Birth of a holstein freemartin calf co-twinned to a schistosomus reflexus fetus

ELSEVIER

BIRTH OF A HOLSTEIN FREEMARTIN CALF CO-TWINNED TO A SCHISTOSOMUS REFLEXUS FETUS J Cavalieri andPW Farina Departmentof Food Animal andEquineMedicine, Collegeof Veterinary Medicine North CarolinaStateUniversity, Raleigh,NC 27606 Receivedfor publication: 12 ~cve,,b~~ 1998 Accepted: 22 January 1999 ABSTRACT An unusualcaseof a live Holsteinfreemartincalf co-twinnedwith schistosomus reflexus fetus is presentedhere. Delivery of the schistosomus reflexus was achievedby fetotomy 24 h after manualdelivery of a live heifer calf. The damsubsequentlyexperiencedconcurrentmet&is and left displacementof the abomasum;however, sheconceivedfollowing insemination85 d post par-turn.Cytogenetic examinationof the live heifer using lymphocyte culture and cutaneous fibroblast cell culture failed to demonstratechromosomal chimerism,whereaspolymerasechain reaction (PCR) detectedthe presenceof the bovine Y-chromosomemarker BRY-I. Low concentrations of testosterone, estradiolandprogesterone at 3, 6, 24 and48 h after administration of hCG were detectedin the serumof the freemartinheifer. Genetic,hormonal,histologicaland clinical findings establishedthe live female co-twin calf was a freemartin, an abnormality of phenotypicsex.Failure to detectany significantperipheralconcentrationsof androgensupports the hypothesisthat masculinizationof the freemartinreproductivetract arisesfrom diftision of androgenand possiblyother substances from the male co-twin rather than from endogenous productionof androgenwithin the freemartin.Thisreportdocumentsthat the freemartincondition canbe inducedby a malefetuswith severedevelopmentalabnormalities. Ql999tyElsder8cimeInc.

Key words:freemartinism,schistosomus reflexus,cytogenetictesting,hCG Acknowledgments The authorsthank the North CarolinaDepartmentof Agriculture, UmsteadFarm Unit,for of Animal Sciencefor the bullsusedin this study; providingthefreemaninheifer;Department Ms. V. HedgpethandMs. J. Paulfor providingtechnicalassistance; Dr. N. Barlow, andDr. B.J. Davis,Laboratoryof ExperimentalPathology,NIEHS for pathology support;andDr. M. Robertsfor critical review of this manuscript.Fundingwasprovided by the Departmentof Food Animal andEquineMedicine, NCSU. %orrespondence:FAX: 919-513-6464,e-mail:[email protected].

Theriogenolqy !52~815428,1999 Ql999byEbeviefScienceInc.

0093.SSlX/99/!$-see front PII s0093-891x(99)0017Q0

matter

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INTRODUCTION Freemartinismdevelopsafter chorionicvascularanastomoses occur early in gestationwhen a genetic femaleembryo is co-twined with a male(14,lS). The exact causeof the freemartin anomalyremainsunknown.Currentconceptscenteraroundthe exchangeof substances suchas androgensand Mtillerian Inhibiting Substance(MIS; 32) andpartial expressionof the testisdetermininggenelocatedon the short armof the Y chromosome, termedSty (sex determining region Y), in the femalegonad(15). Androgensare thought to inducemasculinizationof the femalereproductivetract, while MIS leadsto partialregression of the paramesonephric duct and developmentof a hypoplasticuterus. Sty is thought to normally trigger a cascadeof events resultingin the differentiationof the bipotentialgonadinto a testis,andwhen absent,the gonad differentiates into an ovary (20). However, in the freemartin,partial expressionof Sty in the gonadmay be inhibitory to normaldevelopmentof the ovary. Exchangeof substances from the maleto the femaleembryoapparentlyoccursat a critical time in embryonicdevelopmentduring differentiation of the gonad. Schistosomusreflexus is a congenitalabnormality of ruminants,mainly cattle and less commonlyin sheepandgoats,althoughoccasionallyother speciesareaffected(4). Thesefetuses have severeventral curvatureof the spinalcolumn,the body wallsarebentlaterally anddorsally, the limbs are usually ankylosed,and the viscera areexposed(24). In cattle the prevalenceof schistosomus reflexusrangesfrom 0.01to 1.3%of dystocias(17,29).A schisotosomus reflexus fetushasbeenreportedco-twinnedwith a phenotypicallynormalcalf (17,21,33); however,only 4.4% of twins are associatedwith a schistosomus reflexus fetus (17). The presenceof an apparentlynormaltwin fetusassociated with a schistosomus reflexusfetushasbeenreportedin sheep(30) and goats(1,3). This caseis the first known report of a schistosomus reflexus cotwinned with a live freemartincalf. In this report genetic,hormonal,histologicaland clinical findings characterizedthe live femaleco-twin calf asa freemartin.Comparisonof circulating concentrationsof steroidsin the freemartinheiferwith entiremaleandfemalecontrolssupported the categorizationof the freemartinasanabnormalityof phenotypicsex. MATERIALS AND METHODS A 4-yr-old, multiparousHolsteincow waspresentedat the North CarolinaStateUniversity Veterinary TeachingHospital(NCSU-VTH) with ahistory of dystocia24 h after delivery of a live heifer calf. The femalecalf presentedin anteriorpresentationanddeliveredwith the aid of slight manualtraction. Thecow wasartificially inseminated usingfrozen-thawedsemenfrom a Holstein bull 277 d prior to delivery of the calf, and again9 d later usingfrozen semenfrom a second Holstein bull. Vaginal examinationof the cow at NCSU-VTH revealedthe presenceof an emphysematous fetusin transverse-ventralpresentationwith all 4 legsextendedinto the pelvic cavity and right lateral deviation of the head and neck. Ankylosis of the right metacarpalphalangealjoint wasalsodetected. Delivery of the fetus was achievedusing percutaneousfetotomy by first removing the anteriorly presentedhead,then the left forelimb, transversesectioningof the fetal trunk and finally the applicationof traction to the hindlimbs.During the fetotomy procedureButorphenol

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(25 mg, iv Torbugesic;@Fort-DodgeLaboratories,Inc., Fort Dodge,IA), flunixin meglumine (600 mg, iv Banamine;@Schering-PloughAnimal Health, Kenilworth, NJ) and epidural anesthesia(5 mL, 2% LidocaineHCL, sacrococcygealspace;Phoenix PharmaceuticalInc., St Joseph,MO) wereadministered. Following the fetotomy procedurethe uteruswaswashedwith a 1%betadinesolution,andoxytocin (100 IU, im Oxytocin injection;@Phoenix Pharmaceutical Inc.) wasthen administered.Procainepenicillin (13.2 million IU, im Pfl-PenG;@Q12 h, Pfizer Animal Health,NY, NY) andflunixin meglumine(600mg, im, 424 h) wereadministeredfor the next 5 d. After delivery of the fetusthe cow exhibitedreducedappetiteandhad decreasedrumen protozoal activity; therefore shewas transfaunatedwith 800 mL of rumen fluid. The cow’s appetiteimproved andshewasdischarged3 d after presentation. Ten daysafter delivery of the fetal monsterthe cow presented againto theNCSU-VTH with a history of reduced appetite, low milk production and a yellow vulvar discharge.Left displacementof the abomasumwas diagnosedand corrected surgically using a right flank omentopexy.Concurrentmetritiswasalsodiagnosedbasedon palpationof a partially involuted uteruswith flaccid uterinetone anda viscous,white, malodorousfluid dischargein the vaginal vault. No specifictherapywasinstitutedfor the metritisdueto the absenceof systemicsignsof infection; however,cefiiofur (Naxcel@lg, im; Pharmacia& UpjohnCompany,Kalamazoo,MI) wasadministeredprophylacticallyon the dayof surgeryandon the following day. The cow was dischargedthe day after admissionto the NCSU-VTH. Estruswas first detected85 d after the correction of the dystocia.The cow wasinseminatedoncewith frozen-thawedsemenand was subsequentlydiagnosedpregnant. Cytogeneticwork-upof thefetuswasnot possibledueto the poorconditionof the body fluids andtissues;however,parentageof the live heifer calf wasverified by blood typing the damand calf (Holstein AssociationUSA, Inc., Brattleboro, VT). Chromosomalsex of the heifer was establishedby culture and examinationof lymphocytesfrom blood and gbroblastsfrom skin biopsies.Blood sampleswere collectedin lithium heparin(Vacutainers; Becton-Dickinson, Rutherford,NJ) for cytogentictesting(MinnesotaVeterinary DiagnosticLaboratory, University of Minnesota, St. Paul, MN) whenthe heifer was 1, 10 and 13 mo of age. Skin biopsieswe&e collectedat 10 and 13 mo of ageandplacedin venousblood containingEDTA (Vacutainers ) as an anticoagulant.Blood lymphocytes(100 metaphase cells) were examinedusing methods previously described(5), except that lymphocytes were not separatedfrom whole blood but instead0.25 mL of whole bloodwasaddeddirectly to the RPMI-1640 culture medium(Gibco; Grand Island, NY) readsof cultured epithelialcells were examined(6). In addition, a blood samplefrom the heiferat 15mo of agewasexaminedusingpolymerasechainreaction(PCR) for the presenceof the bovine Y-chromosomemarker BRY-1 (Stormont Laboratories, Inc., Woodland,CA). The heiferwaseuthanizedat 16mo of age(Beuthanasia-DSpecial,60 mL, iv; Schering-PloughAnimal Health, Kenilworth, NJ). Reproductivetissueswere fixed in 10% neutral buffered formalin, thin-sectionedand stainedwith hematoxylin-eosinfor histological examination. Gonadalsexwasdeterminedby a response testwith humanChorionicGonadotropin(hCG) to induce endogenoustestosteronesecretion,indicating the presenceof functional testicular tissue,andby histologicalexaminationof thegonads.The freemartinheiferwas 16mo old at the

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time of blood samplecollection. For the hCG responsetest, 3 reproductively normalbulls (2 Angus and 1 Poll Hereford,12to 13mo of age)and3 nonpregnantcyclic Holsteinheifers(13 to 15 mo of age)were usedasthe male and femalecontrols.A corpusluteum (CL) 114 mm in diameterwasdetectedusingtransrectalultrasonography (7.5 MHZ probe;Sonovet600,Universal Medical Systems,Inc., BedfordHills, NY) within the ovariesof eachof the control heifersprior to collection of blood samplesfrom the coccygealvein into vacutainersat 0,3, 6,24 and48 h. Humanchorionicgonadotropin(6000IU, iv; ScheinPharmaceutical Inc., FlorhamPark,NJ) was administeredto all animalsafter an initial blood samplewastakenat 0 h. Blood sampleswere subsequentlycentrifuged and the serumremoved and stored at -20°C until assayedfor testosterone,progesteroneand [email protected] testosteroneand progesterone weredeterminedusingsolid-phase radioimmunoassay kits (Coat-A-Count@, DiagnosticsProducts Corporation, Los Angeles, CA). Concentrationsof 17P-estradiolwere determinedin our laboratoryusinga previouslyvalidatedmethod(11).All samples wereassayedin duplicate.Intraassay variation of each endocrine assayswas < 12%. Sensitivities of the testosterone, progesteroneand estradiol assayswere 14 pg/mL, 1.0 ng/mL and 2.0 pg/mL, respectively. Phenotypic sexwasdeterminedby examinationof the externalandinternalgenitalia. RESULTS Pathology of the Fetus Examination of the fetus deliveredby fetotomy revealedmultiple congenitalanomalies. Theseincludedprognathia,a right deviatedwry nose,severelordosis,dorsallydirectedribs, lack of fusion of the sternumandventral abdominalwall with total eventration,bifurcation of the scrotumandatresiaani(Figure 1A). The fetusweighed25 kg. Testicleswerenot identified and may have beenremovedduringthe fetotomy procedure;however,the presenceof a bifurcated scrotumand seminalvesiclessuggested that the fetuswasmale. Clinical andCytogeneticResultsof the Heifer Calf Parentageanalysis of a sampleof blood obtained from the calf co-twinned with the schistosomusreflexus fetus verified the dam and sire whosesemenwas used at the first insemination.The length of gestationwasdeterminedto be 277 d. Physicalexaminationof the heifer at 4 wk of agerevealeda prominentclitoris and a long tuft of &ir extending from the ventral commissureof the vulva (Figure 1B). A 7-mL tube (Vacutainer ) wasinsertedinto the vaginalvault to a distanceof 5 cm beforeresistanceto further passage wasencountered(Figure lB), suggestingthat the heifer was a freemartin(19). A sampleof blood was submittedfor cytogenetic testing to confirm this diagnosis.This initial cytogenetic test failed to detect chromosomalchimerism.At 10mo of ageadditionalsamples weretaken for cytogenetictesting of blood lymphocytes and skin fibroblasts.Cytogenetictestingof cultured lymphocytesagain failed to detect chromosomalchimerism. Becausethe fibroblast cell cultures became contaminatedwith bacteriathey could not be usedfor testing.At 13mo of age,examinationof fibroblastcellsfrom cultureof a skinbiopsyandlymphocytesculturedfrom bloodfailedto show any evidence of chromosomalchimerism.Clinical examinationof the heifer at 15 mo of age revealedhypoplasticmammaryteats,a long tuft of vaginal hair anda hypoplasticuterus.

819

Figm72.1 . A) Schistosomus reflexus fetus deliveredby fetotomy. B) Measurementof vaginal lengthusinga 7 cm longevacuatedtubein thefreemartinheiferat 3 weeksof age.TIube is insertedinto thevaginato the maximumdepthof penetration,C) Reproductivetr‘act andgonadsof the freemartinat 16monthsof age.D) Histologicalsectionof gonad1(x 400, hemotoxylin-eosinstain).Note clustersof tubuleslined by Sertoli-like cellsand occasionallycontainingprimordialgermcells(e).

820

Theriogenology

Neither gonads nor a cervix could be located by trans.-rectal examination. However, a sample of blood was positive for the presence of the bovine Y-chromosome marker BRY-1. Steroid Measurements Concentrations of testosterone, 17P-estradiol and progesterone in the heifer born co-twinned to the schistosomus reflexus fetus and the normal bulls and heifers before and after administration of hCG are shown in Table 1. Concentrations of testosterone were at the minimum level of detection by assay for phenotypically normal heifers and for the freemartin at all time intervals, while these concentrations for the bulls were numerically much higher at each sample period and peaked 48 h after administration of hCG. Concentrations of estradiol in the bulls peaked 48 h after administration of hCG. In the normal heifers, estradiol peaked 6 h post administration of hCG then declined within 48 h to a level similar to the mean concentration at Time 0. In the freemartin heifer, concentrations of estradiol in serum were lower than in the control heifers and in the bulls at each sample time, and did not fluctuate beyond 1 pg/mL throughout the sampling period. Concentrations of progesterone in the control heifers remained higher than 8.0 nglmt during the sampling period, peaking at 48 h after administration of hCG. In the bulls, concentrations of progesterone remained at basal levels of around 1.Ong/mL throughout the period of sampling. In the freemartin heifer, concentrations of progesterone were highest prior to the administration of hCG (Time 0), subsequently declining to a.0 ng/mL, which was similar to the concentration detected in the bulls at the subsequent sampling periods. Necropsy and Histological Findings Vaginal length of the freemartin heifer (10.3 cm) was more than 50% shorter than that of the control heifers (23,29 and 30 cm, respectively) used for the hCG response test (Table 2). The uterine horns and gonads were hypoplastic, the cervix was absent, and there was no connection between the uterine horns and the vagina (Figure 1C). Small paired seminal vesicles were also present (Figure 1C). The presence of oviducts lined with ciliated columnar epithelial cells and of small remnants of the mesonephric duct was confirmed histologically. The gonads were surrounded by a relatively thickened tunica albuginea. Rete ovarii lined by cuboidal cells occupied a large portion of the ovarian parenchyma, and nests of undifferentiated sex cords surrounded by interstitial cells were observed throughout the gonad (Figure 1D). The interstitial cells were separated by stromal tissue. Clusters of tubules lined with Sertoli-like cells were identified within the gonads. Primordial female germ cells were only rarely observed within tubules. Vesicular glands consisted of hypoplastic glandular tissue lined by cuboidal cells and surrounded by extensive fibrous connective tissue. The uterus contained numerous endometrial glands within the lamina propria, with many of the glands containing an accumulation of proteinaceous fluid (Figure 1D).

14.9 f 0

14.9 f 0 14.9 f 0

5476 f 1485 7440 f 765 8086 f 1396 8356 f 1329 14658*2240

0

3

6

24

48

14.9

14.9

14.9

14.9

14.9

FM

9.6* 3.5

18.8 f 2.5 26.0 f 7.6

6.4 f 2.1

2.5

12.3 f 0.4

13.9 f 2.3

3.5

2.3

3.1

16.6 f 1.5

11.8* 1.7

FM 3.3

Heifers 5.6 f 0.7

6.8 f 0.8

Bulls

17P-estradiol @g/mL)

‘All samples tested below the minimum detection limit of the assay (14.9 pg/mL).

14.9 f 0

14.9 f 0

Heifersa

Testosterone (pg/mL) Bulls

Time (hours)

1.5 f 0.3

1.1 hO.1

1.1 f 0.1

1.4*0.2

1.1 f 0.2

Bulls

1.0 1.1

13.0 f 5.2

1.1 9.0 f 4.0

11.0*4.6

1.9

4.5

8.2 f 3.2 11.2k3.9

FM

Heifers

Progesterone (ng/mL)

Table 1. Concentrations (Mean f SEM) of testosterone, 17P-estradiol and progesterone, at various time intervals relative to the intravenous administration of 6000 KJ of hCG, in 3 bulls, 3 heifers and a freemartin (FM) born as a co-twin of a male schistosomus reflexus fetus

Theriogenology

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Table 2. Anatomical dimensions of the reproductive tract of a 16-month-old freemartin Anatomical structure

Length (mm)

Width (mm)

Depth (mm)

Right gonad

19

5

4

Left gonad

20

8

4

Right seminal vesicle

48

9

5

Left seminal vesicle

50

8

6

Right uterine horn

141

98

NA

Left uterine horn

147

8’

NA

Vagina

103

22

NA

Clitoris

9

3.5

NA

‘Measured immediately cranial to the bifurcation of the uterus. NA=measurement not applicable. DISCUSSION This study documents the presence of the birth of a live freemattin calf co-twin with a schistosomus reflexus fetus. Necropsy findings of the fetal monster were consistent with a diagnosis of schistosomus reflexus (24). The etiology of the condition is not known, although a genetic cause has been postulated (4). Testes were not located in the schistosomus reflexus fetus; however, gross pathological findings together with the detection of Y-specific chromosomes in the circulation and masculinization of reproductive tract of the female co-twin strongly suggest that it was male. Analysis of chromosomes confirmed the chromosomal sex of the freemartin to be female (60, XX). In addition, low peripheral concentrations of testosterone following the hCG response test indicated that the gonadal sex of the freemartin resembled a female. The detection of Y-specific chromosomal material as well as the gross and histological appearance of the reproductive tract led to a final diagnosis of freemartinism and the categorization of the abnormal reproductive tract as an abnormality of phenotypic sex. Low concentrations of steroid hormones were detected in the freemartin heifer at each sampling period, with exception to an elevated concentration of progesterone at 0 h. Low mean plasma concentrations (CO.4 ng/mL, with occasional surges between 0.5 and 1.6 ng/mL) of progesterone have been reported in lo- to 12-mo-old freemartins (25). Johnson et al. (13) reported concentrations of progesterone in 12-mo-old control and freemartin heifers, with and without gonadal tissue, of 8.6 f 0.5, 1.2 f 0.5 and 0.4 f 0.1 ng/mL, respectively. The high initial serum concentration of progesterone (4.5 ng/mL) in the freemartin heifer reported here was probably due to stress-induced production of progesterone from the adrenal glands, because all subsequent samples were < 2.0 ng/mL (2). Concentrations of progesterone at other sample periods in the freemartin were similar to those reported for freemartins with gonads (13).

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Administration of hCG to the control heifers and the freemartin failed to increase testosterone concentrations. Peak concentrations of testosterone recorded 48 h after administration of hCG to the bulls were comparable to those in another study (31). Basal concentrations of circulating testosterone have been reported in both normal heifers and freemartins (25). In vitro culture of gonadal tissue from freemartins also failed to produce any significant concentrations of testosterone (22). However, an 11-mo-old freemartin heifer with gonads within the inguinal canals, histological evidence of well-developed Leydig cells, had elevated concentrations of testosterone in testicular tissue and in plasma from the spermatic vein (28). Ovaries cultured from freemartin fetuses at 47 to 77 d of gestation yielded higher concentrations of testosterone and lower concentrations of estradiol compared with ovaries from singleton fetuses at the same stage of gestation (27). Differences in the secretion of testosterone by gonads from freemartins may reflect variation in the extent of masculinization of the gonads. Differences between fetal and older freemartins may indicate an age-related alteration in steroid production within the gonad. The gross and histological features observed in the reproductive tract of the freemartin (Figure 1A to D) were similar to those of a previous report (16). Abundant interstitial cells within the gonads and the lack of apparent testosterone secretion suggest a disruption in the synthesis of testosterone. In our present study, hCG would have stimulated testosterone production in the male controls and estradiol production in the female controls independent of pituitary function. The low synthesis of steroids by the freemartin in our study suggests a deficiency in steroid synthesis within the gonad rather than in pituitary secretion of gonadotrophins. A pituitary defect may occur in some freemartins due to differences in LH secretion following administration of estradiol or GnRH (7). Concentrations of estradiol increased in both the control heifers and in bulls following administration of hCG. An increase in serum concentrations of estradiol following administration of hCG has previously been reported in reproductively normal heifers (11) and is probably due to the stimulatory effect of hCG on estradiol synthesis by the granulosa cells. The increase in concentrations of estradiol observed in the bulls which paralleled the rise in testosterone was probably due to the aromatization of testosterone to estradiol. Low concentrations of 17pestradiol in the serum of the freemartin in this study are consistent with the detection of low concentrations of 17P-e&radio1 from the urine of fieemartins 6 to 11 mo of age (23) and from tissue cultures of ovaries of freemartin fetuses between 47 and 77 d of gestation (27). The low steroidogenic capacity of freemartin gonads apparent from this study and others (22) supports the hypothesis of Lillie (18) that androgens from the male fetus twinned with a freemartin lead to masculinization of the female rather than to increased androgen production by the f&martin gonad. Gonadal changes evident in freemartins do not, however, appear to be due to the androgen produced by the male co-twin, as exogenous administration of androgens, while causing masculinization of the external genitalia, has not been shown to induce changes in the gonads that are generally present in freemartins (12). Other contributing factors to the development of freemartins include the transfer of h4IS from the male to the female co-twin (32) and possibly the partial expression of Sry in the female gonad. The means by which Sry may inhibit ovarian development is not known, but may involve differentiation of Sertoli cells and/or complete or partial activation of male-specific genes (20).

824

Theriogenology

Diagnosis of fieemartinism can be made by demonstrating sex chromosome chimerism using leucocyte tissue culture, blood typing (19), PCR assay (9) or clinical examination. In normal calves ~1 mo old, vaginal length is usually 13 to 15 cm, while in adult animals it is 30 cm in length. In freemartins ~1 mo old and in adults vaginal length is generally 5 to 6 cm and 8 to 10 cm long, respectively (19). Vaginal length of the heifer described here (5 cm at 1 mo of age and 10.3 cm at 16 mo of age) is consistent with the length reported in most freemartins (19). Cytogenetic testing of cultured lymphocytes and fibroblast cells established the cbromosomal sex of the heifer as female but failed to detect chromosomal chimer&m. Examination of 100 chromosome spreads accurately identifies approximately 98% of freemartins (8). Mean percentage of chromosomal chimerism in freemartins is approximately 50% XX and XY cells (16,26). However, the ratio of XX to XY cells does not appear to influence the degree to which normal development of the female reproductive tract is disrupted and masculinized (26). Detection of a Y-specific sequence in the heifer described here using PCR methodology demonstrates the greater sensitivity of PCR in identifying freemartins compared with that of cytogenetic tests for chime&m when the XY cell count is low. Fujishiro et al. (9) used the PCR method to detect a Y-specific sequence in blood from a normal bull diluted with blood from a normal cow to a concentration containing 0.1% XY cells. They were also able to detect a Yspecific sequence with no detectable XY cells upon cytogenetic examination in female calves cotwinned with male calves. The schistosomus reflexus fetus in our report was removed by fetotomy; however, in some cases, gentle traction is reported to enable delivery (17,24). Knight (17) reported that a Cesarian section was conducted on 25.6% of cases, fetotomy was performed on 56.7% of cases, while 14.4% of cases were not treated due to the presence of an emphysematous fetus and a poor prognosis. In the case described in this report, the presence of an emphysematous fetus and the desire of the owner’s to rebreed the cow dictated that vaginal delivery probably carried a better prognosis for future fertility. Concurrent conditions such as twinning, dystocia and metritis have been associated with displacement of the abomasum (lo), and so its occurrence in the case presented here is not unusual. Lack of medical intervention in order to therapeutically resolve metritis in the cow presented here resulted in an acceptable interval from parturition to conception, The case described in this report indicates that clinical examination, including a vaginal tube test, was an accurate means of diagnosing freemartinism, while PCR analysis proved to be a more sensitive test for diagnosing freemartinism than cytogenetic testing. A response test to hCG failed to result in an elevation in concentrations of testosterone, progesterone and 17P-estradiol in the serum of the freemartin, thus supporting the categorization of freemartinism as an abnormality of phenotypic sex. This report supports the hypothesis that masculinization of the freemartin reproductive tract occurs by the diffusion of androgens and/or other substances from the male to the female co-twin rather than from endogenous production of steroids within the female. Delivery of female calves that are a co-twin with a male schistosomus reflexus should be investigated for possible freemartinism.

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825 REFERENCES

1. Ayyappan S, Balasubramanian S, Ravisudar G, Srijayanth R, Dhanapalan P. Schistosomus reflexus monster co-twinned with a live kid - a case report. Cheiron 1992;2 1:3 abstr. 2. Balfour WE, Comline RS, Short RV. Secretion of progesterone by the adrenal gland. Nature 1957;180:1480-1481. 3. Bedford PGC. Schistosoma reflexus in a goat: a case report. Vet Ret 1967;80:326 abstr. 4. Bezek DM, Frazer GS. Schistosomus reflexus in large animals. Comp Cont Ed Pratt Vet 1994;16:1393-1396,139s. 5. Buoen LC, Weber AF, Meiske JC, Hooker EC. Cases of l/29 Robertsonian translocation (centric fusion) in Charolais cattle. Can Vet J 1988;29:455-457. 6. Buoen LC, Zhang TQ, Weber AF, Anderson MR, Ruth GR. The requirement of flbroblasts to confirm the identity of cytogenetic centric fusion (CF) carriers in same-sex twin cattle. J Vet Diagn Invest 1992;4:212-214. 7. Cunningham NF, Saba N, Boarer CD. The acute effects of oestradiol- 17p and synthetic LHRI-I on plasma LH levels in freemartin heifers. J Reprod Fertil 1977;51:29-33. 8. Dunn HO, Johnson RH Jr. Sample size for detection of Y-chromosome in lymphocytes for possible freemartins. Cornell Vet 1981;71:297-304. 9. Fujishiro A, Kawakura K, Miyake Y-I, Kaneda Y. A fast, convenient diagnosis of the bovine fieemartin syndrome using polymerase chain reaction. Theriogenology 1995;43:883-891. 10. Geishauser T. Abomasal displacement in the bovine - a review on character, occurrence, aetiology and pathogenesis. J Vet Med A 1995;42: 229-25 1. 11. Howard I-U, Scott RG, Britt JH. Associations among progesterone, estradiol-17p, oxytocin and prostaglandin in cattle treated with hCG during diestrus to extend corpus luteum function. Prostaglandins 1990;40:5 l-70. 12. Jainudeen MR, Hafez ESE. Attempts to induce bovine freemartinism experimentally. J Reprod Fertil 1965;10:281-283. 13. Johnson BH, Lasater DB, Ewing LL, Turman EJ, Stephens DF. Hormonal steroid levels in peripheral plasma of freemartins. J Anim Sci 1970;30:321-322 abstr. 14. Jost A, Vigier B, Prepin J. Freemartins in cattle: The first steps of sexual organogenesis. J Reprod Fertil 1972;29:349-379. 15. Kennedy PC, Miller RB. The Female Genital System. In: Jubb KVF, Kennedy PC, Palmer N (eds), Pathology of Domestic Animals, Vo13. San Diego: Academic Press Inc, 1994;350351. 16. Khan MZ , Foley GL. Retrospective studies on the measurements, karyotyping and pathology of reproductive organs of bovine freemartins. J Comp Path01 1994; 110:25-36. 17. Knight RP. The occurrence of schistosomus reflexus in bovine dystocia. Aust Vet J 1996;73:105-107. 18. Lillie FR. The theory of the free-martin. Science 1916;43:611-613. 19. Long SE. Development and diagnosis of freemartinism in cattle. In Pratt 1990; 12:208-2 10. 20. McElreavey K, Barbaux S, Ion A, Fellous M. The genetic basis of murine and human sex determination: a review. Heredity 1995;75:599-611. 21. Morton DH, Cox JE. Bovine dystocia. A survey of 200 cases met with in general practice. Vet Ret 1968;82:530-537.

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