Embryotoxic effects adjacent and opposite to the early regressing bovine corpus luteum

ELSEVIER

EMBRYOTOXIC

EFFECTS ADJACENT AND OPPOSITE TO THE EARLY REGRESSING BOVINE CORPUS LUTEUM

H. J. Hemandez-Fonseca, B.L. Sayre, R.L. Butcher and E.K. Inskeep Division of Animal and Veterinary Sciences, West Virginia University Morgantown, WV 26506 USA Received for publication: Accepted:

January

February

29,

18,

1999

2000

ABSTRACT Early luteal regression in cattle has an embryotoxic effect that is not overcome by replacement with progesterone, but is prevented by removal of the regressing CL. Two experiments were designed to test the null hypothesis that the luteal component of the embryotoxic effect is delivered by a systemic pathway. Beef heifers and cows (n = 39) received two good quality embryos, one placed into each uterine horn on Day 6 or 7 of the estrous cycle. Treated animals (n = 20) received 15 mg of PGF,, three times per day from Day 7 (n = 11; Experiment 1) or 5 (n = 9; Experiment 2) through 8; controls (n = 19) received saline. Progestogen replacement therapy (12 mg flurogestone acetate daily, SC) was provided from Day 6 (Experiment 1) or 4 (Experiment 2) until ultrasonographic diagnosis of embryo survival on Day 35 after estrus. The effects of treatment, location of the embryo and location by treatment interaction on embryo survival were tested by Chi square. In Experiment 1, there was no significant difference in embryo survival rate between PGF,-treated and control recipients. In Experiment 2, only 6 of 18 embryos survived to Day 35 when transferred to animals treated with PGF, compared to 12 of 18 in control animals (PC 0.05). The survival of embryos did not differ with location (adjacent or opposite to the regressing CL) or location by treatment interaction. Thus no evidence was obtained to support a local effect of the regressing CL. The embryo mortality associated with luteolytic doses of PGF, in cows receiving replacement therapy with progestogen probably involves compounds that either act systemically or are transported via the uterine lumen to the uterine horn contralateral to the regressing CL. 0 2000 by Elsevier Science Inc. Key words: embryo mortality, corpus luteum, cattle Acknowledgments Published with the approval of the director of the West Virginia Agricultural and Forestry Experiment Station as Scientific Paper No. 2708, and supported by Project 129, Animal Health. The authors thank Dr. Robert D. Baker (Baker Embryo Transfer Service, Manchester OH) for invaluable assistance in embryo transfer and Silvertowne Farms (Winchester, Indiana) for donation of the frozen embryos used in Experiment 1. Theriogenology Q 2000 Elsevier

54:63-91, Science

2000 Inc.

0093-691X/00/$-see PII: SOO93-691

front matter X(00)00327-7

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INTRODUCTION In cattle, the first ovulation following parturition usuallyis associated with the developmentof a CL that hasa shortlifespan(~14 days;2, l&23,26,27,28,30). The shortluteal phaseis causedby prematureuterine secretionof PGF, (7, 8,24), observedby Cooperet al. (6) on Days 4 through 9 after ovulation, in responseto the first exposureof the uterusto progesterone. Pregnancyrate wasextremelylow or zero whenthe first postpartumovulationwasaccompanied by estrusandthe cow wasmated(2, 1523, 26, 27, 28), but oocyteswerefertilized. In fact, proportionsof oocytesthat werefertilized, underwentearly embryonicdevelopment,andwere transportedinto the uteruswere similarin cowswith short or normalluteal phases(2,27). It was assumed initially that embryoswerelost whenthe CL regressed prematurely,becauseluteal progesteroneis essentialfor the maintenance of early pregnancy(17). However, replacement therapy with progestogens, at dosages effective in unilaterallyor bilaterallyovariectomizedcows, did not maintainpregnancyin cowswith short lutealphases(2). Therewasa strikingsimilaritybetweenthe apparenttiming of embryoloss(4,30), despite replacementtherapy with progestogens, andthe timingof increaseduterinesecretionof PGF, (6, 30, 35) in cowswith short lutealphases.In addition,there wasa negativecorrelationof quality of the embryoon Day 6 with concentrationsof PGF,, in uterineflushings(r = -0.42; 30). Thus PGF,, may be an embryotoxic factor; it hasbeenshownto reduceviability or developmentof embryosfrom rabbits,rats andcowsin vitro (1, 10, 16). The early regressingCL hasbeenshown to secretePGF, (1 l), which could supplement PGF,, secretedby the uterus. In the early postpartumcow, the combinationof inhibitionof prostaglandinsynthesiswith flu&in meglumineon Days4 through9 andlutectomyimprovedembryonicsurvival; however, neithertreatmentalonewaseffective (3). In contrast,PGF, on eitherDays4 through 7 or 5 through8 after estrusin progestogen-treated, mated,nonlactatingcowsinducedluteolysisand reducedembryonicsurvival, but lutectomy alonerestoredsurvivalof the embryo(3,3 1). Cows that were in the lutealphaseof the estrouscycle releasedoxytocin within 30 minafter the initial injectionof PGF,, (3). Treatmentof matedcowswith oxytocin on Days 5 through 8 lowered pregnancyrates(14). That effect wasovercomeby flunixin meglumineandnot by lutectomy, evidencethat luteal oxytocin wasnot directly embryotoxic,but probablyactedby stimulating uterine secretionof PGF,, ( 14). Possiblepathwaysfor the delivery of an embryotoxicfactor from the ovary to the uterus includeveno-arterialcountercurrentmechanisms betweenthe ovarianvein anduterineartery, uterineluminaltransport,the lymphaticsystem,or the systemiccirculation. If, for example,luteal oxytocin actedonly on the adjacentuterinehornto increasesecretionof PGF,, andPGF,, acted directly asthe embryotoxin,thenthe regressingCL could reduceviability of the embryoby a local effect (in the uterinehorn adjacentto the CL). A systemicactionof PGF, via the blood seems unlikely, becausePGF, hasa very shorthalf life in the systemiccirculationdueto rapid metabolism in the lungs(25). Oxytocin, becauseof its longerhalf life, might act systemicallyto enhancesecretionof PGF,, by the entireuterus. Two experimentsutilized transferof embryosinto eachuterinehorn to testthe null hypothesis that the luteal componentof the embryotoxiceffect wasdeliveredby a systemicpathway. If the

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lutealembryotoxinactedlocally, aninteractionof locationof the embryo(adjacentor oppositeto the corpusluteum)andtreatment(control vs. lutealregression)on embryosurvival would be expected. MATERIALS AND METHODS GeneralProcedure Non-lactatingbeefcowsandheifersof mixedbreeding(includingAngus, Hereford and Simmental)wereusedasrecipientsof transferredembryos.Estruswassynchronizedwith 15or 30 mg of PGF,, im and400 (ug of estradiolbenzoateim 48 h later. Cowswere observedfor estrus(Day 0) twice daily (7 amand6 pm) for a minimumof 30 mmeachtime. Beginning12 h beforethe start of eachtreatment,recipientcowsreceivedtlurogestoneacetate (FGA, 12 mg/day,sc, aseither4 mg threetimesper day duringtreatmentwith PGF, or 6 mg two timesper day thereafter;equivalentto 300 mg progesteroneper day) until diagnosisof embryosurvival on Day 35. Recipientcowswere divided at randominto two groups(control and treated). Treatedrecipientsreceived15 mg (im) of PGF, threetimesper day on Days 7 and 8 (Experiment1; n = 11) or on Days 5 through8 (Experiment2; n = 9). Controlsreceived3 mL of salinethreetimesper day duringthe sameperiodsin eachexperiment(n = 10 and9, respectively). On Day 7 (Experiment1) or Day 6 (Experiment2) good or excellentquality embryoswere transferrednonsurgicallyto recipientcows. A total of 78 embryoswere transferred(42 frozen-thawedembryosin Experiment 1 [donatedby SilvertowneFarms,Winchester,IN] and36 freshembryosin Experiment2). An experiencedET technician (Dr. R. D. Baker, Baker Embryo Service,Manchester,OH) placedone embryoascloseaspossibleto the tip of eachuterinehorn ipsilateralandcontralateralto the CL. Location of the corpusluteumin recipientswas determinedby transrectalultrasonography(Aloka 500,with a 7.5~mHztransducer). Diagnosisof embryosurvival (presenceof an embryowith a heartbeat)andlocation of eachviable embryo were determinedin the samemanneron Day 35. Experiment1 wasconductedduringthe fall of 1995.Treatmentsdescribedabovewere applied on Days 7 and8. Experiment2 wasconductedin two replicatesduring the summerandfall of 1996. Replacement therapywith FGA wasbegunon Day 4 after estrus. Becauseof the low pregnancyrate obtainedin control cowsin Experiment 1 andthe ineffectivenessof 2 daysof treatmentwith PGF, to reduceeitherpregnancyor embryosurvival rates,changesin designwere madefor Experiment2. Fist, only freshembryosweretransferred. Second,baseduponthe known effectsof timing of treatmentwith PGF,, on embryosurvival (3,3 l), treatmentwasbegun earlier(from Days 5 to S), andDay-6 embryoswere transferredon Day 6 to increasethe period of exposureof the embryosto eitherdirect or indirecteffectsof PGF,. Donor Cows Sevencowsthat were on Days7 to 13of the estrouscycle were selectedto serveasdonors for eachreplicateof Experiment2. Eachcow wastreatedwith decreasingmorningandafternoon

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dosesof follicle stimulatinghormone(FSH; Folltropin-V; Vetrepharm,London, Ontario, Canada; total of 260to 330 mg equivalentsNIH-FSH-Pl; superovulatorytreatment)during a 4-day period beginningon Days 9 to12. Donor cowsreceived30 mgof PGF, twice duringthe lastday of the superovulatorytreatment,were placedwith a fertile bull, andwere observedfor estrustwice daily. Threeproven bulls(two Angus andoneMaine-Anjou) were rotatedtwice daily, sothat eachdonor wasbred by one or two bulls. Eachcow wasinseminated artificially (one strawof fertile Angusor Simmentalsemenwasdepositedinto eachuterinehorn) at leastoncebetween12 and24 h after onsetof es&us. On Day 6 after es&us,the uteri of cowswere flushednonsurgicallywith Dulbecco’sphosphate bufferedsaline,andthe flushingmediawere searchedfor embryos(morulaeandblastocysts). Embryosweretransferredinto a sterilizedpetri dishcontainingholdingmedium(Dulbecco’s phosphatebufferedsalinewith 10%newborncalf serum)andevaluatedby experiencedpersonnel (Drs. R.D. Baker and R.L. Butcher). Statisticalanalysis The effectsof treatment,location of the embryo(ipsilateralor contralateralto the CL) and locationby treatmentinteractionon embryosurvival weretestedby Chi squareusingthe CATMOD procedureof SAS (29). RESULTS In Experiment1, in which frozen-thawedDay-7 embryoswere used,40% of control recipients were pregnanton Day 35 after estrus. Pregnancyrate in recipientanimalstreatedwith PGF,, (7/l 1) did not differ from controls(4/10). Treatedrecipientshada low embryosurvivalrate (S/22) that wasnot significantlydifferent from that of control recipients(4/20). Thusthe null hypothesisthat embryotoxic effectswere equalin the two uterinehornscouldnot be tested. The only casein which both embryossurvived(twin pregnancy)after beingtransferredto an experimentalcow wasrecordedin the treatedgroup. In this experiment,exogenousprogestogen wasnot beneficialto survival of frozen-thawedembryosin the uterinehorn contralateralto the CL. Only 2 of 21 embryossurvivedin the contralateraluterinehorn comparedto 10 of 21 embryosin the ipsilateralhorn (P = 0.01; Table 1). Table 1. Effect of treatmentandlocationon embryosurvival (Experiment 1). Treatment Embrvo location

PGF,, Davs 7 and8

Control

Total

Ipsilateraluterine horn

601

400

10/21” (48%)

Contralateraluterinehorn

2111

o/10

2/21b (10%)

Total

8122

4120

12142 (29%)

4bValuesin the samecolumnwith different superscriptsdiffer (P = 0.01).

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In Experiment2, treatmentwith PGF, decreased pregnancyrates(P < 0.05; 4/9) comparedto controls(8/g). Only 6 of 18embryossurvivedto Day 35 (33%) whentransferredto uterinehorns of animalstreatedwith PGF,, comparedwith an embryosurvival rate of 67% in control animals (12 of 18;P< 0.05; Table2). Table2. Effect of treatmentandlocationon embrvosurvival (ExDeriment2). Treatment Embrvo location

PGF,,. Davs 5-8

Control

Total

Ipsilateraluterinehorn

319

719

lo/18 (56%)

Contralateraluterinehorn

319

519

8/18 (44%)

6/18”

12/18b

18136(50%)

Total

a,bValuesin the samecolumnwith different superscripts differ (P < 0.05). The survival of embryosdid not differ with location of the embryo(adjacentor oppositeto the corpusluteum)or with locationby treatmentinteraction. The proportionsof surviving embryos in the uterinehornsadjacentandoppositeto the regressingCL wereexactly the samein animals treatedwith PGF, (33%; 3/9). Embryo survivalrateswere 77% (7/9) ipsilateraland 56% (5/9) contralateralto the CL in control animals(Table2). Five of the 6 cowsthat lost both transferred embryoshadbeentreatedwith PGF,, while 4 of 6 cowsthat maintainedtwin pregnancieswerein the control group. DISCUSSION Data on embryosurvival providedno evidenceto supportthe existenceof a local component of the embryotoxic effect of a regressingCL. This findingwasmadein a situation(Experiment2) in which embryonicsurvivalin the uterinehornscontralateralandipsilateralto the CL did not differ in control animals.It is well known that the uterinehorn ipsilateralto the CL offers a more favorableenvironmentfor embryosurvival thanthe contralateralhorn (9, 12,21,22,32,33). However, supplementation of recipientswith 100mg of progesterone(im) hasbeenshownto improveembryosurvivalin the contralateraluterinehorn (5,9). Replacement therapy with progestogen(equivalentto 300 mg of progesteronedaily) did not supportsurvival of frozen-thawedembryosin the contralateraluterinehorn in Experiment 1. However, survival rates of freshembryosin the ipsilateral(7/9)andcontralateral(5/9) uterinehornsdid not differ in control recipientsin Experiment2. Ifluteolysis exerteda local effect, embryonicsurvival in treatedanimalswould be reducedonly in the horn ipsilateralto the regressingCL. Instead,a reductionin embryosurvival to 33% wasexperiencedin eachuterinehorn, providing no evidence for a local action of the regressingcorpusluteum. The presenceof a regressingCL is necessaryfor treatmentwith PGF, to reducepregnancy rates.The removalof the CL beforeor early duringtreatmentwith luteolytic dosesof PGF, in cows on replacementtherapywith progestogenimprovedor restoredpregnancyrates(3,3 1).

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Therefore,the regressingCL-mustbe eitherproducingor controllingsecretionof anembryotoxic factor(s). The systemiccirculationcouldbe the route for a lutealfactor to reachthe entireuterus. It is alsopossiblethat secretedproductsTom the regressingCL coulduselocal mechanisms in the femalereproductivevasculatureto reachthe ipsilateraluterinehorn in greaterconcentrationsthan havebeendemonstrated for progesterone thosefound in systemiccirculation. Suchmechanisms (34) and for PGF,, (13) in the ewe. Oncein the uterus,embryotoxiccompoundscould be transportedthrough the uterinelumento the contralateralhorn. Luteal compoundsthat reachthe uterusmay not be directly embryotoxic;they could stimulatethe productionof PGF,, whichhas beenshownto interferewith embryonicdevelopmentin vitro ( 1, 10, 16). As shownby Lemaster et al. (14), exogenousoxytocin duringDays 5 to 8 reducedpregnancyrates,but wasnot effective if secretionof PGF,, wasinhibited. They proposedthat luteal oxytocin reducedembryoviability by stimulatingsecretionof uterinePGF,,. Studieswith ligatedor separateduterinehornswill be requiredto determinewhetherthe lutealembryotoxic effect is truly systemicor involves uterine intraluminaltransportof anembryotoxin. In Experiment2, pregnancyrate wasreducedin cowstreatedwith PGF, (44%) relative to control animals(89%), despitereplacementtherapywith a progestogen.This resultfits with previouslyreportedlow pregnancyratesin postpartumcowswith a shortlutealphase(0%; 2,23, 28) andin matedcycling beef cowstreatedwith PGF, from Days 4 or 5 to Days 7 or 8 of the estrouscycle (20% to 25%; 3, 3 1). Treatmentwith PGF,, on only Days 7 and8 (Experiment1) did not reducepregnancyrate (7/l 1; 64%) or embryosurvival (8/22; 36%) whencomparedto controls(4110;40% and4120; 20%, respectively). Two majorfactorscould havebeeninvolved in theseresults;the duration(2 days)andthe initial day (Day 7) of treatmentwith PGF,. Sealset al. (3 1) haveshownthat treatmentfor 4 dayswasnot effective if startedon Days 10 or 15. Schtick et al. (30) demonstratedthat embryotoxic effectswere alreadyunderwaywhenembryosfrom cowswith shortestrouscyclesandhigh cone ntrationsof PGF,, in the uterinelumenwere recoveredand transferredto normalrecipientsaseearly asDay 6. Likewise,survival of normalembryos transferredon Day 7 into postpartumcowswith a shortluteal phasewasreduced(4). Therefore the actionof PGF,, or of the regressingCL, couldbe exertedon the uterineenvironmentandnot necessarilyor exclusivelyon the embryo,but it appearsthat concentrationsof PGF, mustbe increasedbeforeDay 7 to observeembryotoxiceffects, Pregnancyrate in the control group (4/10) in Experiment 1waslower than expected. In this group, noneof the embryosplacedinto the contralateraluterinehorn surviveddespite progestogenreplacementtherapy (O/10). One interpretationof the total absenceof twin pregnancies in control cowsis that the survival potentialof the cozen-thawedembryoswas particularly compromised in contralateraluterinehorns. This interpretationis consistentwith the improvedpregnancyrate (S/9) obtainedwith freshembryosin control cowsin Experiment2. From analysesof numeroussetsof datainvolving transferof singleandtwin embryosto cattle, sheepandgoats,McMillan (18, 19)andMcMillan et al. (20) haveproposeda binomialmodelof embryonicsurvival, with an embryonicand a maternalcontribution. He suggested that improvementof the embryonicdeterminantsof embryosurvivalis necessary to increasetwinning rates,

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In conclusion, no evidence was obtained that the embryotoxic effect of the early regressing CL acts in a strictly local manner. The embryo mortality associated with luteolysis by endogenous or exogenous PGF, probably involves compounds that act systemically or are transported through the uterine lumen after local transfer to the uterus. Alternatively, the luteal component could stimulate secretion of a uterine embryotoxin (such as PGF,,) into the uterine lumen. REFERENCES 1. Breuel KF, Fukuda A, S&rick FN. Effects of prostaglandin F2a on development of g-cell rat embryos in vitro. Biol Reprod 1993; 48(Suppl. 1): 173. 2. Breuel KF, Lewis PE, S&rick FN, Lishman AW, Inskeep EK, Butcher RL. Factors affecting fertility in the postpartum cow: Role of the oocyte and follicle in conception rate. Biol Reprod 1993;48:655-661, 3. Buford WI, Ahmad N, Schrick FN, Butcher RL, Lewis PE, Inskeep EK. Embryotoxicity of a regressing corpus luteum in beef cows supplemented with progestogen. Biol Reprod 1996;54:531-537. 4. Butcher RL, Reber JE, Lishman AW, Breuel KF, Schtick FN, Spitzer JC, Inskeep EK. Maintenance of pregnancy in postpartum beef cows that have short-lived corpora lutea. J Anim Sci 1992;70:3831-3837. 5. Christie WB, Newcomb R, Rowson LEA. Embryo survival in heifers after transfer of an egg to the uterine horn contralateral to the corpus luteum and the effect of treatments with progesterone or hCG on pregnancy rates. J Reprod Fertil 1979;56:701-706. 6. Cooper DA, Carver DA, Villeneuve P, Silvia WJ, Inskeep EK. Effects of progestagen treatment on concentrations of prostaglandins and oxytocin in plasma from the posterior vena cava of post-partum beef cows. J Reprod Fertil 1991;9 1:4 1 l-42 1. 7. Copelin JP, Smith MF, Garverick I-IA, Youngquist RS. Effect of the uterus on subnormal luteal function in anestrous beef cows. J Anim Sci 1987;64: 1506- 15 11. 8. Copelin JP, Smith MF, Keisler DH, Garverick HA. Effect of active immunization of prepartum and post-partum cows against prostaglandin F-2a on lifespan and progesterone secretion of short-lived corpora lutea. J Reprod Fertil 198987: 199-207. 9. Del Camp0 MR, Rowe RF, French LR, Ginther OJ. Unilateral relationship of embryos and the corpus luteum in cattle. Biol Reprod 1977;16:580-585. 10. Fazio RA, Buuck MJ, S&rick FN. Embryonic development of frozen-thawed bovine embryos cultured in vitro in response to elevated concentrations of prostaglandin F,a. Biol Reprod 1997;56(Suppl 1): 187 abstr. 11. Hu Y, Sanders DH, Kurz SG, Ottobre JS, Day ML. In vitro prostaglandin production by corpora lutea destined to be normal or short-lived. Biol Reprod 1990;42:801-807.

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