- Email: [email protected]
anti-PMSG treated heifers
SRY PUEMA EXTRADIOL17~ axJcZmmATIONs AND OVDIATION RATES IN FMSG/ANTI-PMSC TREATED HEIFERS H. Calleser?,T. Greve, P. Hyttel, A. Bak, P. Gotfredsenand P. Helm Departnent of Clinical Sciences,Reprcduction RoyalVeterinaryamdAgriculturalUniversity 13 Biilowsvej, DK-1870 RcederiksbergC, Demark Received for publication:Mq 23, 1989 Accepted: May 30, 1990
Possibilitiesfor early characterizationof the supemvulatory responsewerestudiedin41FEiZSG/FGtreateddairyheifers,ofwhich21 received ah additional tra+mmt of PMSG-antiserum. Plasm was obtained at 33, 36, 41, 47 and 51 h after FG for hornone analyses. of After slaughter at 6 or 7 d after insemination, the nti folliclesand corporalutea (CL) were recorded,and ovawere recovered for mxpholcgical evaluation. Significant correlations were denrmstratedbetween plasm concentrationsof estradiol-17a(EZ) at 33, 36 and 41 h after PC and the ovulationrate (nmber of CL). Each ofthesecorrelations was equal to theone found byusitqthe peak concentrationof E2 achievedduring the preovulatory EZ surge. In heifers with preovulatoryFL? surges, as determinedwith the blood san+ngscheme used,both theovarian response(number 0fCL and follicles)and the guality of ova recovered (numberof transferable e&ryos) was clearly better coqared to heifers without this surge. None of the parametersstudiedwas affected significantlyby treatmnt withmEG-antiserum. It is concludedthat plasma E2 determinationsat fixed times in relation to prostaglamdin treatmentcan be used to characterizethe superovulatoryresponse in donor cattle in term of the ovulationrate and the guality of ova recovered. No evidence was foundin favor of usingF%G-antiserum for improving either the supemvulatory response or such characterization. Key words: cattle, superovulation,anti-PMSG, estradiol-17a,ovarian response
Ihisworkwas supportedbytheDanishVeterinaqandAgricultura1 Research Council,Animal BiotechnologyResearch Center, DANOVA I/S and by Intervet Scandinavia. lhepracticalandtechnicalassistance of B. Avery, K. Ejstxup, I. Heinze, A.M. Olsen, J.O. Pedersen,M. Schmidt, S. smith, N. Svanborg and B. Synnes'tvedtis likewise greatly appreciated. ' Present address: Fmbryotechnolcgical Laboratory,National Institute of Animal Science, Fouls, P.O. Box 39, DK-8830 Tjele, Denmark.
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251
THERIOGENOLOGY INIWDUCTION In cattle, the productionof E2 from the preovulatoryfollicle is reflected in the plasma E2 concentrationwhich increases steadily until the LH surge cccurs (1). In superovulatedcattle, the maximal preovulatoryE2 concentration(the E2 peak) is highly correlated with the number of ovulationsas expressed by the number of corpora lutea present 6 to 7 d after insemination(2-6). Hawever, several blood samples are reguiredtodetermine themaximalE2 concentration,asits appearancevaries in relationto the time of prostaglandin injection (l-3,7). Therefore,determinationof E2 concentrationsat fixed times in relationto the prostaglardintreatmentwould facilitate attempts to charatiize the superovulatoryresponse in donor cattle. The presence and magnitude of the describedcorrelation depends on the normality of the periovulatory period. The use of FTEG for superovulationin cattle is reportedto have a serious impact on this normalityin terms of both preovulatoryfolliculardevelwt (B-11), ovulation rate and number (9,12-15), postovulatory plasma E2 concentrations (1,12,16), as well as embryo number and guality (12-18). In sconeof the cited studies,the adverse effects of FMSG arereportedtobe minimizedbytheuse of FWX-antiserum,while in others such a beneficialeffect has not been demonstratedin terms of the ovulation rate, embryo number and guality (19,20). It would therefore be useful to learn if treatment with EWSG-antiserum influences the periovulatory events and, thus, the relationship between preovulatory E2 production andpostovulatory corpus luteum formation. Ihe aim of this study was a) to examine if fixed-time determinations of plasma JT.2concentrations could be used to characterizethe super-ovulatory response inFMSG-treateddairyheifers and b) to evaluatethe influenceof PMX-antiserum treatmenton such characterization.
MA,Ilmuxs
AND
MEIHODS
Ten days after estrus, 41 dairy heifers from one herd werg stimulatedwith a singledose of pregnantmare serum gonadotrophin (FiYSG; 2500 IU i.m.). At 69 h after the injectionof gona%trophin, the heifers were given a prostaglandin analogue (luprcstiol; 15 rrrg i.m.) to induce luteal regression, and 73 h later they were inseminatedwith two straws of semen. At56 hafter prcstaglandin injection,thecheiferswere given an intravenousinjectionof either EWG-antiserum (n = 21) or saline (n = 20). The heifers were slaughtxxed6 or 7 d after insemination,and the number of follicles arxdcorporalutea wererecorded. The number of folliclescould be Inter-vet Scandinavia,Skovlunde,Denmark. IntervetScandinavia,Skovlunde,Denmark. , IntervetScandinavia,Skovlunde,Dermark.
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AUGUST 1990 VOL. 34 NO. 2
THERIOGENOLOGY accuratelydetemined in 30 heifers,while this was not possible in 11 heifers with numerousconfluent follicles. Corpora lutea were found horns of these in 37 of the 41 heifers, and the oviducts/uterine heiferswere flushedwith sukseguentmorphologicalevaluation of the collectedova (21). Blood samples were obtained frcnnvena jugularisat 33, 36, 41, 47 and 51h after prcstaglardininjection to determim plasma E2 and LH come&rations us&-g methods previouslydescribsl (7,22). The LH and E2 peaks were defined as the highest LH and F.2 concentration, respectively,masureddurimgthesuxge. Differencesin the superovulatoryresponseWmeenthegroupe of heifers were analyzed statisticallyusing Least Squares Means in the GLM procedure for mnparison ofmeanvalues and Fisher'sExact Test for cxqxarw frequencies (23). Significance of the differences between two correlations was tested against zero by t-test after with conversionof each r value to z (24). Meanvalues are exprb the star&r-d error of the mean (SEM).
RESULTS
In 36 of the heifers,preovulatoryE2 and LH surges were found with both peak levels occurring 41 f 1 h after the time of of prcstaglandininjection. The intervalfrmthe LHpeaktothetime thePMSCZantiserm injection averaged 15 t 1 h (range5 to 23 h), whilethe intervalk&ween the Wand them peakaveraged If 1 h (range7 to -2 h). The E2 peak concentrationsaveraged 195 2 19 pg/ml, while the mean concentrationsof E2 in plasma at 33, 36, 41, 47 and 51 h after prostaglandininjectionwere 137 f 13, 150 + 14, 170 + 17, 114 f 17 and 77 + 15 pg/ml, respectively. In the remainingfive heifers, no E2 and LH surges were found, and E2 continuedto increasethroughout the samplingperiod (147 f 48, 157 + 54, 197 f 65, 217 + 70 and 240 + 81 pg/rnlat 33, 36, 41, 47 and 51 h after prostaglandininjection,respectively). the correlation coefficients between 82 Table 1 shms concentrationsat the different samplingpoints and the nunker of wrpcra lutea found in each heifer at slaughter. me similar wrrelation coefficientobtained with the maximal F.2 wncentrations duringthe surge (EL?-peak)for the 36 heifers with LH and E2 surges was +0.6 (EO.01). Tbepresence 0fLH andF2 surgeswas inportantforthe ovarian response and the embryo guality as illustrated by the following differencesbetween the 36 heifers with U-Iand EL2surges and the five heifers without: nuinkerof wrpora lutea (26,3 ? 2,l vs 5,2 2 5,6; RO.O02), incidence of heifers with multiple anovulatory follicles (19% vs 80%, X0.02), incidence of heifers with ovaries with no wrpcra luti (3% vs 60%, PCO.005)and numhzr of transferable embrycs (4 f 1 vs 0 _+4, -0.05).
AUGUST 1990 VOL. 34 NO. 2
253
Table
1.
Correlation coefficients of the differenttimas of blood samplins after prostaglandin injection are shown for concentrations of estradiol-17p(E2) and the number of corpora lutea 6 to 7 d after inseminationfor each heifer with (+t) or without (--) LH and S2 surges. [email protected]~ (h after prostaglandininjection) LHand
msurge Correlation coefficients
No.
of heifers
33
41
36
47
51
++ - -
36 5
+0.7** i-0.6** +0.6* NS NS NS
NS NS
NS NS
SUm
41
+0.6** +0.5*
NS
NS
+0.5*
*pio.o1, **Ro.ool, NS: P>O.20. 4Jo significantdifferences(P>0.20)between correlationccefficients ateachtime. No significantdifferences (P>O.lO;data not sham) were found between heifers treated and those not treatedwith FWXGantiserm in anyoftheparan&ers studied, i.e. IH and E2 patterns (concentration levels,presence of LH and E2 surges),correlations ktweerlE2aJXd number of CL+ as well as ovarian response (aand follicles) and embryo yield (ova recoveredand transferableembryos).
DISCUSSION (xlrstudydemnstratesthat fixed-timeplasmE2 determinations in relation to the prostaglarxlin injectioncan be used to characterize the superovulatoryresponse in dairy heifers. The correlation between preovulatory E2 concentrations in the plasm and the ovulationrate (nmber of corpora lutea) was found to be eguallyhighwhetherthe E2 neasurmentswereperfomedatthepeak level or at fixed times prior to the expeckd time of the I.H peak. These fixed samplirqpoints should be chosen just prior to the time of theHi surge, whenE2 has reachedits highest concentration. A sampling time of up to approximately 33 h after prostaglandin treatmentmeets these requirements(1,2,5,7,9,16,25,26). surgeswasan important Thepresence ofpreovulatory IHandE2 factor in governingovarian developmentand guality of ova. 'Ibobtain this information, a second E2 determination needs to be performed afterthetimeof theUi peak, because E2 concentrationsat this time are lower than the presurge levels. From approximately 51 h after prostaglandintreatmntis a suitable time for such sampling, since most IH surges have appear& by this tine (1,2,25,26).
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THERIOGENOLOGY Thus, two E2 determinations- one performedbefore and one after theUIsurge- may characterizethe superovulatoryresponse in donor cattle in term of the number of corpora lutea present 6 to 7 d after inseminationand the guality of ova recovered. For evaluatirq donor cattle, these measurements should be used in conjunctionwith other hormonal parameters, e.g. progesterone concentrationsin plasm or milk before, during and/or after the superovulatory estrus (6,25,27,28). The E2 levels and ovulationrates and the correlations between these parametersdid not differ significantlybetween heifers treated andthcsenottreatedwith~antiserum. Moreover,therewere only minor and non-significant differencesbetween theovarian response, the recovery rate and the quality ofova betweenthese groups of therefore,gave no evidence in favor of usirq heifers. our study, IWSG-antifor insuriq a more normal periovulatoryperiod with subsequentimprovementof the embryo yield. Similar results have been reported in other studies (19,20,29) in which an effect of FMSG-antiserumwas noted in term of fewer anovulatoryfollicles but not of ova production or quality. Theseresults are in contrast, howevex, with those of other studies (12,14,17,18) in which FNSG-antiserumwas administered after preovulatory changes had been initiatedby the LH surge, i.e. at least 36 h after the prostaglandin treatment (2,5,7-9,16,25,26,30).The importance of the relation between this LHrelease andthetimeof FWSG-antiserum administration was demonstratedin three studies (12,13,18),where superovulationwas preventedby administrationof RG&antisemm earlier than the time of them surge. As a consequence, Dielemn et al. (9-11,15) used frequentblood samplings for rapid LH determination, administered FEZ-antiserum 5to6 hafterthe timeofthe LHpaakand obtained normalizationof the periovulatoryevents. The value of such timed antiserm administrationhas been questionedby Chupin et al. (30), who found no differencein %nyparameter of the ovarian re.sponseUN (ovulationrate, mmker of ova recovered, number of transferable embryos)when JXX-antiserum was given in average 7 or 15 h after the IHpmk,similartoourfindiqs (datanot shown). In comparison to these two studies,Dielemn et al. (g-11,15)still recovereda higher nmber of transferableembryos. Thus, althoughthe advantageof and protocol for EWG-antiserum administration is not clear from the resultsof our study or from most of the literature cited, the principleof neutralizingFWX with antiserumsoon after the IH peak seems a reasonable one. !Co use such a procedure, hmever, an alternativeto the I_Hpeak determination mustbefound,sincesuch a determinationrequires frequent blood samplingsover a period of time. Asdemonstrated inour study, theuse of S2determinations is a possiblemeans for determiningthe optimal timing for administering FNSG-antiserum. In conclusion,this study has shownthat theuse of fixed-time determinationsof E2 concentrations in plasma frm SuperovU~ated heifers can serve as a reliable characterization of the number of corpora lutea present 6 to 7 d after ihsemination. Moreover, it is as practicaland valid as the use of E2 peak levels,and it is a good indirectcharacterizationof the guality of ova recovered. The use of
AUGUST 1990VOL. 34 NO. 2
255
THERIOGENOLOGY
nor did it PMSG-antiserumdid not result in a better characterization, -rove ovulationrate, embryo recoveryrate or embryo guality. REFERENCES 1.
Bevers, M.M. arid Dielman, S.J. Superovulation of ems with PM.%: variations in plasma concentrations of progesterone, oestradiol, IH, cortisol, prolactin ahd PM9G and in nm&x.r of preovulatoryfollicles. Anim. Reprod. Sci. -15:37-52 (1987).
luteinizing hormone, Concentrations of 2. Saumnde, J. oestradiol-17pand progesterone in the plasm of heifers treated to induce superovulation.J. Erdocrinol.84:425-437(1980). Superovulation in the cow: 3. Samande, 3. and Batra, S.K. comparisonof oestradiol-17pand progesteronepatterns in plasma and milk of cows inducedto superovulate; relationshipswith ovarianresponses. J. Endocrinol.107:259-264(1985). 4. ~indsell,C.E., Murphy, B.D. and Mapletoft,R.J. Superovulatory and endocrine responses in heifers treated with FSH-P at differentstages of the estrous cycle. lhericgenolqy 26:209-219 (1986). 5. Savage, N.C., Howell, W. and Mapletoft,R.J. Superovulation in the cow using estradiol-17por GnRH in conjunctionwith FSH-P. lhericgenology27:383-393(1987). 6. Goto, K., ohkutsu, s., Nakanishi,Y., Cgawa, K., Tasaki, M., Inohae, S., Tateym, S., Kawabata,T., Ishii, S., Ohta, H., Erdccrihe Miyamoto,A., Furusawa,T., Umezu, M. and Masaki, J. profiles and embryo guality in superovulated Japanese black 29:615-629(1988). cattle. Thericgenology7. Callesen,H., Greve, T. ahd Hyttel, P. Premture ovulationsin superovulatedcattle. Theriogenolcqy28:155-166(1987). and Hyttel, P. Preovulatory 8. Callesen, H., Greve, T. ehdocrinolcgy and. cxxyte maturation in superovulatsd cattle. Theriogenolcgy-25:71-86 (1986). 9. Dielemah,S.J. and Bevers,M.M. Effects of monoclonalantibody against EMSG administeredshortly after the preovulatoryIH surge on time and number of ovulations in PMSG/FG-treated ems. J. Reprcd. Fertil.-81:533-542(1987). 10. Dielfman,S-J., Bevexs,M.M. and Kruip, T.A.M. Differentiated micrmorpholagy of the preovulatory follicle population at ovulation in FMSG-superovulated cows with or without anti-w 11th Int. Congr. Anim. administeredshortlyafter the IHpeak. Reprod. & AI, Dublin, Ireland,II:152 (1988a).
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THERIOGENOLOGY
11.
Dielemn, S.J., Bevers, M.M., Kruip, T.A.M., van Tel, H.T.M. and Blankenstxin, D-M. Steroid profilesand micronmrphologyof the folliclepopulation before ovulation in PM%-superovulatedcm.m with or without monoclonalanti-w administeredshortly after the preovulatoryLHpeak. 11th Int. Congr. Anim. Reprcd. &AI, Dublin, Ireland, II:154 (1988b).
12. Ekxute.rs, R., Moyaert, I., Coryn, M. and Vandeplassche,M. The useof a PMSGantiserm in superovulatedcattle: endocrinological changes arxl effects on timing of ovulation. Zuchthygiene -18:172-177 (1983). 13. Kim, H.N., Rorie, R.W., Youngs, C.R., White, K.L. and Gxxlke,R.A. Ihe use of anti-w antibodieswith m for superovulatingbeef 27:243 abstr. (1987). cattle. !Iheriogenolcgy 14. Wang, H., Wu, M., Xu, K., Hagele, W.C. and Mapletoft,R.J. Control of superovulation in the cowwith aPMSGantiserum. lheriogenology-27:291 abet-r.(1987). 15. Dieleman, S-J., Bevers, M.M., Worth, Y.A. and Kruip, T.A.M. stage of Develovtal embryos at Day 6 and7i.n PMSG-superovulated ems with or without nmoclonal anti-EMS3 administeredshortly after the preovulatoryIH peak. 11th Int. Cohgr. Ax-&n.Reprod. & AI, Dublin, Ireland,II:156 (1988c). G., Tiemann, U., 16. Kanitz, W., Kanitz, E., Kitzig, M., Bliidaw, Bergfeld,J. and Ronunel, P. Effects of PM.% antiserumon honmne profiles of cows in superovulation. Mh. Vet.-Med. 41:484-487 (1986). 17. Sammnde, J., Procureur,R. and Chupin, D. Effect of injection time of anti-PMSGantiserum on ovulation rate and guality of embryos ihsuperovulatedcows. Thericqenology-21:727-731 (1984). 18. Rehbock, F., Rammel, P., Tier&am,U. and Blodow, G. UseOf PMSG antiserum in superovulation treatmentof cattle. Mh. Vet.-Med. -41:581-583 (1986). 19. Greve, T., EQk, A. and Schmidt, M. Superovulationof dairy cattle: effect of PM%-antiserum treatment. Thericgenolcgy 29:252 abstr. (1988). 20. Callesen,H., Bak, A., Greve, T., Avery, B., Gotfredsen,P., Holm, P., Hyttel, P., Pederseh,J.O., Schmidt,M., Smith, S. and Use of PMS.3 antisera in superovulated dairy svanborg,N. heifers. Iheriogenolcgy-31:179 abetr. (1989a). 21. Greve, T. Bovine Egg Transplantationin Denmark. Dr. Vet. Sci. Dissertation, Royal Veterinary and Agricultural University, copenhasen,Denmark, 1981.
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22. Hoier, R. Radioimunoassay for luteinizinghormone in bovine species based on commercially available reagents. Acta Vet. Scar-d. -29:61-66 (1988). 23. S?iS User's Guide: Statistics. SAS Institute,Inc., Cary, NC, USA (1985). 24. Snedecor,G.W. and Cc&ran, W.G. StatisticalMethods. State UniversityPress, Ames, Iowa, USA (1967).
The Im
Preovulatory evaluation 25. Callesen,H., Greve, T. and Hyttel, P. of the superovulatoryresponse in donor cattle. Thericgenology 30:477-488(1988). 26. Kweon, O.K., Kanagawa,H., Takahashi,Y., Miyamoto,A., Masaki, Plasm J ., Umezu, M., Kagabu, S., Iwazumi,Y. and Aoyagi, Y. endocrineprofiles and total cholesterollevels in superovulated cows. Thericgenolcgy-27:841-857(1987). 27. Allen, S.E. and Foote, R.H. An enzyme-linkedimnmoassay of milk progesterone as a diagnosticaid inembryotransferprqrams. Theriogenology29:893-903(1988). 28. Callesen,H., Bak, A., Greve, T., Avery, B., Gotfredsem, P., Holm, P., Hyttel, P., Pedersen,J.O., Schmidt,M., Smith, S. and svanborq,N. Hormonalparametersfor evaluationof superovulated heifers. Thericgenolcqy-31:180 abstr. (198933). 29. Alfuraiji,M.M., Broadbent, P.J., Hutchinson,J.S.M., Dolman, Effect of time of administrationof D.F. and Atkinson,T. monoclonal anti-EMS+3on superovulatory response and embryo guality in cattle. Theriogenology-31:165 abstr. (1989). 30. Chupin, D., Steiner, M. and Saumande,J. Neutra-PMSGinjected early after the IH peak does not improveovulationrate in PMSG 11th Int. Congr. Anim Reprod. & AI, Dublin, treated heifers. Ireland, II:147 (1988).
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1990 VOL. 34 NO. 2
Possibilitiesfor early characterizationof the supemvulatory responsewerestudiedin41FEiZSG/FGtreateddairyheifers,ofwhich21 received ah additional tra+mmt of PMSG-antiserum. Plasm was obtained at 33, 36, 41, 47 and 51 h after FG for hornone analyses. of After slaughter at 6 or 7 d after insemination, the nti folliclesand corporalutea (CL) were recorded,and ovawere recovered for mxpholcgical evaluation. Significant correlations were denrmstratedbetween plasm concentrationsof estradiol-17a(EZ) at 33, 36 and 41 h after PC and the ovulationrate (nmber of CL). Each ofthesecorrelations was equal to theone found byusitqthe peak concentrationof E2 achievedduring the preovulatory EZ surge. In heifers with preovulatoryFL? surges, as determinedwith the blood san+ngscheme used,both theovarian response(number 0fCL and follicles)and the guality of ova recovered (numberof transferable e&ryos) was clearly better coqared to heifers without this surge. None of the parametersstudiedwas affected significantlyby treatmnt withmEG-antiserum. It is concludedthat plasma E2 determinationsat fixed times in relation to prostaglamdin treatmentcan be used to characterizethe superovulatoryresponse in donor cattle in term of the ovulationrate and the guality of ova recovered. No evidence was foundin favor of usingF%G-antiserum for improving either the supemvulatory response or such characterization. Key words: cattle, superovulation,anti-PMSG, estradiol-17a,ovarian response
Ihisworkwas supportedbytheDanishVeterinaqandAgricultura1 Research Council,Animal BiotechnologyResearch Center, DANOVA I/S and by Intervet Scandinavia. lhepracticalandtechnicalassistance of B. Avery, K. Ejstxup, I. Heinze, A.M. Olsen, J.O. Pedersen,M. Schmidt, S. smith, N. Svanborg and B. Synnes'tvedtis likewise greatly appreciated. ' Present address: Fmbryotechnolcgical Laboratory,National Institute of Animal Science, Fouls, P.O. Box 39, DK-8830 Tjele, Denmark.
AUGUST
1990 VOL. 34 NO. 2
251
THERIOGENOLOGY INIWDUCTION In cattle, the productionof E2 from the preovulatoryfollicle is reflected in the plasma E2 concentrationwhich increases steadily until the LH surge cccurs (1). In superovulatedcattle, the maximal preovulatoryE2 concentration(the E2 peak) is highly correlated with the number of ovulationsas expressed by the number of corpora lutea present 6 to 7 d after insemination(2-6). Hawever, several blood samples are reguiredtodetermine themaximalE2 concentration,asits appearancevaries in relationto the time of prostaglandin injection (l-3,7). Therefore,determinationof E2 concentrationsat fixed times in relationto the prostaglardintreatmentwould facilitate attempts to charatiize the superovulatoryresponse in donor cattle. The presence and magnitude of the describedcorrelation depends on the normality of the periovulatory period. The use of FTEG for superovulationin cattle is reportedto have a serious impact on this normalityin terms of both preovulatoryfolliculardevelwt (B-11), ovulation rate and number (9,12-15), postovulatory plasma E2 concentrations (1,12,16), as well as embryo number and guality (12-18). In sconeof the cited studies,the adverse effects of FMSG arereportedtobe minimizedbytheuse of FWX-antiserum,while in others such a beneficialeffect has not been demonstratedin terms of the ovulation rate, embryo number and guality (19,20). It would therefore be useful to learn if treatment with EWSG-antiserum influences the periovulatory events and, thus, the relationship between preovulatory E2 production andpostovulatory corpus luteum formation. Ihe aim of this study was a) to examine if fixed-time determinations of plasma JT.2concentrations could be used to characterizethe super-ovulatory response inFMSG-treateddairyheifers and b) to evaluatethe influenceof PMX-antiserum treatmenton such characterization.
MA,Ilmuxs
AND
MEIHODS
Ten days after estrus, 41 dairy heifers from one herd werg stimulatedwith a singledose of pregnantmare serum gonadotrophin (FiYSG; 2500 IU i.m.). At 69 h after the injectionof gona%trophin, the heifers were given a prostaglandin analogue (luprcstiol; 15 rrrg i.m.) to induce luteal regression, and 73 h later they were inseminatedwith two straws of semen. At56 hafter prcstaglandin injection,thecheiferswere given an intravenousinjectionof either EWG-antiserum (n = 21) or saline (n = 20). The heifers were slaughtxxed6 or 7 d after insemination,and the number of follicles arxdcorporalutea wererecorded. The number of folliclescould be Inter-vet Scandinavia,Skovlunde,Denmark. IntervetScandinavia,Skovlunde,Denmark. , IntervetScandinavia,Skovlunde,Dermark.
252
AUGUST 1990 VOL. 34 NO. 2
THERIOGENOLOGY accuratelydetemined in 30 heifers,while this was not possible in 11 heifers with numerousconfluent follicles. Corpora lutea were found horns of these in 37 of the 41 heifers, and the oviducts/uterine heiferswere flushedwith sukseguentmorphologicalevaluation of the collectedova (21). Blood samples were obtained frcnnvena jugularisat 33, 36, 41, 47 and 51h after prcstaglardininjection to determim plasma E2 and LH come&rations us&-g methods previouslydescribsl (7,22). The LH and E2 peaks were defined as the highest LH and F.2 concentration, respectively,masureddurimgthesuxge. Differencesin the superovulatoryresponseWmeenthegroupe of heifers were analyzed statisticallyusing Least Squares Means in the GLM procedure for mnparison ofmeanvalues and Fisher'sExact Test for cxqxarw frequencies (23). Significance of the differences between two correlations was tested against zero by t-test after with conversionof each r value to z (24). Meanvalues are exprb the star&r-d error of the mean (SEM).
RESULTS
In 36 of the heifers,preovulatoryE2 and LH surges were found with both peak levels occurring 41 f 1 h after the time of of prcstaglandininjection. The intervalfrmthe LHpeaktothetime thePMSCZantiserm injection averaged 15 t 1 h (range5 to 23 h), whilethe intervalk&ween the Wand them peakaveraged If 1 h (range7 to -2 h). The E2 peak concentrationsaveraged 195 2 19 pg/ml, while the mean concentrationsof E2 in plasma at 33, 36, 41, 47 and 51 h after prostaglandininjectionwere 137 f 13, 150 + 14, 170 + 17, 114 f 17 and 77 + 15 pg/ml, respectively. In the remainingfive heifers, no E2 and LH surges were found, and E2 continuedto increasethroughout the samplingperiod (147 f 48, 157 + 54, 197 f 65, 217 + 70 and 240 + 81 pg/rnlat 33, 36, 41, 47 and 51 h after prostaglandininjection,respectively). the correlation coefficients between 82 Table 1 shms concentrationsat the different samplingpoints and the nunker of wrpcra lutea found in each heifer at slaughter. me similar wrrelation coefficientobtained with the maximal F.2 wncentrations duringthe surge (EL?-peak)for the 36 heifers with LH and E2 surges was +0.6 (EO.01). Tbepresence 0fLH andF2 surgeswas inportantforthe ovarian response and the embryo guality as illustrated by the following differencesbetween the 36 heifers with U-Iand EL2surges and the five heifers without: nuinkerof wrpora lutea (26,3 ? 2,l vs 5,2 2 5,6; RO.O02), incidence of heifers with multiple anovulatory follicles (19% vs 80%, X0.02), incidence of heifers with ovaries with no wrpcra luti (3% vs 60%, PCO.005)and numhzr of transferable embrycs (4 f 1 vs 0 _+4, -0.05).
AUGUST 1990 VOL. 34 NO. 2
253
Table
1.
Correlation coefficients of the differenttimas of blood samplins after prostaglandin injection are shown for concentrations of estradiol-17p(E2) and the number of corpora lutea 6 to 7 d after inseminationfor each heifer with (+t) or without (--) LH and S2 surges. [email protected]~ (h after prostaglandininjection) LHand
msurge Correlation coefficients
No.
of heifers
33
41
36
47
51
++ - -
36 5
+0.7** i-0.6** +0.6* NS NS NS
NS NS
NS NS
SUm
41
+0.6** +0.5*
NS
NS
+0.5*
*pio.o1, **Ro.ool, NS: P>O.20. 4Jo significantdifferences(P>0.20)between correlationccefficients ateachtime. No significantdifferences (P>O.lO;data not sham) were found between heifers treated and those not treatedwith FWXGantiserm in anyoftheparan&ers studied, i.e. IH and E2 patterns (concentration levels,presence of LH and E2 surges),correlations ktweerlE2aJXd number of CL+ as well as ovarian response (aand follicles) and embryo yield (ova recoveredand transferableembryos).
DISCUSSION (xlrstudydemnstratesthat fixed-timeplasmE2 determinations in relation to the prostaglarxlin injectioncan be used to characterize the superovulatoryresponse in dairy heifers. The correlation between preovulatory E2 concentrations in the plasm and the ovulationrate (nmber of corpora lutea) was found to be eguallyhighwhetherthe E2 neasurmentswereperfomedatthepeak level or at fixed times prior to the expeckd time of the I.H peak. These fixed samplirqpoints should be chosen just prior to the time of theHi surge, whenE2 has reachedits highest concentration. A sampling time of up to approximately 33 h after prostaglandin treatmentmeets these requirements(1,2,5,7,9,16,25,26). surgeswasan important Thepresence ofpreovulatory IHandE2 factor in governingovarian developmentand guality of ova. 'Ibobtain this information, a second E2 determination needs to be performed afterthetimeof theUi peak, because E2 concentrationsat this time are lower than the presurge levels. From approximately 51 h after prostaglandintreatmntis a suitable time for such sampling, since most IH surges have appear& by this tine (1,2,25,26).
254
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THERIOGENOLOGY Thus, two E2 determinations- one performedbefore and one after theUIsurge- may characterizethe superovulatoryresponse in donor cattle in term of the number of corpora lutea present 6 to 7 d after inseminationand the guality of ova recovered. For evaluatirq donor cattle, these measurements should be used in conjunctionwith other hormonal parameters, e.g. progesterone concentrationsin plasm or milk before, during and/or after the superovulatory estrus (6,25,27,28). The E2 levels and ovulationrates and the correlations between these parametersdid not differ significantlybetween heifers treated andthcsenottreatedwith~antiserum. Moreover,therewere only minor and non-significant differencesbetween theovarian response, the recovery rate and the quality ofova betweenthese groups of therefore,gave no evidence in favor of usirq heifers. our study, IWSG-antifor insuriq a more normal periovulatoryperiod with subsequentimprovementof the embryo yield. Similar results have been reported in other studies (19,20,29) in which an effect of FMSG-antiserumwas noted in term of fewer anovulatoryfollicles but not of ova production or quality. Theseresults are in contrast, howevex, with those of other studies (12,14,17,18) in which FNSG-antiserumwas administered after preovulatory changes had been initiatedby the LH surge, i.e. at least 36 h after the prostaglandin treatment (2,5,7-9,16,25,26,30).The importance of the relation between this LHrelease andthetimeof FWSG-antiserum administration was demonstratedin three studies (12,13,18),where superovulationwas preventedby administrationof RG&antisemm earlier than the time of them surge. As a consequence, Dielemn et al. (9-11,15) used frequentblood samplings for rapid LH determination, administered FEZ-antiserum 5to6 hafterthe timeofthe LHpaakand obtained normalizationof the periovulatoryevents. The value of such timed antiserm administrationhas been questionedby Chupin et al. (30), who found no differencein %nyparameter of the ovarian re.sponseUN (ovulationrate, mmker of ova recovered, number of transferable embryos)when JXX-antiserum was given in average 7 or 15 h after the IHpmk,similartoourfindiqs (datanot shown). In comparison to these two studies,Dielemn et al. (g-11,15)still recovereda higher nmber of transferableembryos. Thus, althoughthe advantageof and protocol for EWG-antiserum administration is not clear from the resultsof our study or from most of the literature cited, the principleof neutralizingFWX with antiserumsoon after the IH peak seems a reasonable one. !Co use such a procedure, hmever, an alternativeto the I_Hpeak determination mustbefound,sincesuch a determinationrequires frequent blood samplingsover a period of time. Asdemonstrated inour study, theuse of S2determinations is a possiblemeans for determiningthe optimal timing for administering FNSG-antiserum. In conclusion,this study has shownthat theuse of fixed-time determinationsof E2 concentrations in plasma frm SuperovU~ated heifers can serve as a reliable characterization of the number of corpora lutea present 6 to 7 d after ihsemination. Moreover, it is as practicaland valid as the use of E2 peak levels,and it is a good indirectcharacterizationof the guality of ova recovered. The use of
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nor did it PMSG-antiserumdid not result in a better characterization, -rove ovulationrate, embryo recoveryrate or embryo guality. REFERENCES 1.
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Preovulatory evaluation 25. Callesen,H., Greve, T. and Hyttel, P. of the superovulatoryresponse in donor cattle. Thericgenology 30:477-488(1988). 26. Kweon, O.K., Kanagawa,H., Takahashi,Y., Miyamoto,A., Masaki, Plasm J ., Umezu, M., Kagabu, S., Iwazumi,Y. and Aoyagi, Y. endocrineprofiles and total cholesterollevels in superovulated cows. Thericgenolcgy-27:841-857(1987). 27. Allen, S.E. and Foote, R.H. An enzyme-linkedimnmoassay of milk progesterone as a diagnosticaid inembryotransferprqrams. Theriogenology29:893-903(1988). 28. Callesen,H., Bak, A., Greve, T., Avery, B., Gotfredsem, P., Holm, P., Hyttel, P., Pedersen,J.O., Schmidt,M., Smith, S. and svanborq,N. Hormonalparametersfor evaluationof superovulated heifers. Thericgenolcqy-31:180 abstr. (198933). 29. Alfuraiji,M.M., Broadbent, P.J., Hutchinson,J.S.M., Dolman, Effect of time of administrationof D.F. and Atkinson,T. monoclonal anti-EMS+3on superovulatory response and embryo guality in cattle. Theriogenology-31:165 abstr. (1989). 30. Chupin, D., Steiner, M. and Saumande,J. Neutra-PMSGinjected early after the IH peak does not improveovulationrate in PMSG 11th Int. Congr. Anim Reprod. & AI, Dublin, treated heifers. Ireland, II:147 (1988).
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