hiazole
SF: Sepa a ed emale
SLC1A1: Exci a o y amino
acid anspo e 3
SM: Sepa a ed male
SO: Mase a o sep al o gan
SRA: Sho Read A chi e
TPM: T ansc ip s Pe Million
TRPC: T ansien ecep o
po en ial channel
T pc2: T ansien ecep o
po en ial channel 2
UEA-I: Ulex eu opaeus
V1Rs: Vome onasal ype 1
ecep o s (also called VR1 in
chap e I)
V2Rs: Vome onasal ype 2
ecep o s (also called VR2 in
chap e I)
VMH: Ven omedial
hypo halamus
VNL: Ne e laye
VNO: Vome onasal o gan
VNS: Vome onasal sys em
VRs: Vome onasal ecep o s
VSNs: Vome onasal senso y
neu ons
INDEX
3.5 Di e en ial exp ession o he ome onasal o gan be ween emales
and males .......................................................................................... 217
3.6 Di e en ial exp ession o he ome onasal o gan be ween
ju eniles and adul s .......................................................................... 218
4. DISCUSSION ....................................................................................... 221
4.1 The unique gene epe oi e o he abbi ome onasal o gan ..... 221
4.1.1 Vome onasal- ype ecep o s ............................................... 222
4.1.2 Fo myl pep ide ecep o s.................................................... 224
4.1.3 T ansien ecep o po en ial channels ................................. 225
4.1.4 Majo u ina y p o eins. Mup4 in he abbi nasal mucosa . 226
4.2 VNO-media ed ep oduc i e beha iou does no depend on VNO
ansc ip ome sex di e ences ........................................................... 227
4.3 Insigh s in o ome onasal connec i i y ...................................... 230
CONCLUDING REMARKS ........................................................................ 232
ACKNOWLEDGEMENTS .......................................................................... 232
SUPPLEMENTARY MATERIAL ................................................................ 233
CHAPTER IV: Sex sepa a ion un eils he unc ional plas ici y o
he ome onasal o gan in abbi s .................................................. 235
ABSTRACT .......................................................................................... 237
1. INTRODUCTION .................................................................................. 239
2. MATERIAL AND METHODS ................................................................ 242
2.1 Expe imen al design ................................................................... 242
2.2 Sampling .................................................................................... 244
2.3 T ansc ip omic Analysis ............................................................ 244

3. RESULTS ........................................................................................... 246
3.1 Sex-sepa a ion induces sex-speci ic di e ences in gene exp ession
o male and emale abbi s ................................................................ 246
3.2 VNO ansc ip ome changes depending on en i onmen al
condi ions in a sex- speci ic manne ................................................. 248
3.3 Vome onasal ecep o epe oi e unde goes signi ican down-
egula ion in adul emale VNO unde he sex-sepa a ion condi ion 249
3.4 En i onmen al modula ion igge s di e en ial exp ession o VNO
genes in ol ed in ep oduc ion and sexual beha iou , suppo ing i s
plas ic capaci y o ensu e indi idual su i al ................................... 254
3.5 En i onmen al changes modula e VNO unc ional ac i i y, hus
adding complexi y and lexibili y o he VNO senso y code ............ 256
3.6 VNO as a i s ba ie in he de ec ion o ex e nal s imuli,
including ha m ul pa hogens, and i s ela ionship wi h he immune
sys em ............................................................................................... 259
4. DISCUSSION ........................................................................................ 261
4.1 VNO and VR epe oi es show species-speci ic and
en i onmen ally modula ed exp ession: a compa ison be ween abbi s
and mice ............................................................................................ 261
4.2 The lipocalin aph odisin is up- egula ed in sex-sepa a ed adul
male VNO ......................................................................................... 264
4.3 Sex-s e oid ecep o s a e modula ed by he en i onmen and may
a ise as a new ype o VNO phe omone ecep o s ............................ 265
4.4 A po en ial close ela ionship be ween he VNO and emale
pube y .............................................................................................. 267
CONCLUDING REMARKS ......................................................................... 269
ACKNOWLEDGEMENTS ........................................................................... 269
SUPPLEMENTARY MATERIAL .................................................................. 269
CHAPTER V: Assessmen o bios imula ion me hods based on
chemical communica ion in emale doe ep oduc ion ................. 283
ABSTRACT .......................................................................................... 285
1. INTRODUCTION .................................................................................. 287
2. MATERIALS AND METHODS ............................................................... 289
2.1 Animals ...................................................................................... 289
2.2 Sample collec ion ....................................................................... 290
2.3 Semen p ocessing and a i icial insemina ion ............................ 290
2.4 Rep oduc i e managemenen ..................................................... 290
2.5 Expe imen al design ................................................................... 291
2.6 S a is ical analysis ...................................................................... 292
3. RESULTS .......................................................................................... 293
3.1 Fe ili y ....................................................................................... 294
3.2 P oli icacy .................................................................................. 296
3.3 Recep i i y ................................................................................. 297
4. DISCUSSION ................................................................................... 299
4.1 Social in e ac ion seems no in luencing ep oduc i e physiology
in a m emale doe............................................................................ 300
4.2 U ine as a po en ial sou ce o sex phe omones in emale doe
ep oduc ion ..................................................................................... 301
4.3 Seminal plasma migh a ise as a new sou ce o phe omones .... .301
4.4 P ac ical conside a ions when assessing bios imula ion me hods 302
4.5 Could bios imula ion me hods educe o eplace ho monal
ea men s? ....................................................................................... 304
CONCLUDING REMARKS ....................................................................... 305
ACKNOWLEDGMENTS ................................................................................. 305
DISCUSSION .................................................................................. 307
1. IMPORTANCE OF AN INTEGRATIVE APPROACH IN
CHEMOCOMMUNICATION RESEARCH ................................................ 309
2. COMPLEXITY OF VOMERONASAL CHEMORECEPTORS ........................ 313
2.1 V1R and V2R ecep o s .............................................................. 314
2.1.1 Ex anasal exp ession o VRs ............................................. 317
2.2 FPRs ............................................................................................ 318
2.3 Sex-s e oid ecep o s ................................................................... 319
3. IMPLEMENTATION OF PHEROMONES IN THE MARKET ........................ 321
CONCLUDING REMARKS AND FURTHER STEPS ............. 327
CONCLUSIONS ............................................................................. 329
REFERENCES ............................................................................... 335
ANNEX I ......................................................................................... 409
ANNEX II ........................................................................................ 421
ANNEX III ...................................................................................... 427
ANNEX IV ....................................................................................... 431
ANNEX V......................................................................................... 439
ANNEX VI ....................................................................................... 441
All igu es included in his manusc ip a e o iginal and
elabo a ed by Paula Rod íguez Villamayo . Some igu es we e
ed awed based on he con en o e e enced esea ch a icles and his
is indica ed in he co esponding igu e oo no es.
51
SUMMARY
Phe omone communica ion is in ol ed in undamen al inna e socio-
sexual beha io s such as ma ing and igh ing, ha a e essen ial o
animal ep oduc ion and su i al. Phe omonal cues a e mainly
pe cei ed by he accesso y ol ac o y o ome onasal sys em (VNS),
speci ically by senso y neu ons o he ome onasal o gan (VNO)
which in u n send hei inpu s o he accesso y ol ac o y bulb (AOB).
The VNS g ea ly a ies ac oss e olu ion, especially a s uc u al and
genomic le els, o be able o ace a high ange o species-speci ic
beha iou s (i. e. di e en ep oduc i e sys ems, dominances, e c.).
Mos VNS s udies ha e been done in mice, which ended o
ex apola e da a o close phylogene ic species such as abbi s.
Howe e , due o he high specie-speci ici y o he VNS, his may lead
o mis aken conclusions and should always be a oided; acco dingly,
each species should be s udied independen ly and om a compa a i e
poin o iew. Addi ionally, he abbi -O yc olagus cuniculus- is
conside ed a unique model o s udying chemocommunica ion due o
he disco e y o he abbi mamma y phe omone. Howe e , he abbi
VNS emains unexplo ed, especially a mo phological, gene ic and
molecula le els. On he o he hand, he s udy o phe omone
communica ion could ha e a ansla ional applica ion in o animal
p oduc ion (i. e. phe omones could be used as na u al subs ances o
imp o e ep oduc i e pa ame e s and animal wel a e, which in u n
may lead o a educ ion o he use o ho mones and an ibio ics), and
also in o o he indus ies such as pe s (i. e. by educing s ess le els),
o con olling endange ed species o pes s. Acco dingly, i would be
sma add essing esea ch s udies in species in which esul s could be

PAULA RODRÍGUEZ VILLAMAYOR
52
di ec ly ans e ed o he indus ial sec o . Rabbi s a e especially
in e es ing because hey a e a med species, easy o wo k wi h (small,
sho ep oduc i e cycle) and mo e ecen ly hey ha e also become
pe s. The e o e, in es iga ions in o abbi phe omone communica ion
could be used o u he ansla ional wo k aimed a implemen ing
phe omones in abbi ma ke . In his wo k, we aimed a cha ac e izing
he neu oana omical and genomic ea u es o he abbi VNS as well
as e alua ing ce ain phe omone-media ed beha io al and
ep oduc i e pa ame e s in abbi a ms. Fi s , we s udied he
s uc u al and mo pho unc ional p ope ies o he VNO and he AOB.
To do so, dissec ion, mic odissec ion, his ological echniques –
his ochemis y and immunohis ochemis y–, and mo phome y we e
employed. We de e mined ha he abbi VNO is highly de eloped
and equiped o de elop an e ec i e chemocommunica ion. We also
ound ha he AOB is sexually dimo phic and show a complex
s uc u e in e ms o laye o ganiza ion, neu ochemical p ope ies and
cell ypes. We hen explo ed he VNO a ansc ip omic le el o gain
insigh s in o hei unc ional a ionale. Speci ically, an analysis o
gene exp ession was ca ied ou unde di e en condi ions –
p epube al, pube al, male and emale– by using RNAseq. The VNO
gene exp ession did no a y be ween sexes bu showed sha p
di e ences be ween ju enile and adul abbi s. Besides he
comp ehensi e cha ac e iza ion o he abbi VNO ansc ip ome,
including exp ession o ome onasal ecep o s as well as many genes
in ol ed in ep oduc ion, immuni y and unc ional ac i i y, we ha e
also de e mined i s plas ici y – ia ansc ip ional modula ion–. To do
so, we exposed abbi s o di e en en i onmen al condi ions (sex-
combined s sex-sepa a ed) and analysed hei VNO gene exp ession
compa a i ely. We ound sex- and s age- speci ic gene exp ession
di e ences upon such en i onmen al condi ions. Impo an ly, he
ome onasal ecep o s VRs showed signi ican down- and up-
egula ed exp essed genes in sex-sepa a ed adul emale and sex-
sepa a ed ju enile emale, espec i ely. Simila ly, genes in ol ed in
ep oduc ion, immuni y and unc ional ac i i y we e also ound o be
highly plas ic in e ms o hei gene exp ession pa e ns. Finally, a i s
app oach in o ansla ional s udies was ca ied ou di ec ly in abbi
SUMMARY
53
a ms. We de eloped a p o ocol which consis ed o exposing emale
abbi s o ei he u ine o seminal plasma as sou ce o phe omones
ollowed by an e alua ion o hei ep oduc i e pa ame e s compa ed
o a con ol. Al hough we did no ind any imp o emen in he emale
ep oduc i e pa ame e s, ou p o ocol migh be u he used o y on
o he biological compounds such as ex ac s o exoc ine glands, and
de e mine hei po en ial ole in phe omone bios imula ion. All in all,
his s udy shows ha phe omone communica ion and ome onasal
chemope cep ion is highly complex and should always be app oached
species-speci ically and om a mul idisciplina pe spec i e. Ou da a
p o ide ex ensi e knowledge in o abbi chemocommunica ion,
speci ically a ana omical, ansc ip omic, ep oduc i e and
beha iou al le els, which p o ide s ong ounda ion o u he
ansla ional s udies which aim a implemen ing he use o
phe omones in abbi a ms.
Keywo ds: abbi chemocommunica ion, ome onasal sys em,
ome onasal o gan plas ici y, accesso y ol ac o y bulb, sexual
dimo phism, immunohis ochemis y, RNAseq, phe omone
bios imula ion, ep oduc ion, specie-speci ici y, mul idisciplina
app oach.
GENERAL
INTRODUCTION
PAULA RODRÍGUEZ VILLAMAYOR
62
Odo : Re e s o he de ec ion and disc imina ion o all di e en
chemical molecules (odo an s) by he ol ac o y sys em (Wya , 2014a)
Smell: Odo s ha a e ‘consciously pe cei ed’. They a e mainly
de ec ed h ough he MOS.
Semiochemicals/social odo signals: Complex specialized odo s,
also called chemosenso y cues
3
, eleased by any li ing o ganism o
he en i onmen and pe cei ed by o he s, p o iding in o ma ion abou
he ex e nal wo ld. They a e mainly pe cei ed by he VNS and ac as
key playe s in chemical communica ion (Wya , 2014a). They can be
classi ied acco ding o how hey a e eleased s ecei ed among
indi iduals (Figu e 2):
A) Chemical s imuli exchanged be ween membe s o he same
species:
- Phe omones (g eek ‘phe ein’) a e speci ic ol ac o y
cues/chemical s imuli eleased by one indi idual and pe cei ed
by ano he indi idual o he same species, igge ing a
beha iou al o physiological esponse in he ecei e (Wya ,
2014b; Bakke and Leinde s-Zu all, 2016).
- Signa u e mix u es/ins inc i e cues: Subse o a iable
molecules (no phe omones) om he chemical p o ile o a
conspeci ic ha a e lea n by he ecei e o dis inguishing
indi iduals o colonies (Wya , 2014b).
- Scen ma ks: Complex composi ion o chemical molecules,
including he majo his ocompa ibili y complex (MHC) and
majo u ina y p o eins (MUPs), ha p o ide gene ic
in o ma ion abou indi iduals ega ding species, sex,
indi idual iden i y, and me abolic in o ma ion (social
dominance and ep oduc i e and heal h s a us) (Ne ison e al.,
2003; A akawa e al., 2008). They o igina e om mul iple
sou ces, being u ine and anal gland sec e ion he mos
common ones (Robe s, 2007) and hei chemical composi ion
3
Semiochemicals a e usually a mix o a ious molecules: (1) a iable molecules, meaning
hose unique om each indi idual and (2) phe omones ha a e sha ed among g oups o
indi iduals. Al oge he , hey ep esen he chemical p o ile o chemical signa u e o an
indi idual (Wya , 2014a).

GENERAL INTRODUCTION
63
is hough o include bo h phe omones and o he gene al
odou s (Johnson, 1973). The o m o ol ac o y social
communica ion be ween conspeci ics media ed by scen ma ks
(Melo e al., 2008) is de ined as scen -ma king
4
.
B) Chemical s imuli exchanged be ween membe s o di e en
species (Wya , 2014b; Bakke and Leinde s-Zu all, 2016).
These a e called allelochemicals and hey can be di ided in:
- Allomones, when hey bene i he emi e .
- Kai omones, when hey bene i he ecei e .
- Synomones, when hey bene i bo h he ecei e and he
emmi e .
Figu e 2. Diag am showing he di e en ypes o chemosenso y cues
in ol ed in chemical communicac ion. An example o ‘species-wide’
phe omone signals could be male sex phe omones, p oduced by any dominan
male. Signa u e mix u es in ol e he p ocess o lea ning (i.e. ecogni ion o
siblings) based on unique chemical p o iles o each indi idual, and hus he e
is no species-wide molecule(s) o ind. Modi ied om Wya , 2014b.
4
The mos common scen -ma king beha iou is e i o ial ma king (be ween conspeci ics),
closely ela ed o dominance, bu i is also used as an ala m signal and as a h ea o
conspeci ics as well as o o he species. Fo ins ance, gland sec e ions may be deposi ed as a
esponse o a p eda o odou wi h he e ec o signaling ala m o conspeci ics (A akawa e al.,
2008). This beha iou acili a es main enance o a e i o y, ep oduc i e compe i ion, and
sexual ad e isemen (B own, 1979; Wol , 2004).
CHEMICAL COMMUNICATION
(also called semiochemicals, social odo signals)
Signa u e
mix u es
Phe omones
In e ac ion be ween membe s o
he same species
Species-wide
signals
Allelochemicals
Synomones
Bene i he
emi e
Va iable molecules,
speci ic o each
indi idual
In e ac ion be ween membe s o
di e en species
Chemical p o ile / chemical signa u e
Kai omones
Bene i bo h
he emi e
and he
ecei e
Bene i he
ecei e
Allomones
CHEMOSENSORY CUES
PAULA RODRÍGUEZ VILLAMAYOR
64
In pa icula , phe omones a e classi ied in o di e en ypes based
on he esponses hey igge as well as on hei chemical na u e. They
we e i s desc ibed by Ka lson and Lüsche (1959) as chemical
signals eleased by one indi idual o he en i onmen and pe cei ed
by ano he indi idual o he same species, causing a physiological o a
speci ic beha iou al esponse in he ecei e . They sugges ed he e m
would apply o chemical signals in all ypes o animals om
in e eb a es o ish o e es ial mammals, and hey could be single
molecules o a mix o a ious molecules – e en di e en species
could sha e some molecules, p obably causing di e en esponses.
Phe omones can be classi ied acco ding o he esponse hey igge in
he ecei e (Wya , 2014a) (Figu e 3):
- P ime phe omones: They igge a physiological esponse in
he ecei e , which is slow (no immedia e) and usually
modula ed by he endoc ine sys em (i.e. Ea ly e mina ion o
p egnancy, o p egnancy block when a ecen ly ma ed emale
is housed wi h a s ange male, known as B uce e ec (B uce
1959; 1960)).
- Release phe omones: They igge an immedia e beha iou al
esponse upon ecep ion (i.e. Nipple-sea ch and suckling
beha iou by abbi ki s, elici ed by he abbi mamma y
phe omone (2MB2; 2-me hylbu -2-enal) (Schaal e al., 2003)).
Vome onasal senso y neu ons
NEUROCHEMESTRY
P ime
e ec
Release
e ec
BEHAVIOURAL
RESPONSE
(immedia e;
di ec )
PHYSIOLOGICAL
RESPONSE
(slow; indi ec )
AOB s imula ion
Phe omones
CNS (highe
cen e s s imula ion)
GENERAL INTRODUCTION
65
Figu e 3. Diag am showing how phe omones s imula e he VNS. Phe omones
s imula e ome onasal senso y neu ons o he VNO, which send elec ical inpu s o
he AOB and in u n p omp ei he di ec beha iou al esponses ( esease e ec ) o
indi ec physiological esponses ia s imula ion o ho mone sec e ion (p ime
e ec ). The same phe omone compound can ac as p ime and elease a same
ime o in di e en con ex s (Wya , 2009). Modi ied om Wya , 2014a.
As p e iously men ioned, classical s udies sugges ed ha mos
phe omones a e non ola ile, ac i a e he VNO, speci ically VSNs, and
igge inna e and s e eo yped social beha iou s and neu oendoc ine
elease (Conno 1972; O’Connell and Me edi h, 1984). Howe e , his
de ini ion may be oo es ic i e and canno explain all he beha iou s
induced by phe omones. In ac , Halpe n (1987) al eady poin ed ou
ha he sepa a ion in o ola ile and non ola ile compounds may be
oo simplis ic. Acco dingly, in he las decades, esea che s ha e
p o en ha he chemical na u e o phe omones is highly di e se, and
almos any ype o chemical molecule (simple o mixed) has he
po en ial o be a phe omone as long as hey igge he app op ia e
esponse in he ecei e . Con a y o adi ional hough s, hey can be
ei he ola ile o non ola ile depending on he medium hey a e
eleased (B ennan and Ke e ne, 2004). Addi ionally, phe omones can
ac i a e he VNO and also he main ol ac o y epi helium (MOE) o
he MOS. Indeed, Halpe n (1987) al eady a gued ha he MOS and
he VNS may in e ac o yield speci ic esponses o chemical signals,
and adi ional beha iou al and neu oscience s udies had always
sugges ed ha mammals use he VNS and he MOS, ei he
independen ly o bo h wo king oge he , o pe cei e odo s and
semiochemicals, depending on species, signals, and p e ious
expe ience. Impo an ly, inpu s om he VNS and he MOS a e
in eg a ed a highe cen e s in he b ain (i.e. amygdala – see sec ion
2.3 ‘Highe cen e s: limbic sys em and hypo halamus’ o de ail)
(Baxi e al., 2006; Me edi h 1998; Wya , 2014a).
Finally, phe omone e ec s can be al e ed by con ex – hei
esponses a e no as ha dwi ed as p e iously hough – (S owe s and
Ma on, 2005). The e o e, phe omones a e powe ul signals ha
in luence indi iduals’ beha iou , ye phe omone esponses a e no
always s e eo yped. Recen molecula and gene ics ad ances ha e
PAULA RODRÍGUEZ VILLAMAYOR
66
shown ha a iabili y in phe omone esponses among indi iduals may
be modula ed a di e en le els o phe omone-p ocessing ci cui y,
and in pa icula in he ome onasal sys em – om he pe iphe al
(VSNs) o highe cen e s in he cen al ne ous sys em (AOB and
ome onasal amygdala)–. S udying he molecula mechanisms
unde lying a iabili y in phe omone esponses –i.e. VNS plas ici y
(see sec ion ‘3. Plas ici y o he ome onasal sys em’ o de ail)–
should p o ide a needed amewo k o deciphe ing how phe omonal
ou pu s a e lexibly shaped in o neu al ci cui s and al e beha iou
(S owe s and Libe les, 2016).
1.1.1.2 Chemosenso y ecep o s o he ol ac o y sys em
In mammals, he wo majo ol ac o y subsys ems, he
MOS and he VNS, a e p o ided wi h OSNs and VSNs espec i ely,
each wi h hei own se s o ecep o s. Iden i ying such ecep o s as
well as hei mechanisms o ac ion is a needed i s s ep o u he
unde s and how his senso y sys em wo ks. I was no un il he
nine ies ha a i s la ge amily o ol ac o y ecep o s –ol ac o y
ecep o s (OR) in he MOE– was disco e ed by Buck and Axel
(1991), a s udy which ep esen s a big b eak h ough in he ield o
ol ac ion and was awa ded wi h he 2004 Nobel P ize o Physiology
and Medicine. Soon a e , esea che s om Axel’s lab iden i ied he
ome onasal ecep o amily (VRs) in he VNO (Dulac and Axel,
1995). Since hen and hanks o he ad en o new bio echnological
and genomic echniques, g ea ad ances ha e been made in he ield
o ol ac ion and chemocommunica ion, especially ega ding signal
ansduc ion, and he o ganiza ion o he ol ac o y subsys ems ac oss
e olu ion (Bakke and Leinde s-Zu all, 2016; B ennan, 2018).
In his pa , we will b ie ly explain he di e en ypes o
chemosenso y ecep o s ound in he MOS and VNS. In sec ion
‘1.1.1.3 E olu ion o he ol ac o y sys em in e eb a es’, he
chemosenso y ecep o s will be explained in an e olu iona y con ex ,
and in sec ion ‘2.1.2 Senso y ecep o s o he ome onasal o gan’
he ome onasal ecep o s will be explained in de ail.
Ac oss he mammalian chemosenso y sys em, i e ypes o
GENERAL INTRODUCTION
67
chemosenso y G-p o ein coupled ecep o s (GPCRs) ha e been
iden i ied so a : ol ac o y ecep o s (ORs) and ace amine associa ed
ecep o s (TAARs) exp essed in he MOE; and ome onasal ecep o s
(V1Rs and V2Rs) and o myl pep ide ecep o s (FPRs), in he VNO.
Addi ionally, a subse o VSNs in he VNO coexp esses V2R and H2-
M , a sub amily o nine nonclassical class I majo his ocompa ibili y
complex genes.
Ol ac o y ecep o s (ORs) (Buck and Axel, 1991). They a e he
la ges gene amily in mammals, ep esen ing he 2-5% o he o al
genome (Ba nes e al., 2020), and a e loca ed in OSNs in he MOE.
Each ma u e OSN exp esses only one OR –one ecep o -one neu on–
ollowing a monoallelic ashion (Chess e al., 1994). Each OSN is able
o espond o a high di e si y o odo an s; and con e sely, each
odo an can ac i a e OSNs exp essing di e en ecep o s (Malnic e
al., 1999). This combina o ial code explains why we a e able o de ec
mo e han one illion o ol ac o y s imuli (Bushdid e al., 2014) wi h
jus a epe oi e o se e al hund eds o ORs.
T ace amine-associa ed ecep o s (TAARs) (Libe les and Buck,
2006). They ha e a isen as a second ype o chemo ecep o s loca ed in
he MOE. This amily is exp essed in a small subpopula ion o OSNs
and despi e hey do no co-exp ess wi h ORs (S owe s and Logan,
2010), bo h sha e some ea u es such as monoallelic exp ession and
he one-neu on-one- ecep o ule (Dewan e al., 2021). TAARs
ep esen only he 1% (15 genes in mice and 6 in humans (Lindemann
e al., 2005)) o all ol ac o y ecep o s (including ORs and TAARSs),
bu hei e olu iona y main enance poin s o hei undamen al ole in
ol ac o y unc ion (Dewan e al., 2018). Despi e hei p ecise
unc ionali y is ye o be de e mined, hese ecep o s a e ac i a ed by
chemical cues ha show he hallma ks o phe omones (Libe les and
Buck 2006).
Some s udies sugges ed ha he MOS may be composed o wo
unc ional modules: one o he disc imina ion and associa i e
lea ning o complex odou s and he o he o inna e odou esponses
media ed by phe omones (Kobayakawa e al., 2007). La e s udies
sugges ed ha ORs and TAARs could be he ecep o s specialized in

PAULA RODRÍGUEZ VILLAMAYOR
68
he de ec ion o lea ned s inna e esponses, espec i ely (Johnson e
al., 2012). Despi e we know now ha phe omone esponses depend on
con ex and he e o e he e ms ‘inna e’ ‘ha dwi ed’ and ‘s e o yped’
a e no ully co ec (Sa ai a e al., 2016; Dewan e al., 2021), he
in ol emen o TAARs in he de ec ion o speci ic socio-
en i onmen al cues makes hem po en ial candida es o phe omone
de ec ion in he MOS (Libe les, 2009).
The chemo ecep o s o he VNO a e explained in de ail in sec ion
‘2.1.2 Senso y ecep o s o he ome onasal o gan’. B ie ly, he e a e
wo main ypes o ome onasal ecep o s –V1R (Dulac and Axel,
1995) and V2R (Ryba and Ti indelli, 1997)–, which g ea ly a y
ac oss e olu ion, possibly o ace adap a ion o a con inuous changing
en i onmen . Addi ionally, a second ype o ome onasal ecep o s –
o myl pep ide ecep o s (FPRs)– we e iden i ied (Ri iè e e al., 2009;
Libe les e al., 2009) and appea o be exclusi e o he oden lineage.
Finally, a sub amily o nine nonclassical class I majo
his ocompa ibili y complex genes, called H2-M we e iden i ied as
ome onasal ecep o s ha ac join ly wi h V2R (Ishii e al., 2003).
V1R and V2R ecep o s a e di e en ially exp essed in wo VSN
popula ions ha also exp ess he G-p o ein alpha-subuni s: Gαi2 o
Gαo, espec i ely (Jia and Halpe n 1996). In con as , FPRs co-
exp ess wi h bo h Gαi2 and Gαo – hough each indi idual ecep o
only exp esses one G-p o ein– (Libe les e al., 2009; Ri ie e e al.,
2009) (Figu e 4).
GENERAL INTRODUCTION
69
Figu e 4. Schema ic diag am illus a ing he molecula ly dis inc apical (o ange)
and basal (g een) laye s o he ome onasal senso y neu oepi helium –based on
mice exp ession–. The h ee main ome onasal ecep o s (V1Rs, V2Rs and FPRs) as
well as hei associa ed G p o ein coupled ecep o s (GPCR) a e shown. Modi ied
om Mi as-Po ugal, 2006.
1.1.1.3 E olu ion o he ol ac o y sys em in e eb a es
Ol ac ion is usually e e ed as one o he mos
'p imi i e' sense in o ganisms – e en bac e ia a e p o ided wi h a
chemosenso y sys em able o de ec chemical molecules om he
en i onmen . Th oughou e olu ion, di e en species e ol ed
di e en ecep o s, s uc u es and o gans o be able o adap o a
con inuously changing en i onmen and ensu e species su i al. In
Figu e 5 and Table 1 we b ie ly summa ize he dis inc ypes o
ecep o s and ol ac o y sys ems ha e eb a es ha e e ol ed
h oughou e olu ion.
Figu e 5. Ve eb a e phylogeny, showing he ol ac o y sys ems. MOS: main
ol ac o y sys em. VNS: ome onasal sys em. GG: g ünebe g ganglion. MO: mase a
o gan.
PAULA RODRÍGUEZ VILLAMAYOR
70
Table 1. Ol ac o y sys ems and ecep o s o ep esen a i e species
om he di e en Ve eb a e Supe classes. * amphibian V2Rs, 448 a e
pseudogenes –non- unc ional genes–. ** V1Rs and V2Rs epe oi es g ea ly di e
ac oss species in mammals (see sec ion ‘2.1.2 Senso y ecep o s o he
ome onasal o gan’and Nei e al., 2008 o de ails).
Phylogene ic
Supe class
MOS
VNS
ORs
TAA
Rs
V1Rs
V2Rs
Re e .
Agna a
(lamp ey)
Yes
VNS
p ecu
so
~ 40
~ 28
~ 4
~ 2
Beauséjo
u e al.,
2022;
Kowa sc
hew and
Ko schin
g, 2022)
Gna hos oma
a (sha k)
One unique
‘accesso y
sys em’
~ 8
~ 5
~ 6
~ 35
(Sha ma
e al.,
2019)
Os eich hyes
(bony ishes)
One sys em
~ 160
~
112
~ 6
(ORAs)
~ 60
(Ol C)
Nei e
al.,
2018;
Villamay
o e al.,
2021b)
Amphibia
Yes
Yes
> 1000
7
22
697*
Nei e
al.,
2008)
Rep ilia
Yes
Yes
~ 360-
1000
~ 2
~ 2-4
~ 116-
430
Hogan e
al.,
2021)
A es
Yes
No
> 200
~ 3
(2)
0
0
Shi and
Zhang,
2007;
Vandewe
ge e
al., 2016
Mammalia
Yes
Yes
~ 800-
1200
~20
~0-
200**
~0-
200**
Nei e
al.,
2008)
The VNS was la gely conside ed as an adap a ion o e es ial
li e, bu his iew is now ques ioned since VRs and e en a so o
‘VNS’ appea ed be o e he wa e - o-land adap a ion (i.e. lamp ey,
lung ish) (Ubeda-Abañón e al., 2011). A de ailed desc ip ion o he
e olu ion o he ol ac o y sys em in e eb a es is beyond he scope o
his in oduc ion bu use ul e e ences a e added o Table 1.
GENERAL INTRODUCTION
71
Mammals can be classi ied depending on hei abili y o smell in
mic osma ic (well-de eloped sense o smell (i.e. oden s, ca ni o es)),
mic osma ic ( eebly de eloped sense o smell (i.e. p ima es)) o
anosma ic (lacking he sense o smell (i.e. many aqua ic mammals
(Smi h and Bha naga , 2004). In e es ial mammals, he e a e wo
majo and well-cha ac e ized nasal chemosenso y subsys ems –MOS
and VNS–, con aining a high di e si y o gene amilies ha encode
MOE and VNO ecep o s. An addi ional le el o complexi y should
be added o he o ganiza ion o he sense o smell in mammals: 1)
he e a e wo o he s uc u es in he NC which we e in ol ed in
de ec ing odou s, namely he Mase a o sep al o gan (SO) and he
G ünebe g ganglion (GG), mainly s udied in oden s (Salaza and
Sánchez-Quin ei o, 2009; Munge e al., 2009). Since hei axons
p ojec o speci ic a eas o he MOB –SO o he en omedial aspec
o he MOB and GG o he caudal MOB, named he necklace
glome uli (Figu e 1)–, some au ho s conside ed hem as pa o he
MOS (Zimme man and Munge , 2021). E en hough hei unc ional
con ibu ions a e s ill unknown, he e is e idence ha bo h con ibu e
o phe omone de ec ion (Ma e al., 2003; Roppolo e al., 2006;
Ti indelli e al., 2009); and 2) he MOE is u he subdi ided in
a ious ‘subsys ems’ which con ain di e en ol ac o y senso y neu on
subpopula ions, each wi h hei own ecep o s and p ojec ion-a eas.
B ie ly, apa om he p e iously men ioned OSNs ha exp ess ORs
and TAARs (called canonical OSNs), o he non-canonical OSNs
subpopula ions a e ound in he MOE: OR37 neu ons, GC-D+ OSNs
and T pc2+ OSNs (mo e ex ensi e in o ma ion can be ound in Bade
e al., 2012; Klein e al., 2015 o OR37 ecep o s; G ee e al., 2016
o GC-D+ OSNs; Omu a and Mombae s (2014) o T pc2+ OSNs).
Al oge he , he mammalian sense o smell con ains a di e se
a ay o subsys ems which a e cha ac e ized based on he s imuli hey
espond o, he cell ypes and ecep o s hey hold, hei signal
ansduc ion mechanisms and he connec ions hey es ablish o
speci ic a eas o he b ain. This mul i unc ional ask may allow o
pa allel p ocessing o chemical cues, in which each subsys em
p obably se e a speci ic unc ion he e o e con eying di e en ypes
o in o ma ion om a single chemical cue.
PAULA RODRÍGUEZ VILLAMAYOR
78
in e ac ion, and simple compa isons o he V1R gene epe oi e, e en
be ween closely ela ed species, may lead o unexpec ed and
ascina ing indings, which ul ima ely would b ing us one s ep close
o he unde s anding o social communica ion in mammals.
2.1.2.2 Vome onasal ype-2 ecep o s
V2R ecep o s also belong o he se en- ansmemb ane
domain GPCR amily, bu hey sha e no sequence homology wi h
V1Rs. Thus, V1Rs and V2Rs a e gene ally conside ed o ha e
e ol ed independen ly. V2R genes ha e been iden i ied in e eb a es,
and a e supposed o be es ic ed, simila o V1Rs, o his subphylum
(Rod iguez, 2008). In con as o V1Rs, he numbe o unc ional
V2Rs is no co ela ed wi h VNO complexi y (G us e al., 2007). The
la ges V2R gene epe oi e is ound in amphibians (see Table 1). In
mammals such as mice and a s, 61 (upda ed o 122 by Ishii and
Mombae s, 2009) and 57 V2R genes wi h in ac open eading ames
ha e been epo ed, espec i ely (Yang e al., 2005); he e o e, V2R
epe oi e in oden s appea s o be smalle han he V1Rs epe oi e. In
o he species such as opossum and pla ypus, 90 and 15 V2Rs ha e
been ound, espec i ely. Un il ecen ly i was hough ha
a yc iodac ila did no posses V2Rs, bu a ew ac i e V2R genes we e
ound encoded in he genome o ca le, goa s, sheep and pigs (Kondoh
e al., 2022). None heless, no unc ional V2R genes ha e been ound
in dogs, chimpanzee, macaque and humans (G us e al., 2007; Young
e al., 2007) (Table 2). V2Rs also ha e a la ge epe oi e o
pseudogenes in mice (148) and a s (111) (Yang e al., 2005). Pla ypus
and opossum ha e 57 (G us e al., 2007) and 79 (Young e al., 2007),
espec i ely, and be ween 9-20 we e ound in cow, dogs and humans
(Young e al., 2007) (Table 2).
A molecula le el, V2Rs di e om V1Rs by he p esence o
in ons and he long N- e minal ex acellula egion, which is encoded
by i e exons –in con as wi h V1Rs ha only hold one exon– and
likely e lec s he ligand-binding si e (F ancia e al., 2015).
Despi e he as majo i y o V2R genes we e only exp essed in
he VNO, Iba a-So ia e al., (2014) ound exp ession o one V2R in

GENERAL INTRODUCTION
79
he MOE, Vmn2 29, sugges ing p e ious un ecognized mechanisms
o phe omone de ec ion in he MOE.
Func ionally, V2Rs ha e p o en o de ec high molecula weigh
non- ola ile p o eins and pep ides (Robe s e al., 2010). In pa icula ,
he po en ial ligands o V2Rs include pep ide phe omones such as
exoc ine gland-sec e ing pep ide 1 (ESP1) (Kimo o e al., 2005),
mouse u ina y p o eins (MUPs) (Chame o e al., 2007) and MHC
pep ides (Leinde s-Zu all e al., 2004). La e s udies sugges ed ha
V2Rs may be de o ed o encode in o ma ion abou he iden i y o
emi e s (i.e. gende iden i y, he e ospeci c cues such as p eda o s
non-p eda o ), wi h clea beha iou al signi icance (Isogai e al., 2011).
A gene exp ession le els, bo h V1Rs and V2Rs showed a ia ion
ac oss di e en mice s ains. Al hough hese di e ences a e no
equi alen o gene ic di e ences a he nucleo ide le el, hey a e
none heless impo an ai s ha can p o ide in o ma ion abou
e olu iona y di e gence among mice s ains (Duyck e al., 2017).
Table 2. Numbe o V1Rs and V2Rs in di e en mammal species. Numbe s in
pa en hesis indica e pseudogenes. Da a ob ained om Nei e al., 2008; Kondoh e
al., 2022.
Species
V1Rs
V2Rs
Mice
187 (121)
121 (158)
Ra s
106 (66)
79 (142)
Ca le
24 (8)
2 (11)
Goa s
9 (2)
1 (2)
Sheep
16 (4)
1 (2)
Pigs
16 (9)
10 (3)
Dogs
8 (33)
0 (9)
Chimpanzee
0 (116)
0 (17)
Humans
5 (115)
0 (20)
2.1.2.3 Fo myl-pep ide ecep o s
FPRs a e se en ansmemb ane domain GPCR belonging
o he hodopsin-like supe amily and we e i s desc ibed in immune
cells (Le e al., 2002). In mammals, p 1 and p 2 a e belie ed o play
an impo an ole in inna e immune esponse and a e exp essed by
PAULA RODRÍGUEZ VILLAMAYOR
80
immune cells such as g anulocy es and monocy es (He e al., 2013;
Ackels e al., 2014). The e olu iona y his o y o his immune amily is
highly complex (Sil a e al., 2020); while a single copy o p 1 has
been desc ibed in mammals, p 2 has unde gone dynamic episodes o
duplica ion e en s widely a ying among species, ha poin s owa ds
he equen neo unc ionaliza ion o his sub amily. In oden s, a wo
di e en imes, an p coding sequence was in eg a ed in a VR gene
clus e , acqui ing he egula o y elemen s o VRs and leading o hei
exp ession in he VNO. In o he wo ds, wo gene shu ling e en s
o ced an immune pa hogen senso o ansi ion in o an ol ac o y
chemo ecep o (Die schi e al., 2017; Boilla e al., 2021).
Consequen ly, in Roden ia, a hi d class o VNO ecep o s was ound
exclusi ely exp essed in ome onasal issue and ep esen s an
expansion o he immune gene p 2. VNO FPRs include 5 ou o he 7
membe s o he o myl pep ide ecep o amily ( p s) -Fp - s1 (also
known as Fp 3), Fp - s3, Fp - s4, Fp - s6 and Fp - s7, (Ri iè e e al.,
2009; Libe les e al., 2009). The e is no e idence o co-exp ession o
FPRs wi h V1Rs o V2Rs, and i has been a gued ha speci ic subse s
o VSNs exclusi ely exp ess FPRs in a monogenic manne (Libe les
e al., 2009; Ri ie e e al., 2009).
The ligand ypes ha bind VNO FPRs emain la gely unknown
bu hey migh play a ole in VNO pa hogen sensing (Bu e e al.,
2012) and in media ing sickness conspeci ic a oidance (Boilla e al.,
2015;Ti indelli, 2021; Die schi e al., 2017). In ac , Bu e e al., (2019)
ecen ly ound ha he non- ola ile bac e ial Mg B pep ide ac i a es
an FPR sub ype (Fp 3) which is exp essed in a subse o ome onasal
senso y neu ons and which d i es a oidance beha iou (Bu e e al.,
2019). Al hough his ep esen s he only unc ional s udy o any FPR
in he VNO o da e, FPRs cons i u e p omising candida es o he
de ec ion o sickness- ela ed cues. The e o e, VNO FPRs may ha e an
impo an unc ion a con olling he i ulence o in ec ions, placing
his senso y o gan as a po en ial in e ace be ween he ex e nal wo ld
and he immune sys em (Bu e e al., 2012, 2015, 2019).
Finally, al hough mos s udies on FPRs ha e been es ic ed o
oden s and p ima es (Die schi e al., 2017; Yang and Shi, 2010),
ecen inding showed p 2 pa alogs in Chi op e a and Pe issodac yla,
GENERAL INTRODUCTION
81
hough hei pu a i e exp ession in he VNO emains unknown (Sil a
e al., 2020). These esul s sugges ha p 2 expansions may ha e no
been es ic ed o Roden ia, and u he s udies using high quali y
genome assembles will help deciphe ing he e olu iona y his o y o
his unusual genomic e en .
2.1.2.4 H2-M ecep o s
In mice, nine genes belonging o he amily o non-
classical class I majo his ocompa ibili y complex (MHC) genes (H2-
M ), we e ound di e en ially exp essed in subse s o basal VNO
neu ons (Ishii e al., 2003; Locon o e al., 2003; Ishii and Mombae s
2008; Leinde s-Zu all e al., 2009). These ome onasal-speci ic genes
we e ound co-exp essed wi h V2R (Ishii e al., 2003). To ou
knowledge, no in o ma ion o H2-M genes has been epo ed in o he
mammal species. Func ionally, al hough dispensable o igge
physiological esponses in he VNO neu ons, H2-M genes con ibu e
o ul asensi i e de ec ion o phe omones by a subse o VSNs
(Leinde s-Zu all e al., 2014). Howe e , hei speci ic ole in he VNO
needs u he in es iga ion and in ac whe he hese molecules should
be conside ed as phe omone ecep o s emains con o e sial (Ishii and
Mombae s 2008; F ancia e al., 2014).
2.1.3 Signal ansduc ion mechanisms
VNO signal ansduc ion is complex and no ye ully
unde s ood. Mos s udies ha e ocused on he ansduc ion
mechanisms o V1Rs and V2Rs ecep o s, bu new s udies a e now
ques ioning he adi ional iew in which speci ic G p o eins -Gαi2
and Gαo- we e hough o di ec ly co ela e wi h V1R and V2R
exp ession.
In gene al, in he VNO he signal is ini ia ed by a phe omone-like
ligand binding o one o wo ypes o VNS-speci ic GPCRs, V1Rs o
V2Rs. This binding modi ies he ecep o con o ma ion, causing he
elease o he βγ complex o he he e o ime ic G p o eins Gαi2 and
Gαo, o V1Rs and V2Rs, espec i ely. This ac i a es phospholipase
PAULA RODRÍGUEZ VILLAMAYOR
82
C (PLC), which will inc ease le els o wo seconda y messenge s,
diacylglyce ol (DAG) and inosi ol 1,4,5- iphospha e (IP3) (Minke
and Cook, 2002). Bo h DAG and IP3 inc ease he in acellula
calcium le el -DAG by ac i a ing he T pc2 channel allowing a
depola izing in lux o Na+ and Ca2+ and IP3 by allowing he elease o
in acellula calcium s o es- (Figu e 7). T pc2 appea s o be he main
ansduc ion channel in V1R-exp essing neu ons bu i s ole in V2R-
exp essing neu ons is less clea (F ancia e al., 2014). While some o
he signal ansduc ion molecules a e common o o he signal
ansduc ion pa hways, h ee ypes o VNS genes (V1Rs, V2Rs, and
pc2) a e known o unc ion in he VNS-speci ic chemo ecep ion.
Figu e 7. T ansduc ion mechanisms o V1Rs and V2Rs. PLC: phospholipase C;
DAG: diacylglyce ol; IP3: inosi ol 1,4,5- iphospha e. Modi ied om Ti indelli e
al., 2009.
GENERAL INTRODUCTION
83
In some o de s such as Roden ia and Didelphimo phia, VNO
show seg ega ed exp ession o speci ic G-p o ein coupled ecep o s:
Gαo, exp essed a he basal VNO neu oepi helium; and Gαi2,
exp essed a he apical pa , (Halpe n e al., 1998). Howe e , no all
mammalian VNOs ha e his seg ega ed pa e n (i.e. in ox Gia2 and
Gαo we e ound in e mingled in he VNO neu oepi helium (O iz-Leal
e al., 2020). Fu he mo e, as p e iously men ioned, in some mammal
species in ac V2R genes ha e comple ely degene a ed (see sec ion
‘2.1.2.2 Vome onasal ype-2 ecep o s’), and consequen ly, hey
usually lack VNO Gαo exp ession. Acco dingly, wo ypes o VNO G
exp ession pa e ns can be de ined: 1) dicho omous exp ession o Gαo
and Gαi2 in species wi h in ac V1Rs and V2Rs such as mouse, a ,
opossum (Jia and Halpe n, 1996) and 2) exclusi e exp ession o Gαi2
in species which lack V2Rs such as goa (Takigami e al., 2000),
sheep (Salaza e al., 2007), dog (Salaza e al., 2013) and ca (Salaza
and Sanchez-Quin ei o, 2011). No wi hs anding, ecen s udies ha e
added an addi ional le el o complexi y o his o ganiza ion: in ox,
Gαo exp ession has been ound in he VNO bu no V2Rs (in ac o
pseudogenes) ha e been iden i ied in he ox genome (O iz-Leal e
al., 2020). The au ho s sugges ed ha his would be he i s case in
mammals in which no V2Rs pseudogenes exis and poin owa ds low
quali y ox genome assemblies, mo e han a eal scien i ic ac .
In any case, VSNs p ojec hei axons o he AOB glome ula
laye (see Figu e 8 and sec ion ‘2.2.2 Func ional ci cui y o he
AOB’). Species ha exp ess bo h ypes o G p o eins –Gαi2 and Gαo–
show an exp ession pa e n es ic i e o he an e io (Gαi2
exp ession) and pos e io (Gαo exp ession) pa s o he AOB
(glome ula and ne e laye s), espec i ely. I he species only
exp esses Gαi2, i s exp ession co e s he en i e su ace o he
glome ula -ne e laye s o he AOB. In ox, whe e Gαo exp ession
was ound in he VNO, such exp ession was lacking a he AOB le el,
sugges ing ha Gαo-media ed p ojec ions om he VNO could be
eaching he MOB a he han he AOB (O iz-Leal e al., 2022).
Addi ionally, esul s om ou g oup (unpublished) indica e ha abbi
e us show s ong pa e ns o Gαi2 and Gαo in bo h he VNO and he
AOB, bu he exp ession o V1Rs and V2Rs –analyzed by RNAseq–

PAULA RODRÍGUEZ VILLAMAYOR
84
is almos absen .
Finally, VNO FPRs ha e also p o ed o co-exp ess wi h Gαi2 and
Gαo. Speci ically, Fp - s1 coexp esses Gαo and all he es (Fp -
s3,4,6,7) coexp esses Gαi2 (Libe les e al., 2009).
In summa y, hese esul s show ha al hough Gαi2 and Gαo
usually show a co ela ed exp ession wi h V1Rs and V2Rs,
espec i ely, hey do no exclusi ely do; and ha he in e play
be ween he VNS and he MOS could be al eady es ablished a he
i s s ep o he senso y p ocessing. Though i seems clea ha all o
he men ioned elemen s (i.e. V1Rs, V2Rs, G p o eins, T pc2, e c.)
con ibu e o he signal ansduc ion machine y, he logic behind hei
exp ession appea s o be highly complex and o he ye unknown
specie-speci ic G p o eins and ome onasal ecep o s may also be
implica ed in signal de ec ion and ansduc ion.
2.2 Accesso y ol ac o y bulb
VSNs axon bundles o m he ome onasal ne e, which en e he
b ain h ough he c ib i o m pla e and a ge a specialized egion
usually loca ed a he pos e io do sal pa o he MOB -i seems o be
embedded in i -; he AOB. The AOB is he e o e he i s neu al
in eg a i e cen e o he VNS. Al hough his s uc u e was i s
desc ibed in he la e 19 h cen u y by Gudden (1870) and i was one o
he ea lies co ical a eas s udied by classic neu oana omis , i s
appa en absence in humans oge he wi h i s ex emely high
complexi y has hampe ed i s in es iga ion, especially a cy ological
and neu al ci cui le els. Bo h AOB and MOB sha e some simila i ies
including hei laye ed o ganiza ion, classes o neu onal cell ypes and
unc ional connec i i y (Moh ha d e al., 2018). No e, howe e , ha
bo h s uc u es also show impo an di e ences wi h majo
physiological implica ions and he e o e, ex apola ion o he
s uc u al and unc ional p inciples om he MOB o he AOB should
be aken wi h cau ion (Dulac and Wagne 2006; S owe s and Speh
2015). The nex pa o he in oduc ion will ocus on he main
ea u es o he AOB, pa icula ly in compa ison o hose o he MOB.
GENERAL INTRODUCTION
85
2.2.1 S uc u e o he AOB
The AOB is loca ed a he an e io pa o he encephalon and
caudally o he MOB. Bo h MOB and AOB p esen a compa able
s uc u e in e ms o laye s and cell- ypes. Since he MOB has been
mo e b oadly s udied han he AOB, he laye ing and cell- ype
nomencla u e o he AOB ha e been an ex ension o ha p e iously
desc ibed in he MOB. Though his seems a p ac ical and sui able
app oach, i has some imes led o misunde s anding and lack o
accu acy o some AOB speci ic ea u es.
In gene al, bo h MOB and AOB can be unde s ood as elonga ed
onions made up o di e en laye s ha a e o ganized in concen ic
ci cles (Olude e al., 2014). T adi ionally, he laye s ha o m he
MOB and, by ex ension, also he AOB a e – om he ou side o he
inside o he bulb– 1) he ne e laye (VNL), 2) he glome ula laye
(GlL), 3) he ex e nal plexi o m laye (EPL), 4) he mi al cell laye ,
5) he in e nal plexi o m laye (IPL), and 6) he g anula laye (G L)
(Allison, 1953) (Figu e 8). The la e can be spli in o ex e nal and
in e nal g anula laye .
PAULA RODRÍGUEZ VILLAMAYOR
86
Figu e 8. Schema ic ep esen a ion o he VNS pa hway om he en i onmen
o he VNO and o he AOB, showing he di e en AOB laye s. The laye s o he
AOB and he di e en ypes o neu ons a e ep esen ed. VNO: ome onasal o gan;
AOB: accesso y ol ac o y bulb; ECL: ex e nal cellula laye ; ICL: in e nal cellula
laye ; VNE: ome onasal neu oepi helium; VNL: ome onasal ne e laye ; GlL:
ome onasal glome ula laye ; EPL: ex e nal plexi o m laye ; MCL: mi al/cell
laye ; IPL: in e nal plexi o m laye ; eG L: ex e nal g anula cell laye ; iG L:
in e nal g anula laye ; VSNs: ome onasal senso y neu ons. Modi ied om Galliano
Lab a h ps://gallianolab.o g/.
Rega ding cell ypes, he AOB is p o ided wi h exci a o y
p incipal cells ( he MOB homologous a e named as mi al/ u ed cells)
ha ecei e inpu om OSNs and send ou pu o highe b ain cen e s.
They a e ound in he MCL (also in he EPL in some cases) and can
be subdi ided acco ding o hei shape (i.e. a AOB: la ge p incipal
cells, ound p ojec ing cells and u ed cells (La i a-Sahd, 2008).
The e a e also inhibi o y in e neu ons (IN): pe iglome ula cells in he
GlL and g anule cells in he G L.
Due o he high complexi y o he sys em, e en hough ad ances
ha e been made ecen ly in e ms o cell- ypes and specie-speci ic
ea u es, he cu en li e a u e s ill main ain he adi ional
nomencla u e as a e e ence, and e y ew s udies ace he a iabili y
and complexi y o he bulbs. Impo an ly, La i a-Sahd (2008) on his
s udy o he a AOB poin ed owa ds he need o di e en ia ing he
AOB and MOB nomencla u es and p oposed a new nomencla u e o
he AOB. His eam p oposed wo new laye s: he ex e nal cellula
laye (ECL), con aining he plexi o m laye s (EPL and IPL), he
MCL, and he ex e nal pa o he G L; and he in e nal cellula laye
(ICL), eplacing he in e nal G L (Figu e 8).
The AOB appea s o e ain he s uc u al dicho omy obse ed in
he VNO: he wo main subse s o ei he Gαi2 o Gαo exp essing
neu ons a ge wo seg ega ed egions in he VNL and GlL along i s
os o-caudal axis. Gαi2 posi i e neu ons a e loca ed in he os al
egion o he AOB, whe eas Gαo-posi i e a e exp essed in he
pos e io egion o he AOB. Though his is usually co ela ed wi h
he exp ession o V1Rs and V2Rs, espec i ely (Ma inez Ma cos,
2008), his is no always he case (see sec ion ‘2.1.3 Signal
GENERAL INTRODUCTION
87
ansduc ion mechanisms’ o de ail). Also, axons o FPR- s3-
exp essing VSNs con e ge on o mul iple (~8) glome uli in a spa ially
es ic ed egion o he os al AOB, which does no o e lap wi h
V1Rs and V2Rs exp essing VSNs (Die schi e al., 2013; Moh ha d e
al., 2018).
Phylogene ically, he AOB has been subjec ed o highe species-
speci ic s uc u al and a ia ions han he MOB (Meisami and
Bha naga 1998), especially in e ms o AOB loca ion, shape, size,
cell- ypes speci ici y, mo phological ea u es and de elopmen al
pa e ns. AOB is well de eloped in oden s (Rod iguez e al., 1999),
ma supials (Jia and Halpe n 2004), and p osimians (Skeen and Hall,
1977). In some species such as dogs (Salaza e al., 2013), mink
(Salaza e al., 1998) and some ba s (F ahm and Bha naga , 1980), he
AOB is p esen bu appea s o be poo ly de eloped, and in some
o he s such as A ican elephan (Ngwenya e al., 2011), Wes Indian
mana ee (Mackay-Sim e al., 1985) and human (T o ie e al., 2000),
he AOB is absen o has no ye been iden i ied.
Addi ionally, ega ding sexual dimo phism, p e ious s udies on
adul a s showed di e ences in he AOB olume o males and
emales (Sego ia e al., 1984), as well as in he densi y o new-bo n
cells a he an e io pa o he AOB (Pe e o e al., 2001), which
appea s o be speci ically linked o disc imina ing ep oduc i e- ela ed
chemosenso y s imuli (Dulac and To ello, 2003; Obo i e al., 2009).
2.2.2 Func ional ci cui y o he AOB
VSNs p ojec hei axons di ec ly o he AOB, which is he
ini ial si e o he adul mammalian b ain whe e ome onasal senso y
in o ma ion is i s p ocessed. VSNs exp essing VRs p ojec hei
axons o ac i a e mul iple neu opil sac- illed s uc u es, known as
glome uli which eside in he GlL o he AOB. Glome uli a e igh ly
clus e ed and spa sely su ounded by pe iglome ula IN. Con a y o
he MOB, pe iglome ula cells do no de ine a clea bounda y a ound
each glome uli bu a he es ablish a ine line which sepa a es he GlL
om he MCL (Ti indelli e al., 2009). An impo an di e ence
be ween he AOB and he MOB is ha while each OSNs exp essing
PAULA RODRÍGUEZ VILLAMAYOR
94
3.1.1 A li e ime o neu ogenesis in VSNs
Neu ogenesis ep esen s ano he key weapon in he adul
ne ous sys em’s plas ici y a mou y o dealing wi h a cons an ly
changing wo ld (Lledo e al., 2006). B ie ly, hough neu ogenesis was
la gely linked o emb yonic and ea ly pos na al s ages in e eb a es, i
is nowadays well-known ha newly gene a ed neu ons con inue
h oughou li e in speci ic egions o he ne ous sys em. In pa icula ,
h ee main neu ogenic niches ha e been iden i ied: 1) subg anula
zone –which send neu ons o he den a e gy us o he hippocampus –;
2) sub en icula zone –which send in e neu ons (mos ly g anula bu
also pe iglome ula cells) o he main and accesso y ol ac o y bulb–;
and 3) ol ac o y and ome onasal neu oepi helia –which gene a e new
exci a o y ol ac o y and ome onasal senso y neu ons whose axons
connec o he main and accesso y ol ac o y bulb, espec i ely
(Kageyama e al., 2012; B ann and Fi es ein, 2014). The wo la e
supply new neu ons o he i s le el s uc u es o he ol ac o y sys em
–senso y neu ons and ol ac o y bulbs–. No ably, adul neu ogenesis in
he ol ac o y sys em, and in pa icula in he ol ac o y bulb, is o
pa amoun o senso y disc imina ion, odou -based lea ning, and
ep oduc i e social beha iou s (Pigna elli and Belluzzi, 2010; Lledo
and Valley, 2016). Howe e , while he ole o sub en icula zone
neu ogenesis in lea ning seems clea (Mo eno e al., 2014), he
ol ac o y neu oepi helia emains unde s udied (B ann and Fi es ein,
2014).
VSNs o igina e as s em cells in he ma ginal zones nea he do sal
and en al aspec s o he VNO and hen mig a e ho izon ally along
he basal zone whe e hey di e en ia e in o i s imma u e and hen
ma u e unc ional VNO neu ons. I is commonly known ha VSNs
unde go egene a ion h oughou li e bo h physiologically and a e
inju y (Ma ínez-Ma cos e al., 2005; B ann and Fi es ein, 2014).
In e es ingly, hough VSNs egene a i e and p oli e a i e capaci y
seems o dec ease in na u al aged animals (Mechin e al., 2021;
Po alés e al., 2022), i emains obus in esponse o damage e en
wi h ad anced age (B ann and Fi es ein, 2010). In con as , aged
hippocampus lacks o neu on egene a ion ollowing inju y (She y e

GENERAL INTRODUCTION
95
al., 2010). This places VSNs a he o e on o s em cell he apies,
which may equi e isola ion om an aged sys em, as he onse o mos
neu odegene a i e diseases is du ing la e li e s ages (B ann and
Fi es ein, 2010). The unc ional a ionale o VSNs egene a ion is no
clea , bu i seems o be in luenced by epigene ic e ec s (Xia e al.,
2010).
All in all, VSNs neu ogenesis emains an unde explo ed ye
p omising ield o s udy. Fu u e wo k should aim a unde s anding he
p oli e a i e na u e o hese neu al s em cells, especially in he con ex
o hei he apeu ic implica ions (B ann and Fi es ein, 2010; 2014). O
no e, e en hough OSNs neu ogenesis is beyond he scope o his
in oduc ion (see in de ail in Mackay-Sim e al., 2015), i ollows a
compa able pa e n o ha o VSNs. O e all, despi e OSNs
neu ogenesis has been mo e b oadly s udied han VSNs neu ogenesis,
e en wi h ad ance single-cell echnologies (Hancha e e al., 2015;
Du an e e al., 2020), he egula ion o ol ac o y neu ogenesis is no
comple ely unde s ood in any o he wo sys ems.
3.2 Plas ici y o he AOB
MOB and AOB a e s uc u es ha display an impo an plas ici y
ecei ing ol ac o y axons, newbo n in e neu ons and showing
adap i e changes o he in insic and also he cen i ugal a e en s
neu ons (Diaz e al., 2017; Zhang and Meeks, 2020). One o he main
plas ic mechanisms o hese sys ems a ise om he inco po a ion o
new local GABAe gic inhibi o y neu ons –mos ly g anula bu also
pe iglome ula in e neu ons (Obo i and Pe e o, 2014)– o he exis ing
ci cui s h oughou li e (Obo i e al., 2009; Wu e al., 2020). Howe e ,
less is known abou hei neu ochemical p o ile and unc ional
in eg a ion in o he bulb, especially in he case o AOB, whe e
in o ma ion p ocessing is poo ly unde s ood compa ed wi h he MOB
(Obo i e al., 2009; Zhang and Meeks, 2020).
The ma u a ion and su i al o newbo n in e neu ons in he MOB
depends on senso y inpu s, as shown by ol ac o y en ichmen and
dep i a ion s udies (Roche o e al, 2002; Mandai one al, 2006).
Indeed, 24 h senso y dep i a ion – ia na is occlusion– is su icien o
PAULA RODRÍGUEZ VILLAMAYOR
96
induce unc ional plas ici y in a speci ic sub ype o MOB in e neu ons
(Galliano e al., 2018).
Al hough enewing a a slowe a e (Obo i e al., 2009), newbo n
cells in he AOB a e likely o simila ly con ibu e o VNS unc ion.
Senso y ac i i y induced by chemical s imuli p esen in bodily
sec e ions o u ine inc eases he su i al o newbo n AOB
in e neu ons (Obo i e al., 2009, 2011; Nunez-Pa a e al., 2011).
In e es ingly, exposu e o male-soiled bedding signi ican ly inc eased
he numbe o new neu ons in he AOB o emale mice (Obo i e al.,
2009), poin ing owa ds a key ole o newbo n in e neu ons –and in
pa icula g anule cells– in he egula ion o ep oduc i e and social
beha iou (Pe e o e al., 2014). Subsequen s udies showed ha
ma ing beha iou o igina es long- e m senso y memo y o he
phe omones o he s ud male, and ha his is di ec ly linked o mi al
cells plas ici y and AOB sensi i i y (Gao e al., 2017). Fu he mo e,
male-male social in e ac ion has p o en o d i e AOB inhibi o y
plas ici y in which mi al cell ac i a ion is supp essed by inc eased
exci abili y o newbo n g anule cell (Cansle e al., 2017); his senso y
inhibi ion is c i ical o he egula ion o beha iou al esponses o
social chemosignals (Zuk e al., 2022). The e o e, AOB adul
neu ogenesis and mi al cell s in e neu on communica ion seems o
be egula ed by an expe ience-speci ic mechanism and may be
unc ionally linked o he speci ic ole o he VNS in phe omone-
pe cep ion (Obo i e al., 2009), c i ical o animal physiology and
beha iou (Zhang and Meeks, 2020).
Finally, male phe omones ha e p o en o be in ol ed in
egula ing neu ogenesis, no only in he AOB bu also in he MOB and
hippocampus (Mak e al., 2007). Fu he wo k should app oach he
molecula and ci cui logic behind he AOB and he o he neu ogenic
niches –MOB and hippocampus–, o unde s and how lea ning and
social beha iou s a e impac ed by newbo n neu ons.
4. RABBIT
Rabbi ep esen s a sui able model o chemocommunica ion in
mammals. Since abbi s a e a med o animal p oduc ion and also
GENERAL INTRODUCTION
97
la ely became pe s, ou comes o ou s udies in abbi s will no only
ha e impac in he esea ch communi y bu also se he basis o apply
phe omones a indus ial le el, o enhance ep oduc i e pa ame e s
and animal wel a e.
4.1 Taxonomy
Rabbi s ha e been conside ed oden s un il ea ly 20 h cen u y.
Howe e , con a y o such popula belie , abbi s belong o he O de
Lagomo pha and di e om oden s in se e al ai s ela ed o ee h -
hey ha e an ex a pai o inciso s- and o he diges i e and
ep oduc i e ea u es. Lagomo pha and Roden ia ha e di e ged om
a common ances o called Gli es a ound 64.5 millions o yea s ago
(57.3–73.3) (Rose e al., 2008). B and (1855) al eady conside ed
Lagomo phs as a subo de wi hin oden s, bu i was no un il 1912
when JW Gidley o icially s a ed Lagomo pha as a sepa a e O de .
Lagomo pha is di ided in o 2 amilies comp ising 92 species:
Ochononidae (29 species; pikas) and Lepo idae (63 species; abbi s
and ha es) (Ruedas e al., 2018). The Eu opean abbi (wild o
domes ic abbi ) is he mos well-known species o abbi and he only
one belonging o he genus O yc olagus. I s comple e axonomic
classi ica ion is as ollows (ITIS, 2022):
Domain: Euka yo a
Kingdom: Me azoa
Phylum: Cho da a
Subphylum: Ve eb a a
Class: Mammalia (Linnaeus, 1758)
O de : Lagomo pha (B and , 1855)
Family: Lepo idae (Fische , 1817)
Genus: O yc olagus (Lilijebo g, 1873)
Species: O yc olagus cuniculus (Linnaeus,
1758) – Eu opean Rabbi
4.2 Biology
The Eu opean abbi (O yc olagus cuniculus) is na i e o he
PAULA RODRÍGUEZ VILLAMAYOR
98
Ibe ian Peninsula, wes e n F ance and No hwes A ica bu i was
success ully in oduced in many coun ies ac oss he wo ld since
medie al imes, and i s dis ibu ion is nowadays conside ed
wo ldwide (Cooke, 2018). Thei ea and eye a e well-de eloped
allowing ea ly de ec ion o p eda o s. They also use a a ie y o
ol ac o y signals / phe omones eleased by exoc ine glands (i.e. chin
gland o e i o y ma king and mamma y-gland o nipple-sea ch
beha iou ) o communica e among hemsel es, and conduc
in o ma ion ela ed o ep oduc ion, dominances, ma e nal ca e, e c.
(Melo and González-Ma iscal, 2010). Rabbi s a e well-known o
hei ep oduc i e capaci y. Ges a ion las s o abou 30 days, and
li e s ange be ween 6-10 ki ens / li e . Females a e o induced
o ula ion, which means ha eggs a e only eleased a e copula ion
(Nowak, 1999; Vaughan e al., 2010).
Rabbi s ha e been aised comme cially o mea , u , as pe s
(Va ga, 2014) and also o esea ch pu poses. In his la e , in addi ion
o being a model o chemical communica ion s udies (Schaal e al.,
2003), (see sec ion ‘4.3 Rabbi as a model o chemocommunica ion’
o de ail), hey a e well-known o an ibody p oduc ion and also
became an animal model o s udy human physiology and pa hology
such as human p egnancy, e al de elopmen o os eoa h osis (Banks,
1989; Es e es e al., 2018; Xu e al., 2021).
4.3 Rabbi as a model o chemocommunica ion
To da e, mos VNS s udies ha e been ocused on labo a o y
oden s. Due o he phylogene ic p oximi y be ween Roden ia and
Lagomo pha, s udies ha e been emp ing o w ongly ex apola e
in o ma ion o m mice and a s o abbi s (Salaza and Sanchez-
Quin ei o, 2009). As we s a ed h oughou he in oduc ion, he VNS
g ea ly a ies among species and e en be ween s ains and
indi iduals, in e ms o s uc u e, unc ion, and molecula and
genomic ea u es.
Rabbi s a e conside ed one o he bes models o s udying
chemocommunica ion in mammals (Schneide e al., 2018). This
species emains as he only mammal species in which a mamma y
GENERAL INTRODUCTION
99
phe omone, he 2MB2, has been ully cha ac e ised (Schaal e al.,
2003). MP is eleased by lac a ing emales and igge s he nipple-
sucking e lex in abbi neona es. Though p e ious s udies poin ed o
he MOS as MP chemodec o (Cha a e al., 2013), ecep o s in ol ed
in i s de ec ion ha e s ill no been iden i ied. Addi ionally, some
beha iou al s udies ha e also poin ed owa ds he impo ance o
phe omone communica ion in abbi s (i.e. he abbi ‘male e ec ’
poin s o an imp o emen o doe ep oduc i e pe o mance (El-Azzazi
e al., 2017).
The VNS anks as he main phe omone de ec o sys em in
mammals. Howe e , i is a pa adox ha abbi s –conside ed a model
o chemocommunica ion– lack o comp ehensi e VNS s udies.
Speci ically, ana omical and physiological s udies on he abbi VNO
and AOB ha e been sca ce in he li e a u e (see in oduc ions o
chap e s I and II o mo e de ails). Also, abbi VNO has no been
included in mos VNO phylogene ic s udies and no unc ional
genomic s udies ha e been app oached o assess gene exp ession (see
in oduc ion o chap e III). The e o e, beha iou al s udies lack o
in eg a i e app oaches which conside he s uc u al, unc ional and
molecula a ionale o phe omone-communica ion. Rabbi -speci ic
new –OMIC echnological s udies a he le el o he VNS a e u gen ly
needed, o example o deciphe he abbi VNO VR epe oi e as well
as o de ine i s gene exp ession pa e ns.
Fa med abbi s a ise as a sui able model o s udy he s uc u e and
unc ion o he VNS as well as o pe o m phe omone-like exposu e
ials. On he one hand, VNS s udies in a mo e ‘na u al’ en i onmen
(ou side lab condi ions) will p o ide a be e pic u e o how his
senso y sys em in eg a es ex e nal cues and in e nal s a es o ins uc
beha iou . On he o he hand, esul s ob ained could be di ec ly
ansla ed in o indus y. Finally, abbi s a e also a con enien species
o wo k wi h because o i s small size, easy handling, sho
ep oduc i e cycle (1 mon h), and high e ili y and p oli icacy.
4.4 Cunicul u e
Rabbi a ming o cunicul u e is he ag icul u al p ac ice o

PAULA RODRÍGUEZ VILLAMAYOR
100
b eeding abbi s as li es ock, mainly o p oduce mea o human
consump ion. Rabbi mea is highly ecommended, no only o i s
low a con en –simila ly o o he whi e mea s– bu also o i s
ichness in highly-quali y p o eins, omega-3 a y acids, i amin B12
and mine als like calcium and po assium (Culle e and Zo e, 2018).
4.4.1 Cu en s a us o cunicul u e
The Eu opean Union (EU) is he second la ges p oduce o
abbi mea wo ldwide, a e China (EFSA, 2019). Cunicul u e wi hin
he EU is mainly concen a ed in h ee coun ies, ep esen ing he 83%
o he o al EU p oduc ion: Spain (48.5 million o animals/yea ),
F ance (29 million) and I aly (24.5 million) (Eu opean Commission,
2017). The e o e, Spain is conside ed an indus ial e e ence in abbi
mea p oduc ion h oughou he Eu opean e i o y. Speci ically, ou
coun y gene a es mo e han 50,000 ons o abbi mea annually.
Wi hin he Spanish e i o y, Galicia occupies he second posi ion in
he anking, jus a e Ca aluña, wi h a p oduc ion o almos 11,500
ons pe yea –da a om 2018; he e is s ill no a ailable da a o
Galicia om 2019 onwa ds– (MAPAMA, 2021a; 2021b). In
pa icula , he company COGAL (Rodei o, Pon e ed a), which has
been ac i ely collabo a ing wi h his PhD p ojec , is esponsible o
mo e han 50% o he abbi mea p oduc ion in Galicia.
Wo ldwide, om 2010 o 2016 abbi mea ma ke inc eased by
1.16%, om 1,224,186 o 1,428,085 ons, wi h China as he la ges
abbi mea p oduce (Li e al., 2018). Up o 2021, mea ma ke has
con inued expanding modes ly, wi h China ollowed by No h Co ea
and Egyp as he main consuming coun ies (IndexBox, 2021).
Cunicul u e has a key ole on di e si ica ion o he mea
p oduc ion sec o in an inc easingly demanding socie y om he
ood poin o iew. Acco dingly, new a ming s a egies as well as
na ional and in e na ional egula ions on animal wel a e, o ganic
a ming and clima e change a e u gen ly needed o ensu e he
sus ainabili y o he sec o , speci ically in he EU (Culle e and Zo e,
2018). In his PhD hesis, we a e se ing he baseline o he
applica ion o phe omones in cunicul u e as a way o educing he
GENERAL INTRODUCTION
101
use o injec able ho mones, hus c ea ing a mo e ‘na u al image’ o
he sec o . In a b oade scena io, phe omones migh con ibu e o
enhance animal p oduc ion and wel a e, co e ing bo h p oduce s’
and consume s’ expec a ions.
4.4.2 Rep oduc i e pe o mance in abbi a ms
Since he end o he las cen u y, he a i icial insemina ion
(AI) has been es ablished as he mos commonly used ep oduc i e
sys em in abbi a ms. Female abbi s a e induced o ula ing animals
and lack o a de ined and egula es ous cycle (A ias-Ál a ez e al.,
2010). The e o e, o pe o m AI animals need o be synch onized in
hei maximum ecep i i y s age. Cu en ly, his is induced by
ho mone ea men s o GnRH and eCG (equine cho ionic
gonado opin). Rega ding GnRH, ad ances ha e been made and
nowadays his ho mone is included in he insemina ion s aw, hus
educing animal handling (Vega e al., 2012). As o eCG, despi e i
can be adminis a ed in se e al consecu i e cycles wi hou ha ing
unwan ed e ec s (Mae en e al., 1995), i implies animal handling
and g ea e wo k o ce. Addi ionally, despi e ho mones a e applied o
mo he s and no en e in he ood chain, he e has been an impo an
discussion abou he possibili y o eplacing ho mones wi h di e en
bio-s imula ion me hods o p ese ing he ‘na u al image’ o abbi
mea .
4.4.3 Bios imula ion me hods
Bios imula ion is an animal managemen p ac ice which
helps imp o ing ep oduc i e e iciency by modula ing animal’s
in e ac ion wi h ex e nal senso y s imuli ( isual, ol ac o y,
phe omone, ac ile, audi o y, social and nu i ional cues, among
o he s). In mos cases i allows indi iduals o de elop hei own
na u al beha iou , also con ibu ing o he imp o emen o animal
wel a e (Theau-Clémen e al., 1998). In abbi a ms, di e en
bios imula ion me hods a e usually employed –mos ly in
conjunc ion– o imp o e animal p oduc ion: mo he -li e sepa a ion
(Ga cia‐Dalmán and González‐Ma iscal, 2012), eeding con ol
PAULA RODRÍGUEZ VILLAMAYOR
102
(Quin ela e al., 2001), pho ope iod p og ams (Mousa-Balabel,
2011), animal manipula ion and, he called ‘male e ec ’ (Theau-
Clemen , 2008). Addi ionally, a ious bios imula ion me hods a e
gene ally used in conjunc ion wi h ho mone ea men in doe abbi
a ms o ensu e ep oduc i e e iciency (Keeling e al., 2019).
4.4.3.1 Bios imula ion me hods based on phe omone
communica ion
The mos powe ul me hod o bios imula ion is p obably
ha which uses chemical o phe omone communica ion as a sou ce o
senso y s imuli. In ac , he e ms ‘phe omone communica ion’ and
‘bios imula ion’ ha e been w ongly in e changed by he li e a u e
(Rekwo e al., 2001; Ke ke a e al., 2017). We should hen cla i y ha
phe omone communica ion is used as one ype o bios imula ion, and
elies on chemosenso y cues o modula e indi idual beha iou ,
usually o enhance hei ep oduc i e pe o mance.
One example o bios imula ion me hod based on phe omone
communica ion –p obably in conjunc ion wi h o he isual and
audi o y cues– is he commonly called ‘male e ec ’, in which
emales exposed o sexually ac i e males igge ac i a ion o
lu einizing ho mone (LH) sec e ion and synch onized o ula ion
(Gelez and Fab e-Nys, 2004). This p ac ice is a aluable managemen
ool exploi ed in small uminan (Walkden-B own e al., 1999; Ma in
e al., 2004; Gelez and Fab e-Nys, 2004) and swine (Chenowe h e al.,
2014) husband y o s imula e he onse o pube y and o educe he
pos pa um pe iod. In abbi s, he e is e idence ha he p esence o
males inc eases he ecep i i y o emales (Le ê e e al., 1976), i s
e ili y (Be epudo e al., 1993) and also induces sexual ma u i y in
p epube al abbi s (F ank, 1966). Mo e ecen s udies poin o an
imp o emen o doe ep oduc i e pe o mance (El-Azzazi e al.,
2017), especially in does a i s lac a ion (Bonnano e al., 2003) when
hey a e exposed o male odo s jus be o e AI. Howe e , published
da a a e con o e sial (Kus os e al., 2000; Ola e al., 2012),
hampe ing clea conclusions. Addi ionally, abbi a ms ha e
implemen ed a bios imula ion me hod based in emale- emale
GENERAL INTRODUCTION
103
in e ac ion p io o AI, which seems o inc ease hei ecep i i y, bu
hei ac ual e iciency in e ms o e ili y and p oli ici y emain o be
elucida ed.
All in all, phe omone communica ion has been la gely
app oached om a ious independen disciplines bu wi hou an
in eg a i e app oach be ween hem, which would de ini ely o e a
deepe unde s anding no only o he knowledge o chemical
communica ion bu also ega ding phe omone applica ions in he
indus ial sec o . Fo his eason, his wo k has in ol ed a
mul idisciplina y app oach, in ol ing ana omy, gene ics ep oduc ion
and beha iou .

111
MATERIAL AND
METHODS (In b ie )
We p o ide he e a b ie desc ip ion o he ma e ial and me hods
employed in his wo k. Mo e de ailed in o ma ion can be ound in he
ma e ial and me hods sec ion o each chap e .
All animals pe ained o a comme cial hyb id -Hyplus s ains PS19
and PS40 o emale and male, espec i ely- and we e main ained on a
a m (Cogal SL, Rodei o, Spain) unde he same empe a u e
condi ions (18-24 °C), da k-ligh cycles o 12:12 hou s and ad libi um
eeding and d inking. All indi iduals we e humanely sac i iced by an
aba oi o he same company, in acco dance wi h he cu en
legisla ion.
1. ANATOMICAL STUDY
1.1 Animals
- Vome onasal o gan: 20 indi iduals (males and emales) o 60-
70 days old.
- Accesso y ol ac o y bulb: 10 indi iduals (males and emales)
o 3-10 mon hs old. Addi ionally, 12 indi iduals (6 males and
6 emales) o 70 days old o mo phome ic s udies.
1.2 Techniques
- Dissec ion and mic odissec ion: Iden i ica ion and ex ac ion
o he VNO and he ol ac o y bulb (OB), exposing he
inne a ion om he VNO o he OB. Aspec s such as he
PAULA RODRÍGUEZ VILLAMAYOR
112
i iga ion and inne a ion o he VNO and i s communica ion
wi h he en i onmen we e s udied, de e mining whe he his is
done di ec ly h ough he nasal ca i y o indi ec ly and doubly
h ough bo h nasal and o al ca i ies.
- His ological p ocessing o he samples, included in pa a in o
cu ing in o mic o ome (5-10 μm hickness). Bo h ou ine
(Hema oxylin-eosin / Nissl s ain) and speci ic his ological
s ains (PAS, Alcian Blue, Gallego’s T ich ome, Toli ia and
Bielschowsky) we e used.
- Immunohis ochemical and his ochemical echniques o ob ain
mo pho unc ional in o ma ion. Lec ins such as LEA, BSI-B4
and UEA and an ibodies agains p o eins such as Gαo and
Gαi2, OMP, GFAP, GAP-43, MAP-2, glu aminase and
calcium binding p o eins (CB, CR) allowed us no only o
disc imina e he di e en cellula componen s o bo h
o ma ions (VNO and OB), bu also o assess he exp ession o
he wo mos impo an sub amilies o ome onasal ecep o , -
V1R and V2R- known o be associa ed o Gαi2 and Gαo
exp ession, espec i ely.
- Mo phome ic and s e eological analysis o he abbi AOB in
bo h males and emales o s udy sexual dimo phism.
2. GENOMIC STUDY
2.1 Animals
24 animals o s udying he abbi VNO ansc ip ome, e alua ing
di e en ial gene exp ession be ween di e en condi ions, and
assessing VNO plas ici y. The e we e 3 animals pe expe imen al
condi ion, as ollows: ju enile (40 days) and adul (180 days) males
and emales, and wo di e en en i onmen al scena ios: sex-sepa a ed
and sex-combined.
2.2 Technique
We employed RNAseq.
A e sac i icing he animals, swi dissec ion o he VNO was
MATERIAL AND METHODS (IN BRIEF)
113
needed o a oid RNA deg ada ion. The double VNO s uc u e was
immedia ely dissec ed ou a e opening he la e al walls o he nasal
ca i y and emo ing he pala e and nasal u bina es. Samples we e
imme sed in T izol and kep in ice (~4°C). Tissue was homogenized a
he sampling poin using a mixe o gua an ee he whole issue sample
is soaked by T izol -due o he double bone and ca ilage en elope o
he abbi VNO-. A e 20 minu es, samples we e s o ed a -80°C o
u he RNA ex ac ion (RNeasy mini ki (Qiagen) wi h DNase
ea men ). RNA was sen o No ogene UK Company SL
(Camb idge) o lib a y cons uc ion and sequencing. The
bioin o ma ics analysis included quali y il e ing (Fas QC) and
elimina ion o adap e s and ba codes (T immoma ic 3.0). Fil e ed
eads we e aligned agains he abbi genome (O yCun2.0) and
assigned o genes based on he la es anno a ion o he abbi genome
using STAR .2.7.0e wo-pass mode. Addi ionally, Kallis o, a
so wa e ha pseudoaligns eads o a e e ence genome p oducing a
lis o ansc ip s ha a e compa ible wi h each ead while a oiding
alignmen o indi idual bases, was also used o compa e he esul s
be ween STAR and Kallis o.
3. FIELD STUDY
3.1 Animals
The beha iou al s udy was done in g oups o 60 emales.
Acco ding o he expe imen al design, he e we e i e expe imen al
g oups and he expe imen was epea ed h ee imes o e a 120-day
pe iod (once each 40 days, acco ding o he emale ep oduc i e
cycle). E en hough some indi iduals we e he same a di e en
expe imen al ime-poin s, we conside hem as ‘di e en indi iduals’
because he da a ob ained was new each ime. In o al, o he
beha iou al s udy we employed 60 x 5 x 3 = 900 emales in
ep oduc i e s age.
U ine was ob ained om 25 ma u e males and 25 ma u e emales
(> 180 days) by ul asound-guided cys ocen esis 24 h be o e he
beha iou al expe imen (and kep a 4 ºC o e nigh ). Conside ing ha
PAULA RODRÍGUEZ VILLAMAYOR
114
he expe imen was epea ed h ee imes, we employed a o al o 75
males and 75 emales.
Seminal plasma was p o ided by an AI cen e om 60 ma u e
males (>180 days) 24 h p io o he beha io al expe imen . Ejacula es
we e mixed oge he and cen i uged a 3000 pm, 10 min, o ob ain
he seminal plasma, which we e hen kep a 4 ºC o e nigh . Simila ly,
60 x 3 expe imen al ime-poin s = 180 ma u e males.
3.2 Expe imen al design
E alua ion o speci ic emale abbi (doe) ep oduc i e
pa ame e s when hey a e exposed o biological luids –u ine and
seminal plasma o adul indi iduals (6 mon hs old)–. G oups o 60
emales/each (be ween hi d and se en h bi h) we e exposed o (1)
emale u ine, (2) male u ine, (3) seminal plasma and (4) emale–
emale (F–F) sepa a ed, jus be o e AI, and compa ed o a ‘golden
me hod’ F-F in e ac ion. The ollowing ep oduc i e pa ame e s we e
analyzed o each g oup: ecep i i y ( ul a colo ), e ili y (kindling
a e), p oli icacy and numbe o bo n ali e and dead ki s/li e . Fo
condi ions 1, 2 and 3, he co esponding s imulan was sp ayed a ound
he nose a ea, 1 h, 15 min, and 1 min be o e insemina ion.
Speci ically, 1 mL nasal sp ay was used in each exposu e pe animal,
in o al, 3 mL / indi idual. AI and he co esponding handling we e
always pe o med by he same a m wo ke s o educe s a is ical
noise.
CHAPTER I
Mo phological and
immunohis ochemical s udy o
he abbi ome onasal o gan
Paula R Villamayo , José Manuel Ci uen es, Pa icia Fe nández de
T oconiz, Pablo Sánchez-Quin ei o* (2018) Mo phological and
immunohis ochemical s udy o he abbi ome onasal o gan. Jou nal
o Ana omy 233:814—827. doi: 10.1111/joa.12884.
Depa men o Ana omy, Animal P oduc ion and Clinical Ve e ina y
Sciences, Facul y o Ve e ina y, Uni e si y o San iago de
Compos ela, Lugo, Spain. *Co esponding au ho .

CHAPTER II
S uc u al, mo phome ic and
immunohis ochemical s udy o
he abbi accesso y ol ac o y
bulb
Paula R Villamayo 1, José Manuel Ci uen es1, Luis Quin ela2,
Rami o Ba cia3, Pablo Sánchez-Quin ei o1*(2020) S uc u al,
mo phome ic and immunohis ochemical s udy o he abbi accesso y
ol ac o y bulb. B ain S uc u e and Func ion 225:203–222. doi:
10.1007/s00429-019-01997-4.
1Depa men o Ana omy, Animal P oduc ion and Clinical Ve e ina y
Sciences, Facul y o Ve e ina y, Uni e si y o San iago de
Compos ela, Lugo, Spain. 2Depa men o Animal Pa hology, Facul y
o Ve e ina y, Uni e si y o San iago de Compos ela, Lugo, Spain; 3
Depa men o Biochemis y and Molecula Biology, Facul y o
Ve e ina y, Uni e si y o San iago de Compos ela, Lugo, Spain.
*Co esponding au ho .
CHAPTER III
Analysis o he ome onasal
o gan ansc ip ome e eals
a iable gene exp ession
depending on age and unc ion
in abbi s
Paula R Villamayo 1,2, Diego Robledo3, Ca los Fe nández1, Julián
Gullón4, Luis Quin ela5, Pablo Sánchez-Quin ei o2*, Paulino
Ma ínez1 (2021) Analysis o he ome onasal o gan ansc ip ome
e eals a iable gene exp ession depending on age and unc ion in
abbi s. Genomics 113:2240–2252. doi:
h ps://doi.o g/10.1016/j.ygeno.2021.05.007. (Open Access).
1Depa men o Zoology Gene ics and Physical An h opology, Facul y
o Ve e ina y, Uni e si y o San iago de Compos ela, Lugo, Spain;
2Depa men o Ana omy, Animal P oduc ion and Clinical Ve e ina y
Sciences, Facul y o Ve e ina y, Uni e si y o San iago de
Compos ela, Lugo, Spain; 3The Roslin Ins i u e and Royal (Dick)
School o Ve e ina y S udies, Uni e si y o Edinbu gh, Midlo hian,
UK; 4Conejos Gallegos, COGAL SL, Rodei o, Pon e ed a, Spain;
5Depa men o Animal Pa hology, Facul y o Ve e ina y, Uni e si y
o San iago de Compos ela, Lugo, Spain. *Co esponding au ho .

309
DISCUSSION
This discussion highligh s he impo ance o app oaching phe omone
communica ion om an in eg a i e pe spec i e, conside ing bo h
basic esea ch (i. e. s uc u al, molecula , beha iou al app oaches,
e c.) and applied esea ch o he indus ial sec o . This means ha
phe omone ma ke should always go hand wi h hand wi h esea ch
s udies o ensu e inal p oduc e iciency and secu i y. Addi ionally,
due o hei undamen al ole in phe omone pe cep ion, ome onasal
ecep o s –VRs, FPRs and sex-s e oid ecep o s– a e also ex ensi ely
app oached in his discussion. We will pay special a en ion o he
plas ic capaci y o sex-speci ic VRs upon en i onmen al modula ion
and discuss hei po en ial implica ion in he ‘male e ec ’. We will
also highligh he ex anasal VRs gene exp ession and hei po en ial
unc ional implica ions. Finally, we will poin owa ds new
ome onasal ecep o s ye o be disco e ed and p o ide e idence o
sex-s e oid ecep o s as possible phe omone ecep o candida es.
O e all, he high complexi y o phe omone pe cep ion and in
pa icula o ome onasal ecep o s, calls o u he in es iga ions
in o hei physiological and molecula a ionale.
1. IMPORTANCE OF AN INTEGRATIVE APPROACH IN
CHEMOCOMMUNICATION RESEARCH
As we s a ed h oughou his manusc ip , phe omone-media ed
chemical communica ion plays a undamen al ole in animal
ep oduc ion and physiology. Ou main goal was o gain an in-dep h
unde s anding o chemocommunica ion media ed by he VNS in
abbi s h ough a mul idisciplina y app oach ha combines adi ional
mo pho unc ional analysis (chap e I and II) wi h inno a i e
PAULA RODRÍGUEZ VILLAMAYOR
ansc ip omic and molecula me hodologies (chap e III and IV), bu
also, beha iou al ield s udies ha measu e ep oduc i e pa ame e s
ela ed o phe omone exposu e (chap e V). A main ou come o his
app oach would be ans e ing esul s o he p oduc i e sys em by
applying phe omonal compounds o imp o ing abbi a ming,
amelio a ing ep oduc i e pa ame e s and animal wel a e, as well as
educing he usage o ho mones in a ms.
Ge ing in o he de ail, he s uc u al and mo pho unc ional s udy
o he abbi VNO (chap e I) shows a well-de eloped s uc u e, wi h
an ex ensi e enous ascula u e, which di e s om any o he g oup
o mammals (Vacca ezza e al., 1981; Ba ios e al., 2014). This,
along wi h a double bone and ca ilaginous en elope, s ongly
sugges s ha he abbi VNO is e y ac i e a ecei ing phe omones
by pumping VNO mechanisms (see discussion o chap e I o de ail).
Also, he exp ession o he wo ome onasal G-p o ein amilies –gαi2
and Gαo– in he abbi VNO neu oepi helium (chap e I) and in wo
seg ega ed egions o he AOB –Gαi2 a he an e io pa and Gαo a
he pos e io pa (chap e II)–, likely co ela es o he exp ession o
VRs, since a link be ween he exp ession o V1Rs wi h Gαi2 and
V2Rs wi h Gαo is gene ally assumed–. Rema kably, p elimina y da a
om ou g oup ha e shown ha in abbi s a pe ina al s age he e is no
co ela ion be ween he exp ession o VRs and hei co esponding G-
p o ein (Villamayo e al., 2019; 2022a). This is a he su p ising and
con adic s mos o he VNO adi ional s udies o da e (Halpe n and
Ma inez-Ma cos, 2003; Sua ez e al., 2011a). No wi hs anding, i
demons a es he impo ance o app oaching VNS s udies om an
in eg a i e pe spec i e (mo pho ucn ional s gene exp ession s udies
in his case) and poin s owa ds he necessi y o mo e in-dep h s udies
ega ding he eal associa ion be ween V1R and V2R wi h Gαi2 and
Gαo, espec i ely.
The b oad di e si y o VRs oge he wi h he lack o speci ic
an ibodies hampe s hei mopho unc ional cha ac e iza ion. Genomic
da a complemen e y well s uc u al s udies. We employed gene
exp ession analysis (RNAseq) o iden i y and upda e he ac i e VR
epe oi e in abbi s –we ound 128 V1Rs and 67 V2Rs– (chap e III).
The numbe o VRs g ea ly a ies among species ( om > 250 in mice
DISCUSSION
o ~ 8 in dogs) (G us e al., 2005; Young and T ask, 2007), and
compa isons o VR epe oi es and hei gene exp ession pa e ns ha e
been done ac oss a ange o mammals, demons a ing ha each
species has a ‘quasi-p i a e’ epe oi e o de ec and espond o a wide
a ie y o ex e nal signals (Wynn e al., 2012).
We also analyzed he abbi VNO gene exp ession a ia ion
ac oss di e en socio-en i onmen al condi ions (chap e IV). Indeed,
we iden i ied ha VRs we e especially up- and down- egula ed in
sex-sepa a ed ju enile emales and sex-sepa a ed adul emales,
espec i ely. Ou da a e ealed ha VRs exp ession epe oi e in
abbi s is en i onmen ally modula ed which con as s wi h esul s
ob ained om a simila expe imen al s udy in adul mice (Van de
Linden e al., 2018) (see discussion o chap e IV o de ails). I we
ansla ed ou abbi da a in o a abbi a m pe spec i e, whe e males
and emale a e usually loca ed in sepa a ed acili ies, we would expec
ha emales no exposed o males – abbi a m ou ine– would display
down- egula ion o hei VRs epe oi e –acco ding o ou sex-
sepa a ion s udy (chap e IV)–, and his could be linked o a lowe
ep oduc i e pe o mance. Then, i we we e o expose emales o
sexually ma u e males – he so-called ‘male e ec ’, which is known o
inc ease emale ecep i i y and e ili y (Gelez and Fab e-Nys, 2004;
El-Azzazi e al., 2017)–, how would VRs exp ession beha e? In o he
wo ds, a e VRs implica ed in he ‘male e ec ’ and i so, how would
hei gene exp ession pa e ns be like?
The physiological a ionale unde lying ‘ he male e ec ’ emains
la gely unknown, bu phe omones a e likely o be he main key
playe s a d i ing such beha iou . The e o e, we would expec a di ec
unc ional implica ion o he VNO and VRs in he ‘male e ec ’.
Knowing ha adul emales show o e all down- egula ion o VRs
when sex-sepa a ed (chap e IV) and assuming, ha exposu e o males
(‘male e ec ’) would lead o an inc ease in ep oduc i e pe o mance,
he ‘male e ec ’ could be guided by an inc ease o VRs exp ession in
emales when exposed o sexually ac i e males. Impo an ly, since he
VNO has shown expe ience dependen plas ici y (Ma om e al., 2019),
his would likely explain why he ‘male e ec ’ is ‘acu e’ –meaning
ha i is e ec i e only i he exposu e akes place du ing a sho
PAULA RODRÍGUEZ VILLAMAYOR
pe iod o ime–. Ins ead, ch onic exposu e o males e en ually leads o
a d op in emale ep oduc i e pe o mance (Gelez and Fab e-Nys,
2004). This ac could be explained by he sa u a ion o VRs and he
consequen educ ion o hei exp ession (see heo y use-i -and-lose-i
in discussion o chap e IV). Fu he beha iou al and gene exp ession
analysis a e needed o e i y his hypo hesis. Since we ha e al eady
es ablished a p o ocol o es ing emale ep oduc i e pe o mance
ollowing ‘phe omone exposu e’ ia biological luids (chap e V), and
we also ha e long expe ience wi h gene exp ession analyses (chap e
III, IV), we a e equiped wi h he expe ience o design expe imen s
aimed a es ing he unc ional implica ion o VRs in ‘male e ec ’.
A a mo e basic scien i ic le el ye essen ial o know how he
VNS d i es beha iou , a u he s ep would be o iden i y how hose
ome onasal inpu s a e p ocessed in he b ain. We ha e p o ided an
ex ensi e s uc u al and mo pho unc ional analysis o he abbi AOB,
bu also de e mined i s s uc u al sexual dimo phism - emale abbi
AOB p esen s highe mo phome ic alues han male (chap e II)-.
This is in acco dance wi h p e ious da a om Guillamón and Sego ia
(1997), who also iden i ied sexual dimo phism in a s, bu wi h highe
mo phome ic alues in male han in emale. We ha e no explana ion
o hese opposi e pa e ns be ween he wo species, and he unc ional
ole o such AOB dimo phism emains unknown.
In any case, we a gue ha he s uc u al AOB sexual dimo phism
may play a ole a media ing sex-speci ic beha iou s. The na u e o
he AOB is highly complex due o con inuous neu o egene a ion o
inhibi o y in e neu ons ha somehow egula e he signaling o
p incipal cells. Conside ing he impo ance o hese newbo n neu ons
in he egula ion o ep oduc i e and social beha iou , u he s udies
in abbi s should aim a deciphe ing hei unc ional implica ion in he
AOB ci cui , and i migh be wise o add ess he s udies in a sex-
speci ic manne . In o de o do so, we could ake ad an age o a ecen
s udy ha has p o ided insigh s in o he complex physiology o mice
AOB in e neu ons, and which se he basis o u u e s udies o AOB
unc ion, ci cui s and plas ici y mechanisms (Maksimo a e al., 2019).
Also, new OMICs echniques, such as single-cell RNAseq and spa ial
ansc ip omics, a e p o iding new insigh s in o he cellula
DISCUSSION
he e ogenei y o adul bo n MOB in e neu ons as well as he complex
o ganiza ion o he MOB glome ula map in mice (Tepe e al., 2018;
Wang e al., 2022). These cu ing-edge echniques ha e no ye been
applied o he AOB, bu hey cons i u e p omising oppo uni ies o
ackle he molecula a ionale o he AOB. The e o e,
mul idisciplina y app oaches including new –OMICs bu also new
imaging echniques, as well as elec ophysiological eco dings would
be c ucial o unde s and he unc ional and s uc u al in e play o his
inny s uc u e, ex emely necessa y o animal ep oduc ion and
su i al.
All in all, phe omone communica ion has been la gely
app oached om a ious independen disciplines, bu wi hou an
in eg a i e s a egy, which would de ini ely p o ide a deepe
unde s anding, no only o he knowledge o chemical communica ion,
bu also ega ding phe omone applica ions o he indus ial sec o .
Fo his eason, his doc o al hesis app oaches he VNS om
ana omical, genomic, beha iou al and ep oduc i e poin s o iew,
and o he new app oaches such as p o eomics and ola olomics a e
al eady in he ack. As a esul , i is expec ed an imp o emen in he
ep oduc i e pa ame e s and well-being o a med abbi s h ough
phe omone implemen a ion.
2. COMPLEXITY OF VOMERONASAL CHEMORECEPTORS
Vome onasal ecep o s ha e e ol ed independen ly in he
di e en axa and species o de ec a b oad ange o chemical s imuli
and ins uc specie-speci ic beha iou s. Despi e many ad ances in he
ield ha e been made in he pas ew yea s ( e iewed in Ti indelli,
2021), we s ill do no know how phe omones / chemical cues bind
ome onasal ecep o s, and he e o e app oaching he s udy o hese
ecep o s in di e en species and om a mul idisciplina y pe spec i e
will help aming how his sys em wo ks and ul ima ely how i a ec s
species-speci ic beha iou s. This pa o he discussion exposes he
high complexi y o ome onasal ecep o s, wi h special ocus on he
VRs gene exp ession a ia ion depending on socio-en i onmen
condi ions as well as i s exp ession in ex anasal issues. Also, due o

PAULA RODRÍGUEZ VILLAMAYOR
he iden i ica ion o a new amily o ome onasal ecep o s –FPRs–
which was exclusi ely ound in mice (Libe les e al., 2009; Ri iè e e
al., 2009), we aise he ques ion o whe he he e may be species-
speci ic amilies o ome onasal chemo ecep o s ye o be disco e ed.
Finally, he exp ession o sex-s e oid ecep o s in VSNs oge he wi h
he ac ha s e oids ac as VNO s imuli a e a he new concep s
which open new a enues o s udying whe he such sex-s e oid
ecep o s could be iden i ied as a new ype o ome onasal ecep o s.
2.1 V1R and V2R ecep o s
V1R and V2R ecep o s a e among he gene amilies showing
b oade a ia ion in gene numbe ac oss e eb a e lineages (Nei e al.,
2008; Shi and Zhang, 2007). Due o his species-speci ici y,
ome onasal s udies should always be add essed in each species
independen ly and om a compa a i e poin o iew among species.
Ex apola ions om one species o ano he migh lead in mos cases o
mis aken conclusions (Salaza and Sanchez-Quin ei o, 2009).
Addi ionally, we should keep in mind in aspeci ic a ia ion as a
sou ce o local adap a ion o popula ions and u he as he aw
ma e ial o e olu iona y s udies.
We pe o med a phylogene ic s udy o he abbi VRs and
compa ed i o ha o mice –a phyloge ically close species– (chap e
III). Ou da a e ealed ha abbi s ha e unde gone se e al expansions
om ances al genes, which ha e been comple ely los in mice, and
ha a e dis ibu ed in a ew independen clades. Addi ionally, VRs
seemed o g oup in clus e s in he abbi genome. Fo example, we
ound 30 V1R genes in ch omosome 9. Howe e , mos o he VRs
appea ed in sca olds no ancho ed o ch omosomes, and al hough
hey may also be g ouped, a new abbi genome assembly will be
necessa y o app op ia ely add ess his ques ion. In con as o mice, in
which VRs ha e been ex ensi ely classi ied in he li e a u e (Yang e
al., 2005; Mille e al., 2020), abbi VRs ha e no ye been g ouped in
di e en sub amilies. In an a emp o classi y hem, we con ac ed
P o . Lei Ande son´s g oup om Uppsala Uni e si y, which ecen ly
eleased a new abbi genome assembly, O yCun 3.0 (2020), bu his
DISCUSSION
genome is no anno a ed and he e o e we could no use i in ou
analysis. Acco ding o P o . Ande son, his assembly would no be
anno a ed due o speci ic issues ela ed o old PacBio chemis y,
which p oduced small gaps in he assembly, bu a new O yCun 4.0
assembly is in he way, which will be c i ical o u u e VNO s udies,
especially conside ing he e olu iona y his o y o ome onasal
ecep o s.
I is well documen ed ha VNO-media ed beha iou is sexually
dimo phic (i. e. male phe omones such as MUPs o ESP1 e oke
e i o ial agg ession when de ec ed by ano he male, bu p omo e
sexual a ac ion and ecep i i y when de ec ed by emales) (Tan and
S owe s, 2020). Mo phological sexual dimo phism has been ound a
e e y s age o he ome onasal pa hway, including he VNO, AOB,
amygdala, and hypo halamic a eas (Guillamón and Sego ia, 1997;
Sego ia e al., 2006; chap e II). Howe e , he i s e idence o
unc ional sexual dimo phism occu s in he MeA (Be gan e al., 2014;
Tan and S owe s, 2020), hus sugges ing ha bo h sexes may equally
de ec cues, which in u n will be di e en ially p ocessed in each sex
a highe b ain cen e s. This hypo hesis is also suppo ed by he ac
ha VRs exp ession was ound o be simila in male and emale mice
exposed o he same en i onmen al condi ions (Iba a-So ia e al.,
2014b). Consis en wi h his, ou da a demons a ed ha , in abbi s,
VNO and VRs exp ession pa e ns ollow a simila logic in males and
emales exposed o he same en i onmen al scena io (chap e III).
Ne e heless, conside ing ha he VNO is a senso y o gan which
akes o e sensing a b oad ange o chemical s imuli om he ex e nal
wo ld, his landscape –meaning he simila VNO and VRs exp ession
pa e ns ound in males and emales exposed o simila en i onmen s–
could change i animals a e subjec ed o di e en en i onmen al
condi ions. Indeed, s udies in mice showed ha he VNO is able o
adap esponses o a gi en scena io, demons a ing i s expe ience- and
s a e- dependen plas ici y (Lanuza e al., 2014; Moh ha d e al.,
2018; Ma om e al., 2019; T ouille e al., 2021; Villa anca-Faus e
al., 2021). Also, VRs gene exp ession has p o en o be a ec ed sex-
speci ically by pa icula en i onmen s in mice (Van de Linden e al.,
2018). In ou s udy, we showed o he i s ime he abbi VNO
PAULA RODRÍGUEZ VILLAMAYOR
plas ici y unde di e en socio-en i onmen al condi ions (chap e IV).
Speci ically, ou sex-sepa a ion s sex-combined expe imen al
app oach, whe e animals do no con ac wi h membe s o he opposi e
sex since bi h s in close con ac since bi h, e ealed ha VNO and
VRs gene exp ession di e s be ween males and emales unde he wo
s udied en i onmen al condi ions. This esul p o ed ha despi e sex
i sel does no in luence VRs exp ession as long as animals a e
exposed o same en i onmen (demons a ed in abbi s in chap e III),
such exp ession d ama ically changes when animals a e exposed o
di e en scena ios (chap e IV). We a gue ha sexual dimo phism o
he VNS and speci ically o he VRs should always be conside ed
acco ding o he en i onmen o which animals a e exposed o,
because despi e sex migh no be a condi ion i sel , esponses o a
gi en en i onmen al condi ion a e de ini ely shaped in a sex-speci ic
manne .
Addi ionally, conside ing ha he VNO has been sugges ed o
play a ole a pube y onse (Szymanski and Kelle , 2014; C oss e al.,
2021), we ex ended ou s udy o ju enile indi iduals. When
compa ing he gene exp ession o ju enile abbi VNO o ha o
adul s –unde same en i onmen al condi ions–, s iking di e ences
we e de ec ed be ween he wo s ages. Howe e , he VR epe oi e
showed li le gene exp ession di e ences be ween he wo s ages
(chap e III). This would ollow a simila logic as he one desc ibed
abo e o male and emale adul s unde he same en i onmen al
condi ions –equal capaci y o de ec ing cues h ough he VNO, bu
di e en ways o p ocessing he in o ma ion a highe b ain cen e s–.
Ou nex ques ion was o de e mine whe he ju enile VNO and VRs
exp ession would also be a ec ed by exposu e o a pa icula
en i onmen al, and i so, whe he his would also be sex-speci ic.
Following a simila app oach as ou lined abo e, we de e mined ha
ju enile VNO and VRs gene exp ession epe oi es sha ply a y upon
en i onmen al condi ions in emales bu no in males, hus poin ing o
a unc ional ole o he VNO a he onse o pube y (see discussion o
chap e IV o de ails). Finally, he DEGs epe oi es ound be ween
sex-sepa a ed and sex-combined condi ions g ea ly di e be ween
ju eniles and adul s, hus p o ing ha he VNO and VRs no only
DISCUSSION
show sex- bu also s age-speci ic gene exp ession, and his is
condi ioned by a gi en en i onmen . All in all, conside ing ha un il
now chemical cues / phe omones we e hough o be equally de ec ed
in males and emales by he VNO and VRs, ou da a s ongly sugges
ha he VNO and VRs a e highly sensi i e chemodec o s wi h an
ou s anding plas ici y o adap o a con inuous changing en i onmen ,
and his is sex- and s age-speci ic.
2.1.1 Ex anasal exp ession o VRs
An addi ional le el o complexi y o ome onasal
ecep o s comes om hei ex anasal exp ession. Ou side ol ac o y
o gans, V1Rs exp ession was ound in es is in mice and swine
(Ta su a e al., 2001; Dinka e al., 2016), bu also in b ain, bulb, hea ,
kidney, and li e (< 7 genes / issues) (Zhang e al., 2010), hough
hei unc ion emains unknown. As o V2Rs, despi e he as
majo i y o V2R genes we e only exp essed in he VNO, Iba a-So ia
e al., (2014) ound exp ession o one V2R in he MOE, Vmn2 29,
sugges ing p e ious un ecognized mechanisms o phe omone
de ec ion in he MOE. In o he non-senso y issues, only e y ew
V2Rs we e ound, speci ically in mice b ain, bulb, hea , kidney, li e ,
and lung (< 5 V2Rs / issue) (Zhang e al., 2010), bu as o V1R, hei
po en ial unc ionali y emains comple ely unexplo ed. In abbi s, we
compa ed he VRs VNO exp ession o ha o o he se en abbi
issues (hindb ain, o eb ain, o a y, es is, li e , hea and kidney)
a ailable in he Rabbi Exp ession A las (Ca doso-Mo ei a e al.,
2019). We ound high exp ession o ex anasal VRs in es is, low
exp ession in o a y and b ain and no exp ession was ound in hea ,
li e and kidney (see chap e III o de ail). Despi e VRs ex anasal
exp ession has no been comp ehensi ely add essed in he li e a u e,
s udies o ex anasal ORs exp ession poin o he necessi y o u he
app oaching his opic. B ie ly, he adi ional de ini ion om Buck
and Axel (1991) s a ed ha ORs we e only ound in he main ol ac o y
epi helium. Howe e , a small subse o ORs was la e ound exp essed
in o he chemosenso y issues such as he VNO (Lé ai e al., 2006)
and he sep al o gan (Kaluza e al., 2004; Tian and Ma, 2004).
Addi ionally, soon a e he disco e y o ORs, hei exp ession was
PAULA RODRÍGUEZ VILLAMAYOR
he ounda ion o con inue his wo k in o he inal goal: phe omone
comme cializa ion.
Up o da e, a ew ‘pu a i e phe omones’ ha e been
comme cialized, especially o dogs and ca s ( o con ol beha iou )
bu also o some a med species ( o con ol ep oduc ion). Howe e ,
mos o hem lacked scien i ic backg ound, leading o ine icien
p oduc s. Fo example, in abbi s he Rabbi Mamma y Phe omone –
2MB2– is known o exclusi ely induce pup suckling beha iou
(Cou eaud e al., 2010; Cha a e al., 2013; Schneide e al., 2016).
Indus ial ma ke has aken ad an age o his only cha ac e ized abbi
phe omone o da e -2MB2-, by comme cializing an analog called
Rabbi Appeasing Phe omone, desc ibed as ‘phe omone p oduc s
based on a combina ion o phe omones’ including he abbi
mamma y phe omone 2MB2 (i.e. 1); Ce a San é Animale c ea ed he
i s ‘Rabbi Appeasing Phe omone’ (h p://www.as c-
lapin.com/Docs/Ac i i e/cunino /Cunino 2007/Cunino 2007-
CEVA.pd , 2007
3
; 2); and Sibpma and SIGNS labo a o ies ha e
comme cialized he Secu eRabbi ®, a syn he ic analog o he ma e nal
appeasing phe omone (licence IRSEA – US Pa en 6-077-867, 6-054-
481 y 6-169-113) (h ps://sibpma.es/wp-
con en /uploads/2020/12/Ficha-Secu eRabbi %C2%AE-SIGNS-
2020.pd )
5
. Howe e , he e a e no published da a (nei he VNS
neu oana omical s udies (senso y pa hways in ol ed, ecep o
IdeaLugo p ize: h ps://www.elp og eso.es/gl/a iculo/lugo/p oxec o-mello a-benes a -animal-
e omonas- ai-p emio-idealugo/202011251340161471891.h ml
3
CEVA. Acco ding o hei esul s, emale abbi s we e less s essed, and echnical ac ions
we e easie . Mo eo e , e ili y (pe cen age o pa u i ions pe a i icial insemina ion (AI),
li e bo n abbi s pe li e and abbi s’ iabili y a bi h imp o ed (Bou ie e al., 2008,
con e ence communica ion). This p oduc is no a ailable in he CEVA websi e, sugges ing
ha is no being comme cialized – we ha e no in o ma ion whe he i has e e been
comme cialized.
4
SIBPMA phe omones p e en he nega i e e ec s o s ess, imp o e animal p oduc ion and
enhance animal wel a e. Howe e , no scien i ic da a has been epo ed and i s e iciency
should be conside ed wi h cau ion.
5
SIBPMA phe omones p e en he nega i e e ec s o s ess, imp o e animal p oduc ion and
enhance animal wel a e. Howe e , no scien i ic da a has been epo ed and i s e iciency
should be conside ed wi h cau ion.

DISCUSSION
loca ions, e c.) no any unc ional app oach (gene exp ession,
elec ophysiology, neu oendoc ine s udies, e c.)) ha jus i y he use o
a ‘newbo n suckling phe omone’ o imp o e he well-being o adul
abbi s.
In his con ex , i is impo an o highligh ha phe omone ma ke
is no egula ed by law. Indeed, a di ec consul o he Spanish Agency
o Medicines and Medical De ices (AEMPS) in 2021 indica ed ha
since phe omone ma ke is qui e small and lacks speci ic legisla ion,
his ype o compounds could be conside ed unde di e en laws
depending on hei cha ac e iza ion / iden i ica ion p ocesses (i. e. in
Spain, ‘Law 29/2006’ i we conside phe omones as compounds
ob ained om glands). Some companies ha e aken ad an age o his
gap o sell ‘phe omone p oduc s’ wi hou any scien i ic backg ound,
which –no su p isingly– u n ou being ex emely ine icien . I is
he e o e o u mos impo ance ha he scien i ic communi y wi hin
he ield ackles his issue, asking o he implemen a ion o speci ic
egula ions in which phe omone p oduc s mus p o e a solid
backg ound which jus i ies i s e ec i eness be o e being sold.
CONCLUDING REMARKS
AND FURTHER STEPS
This doc o al hesis se he basis o chemocommunica ion
esea ch in abbi . I p o ides s ong ounda ion o he s uc u al and
molecula basis o he VNS and speci ically o VRs, as well as
es ablishes a p o ocol o u he ield s udies on phe omone-induced
beha iou s. S a ed om sc a ch i e yea s ago, his p ojec has
al eady gene a ed a g ea deal o ele an da a which may ul ima ely
con ibu e owa ds he compe i i e implemen a ion o phe omones in
he ma ke .
This wo k is included in a b oade p ojec o ou g oup. The nex
s eps include 1) molecula s udies (p o eomics, ola olomics, e c.) o
biological luids o cha ac e ize abbi phe omones; 2) beha iou al
analysis o biological compounds o he han u ine and seminal
plasma, such as ex ac s om exoc ine glands, o s udy hei impac in
ep oduc i e pa ame e s o emale and also male abbi s; 3)
es ablishing he link phe omone- ecep o by molecula s udies (qPCR,
in si u hyb idiza ion); and 4) ield s udies wi h po en ial phe omone
candida es. All hese analyses will bene i om he da a ob ained in
his PhD hesis. The inal goal o his app oach is o imp o e animal
p oduc ion and wel a e by he implemen a ion o phe omones as
na u al molecules ha con ibu e o he ecological sus ainabili y o
li es ock p oduc ion. Indeed, he alidi y o hese esul s as well as o
he coming esea ch app oaches ha e been ecen ly suppo ed by he
Spanish Science and Inno a ion Minis y, wi h a 3-yea p ojec o
hei con inua ion.
CONCLUSIONS

331
CONCLUSIONS
1. Vome onasal chemope cep ion is highly complex and should
always be app oached om a mul idisciplina pe spec i e. Ou
da a p o ide ex ensi e knowledge in o abbi
chemocommunica ion, speci ically a ana omical, genomic,
ep oduc i e and beha iou al le els, p o iding he baseline o
u he ansla ional s udies aimed a implemen ing he use o
phe omones in abbi a ms.
2. A ana omical le el, he abbi , O yc olagus cuniculus, holds a
well-de eloped VNS –VNO and AOB–, which con ains all
necessa y elemen s o de elop an e ec i e
chemocommunica ion be ween indi iduals o he same
species.
3. The deg ee o s uc u al de elopmen o he adul abbi VNO
is compa able o ha o mammals wi h he highes le el o
chemocommunica ion. The VNO displays many species-
speci ic mo phological ea u es, such as a double di ec and
indi ec communica ion – ia nasal and o al ca i ies,
espec i ely– o he ex e nal wo ld. Phe omone ci cula ion
along he VNO leng h is ensu ed by i s double bone and
ca ilaginous en eloped as well as i s unique blood essels.
4. The adul abbi AOB has a complex s uc u e wi h speci ic
opog aphic, lamina ion and neu ochemical p ope ies. I also
con ains h ee ypes o p incipal cells ha signi ican ly di e
PAULA RODRÍGUEZ VILLAMAYOR
in quan i y be ween males and emales, indica ing s uc u al
sexual dimo phism. Addi ionally, ou neu onal clus e s
composed o pi amidal-like cells and ound a he accesso y
bulba whi e ma e sugges an addi ional le el o complexi y
o he AOB ci cui y.
5. Immunohis ochemical and his ochemical ma ke s a e aluable
ools o cha ac e izing he mo pho unc ional ea u es o he
abbi VNS, especially he AOB. Fo ins ance, G-p o ein Gαi2
and Gαo allowed he iden i ica ion o wo well-di e encia ed
AOB egions –an e io and pos e io , espec i ely–.
6. A genomic le el, he abbi VNO ansc ip ome does no
di e be ween males and emales, bu i signi ican ly di e s
be ween adul s and ju eniles. I con ains 128 and 67 V1Rs and
V2Rs, espec i ely, as well as many genes in ol ed in
ep oduc ion, immuni y and VNO unc ional ac i i y.
7. The abbi VNO is highly plas ic and shows sex- and s age-
speci ic gene exp ession di e ences upon socio-en i onmen al
condi ions – ia sex-sepa a ion and sex-combined scena ios-.
VRs gene exp ession is signi ican ly down- and up- egula ed
in sex-sepa a ed adul emale and sex-sepa a ed ju enile
emale espec i ely. Simila ly, genes in ol ed in ep oduc ion,
immuni y and unc ional ac i i y a e also highly plas ic in
e ms o hei gene exp ession pa e ns.
8. Bios imula ion me hods employing u ine and seminal plasma
as sou ce o phe omones did no a ec he ep oduc i e
pe o mance o emale abbi s. Female- emale in e ac ion
be o e a i icial insemina ion, which is a common a m
ou ine, displayed he same ep oduc i e pa ame e s o emale-
emale sepa a ion, and he e o e his p ac ice could be
emo ed om abbi a ms o a oid unnecessa y animal
handling.
CONCLUSIONS
333
9. This s udy shows many VNS abbi speci ic ea u es and
indi idual a iabili y which indica es ha ex apola ion o da a
be ween species and e en be ween indi iduals o he same
species can lead o mis aken conclusion. Acco dingly, species-
speci ic di e ences and indi idual a iabili y should always be
conside ed in VNO s udies and o e all chemocommunica ion
esea ch.
PAULA RODRÍGUEZ VILLAMAYOR
340
Rep oduc i e esponse o p epube al emale abbi o
pho ope iod and/o male p esence. Wo ld Rabbi Sci 1(2):83-87.
Be gan JF, Ben-Shaul Y, Dulac C (2014) Sex-speci ic p ocessing
o social cues in he medial amygdala. eLi e 3:e02743. doi:
h ps://doi.o g/10.7554/eLi e.02743.
Be gha d A, Buck LB (1996) Senso y ansduc ion in
ome onasal neu ons: e idence o G alpha o, G alpha i2, and
adenylyl cyclase II as majo componen s o a phe omone signaling
cascade. J Neu osci 16(3):909-18. doi: 10.1523/JNEUROSCI.16-03-
00909.1996.
Be nha d R, Ma us A (1984) Ligh and elec on mic oscopic
s udies o he dis ibu ion o mic o ubule-associa ed p o ein 2 in
a b ain: a di e ence be ween dend i ic and axonal
cy oskele ons. J Comp Neu ol 226:203–221.
Bha naga KP, Meisami E (1998) Vome onasal o gan in ba s
and p ima es: ex emes o s uc u al a iabili y and i s
phylogene ic implica ions. Mic osc Res Tech 43(6):465-75. doi:
10.1002/(SICI)1097-0029(19981215)43:6<465::AID-
JEMT1>3.0.CO;2-1.
Bock P, Rohn K, Beineke A, Baumgä ne W, Wewe ze K
(2006) Si e-speci ic popula ion dynamics and a iable ol ac o y
ma ke p o ein exp ession in he pos na al canine ol ac o y
epi helium. J Ana 215:522–535. doi: 10.1111/j.1469-
7580.2009.01147.x.
Boehm U (2006) The ome onasal sys em in mice: om he
nose o he hypo halamus- and back! Semin Cell De Biol 17:471–
479. doi: 10.1016/j.semcdb.2006.04.013.
Boehm U, Zou Z, Buck LB (2005) Feedback loops link odo
and phe omone signaling wi h ep oduc ion. Cell 123:683–695.
doi: 10.1016/j.cell.2005.09.027.
Boilla M, Ca le on A, Rod iguez I (2021) F om immune o
ol ac o y exp ession: neo unc ionaliza ion o o myl pep ide
ecep o s. Cell Tissue Res 383(1):387-393. doi: 10.1007/s00441-020-

REFERENCES
341
03393-5.
Boilla M, Challe L, Rossie D, Kan C, Ca le on A, Rod iguez I
(2015) The ome onasal sys em media es sick conspeci ic
a oidance. Cu Biol 25(2):251-255. doi: 10.1016/j.cub.2014.11.061.
Bolge AM, Lohse M, Usadel B (2014) T immoma ic: a lexible
imme o Illumina sequence da a. Bioin o ma ics 30:2114–20.
doi: h ps://doi.o g/10.1093/bioin o ma ics/b u170.
Bonanno A, Mazza F, Alabiso M, Di G igoli A, Alica a ML
(2003) E ec s o bio-s imula ion induced by he p esence o buck
on ep oduc i e pe o mance o abbi does. I alian J Anim Sci
2:133–135. doi: h ps://doi.o g/10.4081/ijas.2003.11675938.
Bou ie AC, Jacquine C (2008) Phe omone in abbi s:
p elimina y echnical esul s on a m use in ance. 9 h Wo ld
Rabbi Cong ess – P oceedings. Xicca o G, T ocino A, Luke ah SD,
eds. Ve ona (I aly) pp. 303–308. Online, accessed July 31, 2022 a
h p://wo ld- abbi -science.com/WRSA-P oceedings/Cong ess-2008-
Ve ona/Pape s/R-Bou ie .pd .
B ann JH, Fi es ein S (2010) Regene a ion o new neu ons is
p e- se ed in aged ome onasal epi helia. J Neu osci 30:15686–
15694. doi: 10.1523/JNEUROSCI.4316-10.2010.
B ann JH, Fi es ein SJ (2014) A li e ime o neu ogenesis in he
ol ac o y sys em. F on Neu osci 8(182):1-11. doi:
10.3389/ nins.2014.00182.
B ay NL, Pimen el H, Mels ed P, Pach e L (2016) Nea -op imal
p obabilis ic RNA-seq quan i ica ion. Na Bio ech 34:525–527. doi:
10.1038/nb .3519.
B ee H, Fleische J, S o mann J (2006) The sense o smell:
mul iple ol ac o y subsys ems. Cell Mol Li e Sci 63:1465–1475. doi:
10.1007/s00018-006-6108-5.
B ennan PA (2004) The nose knows who's who: chemosenso y
indi iduali y and ma e ecogni ion in mice. Ho m Beha 46(3):231-
40. doi: 10.1016/j.yhbeh.2004.01.010.
PAULA RODRÍGUEZ VILLAMAYOR
342
B ennan PA (2010) On he scen o sexual a ac ion. BMC Biol
8:71. doi:10.1186/1741-7007-8-71.
B ennan PA (2018) 50 yea s o decoding ol ac ion. B ain and
Neu osci Ad 2:1-5. doi: 10.1177/2398212818817496.
B ennan PA, Ke e ne EB (2004) Some hing in he ai ? new
insigh s in o mammalian phe omones. Cu Biol 14:R81–R89. doi:
10.1016/j.cub.2003.12.052.
B ennan PA, Zu all F (2006) Phe omonal communica ion in
e eb a es. Na u e 444(7117):308-315. doi: 10.1038/na u e05404.
B iand L, T o ie D, Pe nolle JC (2004b) Aph odisin, an
aph odisiac lipocalin sec e ed in hams e aginal sec e ions.
Pep ides 25(9):1545-52. doi: 10.1016/j.pep ides.2003.10.026.
B iand L, Blon F, T o ie D, Pe nolle JC (2004a) Na u al
ligands o hams e aph odisin. Chem senses 29(5):425–430. doi:
h ps://doi.o g/10.1093/chemse/bjh044.
B ignall AC, Clou ie JF (2015) Neu al map o ma ion and
senso y coding in he ome onasal sys em. Cell Mol Li e Sci
72(24):4697-709. doi:10.1007/s00018-015-2029-5.
B ignall AC, Raja R, Phen A, P ince JEA, Dumon ie E, Clou ie
JF (2018) Loss o Ki el amily membe s al e s glome ula
s uc u e and synapse numbe s in he accesso y ol ac o y bulb.
B ain S uc Func 233:307-19. doi: 10.1007/s00429-017-1485-0.
B iñón JG, We uaga E, C espo C, Po e os A, A é alo R, Aijón J,
Alonso JR (2001) Cal e inin-, neu ocalcin-, and pa albumin-
immuno eac i e elemen s in he ol ac o y bulb o he hedgehog
(E inaceus eu opaeus). J Comp Neu ol 429:554–570. doi:
h ps://doi.o g/10.1002/1096-9861(20010122)429:4<554::AID-
CNE4>3.0.CO;2-T
B oad KD, Ke e ne EB (2012) The pos -na al chemosenso y
en i onmen induces epigene ic changes in ome onasal ecep o
gene exp ession and a bias in ol ac o y p e e ence. Beha Gene
42:461-471. doi: 10.1007/s10519-011-9523-9.
REFERENCES
343
B own RE (1979) Mammalian social odou s: a c i ical e iew.
Ad S udy Beha 10:103–162.
B own RE (1985) E ec s o social isola ion in adul hood on
odo p e e ences and u ine-ma king in male a s. Beha Neu al
Biol 44:139–143. doi: 10.1016/s0163-1047(85)91301-9.
B uce HM (1959) Ex e ocep i e block o p egnancy in he
mouse. Na u e 184:105. doi: 10.1038/184105a0.
B uce HM (1960) A block o p egnancy in he mouse caused
by p oximi y o s ange males. J Rep od Fe il. 1:96–103.
doi: 10.1530/j .0.0010096.
B ykczynska U, Tzika AC, Rod iguez I, Milinko i ch MC
(2013) Con as ed e olu ion o he ome onasal ecep o
epe oi es in mammals and squama e ep iles. Genome Biol E ol
5(2):389-401. doi: 10.1093/gbe/e 013.
Buck L, Axel R (1991) A no el mul igene amily may encode
odo an ecep o s: a molecula basis o odo ecogni ion. Cell
65(1):175-87. doi: 10.1016/0092-8674(91)90418-x.
Buck LB (2000a) Ol ac o y ecep o s and odo coding in
mammals. Nu Re 62 (11 P 2):S184–S188. doi: 10.1111/j.1753-
4887.2004. b00097.x.
Buck LB (2000b) The molecula a chi ec u e o odo and
phe omone sensing in mammals. Cell 100:611–618. doi:
10.1016/s0092-8674(00)80698-4.
Bu e B, Schumann T, Kappl R, Bogeski I, Kumme ow C,
Podgo ska M, Smola S, Ho h M, Zu all F (2015) Recogni ion o
bac e ial signal pep ides by mammalian o myl pep ide ecep o s:
a new mechanism o sensing pa hogens. J Biol Chem 290:7369–
7387. doi: 10.1074/jbc.M114.626747.
Bu e B, Schumann T, Zu all F (2012) Fo myl pep ide ecep o s
om immune and ome onasal sys em exhibi dis inc agonis
p ope ies. J Biol Chem 287(40):33644–33655. doi:
10.1074/jbc.M112.375774.
PAULA RODRÍGUEZ VILLAMAYOR
344
Bu e B, Teuche Y, Schmid A, Py ski M, Pé ez-Gómez A,
Eisenbeis J e al. (2019) Bac e ial Mg B pep ide ac i a es
chemo ecep o Fp 3 in mouse accesso y ol ac o y sys em and
d i es a oidance beha iou . Na Comm, 10:4889. doi:
h ps://doi.o g/10.1038/s41467-019-12842-x.
Bushdid C, Magnasco MO, Vosshall LB, Kelle A (2014)
Humans can disc imina e mo e han one illion ol ac o y s imuli.
Science 343(6177): 1370–1372. doi: 10.1126/science.1249168.
C, Axel R (1995) A no el amily o genes encoding pu a i e
phe omone ecep o s in mammals. Cell 83(2):195-206. doi:
10.1016/0092-8674(95)90161-2.
Caldwella JD, Londea K, Ochsa SD, Hajdua Z, Rodewaldb A,
Gebha b VM, e al. (2019) Th ee s e oid-binding globulins, hei
localiza ion in he b ain and nose, and wha hey migh be doing
he e. S e oids 142:48-54.
Cama go AP, Nakaha a TS, Fi mino LER, Ne o PHM, do
Nascimen o JBP, Donna d ER e al. (2019) Unco e ing he mouse
ol ac o y long non-coding ansc ip ome wi h a no el machine-
lea ning model. DNA Resea ch 26(4):365–378. doi:
h ps://doi.o g/10.1093/dna es/dsz015.
Cansle HL, Maksimo a MA, Meeks JP (2017) Expe ience-
dependen plas ici y in accesso y ol ac o y bulb in e neu ons
ollowing male-male social in e ac ion. J Neu osci 37(30):7240-
7252. doi: 10.1523/JNEUROSCI.1031-17.2017.
Ca doso-Mo ei a M, Halbe J, Vallo on D, Vel en B, Chen C,
Shao Y e al. (2019) Gene exp ession ac oss mammalian o gan
de elopmen . Na u e 571(7766):505–509. doi: 10.1038/s41586-019-
1338-5.
Ca nei o M, Rubin CJ, Di Palma F, Albe FW, Al öldi J,
Ma inez Ba ia A e al. (2014) Rabbi genome analysis e eals a
polygenic basis o pheno ypic change du ing domes ica ion.
Science 345(6200):1074–1079. doi: 10.1126/science.1253714.
Cas ellini C, Dal-Bosco A, A ias-Ál a ez M, Lo enzo PL,
REFERENCES
345
Ca dinali R, Ga cia-Rebolla P (2010) The main ac o s a ec ing
he ep oduc i e pe o mance o abbi does: A e iew. An Rep
Sci 122:174 182. doi: 10.1016/j.ani ep osci.2010.10.003.
Ce an es MP, Palomino JM, Adams GP (2015) In i o imaging
in he abbi as a model o he s udy o o ula ion-inducing
ac o s. Labo a o y Animals 49(1):1–9. doi:
10.1177/0023677214547406.
Chame o P, Leinde s-Zu all T, Zu all F (2012) F om genes o
social communica ion: molecula sensing by he ome onasal
o gan. T ends in Neu osci 35(10): 597-606. doi:
10.1016/j. ins.2012.04.011.
Chame o P, Ma on TF, Logan DW, Flanagan K, C uz JR,
Sagha elian A e al. (2007) Iden i ica ion o p o ein phe omones
ha p omo e agg essi e beha iou . Na u e 450: 899–902. doi:
10.1038/na u e05997.
Cha ko aki G, Wang Y, McAnd ews M, B u o d EA, Thompson
DC, Vasiliou V e al. (2019) Upda e on he human and mouse
lipocalin (LCN) gene amily, including e idence he mouse Mup
clus e is esul o an “e olu iona y bloom”. Hum Genomics 13:11.
doi: 10.1186/s40246-019-0191-9.
Cha a R, Da iche F, Cas hano A, Gigo V, Schaal B, Cou eaud G
(2012) B ain p ocessing o he mamma y phe omone in newbo n
abbi s. Beha B ain Res 226:179–188. doi:
h ps://doi.o g/10.1016/j.bb .2011.09.008.
Cha a R, Da iche F, Gigo V, Schaal B, Cou eaud G (2013)
Phe omone-induced odo lea ning modi ies Fos exp ession in he
newbo n abbi b ain. Beha B ain Res 237, 129–140.
doi: 10.1016/j.bb .2012.09.017.
Cheh ehasa F, Ekbe g JA, S John JA (2014) A no el me hod
using in anasal deli e y o EdU demons a es ha accesso y
ol ac o y enshea hing cells espond o inju y by p oli e a ion.
Neu osci Le 563:90–95. doi:
h ps://doi.o g/10.1016/j.neule .2014.01.043.

PAULA RODRÍGUEZ VILLAMAYOR
346
Chen P, Hong W (2018) Neu al ci cui mechanisms o social
beha iou . Neu on 98(1):16-30. doi: 10.1016/j.neu on.2018.02.026.
Chenowe h PJ, Landae a-He nández AJ, Flöe cke C (2014)
Rep oduc i e and ma e nal beha iou o li es ock (chap e 5). In:
Gene Beha Domes Anim, 2nd Ed. Academic P ess, 5:158–194. doi:
h p://dx.doi.o g/10.1016/B978-0-12-394586-0.00005-6.
Che ian S, Lam YW, McDaniels I, S uziak M, Delay RJ (2014)
Es adiol apidly modula es odo esponses in mouse ome onasal
senso y neu ons. Neu oscience, 269:43-58. doi:
10.1016/j.neu oscience.2014.03.011.
Chess A, Simon I, Ceda H, Axel R (1994) Allelic inac i a ion
egula es ol ac o y ecep o gene exp ession. Cell 78:823-834. doi:
10.1016/s0092-8674(94)90562-2.
Choudha y S, Lal-Kamboj M (2019) E ec o bull
bios imula ion on he oes ous beha iou o pube al Sahiwal (Bos
indicus) hei e s. An Rep Sci 209:106149. doi:
h ps://doi.o g/10.1016/j.ani ep osci.2019.106149.
Clancy AN, Mac ides F, Singe A, Agos a WC (1984) Male
hams e copula o y esponses o a high molecula weigh ac ion
o aginal discha ge: e ec s o ome onasal o gan emo al.
Physiol Beha 33:653–60. doi: 10.1016/0031-9384(84)90386-x.
Clou ie JF, Sahay A, Chang EC, Tessie -La igne M, Dulac C,
Kolodkin AL e al. (2004) di e en ial equi emen s o semapho in
3F and sli -1 in axonal a ge ing, ascicula ion, and seg ega ion o
ol ac o y senso y neu on p ojec ions. J Neu osci 24(41): 9087-96.
doi: 10.1523/JNEUROSCI.2786-04.2004.
Commi ee on Ag icul u e and Ru al De elopmen (2017)
Eu opean Pa liamen . 2016/2077(INI). Repo on minimum
s anda ds o he p o ec ion o a m abbi s.
h ps://www.eu opa l.eu opa.eu/doceo/documen /A-8-2017-
0011_EN.pd .
Conno J (1972) Ol ac o y con ol o agg essi e and sexual
beha io in he mouse (Mus musculus L.). Psychon Sci 27(1):1–3.
REFERENCES
347
Coombes HA, S ockley P, Hu s , JL (2018) Female chemical
signalling unde lying ep oduc ion in mammals. J Chem Ecol
44:851–873. doi: h ps://doi.o g/10.1007/s10886-018-0981-x.
Coope WE, Bu gha d GM (1990) Vome ol ac ion and
omodo . J Chem Ecol 16(1):103-5. doi: 10.1007/BF01021271.
Cou eaud G, Cha a R, Da iche F, Sinding C, Thomas-Danguin T,
Languille S e al. (2010) A phe omone o beha e, a phe omone o
lea n: he abbi mamma y phe omone. J Comp Physiol A 196:
779-790. doi: 10.1007/s00359-010-0548-y.
C oss SKJ, Ma in YH, Salia S, Gamba I, Majo CA, Hassan S, e
al. (2021). Pube y is a c i ical pe iod o ome onasal o gan
media ion o socio-sexual beha io in mice. F on Beha Neu osci
14:606788. doi: 10.3389/ nbeh.2020.606788.
Culle e M, Zo e AD (2018) Rabbi mea p oduc ion and
consump ion: s a e o knowledge and u u e pe spec i es. Mea Sci
143:137-146. doi: h ps://doi.o g/10.1016/j.mea sci.2018.04.029.
D'Aniello B, Semin GR, Scandu a A, Pinelli C (2017) The
ome onasal o gan: a neglec ed o gan. F on Neu oana 11:70.
doi: 10.3389/ nana.2017.00070.
Daune K, Liβmann J, Je idi S, F ings S, Möh len (2012)
Exp ession pa e ns o anoc amin 1 and anoc amin 2 chlo ide
channels in he mammalian nose. Cell Tissue Res 347: 327-41. doi:
10.1007/s00441-012-1324-9.
DeCa anza o, D (2015) Sex s e oids as phe omones in
mammals: The excep ional ole o es adiol. Ho m Beha 68:103–
116. doi: h p://dx.doi.o g/10.1016/j.yhbeh.2014.08.003.
Degl’Innocen i A, Meloni G, Mazzolai B, Cio ani G (2019) A
pu ely bioin o ma ic pipeline o he p edic ion o mammalian
odo an ecep o gene enhance s. BMC Bioin o ma ics 20:474. doi:
h ps://doi.o g/10.1186/s12859-019-3012-1.
Dehmel L, Halpain S (2005) The MAP2/Tau amily o
mic o ubuleassocia ed p o eins. Genome Biol 6:204. doi:
10.1186/gb-2004-6-1-204.
PAULA RODRÍGUEZ VILLAMAYOR
348
Del Pun a K, Leinde s-Zu all T, Rod iguez I, Jukam D, Wysocki
CJ, Ogawa S, e al. (2002) De icien phe omone esponses in mice
lacking a clus e o ome onasal ecep o genes. Na u e 419:70–74.
doi: 10.1038/na u e00955.
Del Pun a K, Ro hman A, Rod iguez I, Mombae s, P (2000)
Sequence di e si y and genomic o ganiza ion o ome onasal
ecep o genes in he mouse. Genome Res 10:1958–1967. doi:
10.1101/g .10.12.1958.
Delc oix I, Mauge R, Signo e JP (1990) Exis ence o
synch oniza ion o ep oduc ion a he le el o he social g oup o
he Eu opean wild boa (Sus sc o a). J Rep od Fe il 89:613–617.
doi: 10.1530/j .0.0890613.
Demi E, Li K, Bob owski-Khou y N, Sande s JI, Beynon RJ,
Hu s JL e al. (2020) The phe omone da cin d i es a ci cui o
inna e and ein o ced beha iou s. Na u e 578:137-159. doi:
10.1038/s41586-020-1967-8.
Dennis JC, Smi h TD, Bha naga KP, Bona CJ, Bu ows AM,
Mo ison EE (2004) Exp ession o neu onspeci ic ma ke s by he
ome onasal neu oepi helium in six species o p ima es. Ana Rec
281:1190–1200. doi: 10.1002/a .a.20124.
Dennis JC, S ilwell NK, Smi h TD, Pa k TJ, Bha naga KP,
Mo ison EE (2019) Is he mole a ome onasal o gan unc ional?
Ana Rec 303(2):318-329. doi: 10.1002/a .24060.
Dewan A (2021) Ol ac o y signaling ia ace amine-
associa ed ecep o s. Cell Tissue Res 383(1):395–407.
doi:10.1007/s00441-020-03331-5.
Dewan A, Cichy A, Zhang J, Miguel K, Feins ein P, Rinbe g D,
e al. (2018) Single ol ac o y ecep o s se odo de ec ion
h esholds. Na Comm 9:2887. doi: 10.1038/s41467-018-05129-0.
Dey S, Chame o P, P u JK, Chien M-S, Iba a-So ia X, Spence
KR, e al. (2015) Cyclic egula ion o senso y pe cep ion by a
emale ho mone al e s beha iou . Cell 161: 1334–44. doi:
10.1016/j.cell.2015.04.052.
REFERENCES
349
Díaz D, Muñoz-Cas añeda R, Á ila-Za za C, Ca e e o J, Alonso
JR, We uaga E (2017) Ol ac o y bulb plas ici y ensu es p ope
ol ac ion a e se e e impai men in pos na al neu ogenesis. Sci
Rep 7(1):5654. doi: 10.1038/s41598-017-05970-1.
Die schi Q, Assens A, Challe L, Ca le on A, Rod iguez I (2013)
Con e gence o FPR- s3-exp essing neu ons in he mouse
accesso y ol ac o y bulb. Mol Cell Neu osci 56:140–147. doi:
10.1016/j.mcn.2013.04.008.
Die schi Q, Tube osa J, Rösingh L, Loicho G, Ruedi M, Ca le on
A (2017) E olu ion o immune chemo ecep o s in o senso s o he
ou side wo ld. PNAS 114(28):7397–7402. doi:
h ps://doi.o g/10.1073/pnas.1704009114.
Dinka H, Le MT, Ha H, Cho H, Choi MK, Choi H e al. (2016)
Analysis o he ome onasal ecep o epe oi e, exp ession and
allelic di e si y in swine. Genomics 107: 208-15. doi:
10.1016/j.ygeno.2015.10.003.
Di ich K, Ku le J, Hassenklo e T, Manzini I (2016)
Me amo phic emodeling o he ol ac o y o gan o he A ican
clawed og, Xenopus lae is. J Comp Neu ol 524:986–998. doi:
10.1002/cne.23887.
Dobin A, Da is CA, Schlesinge F, D enkow J, Zaleski C, Jha S
e al. (2013) STAR: ul a as uni e sal RNA-seq aligne .
Bioin o ma ics 29:15–21. doi:
h ps://doi.o g/10.1093/bioin o ma ics/b s635.
Dominguez-Pé ez D, Du ban J, Agüe o-Chapin G, To es-López
J, Molina-Ruiz R e al. (2019) The Ha de ian gland ansc ip omes
o Ca aiba and eae, Cubophis can he ige us and T e ano hinus
a iabilis, h ee colub oid snakes om Cuba. Genomics
111(6):1720-1727. doi: h ps://doi.o g/10.1016/j.ygeno.2018.11.026.
Dong HW, Pe o ich GD, Swanson LW (2001) Topog aphy o
p ojec ions om amygdala o bed nuclei o he s ia e minalis.
B ain Res B ain Res Re 38:192–246. doi: 10.1016/s0165-
0173(01)00079-0.