Intersexuality in pigs: Impact on veterinary public health and food safety

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©1994‐2009#All#Rights#Reserved.#Online#Journal#of#Veterinary#Research
OJVRTM
Online#Journal#of#Veterinary#Research©!
!
Volume#13#(1):63‐75,#2009
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Intersexuality#in#pigs:Impact#on#veterinary#public#health#and#food#safety#
!
Cornillie#P1#(DVM,#PhD),#Mortier#V2#(Lic),#Verheyden#K2#(MEng),#Simoens#P1#(Prof,#PhD,#HD),#De#
Brabander#HF2#(Prof,#PhD,#PhD),#Vanhaecke#L2#(MEng,#PhD)#
#
1Department#of#Morphology,#Faculty#of#Veterinary#Medicine,#Ghent#University,2Research#group#Veterinary#Public#
Health#and#Zoonoses,#Department#of#Veterinary#Public#Health#and#Food#Safety,#Lab#chemical#analysis,#Faculty#of#
Veterinary#Medicine,#Ghent#University,#Salisburylaan#133,#B‐9820#Merelbeke#Pieter.Cornillie@UGent.be#
!
ABSTRACT#
#
!"#$%&&%'()*( +"#,%'#(-*( -'#.'/0'$(1*( 2%3"'$4()*( 5'(6#787$0'#( 9:*(-7$.7';<'( =*(>$,'#4'?@7&%,/( %$(
A%B4C>3A7;,( "$( D','#%$7#/( A@8&%;( .'7&,.( 7$0( E""0( 47E',/*(F$&%$'( G( -',( H'4( IJKILCMJNOP*( QRRST(
During#a#routine#inspection#on#a#pig#farm,#nortestosterone#was#detected#unexpectedly#in#faecal#
samples# of# sows.# An# intersexual# pig,# held# responsible# for# this# finding,# was# slaughtered# and#
investigated.#Macroscopic#inspection#revealed#a#mainly#female#reproductive#tract#consisting#of#a#
normally# developed# caudal# part# including# a# vagina,# cervix# and# uterus,# and# on# the# left# side# a#
normal#ovary#and# uterine#tube.#However,#on# the#right#side#a# hypoplastic#testis#and# epididymis#
were#found#together#with# a#well‐developed#plexus#pampiniformis‐like#structure.# Histologically,#
no# spermatozoa# were# found# in# this# testis,# but# Leydig# cells# were# numerous.# Chemical# analysis#
showed#the#presence#of#17β‐nortestosterone#in#the#urine,#fat,#kidneys#and#testicular# tissue,#as#
well#as#the#precursor#noradrenostenedione#in#the#urine,#kidneys#and#testis.#As#intersexuality#is#a#
frequent# phenomenon# in# pigs,# veterinarians# who# are# responsible# for# veterinary# public# health#
and#food#safety#must#be#aware#of#this#phenomenon#which#may#interfere#with#the#control#on#the#
abuse#of#illegal#growth#promoters.##
#
Key#words:!Nortestosterone,#Endogenous#source,#Hermafroditism,#Intersex,#Sus#scrofa#
!
INTRODUCTION#
#
The# European# Commission# regulates# the# screening# measure# for# the# presence# of# residues# and#
contaminants#in#slaughter#animals#of#the#European#Union#(Directive#96/23/EC).#This#regulation#
was#already# implemented#in#Belgium#by#the#federal#act#of#15th#of#July#1985#concerning#the#use#
of# drugs# having# a# hormonal,# anti‐hormonal,# β‐adrenergic# or# production# stimulating# activity# in#
animals.#In#contrast#to# most#other#EU#countries,#Belgium# has#a#regular#monitoring#program# of#
pig#farms#on#which#faeces#samples#are#collected#instead#of#urine.#During#a#routine#examination#
on#a#pig#farm,#17β‐nortestosteron#(17β‐NT)#was#identified#in#faeces# samples# of# an# apparently#
female#animal.#Since# the#presence#of#17β‐NT# in# pigs#other#than#boars# was#not#documented#at#

64#
that#time,#a#possible#illegal#use#of#17β‐NT#as#growth#promoter#was#suspected.#Further#research#
however,#showed# the#presence#of#an#intersexual#pig#on#the#farm.#The#first#results#of#this#study#
were#published#in# the#Flemish#Veterinary#Journal#(in#Dutch)#(Van# Cruchten#et#al# 2002)#and#are#
now#completed#with#findings#resulting#from#additional#experiments.#
!
U"#,'4,"4,'#"$'C(NT# is#a#steroid#with#two#isoforms#(17α‐NT#and#17βNT),#which#belongs#
to# the# group# A‐substances# (banned# substances)# according# to# the# Council# Directive#
96/23/EC.# The# 17β# form# of# NT# is# also# one# of# the# most# powerful# androgenic# anabolic#
steroids.# In# the# past# 17β‐NT# was# often# used# for# fattening# purposes,# because# of# its#
positive#effects#on#improved#weight#gain#and#feed#efficiency#in#animals.#Its#use#has#also#
been#reported#in#human#sports#doping.
Until#the#early#eighties,#whenever#residues#of#17β‐NT#or#its#metabolites#were#identified#in#urine#
of#oxes,#race#horses,#or#human# athletes#including#body‐builders,# the#exogenous#administration#
was# considered# to# be# proven,# since# literature# did# not# contain# evidence# of# the# endogenous#
character# of# this# molecule# in# cattle,# horses# and# men.# The# chemical# structures# of# 17β‐NT,# its#
precursor#19‐norandrostenedione#(NAED)#and#its#metabolites#are#illustrated#in#Image#1,#below.##
#
#
These#products#were#previously#only#detected#in#animals#which#have#been#treated#with#17β‐NT#
or#one#of#its#derivates#(e.g.#esters#of#NT#or#nandrolone#(Laurabolin®#or#Durabolin®))#(Delahaut#et#
al#1988,#Daeseleire#et#al#1993,#McEvoy#1998a,#McEvoy#1999a).#
#
However,#in#1984,#Houghton#demonstrated#that#17β‐NT#was#naturally#present#in#the#urine#of#
stallions.#A#short#time#afterwards,#Belgian#and#Dutch#investigators#found#that#17β‐NT#is#present#
in#urine# and#edible#tissues#of#boars.#These#results#were#first#discussed#in#workgroups#and#were#
Image!1!‐#Chemical#structures#of#17β‐NT,#its#precursor#NAED#and#its#metabolites#

65#
published#afterwards#by#Maghuin‐Rogister#(1988),#van#Ginkel#et#al#(1989)#and#Debruyckere#et#al#
(1990).#In# this#context,#it#is#important#to#mention#that#an# isomerase#to#convert#17β‐NT#to#17α‐
NT#is#absent#in#the#pig.#In#cattle#on#the#other#hand,#this#isomerase#is#present#and#the#17α‐form#is#
the#most#abundant#isoform#found#in#bovine#urine.#In#horses,#both#isomers#are#detected.#In#1993#
Rizzo# and# others# reported# the# endogenous# origin# of# 17β‐NT# by# the# analysis# of# boar# testicles.#
Since#then,#the#presence#of#17β‐NT#in#boar#matrices#is#no#longer#considered#as#of#exogenous#
origin.##
#
The#problematic#nature#of#NT#in#bovines#has#also#gained#much#attention.#The#first#observations#
on# the# endogenous# presence# of# 17β‐NT# in# calves# were# based# on# an# analytical# artefact# and#
confusion#between# 17β‐testosterone#and#17β‐NT#(De#Ridder#1989).#Rapp#and#Meyer#described#
in#1989#the#possibility#of#17β‐NT#being#excreted#by#non‐treated#calves#fed#with#milk#substitutes#
contaminated#with#17β‐NT.#Vandenbroeck#et#al#(1991)#were#the#first#to#suggest#the#endogenous#
presence#of#17β‐NT#(and#not#17α‐NT)#in#the#urine#of#pregnant#cows.#Meyer#et#al#(1992)#reported#
the#presence#of#relatively#high#concentrations#of#17α‐NT#in#the#urine#of#a#periparturient#cow#and#
in#the#newborn#calf.#17α‐NT#has#also#been#detected#in#pregnant#animals#of#other#species#(Clouet#
et# al# 1997,# McEvoy# et# al# 1998b).# The# testicular# production# of# nortestosterone# has# also# been#
demonstrated#in#several#animal#species,#including#the#boar#and#stallion,#but#not#yet#in#the#bull#
(McEvoy# et# al# 1999b).# Most# of# these# findings# could# be# confirmed# and# supplemented# with#
additional#data#during#a#large‐scale#experiment#involving#several#European#residue#laboratories#
(De#Brabander#et#al#1994,#2004;#Poelmans#et#al#2005).##
#
During# an# antidoping# control# in# sport# in# 1997,# evidence# was# gained# for# the# presence# of#
endogenous# 19‐nortestosterone# and# its# metabolites# 19‐norandrosterone# and# 19‐
noretiocholanolone#in#human# urine# (Le#Bizec#et#al# 1999).#A#new#NT#problem# emerged#in#1999#
when#two#long# distance#swimmers#were#accused# of#abusing#NT# during#a#competition#in# Brazil.#
Both#athletes# claimed#not#to#have#abused#drugs#and#ascribed#the#positive#results#of#their#urine#
sample# to# the# consumption# of# “Sarapatel”,# a# typical# Brazilian# dish# based# on# pork# which# may#
contain#intact# testicular# tissue# of# boars.# The# attorneys# of# the# swimmers# called# in# advice# from#
experts,#who#carried#out#some#experiments#including#the#consumption#of#meat#and#organs#of#a#
mature# boar# by# three# volunteers# (Le# Bizec# et# al# 2000).# During# approximately# one# day# the#
metabolites#17β‐NT,#19‐norandrosterone# and#19‐noretiocholanolone#could#be# identified#in#the#
urine# samples# of# the# three# test# subjects# in# a# concentration# ># 2# µg.kg‐1# (official# International#
Olympic# Committee# cut‐off# level# for# norandrosterone).# In# the# meat# and# organs# of# boars,#
including#the#consumed# animal# 17β‐NT#and#its#precursor# 19‐norandrostenedione!(NAED)#were#
identified#(De#Wasch#et#al#2001).#NAED#is#a#reduced#testosterone#precursor#which#does#not#carry#
a# methyl# group# in# the# 19th# position.# When# the# NAED# molecule# is# processed# by# the# liver,# a#
reduction#of#the#17‐keto#group#occurs# leading#to#17β‐NT# (17β‐hydroxysteroid#dehydrogenase).#
Recently,#new#data#on#NT#in#the#horse#were#described#(Houghton#et#al#2004).#19‐Norandrost‐4‐
ene‐3,17‐dione# detected# in# urine# from# a# fertile# stallion#following# a# solvolysis# procedure# may#
arise# as# an# artefact# of# decarboxylation# of# a# precursor# 19‐carboxylic# acid#(Image# 2).# A# similar#
conclusion#was#drawn#for#the#presence#of#19‐NT.##
#

66#
#
>$,'#4'?@7&%,/C( An# intersex# is# defined# as# an# individual# having# both# male# and# female#
sexual# characteristics# and# organs.# This# phenomenon# occurs# in# all# domestic# animal#
species,# but# is# most# frequently# encountered# in# cattle,# goats# and# pigs# (Koch# 1961).# A#
distinction#can#be# made#between#true#hermaphrodites#(9'#37A.#"0%,%43@4( D'#@4)#and#
pseudo‐hermaphrodites# ()4'@0".'#37A.#"0%,%43@4).# True# hermaphrodites# possess#
gonadal#tissue#of#both#genders,#viz.#one#ovary#and#one#testis#(lateral#hermaphroditism),#
or# two# ovotestes# both# containing# testicular# as# well# as# ovarian# tissue# (bilateral#
hermafroditism),# or# a# combination# of# one# testis# or# ovary# with# an# ovotestis# (unilateral#
hermafroditism)#(Halina# et# al# 1984,# Bansal# et# al# 2005).# Pseudohermaphrodites# only#
contain# gonads# of# one# single# gender,# mostly# opposite# to# the# animal’s# external# sexual#
characteristics.# Depending# on# whether# these# gonads# are# testes# or# ovaries,# such#
individuals# are# called# )4'@0".'#37A.#"0%,%43@4( 374;@&%$@4( or#
)4'@0".'#37A.#"0%,%43@4(E'3%$%$@4*#respectively#(Cribiu#and#Chaffaux#1990).#The#term#
“:#''37#,%$%43”# is# also# often# used# in# literature.# This# form# of# intersexuality# is# almost#
exclusively#found#in#cattle,#but#has#also#sporadically#been#reported#in#sheep,#goats,#deer,#
pigs,# horses# and# camels# (Padula# 2005).# Freemartins# are# virilized# female# animals# and#
originate#when#a#female# foetus# gets# XY‐leukocytes# and# male# hormones# from# her# twin#
brother# by# vascular# anastomoses# in# the# chorioallantois# (Ladds# 1993).# When# single#
freemartins#are#born#without#twin#sibling,#they#are#supposed#to#be#the#result#of#an#early#
foetal#loss#of#the#male#calf#co‐twin#(Padula#2005).#
#
Intersexuality# is# a# frequent# phenomenon# in# pigs# and# affects# 0.5%# of# the# European# porcine#
population#(Pinton# et# al# 2002).# Most# intersexual# pigs# have# a# female# phenotype# (XX# /# SRY#
negative)#whilst#XX/XY#chimeric#pigs#are#rare#(less#than#5%#of#the#intersexes)#(Hunter#and#Greve#
1996).#Their#morphological#appearance#is#characterised#by#the#presence#of#an#enlarged#clitoris,#
often#enveloped#by#a#prepuce,#and#a#dorsally#directed#ventral#commissure#of#the#vulva#(Image#3,#
next#page).##
Image!2#‐#Decarboxylation#of#OxAED#with#formation#of#NAED#and#17αNT#

67#
#
#
While#the#gonads#may# show# all# variations#as#described#above,#the# morphology# of# the#internal#
reproductive#organs#in#intersexual#pigs#can#assume#all#intermediate#stages#between#females#and#
males.# As# a# result,# the# uterine# horns# and# body# can# either# be# normal,# hypoplastic# or# even#
aplastic,#and#they#can#co‐exist#with#or#be#replaced#by#a#pair#of#deferent#ducts#merging#with#the#
caudal#part#of#the#vagina.#In#the#case#of#male#pseudohermaphroditism,#both#testes#can#either#be#
located#in#their#original#intra‐abdominal#position#(cryptorchidism)#or#can#have#descended#into#an#
inguinal#or#scrotal#position#(personal#observations#and#Halina#et#al#1984,#Świtoński#et#al#2002).##
#
Intersexuality#in#pigs#is#supposed# to# be# a# hereditary#condition.#In#isolated#herds#of# pigs# in# the#
northern# islands# of# the# Vanuatu# archipel,# where# intersexual# pigs# were# considered# to# be# holy#
animals#by#the# native#Melanesians,#the#incidence# of#male#pseudohermaphroditism# reached#up#
to# 10# to# 20# %# of# all# boars# due# to# selective# breeding# (Pacchioli# 1997).# An# autosomal#recessive#
gene# is# suggested# to# be# the# main# cause# of# intersexuality# in# pigs# (Parma# 1999,# Pailhoux# et# al#
2001,#Pinton#et#al#2002,#Świtoński#et#al#2002).#Pinton#et#al#(2002)#showed#that#intersexuality#in#
pigs#is#correlated#to#an#anomaly#on#chromosome#9.#However,#more#research#is#still#required#to#
further#elucidate#this#genetic#component.##
#
Most#intersexual#pigs#are#infertile.#They#often#show#aggressive#behaviour,#have#a#more#rigid#and#
hairy#skin#and#can#spread#the#typical#boar#odour#from#the#age#of#puberty#onwards#(Hunter#and#
Greve#1996).#These#qualities#can#form#a#major#economic#loss#in#the#pig#industry.#Furthermore,#
these# animals# can# show# increased# levels# of# steroidal# hormones# that# can# be# mistaken# for#
exogenously#administered#hormones,#resulting#in#severe#sanctions#for#the#farmer.#
#
Image!3#‐#Typical#appearance#of#the#external#genital#organs#of#an#intersex#pig,#a:#left#
lateral# view# showing# the# enlarged# ventral# commissure# of# the# vulva# orientated# in#
dorsal# direction,# b:# The# enlarged# clitoris# is# clearly# visible# after# pulling# the# ventral#
commissure#of#the#vulva#downwards#(caudal#view).#

68#
MATERIAL#AND#METHODS#
(
+"#A."&"B%;7&( 7$7&/4%4C( The# intersexual# pig# was# slaughtered# at# the# local# farm# and# its# internal#
genital# organs,# together# with# samples# of# fat,# muscle,# liver,# kidneys,# urine# and# faeces,# were#
collected# for# further# morphological# and# chemical# analysis# respectively.# The# vulva# and# clitoris#
could#not#be#investigated#because# these#structures#were# resected#early#during#slaughter.#After#
macroscopic# photography# of# the# reproductive# system,# all# genital# organs# were# sampled# for#
histological# analysis.# The# tissue# samples# were# fixated# in# a# phosphate‐buffered# 3.5# %#
formaldehyde#solution#during#24# hours.#After#fixation#the#samples# were#embedded#in#paraffin#
by#an#automated#system#(Shandon#Citadel#Tissue#Processor,#Cheshire,#UK)#and#subsequently#cut#
in#8#µm#thick#sections#and#stained#with#haematoxylin#and#eosin#(H.E.),#PAS#and#Van#Gieson.#The#
sections#were#analysed#with#an#Olympus#BX61#light#microscope#provided#with#an#Olympus#DP50#
digital#camera#(Olympus,#Tokyo,#Japan).#
#
!.'3%;7&( 7$7&/4%4C( Different# methods,# depending# on# the# matrix,# were# developed# for# the#
extraction# and# purification# steps# prior# to# chemical# analysis.# Five# grams# of# meat# and# testicle#
matrices# were# digested# with# subtilisine,# extracted# with# n‐hexane# and# purified# onto# an#
aminopropyl#solid# phase#extraction#(SPE)#cartridge.#Twenty‐five#grams#of#liver#and# kidney#were#
directly# extracted# by# solvent# and# purified# onto# an# aminopropyl# SPE# cartridge.# Twenty‐five#
millilitres# of# urine# were# hydroysed# with# glucuronidase‐sulfatase# before# performing# a# liquid‐
liquid#extraction# (LLE).#Purification#was#performed#onto#an#aminopropyl#SPE#column#and#finally#
fractionated# onto# a# HPLC# system.# Derivatization# was# achieved# with# twenty# µl# N‐trimethyl‐
silyltrifluoroacteamide# (MSTFA)/trimethyliodosilane# (TMIS)/# dithioerythriol# (DTE)#
(1000:5:5,v/v/w).##
#
The#chemical#analysis#of#nortestosterone#and#noradrenostenedione#in#fat,#urine#and#faeces#was#
carried# out# by# gas# chromatography# followed# by# multiple# mass# spectrometry# (GC‐MSn).# The#
chromatographic# and# spectrometric# analyses# were# performed# using# a# Trace# GC# 2000# Gas#
Chromatograph#fitted#with# a#Polaris#ion#trap# mass#spectrometer#(Thermo#Finnigan,# Austin,#TX,#
USA)#with#a#Carlo#Erba#autosampler#AS2000# (Thermo#Finnigan,#Austin,#TX,#USA).# Helium#(99.99#
%# purity,# Air# Liquide,# France)# was# used# as# carrier# gas# at# a# flow# rate# of# 1# ml.min‐1.# FC43#
(Perfluorotributylamine)#(Ultra#Scientific,#North#Kingstown,# USA)#was#used# as#calibration#gas.# A#
volume#of#1#µl#was#injected#(spit#flow#60#ml/min,#splitless#time#1#min).#Separation#of#the#target#
analytes#was#performed#on#a#BPX‐5#(SGE#Inc.,#Austin,#TX,#USA)#(25#m#x#0.22#mm#I.D.)#fused#silica#
capillary# column# with# 5# %# phenyl‐polysilphenylene‐siloxane# liquid# phase# (film# thickness# 0.25#
µm).#Injector,#ion#source#and#transfer#line#temperature#were#respectively#250#°C,#200#°C#and#275#
°C.#Temperature#program:#initial#100#°C;#ramp#at#17#°C.min‐1#to#250#°C;#ramp#at#2#°C.min‐1#to#300#
°C#(hold#1.30#min).#The#spectra#were#obtained#in#Electron#Impact#(EI)#mode#at#70#eV.#
#
Analysis# of# liver,# kidneys,# muscles# and# testes# for# these# substances# was# performed# by# liquid#
chromatography# again# coupled# to# a# multiple# mass# spectrometric# analysis# (LC‐MSn).##
Chromatographic#separation#was#achieved#using#a#Symmetry#C18#column#(Waters,#5#µm,#150#mm#
x#2.1#mm).#A#1100#series#quaternary#gradient#pump#(Agilent,#Palo#Alto,#CA,#USA)#was#used.#The#
mobile#phase#(methanol:#A/0.02# M# formic# acid#in#water:#B;#55:45)# was# maintained# for#20#min#
and#increased#to#A/B;#100:0#(v;v)#in#4#min.#The#flow#rate#was#0.3#ml/min.#The#MS#detector#was#a#
Finnigan# LCQ# deca# ion# trap# (ThermoFinnigan,# San# Jose,# CA,# USA)# equipped# with# an# APCI#
interface#in#the#positive#ion#mode#MS/MS#full#scan.#
!

69#
RESULTS#
!
Anatomical# and# histological# characteristics:# Macroscopical# examination# of# the# female#
reproductive#system#(Image#4,#below)#showed#a#well#developed#vestibulum#vaginae#and#
vagina#as#well#as#a#normal#uterus#comprising#the#uterine#cervix,#body#and#two#horns.#On#
the#left#side,#a#well#developed#ovary#with#a#normal#uterine#tube#were#identified,#but#on#
the#right#side#a#testis,#epididymis#and#a#deferent#duct#with#a#large#plexus#pampiniformis‐
like#structure#could#be#observed.#A#thin,#tubular#structure#was#found#between#the#testis#
and#epididymis,#but#could#not#be#further#identified#macroscopically.#
#
#
The# vagina,# uterus# and# the# single# uterine# tube# all# had# a# normal# histological# constitution.# The#
left‐sided#ovary#consisted# on# histological#examination#of#a# peripheral#cortex#with#many#atretic#
Image! 4! ‐# Dorsal# view# of# the# internal# genital# organs# of# the# intersex# pig.# The# female#
components#consist#of#the# vestibulum# vaginae#(1),#vagina#(2),#cervix# uteri# (3),# corpus#uteri#
(4),#both#cornua#uteri#(5),#left#ovary#(6)#and#left#uterine#tube#(7).#The#male#components#are#a#
hypoplastic# right# testis# (8)# and# epididymis# (9),# provided# with# a# plexus# pampiniform‐like#
structure# (10).# The# urinary# bladder# (11)# is# also# present.# Insert# a:# detail# of# the# hypoplastic#
testis# and# epididymis.# The# arrowheads# points# to# a# thin,# undefined# tubular# structure# in#
between#both#organs.#Insert# b:#detail#of#the#ovary.#Some#vesicular#follicles#are#indicated#by#
white#arrows.#

70#
and#some#normal#vesicular#follicles,#and#a#central#medulla#with#a#normal#histological#appearance#
(Image#5,#below).##
#
#
On#the#histological#sections#of#the#right‐sided#testicle,#some#seminiferous#tubules# could#clearly#
be#identified,#but#they# were#less#densely#distributed#within#the#testicular#tissue#compared#with#
sections#of#normal#adult#boar#testicles#(Image#6).# The# walls# of# the# tubules# consisted# only#of#a#
single# layer# of# cells,# in# which# sustentacular# Sertoli# cells# could# not# be# distinguished# from#
spermatogonia.# The# seminiferous# tubules# did# not# contain# any# spermatozoa.# Interstitial# Leydig#
cells#were#clearly# identifiable# in#the#stroma#between# the# seminiferous#tubules#and#were# often#
clustered#(Image#6).##
Image#5:#Histological#appearance#of#the#ovarian# cortex# of# the# intersex#pig#showing#several#
atretic#follicles#(*).#Haematoxylin#&#eosin#staining,#scale#bar#=#100#µm.#

71#
#
Histology# of# the# ductus# epididymidis# showed# a# normal# pseudostratified# columnar# epithelium,#
but# no# spermatozoa# could# be# detected# in# the# central# lumen.# The# thin# undefined# tubular#
structure#between#the#testis#and#epididymis#consisted#of#an#inner#squamous#epithelium,#a#thick#
layer#of#loose#subepithelial#connective#tissue,#with#many#twisting#PAS‐negative#cell#strings#which#
sometimes#were#surrounding#a#lumen,#and#an#external#smooth#muscle#layer.######
(
!.'3%;7&( 7$7&/4%4:17β‐nortestosterone# was# detected# in# the# fat,# urine,# kidneys# and#
testes# (Table# I,# next# page).# The# concentration# (27# µg.l‐1)# was# highest# in# the# urine# and#
also#relatively#elevated#in#the#testes#(ca.#5#µg.kg‐1).#The#concentrations#in#fat#and#kidneys#
were# lower# and# near# to# the# limit# of# detection.# No# nortestosterone# was# detected# in#
faeces,# liver# and# muscles# (Table# II).# The# precursor# of# nortestosterone,#
norandrostenedione,#was#present#in#low#concentrations#in#the#urine,#kidneys#and#testes.#
The# fat# surrounding# the# kidneys# as# well# as# the# faeces# and# muscles# contained# no#
detectable#concentrations#of#norandrostenedione.
Image# 6:# Histological# appearance# of# the# hypoplastic# testis# of# the# intersex# pig.# The# tubuli#
seminiferi# (*)# consist# of# a# single# layered# epithelium# in# which# spermatogonia# cannot# be#
distinguished#from#Sertoli#cells.#Interstitial#Leydig#cells# (arrows)#are#numerous.#Haematoxylin#
&#eosin#staining,#scale#bar#=#50#µm.

72#
#
DISCUSSION#
!
The# gross# anatomical# and# histological# appearances# of# the# internal# reproductive# organs# of# the#
investigated# intersex# concord# with# that# of# a# real# hermaphrodite.# In# this# intersexual# pig,#
detectable#levels#of#NT#and#NAED#were#demonstrated#in#different#matrices#by#chemical#analysis.#
The# highest# concentration# nortestosterone# was# found# in# urine# (ca.# 27# µg.l‐1),# but# the#
concentration#was#also#high#in#the#testes# (ca.# 5# µg.kg‐1).# The# precursor# of#NT,#NAED,#was#also#
found#in#the#urine,#kidneys#and#testes,#but#the#concentrations#were#lower#than#those#of#NT.#The#
kidney#fat,#the#faeces#and#the#muscle#tissue#didn’t#contain#any#traceable#levels#of#both#products,#
probably# because# the# concentrations# were# lower# than# the# detection# limit# of# the# analytical#
method.##
#
In#an#international#study#performed#by#3#European#laboratories#in#cooperation#with#the#USDA,#a#
well‐documented#overview#of#the#status#of#the#presence#of#17β‐NT,#NAED#and#17β‐boldenone#in#
swine#(i.e.#intersex,#sow#and#boar)#was#made.#Clear#differences#between#the#investigated#tissues#
could# be# observed# in# all# cases# (De# Wasch# et# al# 2001,# Poelmans# et# al# 2005).# The# highest#
concentrations# were# always# measured# in# the# urine,# kidneys# and# testes.# Therefore# it# is#
recommended#to#use#those#matrices#for#the#detection#of#17β‐NT#and#NAED.#Concentrations#are#
much# lower# and# even# below# the# limit#of# detection# in# fat,# faeces,# liver# and# muscle# tissue.#The#
data# illustrate# that# boars# produce# higher# concentrations# of# 17β‐NT# and# NAED# in# comparison#
with# intersexes# and# sows.# NAED# concentrations# were# higher# in# all# investigated# sow# matrices#
than# in# the# matrices# from# intersexual# pigs.# On# the# other# hand,# samples# from# intersexual# pigs#
contain#a#higher#concentration#of#17β‐NT#than#the#17β‐NT#concentration#range#of#sows.##
#
Whenever#the#presence#of# one# or# more#intersexual#pigs#within#a# female# pig# population# is#not#
discovered# during# sampling# for# the# analysis# of# illegal# growth# promoters,# the# pig# breeder# can#
Table!I:#Results#of#the#chemical#detection#of#17‐beta‐19‐nortestosteron#(µg/kg)#in#porcine#matrices##
Sample#
Intersex#
Sow#
Old#boar#
Fat#
ca#0.3##
/#
/#
Urine#
ca#27##
1.3#‐#1.6#
51#‐#344#
Faeces#
‐#
/#
/#
Liver#
‐#
‐#
1#‐#63#
Kidney#
ca#1.6#
0.2#‐#1.5#
2.5#‐#232#
Meat#
‐#
0.5#
0.7#‐#13.4#
Testes#
ca#5.3#
/#
24#‐#144#
‐#=#below#the#limit#of#detection,#/#=#no#material#available#
!
Table!II:#Results#of#the#chemical#detection#of#19‐norandrostenedione#(µg/kg)#in#porcine#matrices##
Sample#
Intersex#
Sow#
Old#boar#
Fat#
‐#
/#
/#
Urine#
ca#0,5##
0.9#‐#18#
5#‐#109#
Faeces#
‐#
/#
/#
Liver#
trace#
3.1#‐#8.3#
0.1#‐#24#
Kidney#
ca#1.1#
2.7#‐#18#
2.3#‐#535#
Meat#
‐#
0.04#
0.1#‐#5.5#
Testes#
ca#0.9#
/#
6.2#‐#110#
‐#=#below#the#limit#of#detection,#/#=#no#material#available#

73#
wrongly#be# suspected#of#illegal#use#of# these#hormones#in#his#lifestock.#Therefore,#a# proper#and#
early# identification# of# intersexual# pigs# is# essential# for# the# correct# interpretation# of# the# results#
drawn#from#the#chemical#and#serological#screening.##
#
One#of#the#initial#indications#of#an#intersex#pig#is# the#typical#anatomy# of#the#vulva.# The#ventral#
commissure#of#the#vulva#is#enlarged#and#in# 80%# of# the# cases# it# is# pointing# in# dorsal#direction,#
making#these#animals#urinate#skywards# (Hunter# and# Greve#1996).#By#spreading#the#lips# of# the#
vulva,# an# enlarged# clitoris# can# be# observed.# Other# obvious# anatomical# characteristics# are# the#
more# hairy# and# rigid# skin.# A# wide# range# of# variations# can# be# observed# in# the# behaviour# of#
intersexes.# Some# animals# have# a# standing# oestrus# like# sows,# while# other# intersexes# exhibit# a#
masculine#behaviour,#act#more#aggressively,#produce#high#amounts#of#saliva#in#the#presence#of#a#
male,#and#show#copulation#behaviour#in#the#presence#of#a#female.#As#hypertrophy#of#the#clitoris#
and#other#male# characteristics#in#presumptive# female#pigs#also#can# be#the#result# of#exogenous#
administration# of# androgens# such# as# testosterone,# methyltestosterone# and# trenbolone# to#
normal#sows,#chemical#analysis#results#must#be#interpreted#cautiously#in#case#of#high#prevalence#
of#the#described#phenotype#in#a#pig#population.##
#
The#detection#of#an#elevated#level# of# nortestosterone# in# presumptive#female#pigs#can#only#be#
suggestive#for# the#(pseudo)hermaphroditic#nature#of#the#animal,#but#it#cannot# entirely#rule#out#
the# exogenous# administration# of# growth# promoters.# In# such# cases,# only# a# morphologic#
examination#of# the#internal#genital#organs#obtained#after#slaughter#can#conclusively#distinguish#
an#intersexual#pig#from#a#sow#exogenously#supplied#with#androgens.##
It#can#be#concluded#that#public#health#inspectors#responsible#for#the#screening#of#illegally#used#
growth#promoters#on#pig#farms,#must#be#aware#of#the#possible#presence#of#intersexes#and#must#
have#knowledge#of#the#anatomical#characteristics#and#behaviour#pattern#of#these#animals,#as#to#
be#able#to#recognise#them.#Every#suspected#sample# should#be#correlated# to#gender#and# age#of#
the# animal# in# question.# If# intersexuality# is# suspected# from# laboratory# analysis,# it# should# be#
confirmed#by#further#morphological#analysis#at#slaughter.#A#high#prevalence# of#intersexual#pigs#
in#a#farm#can#be#caused#by#one#of#the#breeding#boars.#In#that#case,#zoötechnical#measurements#
should#be#taken#as#to#prevent#great#economic#losses.#
!
ACKNOWLEDGEMENTS#
!
We#thank#doctors#P.#Lobeau#and#P.#De#Smet#for#their#contribution#and#B.#De#Pauw#for#technical#
assistance#
#
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!