Reprod Dom Anim 24\ 54Ð57 "1999#Þ 1999 Blackwell Wissenschafts!Verlag\ BerlinISSN 9825!5657

Relationship between Ultrasonographic Assessment of the Corpus Luteum and Plasma

Progesterone Concentration during the Oestrous Cycle in Monovular Ewes

A Gonzalez de Bulnes\ J Santiago Moreno\ A Gomez Brunet and A Lopez Sebastian

Dpto de Reproduccion Animal\ SGIT!INIA\ Avda[ Puerta de Hierro s:n\ Madrid\ Spain

Contents

Ultrasonographic observations of the corpus luteum "CL# andcollection of blood samples for progesterone radioimmuno!assay were performed daily during 04 oestrous cycles in SpanishMerino ewes\ a consistently monovular breed[ Ultra!sonographic image of the CL changed during the oestrous cycle\increasing its echogenic pattern from ovulation to luteolysis[The size of the CL and mean progesterone levels were sig!ni_cantly a}ected by day of cycle "p ³ 9[94 and p ³ 9[990\respectively#[ Both increased their values from day 0 to day 01"from 38[5 2 6[3 to 043[5 2 00[7 mm1 and from 9[1 2 9[9 to1[7 2 9[4 ng:ml\ respectively# and then declined sharply untilday 9 "17[1 2 4[2 mm1 and 9[02 9[9 ng:ml\ respectively#[ Therewas a signi_cant correlation between CL area and plasma pro!gesterone concentrations during the entire oestrous cycle\ tak!ing the developing and regressing phases of the CL separately"p ³ 9[94#[ A central cavity was observed in 22[2) of the CLstudied[ The presence of this cavity had no e}ect in total luteal!tissue area of the CL nor on oestrous cycle length or on pro!gesterone concentrations[ Likewise\ the cavity did not a}ect thecorrelations observed between CL size and progesterone levels\CL size and day of cycle and progesterone levels and day ofcycle[ It is concluded that ultrasonographic assessment of CLarea is a reliable method for estimating peripheral plasma pro!gesterone levels\ regardless to the presence or absence of a cavityin the CL[

Introduction

Ovarian ultrasonographic observation was initiallyreported in cattle during the 0879s by Pierson and Gin!ther "0873# and Reeves et al[ "0873#[ Since then\ thisnoninvasive technique has been widely used to visualizeeither physiological or pathological ovarian structures"Gri.n and Ginther 0881^ Kahn 0881#[ Ovarian ultra!sonography is very useful among other things to assessovulation and corpus luteum "CL# size[ Ultrasoundmeasurement of the CL size has proven to be a veryreliable method to estimate peripheral progesterone con!centrations in cattle since luteal!tissue area was correlatedwith progesterone concentrations in plasma "Sprecher etal[ 0878^ Kastelic et al[ 0889a# and milk "Son et al[ 0884#[The presence of a central cavity of variable size in 26)"Kito et al[ 0875# to 68) "Kastelic et al[ 0889b# of theCL in heifers did not a}ect the total luteal!tissue areanor progesterone concentration in plasma "Kastelic et al[0889a# or milk "Son et al[ 0884#[

Unlike the cow\ ovarian examination by ultra!sonographic techniques in sheep has not been welldeveloped[ Common trans!abdominal probes used insheep allows imaging of the ovaries\ but not the structures

U[ S[ Copyright Clearance Center Code Statement] 9825Ð5657:1999:2491Ð9954,04[99:9

within them "Bor et al[ 0881#[ Schrick et al[ "0882# _rstused a 6[4MHz transrectal probe to image follicles andCL[ The accuracy for estimating the presence and numberof CL by transrectal probes in polyovular ewes was estab!lished to be between 78 and 099) "Gonzalez de Bulneset al[ 0883#[ In this study\ 07) of the detected CL exhi!bited a central cavity\ which did not a}ect the e.ciencyof detection[ However\ there is not yet any informationon the relationship between CL area measured by ultra!sonography and plasma progesterone levels during theoestrous cycle[ Such information would allow ultra!sonography to be used as an alternative method for esti!mating luteal function in sheep\ as described in heifers[

The objectives of this study were to determine "0# aconceivable relationship between CL area and plasmaprogesterone concentrations during the oestrous cycle inewes^ and "1# the in~uence of a central cavity in the CLon progesterone levels[ The Spanish Merino sheep waschosen for this study as it is known to be consistentlymonovular[ This should avoid possible mistakes in thestudy due to the presence of two or more CL[

Materials and Methods

Animals

The trial was conducted towards the end of the late breed!ing season and involved a total of 04 nonlactating 2! to5!year!old ewes[ They were kept outdoors with access toindoor facilities at the SGIT:INIA experimental farm inMadrid\ Spain\ where the latitude is 39>N[ For each ewe\the study began the day after spontaneous oestrus wasdetected and _nished on the day of the following oestrus[Oestrus detection was carried out using vasectomizedrams[

Ultrasonographic equipment and procedure

Ultrasonographies were made daily with an Aloka 499SSD "Ecotron\ Madrid\ Spain# using a 6[4MHz lineararray transrectal transducer\ designed for examination ofthe human prostate[ Observations were conducted withthe ewes placed in dorsal recumbence in a metallic cradleas used for laparoscopy[ After introducing an hydro!soluble contact gel into the rectum to enhance the ultra!sound transmission\ the probe was placed in the rectumwith the transducer orientated perpendicularly to the ven!tral abdominal wall[ When the urinary bladder was sur!passed and the uterine horns located\ the probe was

55 A Gonzalez de Bulnes\ J Santiago Moreno\ A Gomez Brunet and A Lopez Sebastian

rotated laterally 89> clockwise and 079> counterclockwiseto observe both ovaries and their structures[

Each ovary was scanned several times in di}erentplanes in order to obtain the largest diameter of the CL\and the image was frozen at that stage[ Using electroniccalipers\ the CL area was then determined by measuringthe interface of luteal tissue with ovarian stroma in caseswhere the shape was spherical\ or multiplying "9[4×height#× "9[4×width#×p "�2[03# in cases whereshape was not spherical[ The same procedure was usedto measure the central CL cavity\ by placing the calipersin the interface between the luteal tissue and the cavity[

Blood collection and progesterone assay

Samples "4ml# of jugular blood were drawn before everyultrasonographic observation using vacuum bloodevacuation tubes with heparin "VacutainerR SystemsEurope 4ml\ Becton Dickinson\ Meylan Cedex\ France#[Blood samples were centrifuged at 2499 r[p[m[ "0499 g#for 04min and the plasma was stored at −19>C untilassayed[ Progesterone concentration was measured byradioimmunoassay as described by Lopez!Sebastian etal[ "0873#[ Sensitivity was 9[95 ng:ml\ e.ciency was73[2)\ and the inter! and intra!assay variationcoe.cients were 09[3 and 02[5)\ respectively[

Statistical analysis

The statistical model determined the e}ect of day on CLarea and on plasma progesterone concentrations[ Threegroups\ namely all ewes together\ ewes with a CL with acavity and ewes with a CL without a cavity\ were usedfor comparisons[ Because of considerable variation inlength of cycle\ the developing "days 0 to 01# and regress!ing "days −3 to 9# periods of CL were examined separ!ately[ Data for day −3 to the day of the new oestrus "day9# were normalized to the day of the new oestrus[ Thee}ects of day of oestrous cycle were partitioned into alinear component and remainder "lack of _t# in re!analy!sis of variables for which day of cycle di}ered from day0 to day 01 or day −3 to day 9[ All comparisons weremade by analysis of variance "Steel and Torrie 0879# usingthe {analysis of variance and covariance with repeatedmeasures| and {one! and two!way analysis of variancewith data screening| procedures of BMDP "BMDP Sta!tistical Software Inc[\ Los Angeles\ CA\ USA#[ Data wereexpressed as mean2SEM and di}erences were con!sidered to be statistically signi_cant at p³ 9[94[ The cor!relation coe.cients between day of oestrous cycle\ CLsize and progesterone levels was analysed for the threegroups of ewes by the Pearson correlation procedure ofBMDP Statistical Software "{linear regression by groups|and {bivariate scatter plots|# and correlations were con!sidered to be statistically signi_cant at p³ 9[94[

Results

The ovulation rate was 0 for the 04 oestrous cycles stud!ied[ Presence of ovulation was _rst identi_ed on day 0"day 9�day of oestrus#\ and mean _rst day of detectionof ovulation was 1[32 9[5[ Ultrasonographic images

were very in~uenced by the stage of the cycle\ showing anhypoechogenic pattern just after ovulation[ Echogenicityincreased from day 2 to 3 of the oestrous cycle\ beinghomogeneous and normoechogenic or slightly hyper!echogenic during luteal phase\ and hyperechogenic afterluteolysis[ Margins between CL and ovarian tissue wereirregular in both follicular and early luteal phase but well!de_ned during mesoluteal phase[

The mean inter!ovulatory interval for the 04 oestrouscycles was 06[42 9[4 days\ with a range from 03 to 08days[ No relationship was observed between inter!ovu!latory interval and ewe|s age CL size and mean pro!gesterone levels were signi_cantly a}ected by day of cycle"p³ 9[94 and p³ 9[990\ respectively^ Fig[ 0#[ The CLarea was smaller than 099mm1 during the _rst 2 days ofcycle\ and then it increased linearly until day 01"043[52 00[7mm1#\ after which it decreased pro!gressively to 17[12 4[2mm1 on day 9[ Progesterone con!centrations increased sharply from day 0 to day 01"9[12 9[9 to 1[72 9[4 ng:ml#\ and then decreased to9[02 9[9 ng:ml on day 9[ There was a signi_cant cor!relation between CL area and plasma progesterone con!centrations throughout the entire oestrous cycle"p³ 9[94#\ both during the developing "p³ 9[90# andduring regressing phase "p³ 9[94# of the CL[

The presence of a central cavity showing an irregularshape and hypoechogenic pattern was observed in _veof the 04CL "22[2)#[ Mean days for the _rst and lastdetection of the cavity were 1[92 9[2 "0Ð2# and 8[52 0[1"5Ð01#\ respectively[ No relationship was found betweenthe existence of cavity and oestrous cycle length"06[72 9[3 days for ewes that had a CL with cavity versus06[12 9[4 days for ewes that had no cavity within theCL#\ nor with plasma progesterone levels throughout thecycle[ Measurements of the luteal tissue area showed nosigni_cant di}erences between CL with or without cavity"Fig[ 1#[ Presence or absence of the cavity did not a}ectthe relationship between total luteal!tissue area and dayof cycle or between progesterone concentrations andluteinized area[

Discussion

The results of this study indicate that transrectal ultra!sonography allows observation and evaluation of theovine CL along the oestrous cycle[ Both the ultra!sonographic image and size varied from ovulation toluteolysis\ the size in mm1 being correlated with day ofcycle and plasma progesterone levels[ The presence of acentral cavity within the CL did not have any e}ect onoestrous cycle length\ nor total luteal!tissue area orplasma progesterone concentrations[ Therefore\ ultra!sonographic assessment of CL area is a reliable methodfor estimating peripheral plasma progesterone levels\regardless to the presence or absence of a cavity into theCL[

Our observations showed that it is possible to detectthe corpus haemorrhagicum from the moment ofovulation[ However\ it is better to wait until days 2Ð3 ofthe cycle in practice\ when the CL is well!luteinized[ Asdescribed in a prior study "Gonzalez de Bulnes et al[0883#\ e.ciency of detection without prior scanning is atits lowest on the day after ovulation "49)#^ it increases

56Corpus Luteum and Progesterone in Ewes

Fig[ 0[ Mean diameter of CL areain mm1 "Ž# and mean plasmaprogesterone concentration inng:ml "ž# on each day ofoestrous cycle of monovularSpanish Merino ewes

Fig[ 1[ Mean diameter of CL areain mm1 "Ž# and mean plasmaprogesterone concentration inng:ml "ž# on each day ofoestrous cycle of monovularSpanish Merino ewes that had aCL with "A# or without cavity "B#

to 51[2) on day 3 and rises to 099) from day 4[ Thepresent data indicate that ultrasound e.ciency for detec!tion of luteal tissue depends on its ultrasonographiccharacteristics[ The corpus haemorrhagicum is hypo!echogenic\ irregular in shape and di.cult to di}erentiatefrom an ovulatory!size follicle[ On the other hand\ theCL is homogeneous\ normoechogenic and well!de_nedfrom days 2Ð3 and during the early luteal phase[ Whenthe cycle advances\ luteal tissue becomes slightly hyper!echogenic in the mesoluteal phase and markedly hyper!echogenic after luteolysis\ whereas shape outlining ishighest in midcycle and decreases during the follicularphase[ These variations in ultrasonographic morphology

along the cycle are in agreement with previous descrip!tions in cows "Pierson and Ginther 0873^ Chevalier 0877#[

Moreover\ the increased e.ciency for detecting theCL as the cycle advances "Gonza�lez de Bulnes et al[ 0883#is also favoured by the luteal!tissue growth\ which wassigni_cantly correlated with day of cycle and showed itslargest area during mesoluteal phase in current study[Total luteal!tissue area in Spanish Merino ewes was alsocorrelated with plasma progesterone concentrations[ Therelationship between ultrasonographic assessment of CLand peripheral progesterone levels has been establishedin cattle\ both in plasma "Sprecher et al[ 0878^ Kastelic etal[ 0889a# and in milk "Son et al[ 0884#[ The present

57 A Gonzalez de Bulnes\ J Santiago Moreno\ A Gomez Brunet and A Lopez Sebastian

observations strongly support the usefulness of ultra!sonography as a tool to assess functionality of CL inruminants\ particularly during the developing lutealphase\ when our results showed a high correlationbetween luteal area and progesterone levels[ Similarresults were previously observed by Kastelic et al[ "0889a#in cattle\ who reported that luteal tissue area and plasmaprogesterone concentrations increased at a similar rateduring luteal growth[ However\ they also noticed thatwhen luteal regression began\ progesterone levels de!creased faster than the luteal area "Kastelic et al[ 0889a#[That observation\ con_rmed in this study\ could indicatethat functional regression is earlier than morphologicregression of the CL\ as suggested by Assey et al[ "0882#[

Presence of a central cavity of variable size wasdetected in 22[2) of the CL studied\ a higher percentagethan the 07) previously reported in the same Merinobreed after ovariectomy "Gonza�lez de Bulnes et al[ 0883#[In accordance with the observations of Kastelic et al["0889b# in heifers\ this discrepancy may be due to anunderestimation in prior data\ since observations wereperformed at di}erent stages of the oestrous cycle[ Thus\it is possible that some failures to detect the cavityoccurred in CL evaluated after day 02\ since this studyshows that cavities can disappear between days 5 and 01[Presence of a cavity in the CL could be a source of errorwhen detecting the CL due to confusion with luteinizedanovulatory follicles[ The CL cavity is usually hypo!echoic\ but occasionally exhibits a slightly increased anddi}use pattern or the presence of echogenic lines[ Analy!ses performed in heifers showed that the former was theultrasonic image of haemolysed blood accumulations\whereas the latter was _brin!like strands "Pierson andGinther 0876#[ Luteinized follicles were not identi_ed inthis study\ but unpublished data obtained in our lab!oratory with gonadotrophin!treated ewes indicated thatluteinized follicles show a thicker\ larger as well as more!de_ned and anechogenic cavity than CL[

When the presence of a central cavity in the ovine CLwas considered\ there was no relationship between luteal!tissue area and progesterone levels\ as described in pre!vious studies on heifers "Kito et al[ 0875^ Kastelic et al[0889b^ Son et al[ 0884#[ This fact\ together with the lackof in~uence of the cavity on cycle length\ as seen in cows"Kito et al[ 0875^ Kastelic et al[ 0889b# and mares "Town!son and Ginther 0878#\ could indicate that cavities do nota}ect luteal!tissue functionality nor half!life in sheep[ Thisassumption was con_rmed by unpublished preliminarystudies performed in our laboratory in which the presenceof cavity did not in~uence pregnancy rates\ as previouslyreported in cattle "Kito et al[ 0875^ Kastelic et al[ 0889b#[

Acknowledgements

This research was supported by a C[I[C[Y[T[ project "AGF83Ð0359\ Spain#[ AGde B and JSM were supported by a researchgrant of the Ministry of Agriculture of Spain[ Special thanks toBlanca Cabellos for progesterone radioimmunoassays and DrRC Mainar for kind revision of the manuscript[

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Submitted] 3[2[0888

Authors| address] A Gonzales de Bulnes "for correspondence#\ Dpto[de Reproduccion Animal\ Subdireccion General de Investigacion yTecnologia Istituto Nacional de Investigaciones Agrarias y Ali!mentarias "SGIT:INIA#\ Avda[ Puerta de Hierro s:n\ 17939!Madrid\Spain[ E!mail] bulnesÝinia[es