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Fertilization rate after synchronization of the oestrous cycle in heifers with prostaglandin F2α
X?RTILIZATIONHATE AFYXR SYNCHRONIZATIONOF THH OESTHOUS CYCLR IN HZXFZRS WITH PHOSTAGLWDIN F2d J. Fulka, J. Yotlik,A. Pavlok Department of Genetics, Institute of Animal_Physioiogy and Genetics, CzechoslovakAcademy of Sciences Lib&chov, Czechoslovakia (Receivedfor publicationFebruary 4, 1975)
Oestrus was synchronizedin 31 heifers by the intrauterine administrationof PGF dtham salt. Nineteen were given 2 doses of 0.5~ 24 hr gpart and 10 of these received 1500 I.U. of P&SG i.m. 24 hr befor& the treatment with -PGF2"L. The remaining 12 heifers in the experimentwere given a single dose of znlgfollowed at the beginning of oestrus by 1500 I.U. of HCG i.m. Of 9 heifers which received only the two doses of O.&g (Group 11, 7 were observed to have corpora lutea when slaughtered56-72 hr after the onset of oestrus, and four fertilized eggs were recovered. In those which received PEG before the double injection of PGF o+!(Group 21, 118 corpora lutea were observed at slaughter a%d 34 fertilized eggs were recovered. Each heifer which received a single injection of R3F &and HCG had a corpus luteum, and 9 fertilizedeggs were i: ecovered. Unovulated follicles were most commonly observed in the PkEGtreated heifers but they were also observed in the heifers given the double injection treatment. It was observed that in the two-injectiontreatments, whether or not given PXSG, time of ovulation relative to the onset of oestrus was variable, and eggs were found in the uterus before the expected time. ACKNOV~zEM;~~X?~T ProstaglandinF dtham salt was a generous gift of Drs. J.B. Pike and J.Y. L&derdale, the Upjohn Company.
MARCH
1975 VOL. 3 NO. 3
107
THERIOGENOLOGY
INTXODUCTION
ProstaglandinF oL(FGF d> given either locally into
the uterine horn ipsQlateraf to the CL (1 2 3 4) or sys-
temically (5,6) is effective in causing &tiai re ression in the cow. The administrationof EGF2&between ehe 5th and 16th days of the cycle is followed by a significant decrease of serum progesterone,increaseof serum estro en and occurrence of LH ovulatory surge and oestrus (2,3,4f . At present, there are limited data on the use of FGF cLin connectionwith fertilizationand conception rate aft& artificial insemination(1,5,6,7). !Pheaim of the present experimentswas to study some details concerning oestrus and fertilizationrate after dffferent application of FGF2&alone, or in combinationwith PMS3 or HCG.
31 heifers of the Red-Spotted breed, aged 14 to 20 months, and weighing 390 to 500 kg, were used in the erperiments. The stage of the oestrous cycle was determined by repeated rectal palpation. WF Oc- tham salt -8 injected either twice, 0.5mg on each ef%c consecutivedays (19 heifers - groups 1 and 21, or as a ringle dose of @ (l2 heifers - group 3) into the ipsilateraluterine horn, between the 7th and 14th day of the cycle. To ensure mre reliable administrationof FGF o$ Bunpun was injected i.m. beforehand i% a dose of 0. Before application of RP &through the wall rectiinto the uterine lden with a long K ulla recti rraeirrigated rith 0.W of Ajatin (Slore~ kofs$~ of 1,500 LIT. R86 (Antex, Lao, Copsnbagen) ~oskg~iv&i.m. 24 hours before the treatipent rith wF9oLtO A dose of 1,509 I.U. HCG (FPaadyn,Spofn, I?~qpa) was injectid in 12 heifene (group 3) at the ornet af ee#tFus (Table 1). The onset of oestrus was determinedaccordiag t0 behavior and rectal palpation.All heifer6 were lBmminated twice at 15 hour intervals with frozen semen frtxebulls belonging to the &ate A.I. station. 56 to 72 how titer the onset of oestrus the heifers were killad at the alaughterhouse and their reproductive organs taken to the laborAfter inspectionof ovaries the oriducts ax& %%e horns were flushed with salbe containiag-u&n. The ova were first evaluated urder the stereorricreocope. Phase-contrastmicroscopy was wed for detailed exaaimtian of ova fixed in acetalcoholand stained with acetic OXWin.
108
MARCH 1975 VOL. 3 NO. 3
THERIOGENOLOGY
MARCH
1975
VOL. 3 NO. 3
109
THJZRIOGENOLOGY
Cleaved ova with nuclei in all blastomeresand distinct polar bodies were evaluatedas fertilized. The presence of pronuclei,the occurrenceof sperm tails in the cytoplasm, and extrusion of the seco_ndpolar body, were criteria for fertilizationof uncleaved ova. RESULTS The onset of oestrus was observed in all experimental heifers within 56 to 96 hours. In most animals the first symptoms of oestrusappeared on the third day, regardless of whether a single or two injections of FGF &were given. In Groups 1 and 2 abnormally long and intensvveoestrus was observed. In some heifers uterine cervix was open for more than 48 hours. Ovulation occurred in all heifers, with two exceptions in Group 1 (Table I>. Even in these cases, well developed preovulatoryfollicleswere found on the ovaries. Treatment with P?&SGat-ted the developmentof a higher number of follicles in all animals but individual response was variable. The mean number o# follicles cer heifer was 15, of which 11.8 per heifer ovulated. The number of ova obtained from Group 1 (2 x 0.5mg FGF d was relatively low, since from 'Iovulated follicles onlf4 ova were found. With reference to the abnormally long oestrus, it seems likely that at least in some animals the ova escaped into the uterine horns at a time when they were expected to be in the oviducts. The proportion of ova obtained from PBG-stimulated heifers was also low (66.9%). Here again, some ova were flushed from the uterine horns, although according to the onset of oestrus and ~~h$ime of slaughteringthey should have been in the ovi.
The fertilizationrate of ova flushed from heifers injected with HCG was relatively high ('75%).On the other hand, fertilization in FHSGtreated heifers m low - 43% These unsatisfactoryresults were caused mainly by complete failure of fertilizationin some aniPPelswith relktively high ovulationrate. DISCUSSION The results concerningoestrus synchronizationare comparable with those referred by other authors l&2,3,4, 5). The main aim of our study was to follow the ovulation and fertilizationrate in the first synchronizedoestrus. Ovulation failed to occur onl in two heifers, both treated with/2 x 0.5 mg HGF2a? The reason for this failure
MARCH
1975 VOL. 3 NO. 3
THERIOGENOLOGY
is difficult to explain, especially as the oestrus behavior was completely norms1 in both animals. Except for the &%-treated group, the fertilization rate attained a satisfactorylevel higher than after synchronization with tilengestrolaceiate (MGA)(8). In superovulated heifers, on the other hand, the fertilizationrate was relatively low and did not reach the values described by other authors (9). This may have been due to wrong choice of the inseminationtime, the quality of the spermatozoa or unfavorable conditionsfor transport and capacitation of the spermatozoacaused by the abnormal hormonal conditions. This view would be also in agreement with the absence of spermatozoa on the zona pellucida of unfertilized ova. The relative high proportionof the ova flushed from the uterine horns in some animals would support this opinion. The purpose of HCG administrationin the last group was to fix the time of ovulation. In the normal oestrous cycle a transient Ui surge occurs soon after the onset of oestrus (10). In W-synchronized heifer8 ovulation occurred 32 to 40 hours after IiCGwection (11). Conception after inseminationcarried out with respect to this time interval was far below expectation (12). On the contrary, in our experiments the proportionof fertilized ova was relatively high after BCG treatment. Cur knowledge of the level of pre- and postimplantationlosses in relation to fertility in the given treatment, is likewise altogether inadequate. We can conclude, however, from preliminary,unpublished results, that using ;PGF2+for synchronizationand HCG for induction of ovulationgive conception rates which are comparable with figures obtained in normal oestrus (57.2% pre nant in normal oestrus and 68.6% pregnant after FGF2dand fliCG treatment). ~BZIKXS 1. Bowson, L.E.A., Tervit, R., and &a&, A. Synchronization of oeetrus in cattle by means of prostaglandin F d Froc. VII. Int. Congr. Anim. Reprod. and A.1. M&hen. vol. IIt 866469 (19'72). 2.
Louis, T.Y. Bafs, H.D. and Morrow, D.A. Estrus_and ovulation after uterine &z 2din cows* J. Anim. Scl. 35: 247-248 (1972).
3. Louis, T.Y., Bafs, H.D., and Se&n, B.E. Progesterone, LH eetrus and ovulationafter pros landin F din hehers. Proc. Sot. Exp. Biol. 152-266 (1973).
MARCH
1975 VOL. 3 NO. 3
111
THERIOGENOLOGY
4.
Louis, T.M., Hafs, H.D., and Morrow, D.A. Intrauterine administrationof prostaglandinF &in cows: progesterestrogen,LH estrus and ovuPation. J. Anim. Sci. g '347.353 (39743
5.
Philipsen H., and Rasbech, N.O. The use of prostalandin l&F aC>for heat synchronizationin heifers. L sberetn. ?nst. f. Sterilitetsforskn.,Kobenhavn: 157-162 (1973).
6.
Inskeep, ES. Potentialuses of prostaglandinsin control of reproductivecycles of domestic animals. J. Anim. Sci. 36: 1149-ll59 (1973).
7.
Lauderdale, J.W., Seguin, B.E., Stellflug,J.N., Chenault, JR., Thatcher, W.W., Vincent, C.K., and Loyancano,A.F. Fertility of cattle following PGF2d injection.J. Anim. Sci. g_t 964-967 (1974).
8.
Hill, JR., Jr., Lamond, D.R., Henricks, D.M., Dickey, J.F., and Niswender,G.D. The effect of melengestrol acetate (laca)on ovarianfunction and fertilization in beef heifers. Biol. Repcod. St 16-22 (197l)
9.
gleden, B.P., Lewis, Susan, Gumming, I&., and Lawson, R.A.S. Superovulationin the cow following treatment with PMSG and prostaglandinF2d J. Reprod. Fert. 36: 435-456 (1974).
D., and Karg, H. RadioimmunologischeLH10. SC-, Bestin Blutserumvom Rind unter besondererBerllcbichtigungde8 Brunstzyklus.Acta endocr., Copenh. 6& 96-102 (1969). 11.
Roche, J.F., and Crowley, J.P. Control of oestrus and owulation in heifers. Proc. VII. Int. Congr. Bnim. Repod. and A.I. N&chen, Vol. II: 860-863 (1972).
12. Roche, J.F., and Crowley, J.P. The fertility of heifers 3memlnated at predeterminedintervalsfollowing treatment with m and HCG to control ovulation. J. Reprod. Fert. -358 211-216 (1973).
112
MARCH
1975 VOL. 3 NO. 3
Oestrus was synchronizedin 31 heifers by the intrauterine administrationof PGF dtham salt. Nineteen were given 2 doses of 0.5~ 24 hr gpart and 10 of these received 1500 I.U. of P&SG i.m. 24 hr befor& the treatment with -PGF2"L. The remaining 12 heifers in the experimentwere given a single dose of znlgfollowed at the beginning of oestrus by 1500 I.U. of HCG i.m. Of 9 heifers which received only the two doses of O.&g (Group 11, 7 were observed to have corpora lutea when slaughtered56-72 hr after the onset of oestrus, and four fertilized eggs were recovered. In those which received PEG before the double injection of PGF o+!(Group 21, 118 corpora lutea were observed at slaughter a%d 34 fertilized eggs were recovered. Each heifer which received a single injection of R3F &and HCG had a corpus luteum, and 9 fertilizedeggs were i: ecovered. Unovulated follicles were most commonly observed in the PkEGtreated heifers but they were also observed in the heifers given the double injection treatment. It was observed that in the two-injectiontreatments, whether or not given PXSG, time of ovulation relative to the onset of oestrus was variable, and eggs were found in the uterus before the expected time. ACKNOV~zEM;~~X?~T ProstaglandinF dtham salt was a generous gift of Drs. J.B. Pike and J.Y. L&derdale, the Upjohn Company.
MARCH
1975 VOL. 3 NO. 3
107
THERIOGENOLOGY
INTXODUCTION
ProstaglandinF oL(FGF d> given either locally into
the uterine horn ipsQlateraf to the CL (1 2 3 4) or sys-
temically (5,6) is effective in causing &tiai re ression in the cow. The administrationof EGF2&between ehe 5th and 16th days of the cycle is followed by a significant decrease of serum progesterone,increaseof serum estro en and occurrence of LH ovulatory surge and oestrus (2,3,4f . At present, there are limited data on the use of FGF cLin connectionwith fertilizationand conception rate aft& artificial insemination(1,5,6,7). !Pheaim of the present experimentswas to study some details concerning oestrus and fertilizationrate after dffferent application of FGF2&alone, or in combinationwith PMS3 or HCG.
31 heifers of the Red-Spotted breed, aged 14 to 20 months, and weighing 390 to 500 kg, were used in the erperiments. The stage of the oestrous cycle was determined by repeated rectal palpation. WF Oc- tham salt -8 injected either twice, 0.5mg on each ef%c consecutivedays (19 heifers - groups 1 and 21, or as a ringle dose of @ (l2 heifers - group 3) into the ipsilateraluterine horn, between the 7th and 14th day of the cycle. To ensure mre reliable administrationof FGF o$ Bunpun was injected i.m. beforehand i% a dose of 0. Before application of RP &through the wall rectiinto the uterine lden with a long K ulla recti rraeirrigated rith 0.W of Ajatin (Slore~ kofs$~ of 1,500 LIT. R86 (Antex, Lao, Copsnbagen) ~oskg~iv&i.m. 24 hours before the treatipent rith wF9oLtO A dose of 1,509 I.U. HCG (FPaadyn,Spofn, I?~qpa) was injectid in 12 heifene (group 3) at the ornet af ee#tFus (Table 1). The onset of oestrus was determinedaccordiag t0 behavior and rectal palpation.All heifer6 were lBmminated twice at 15 hour intervals with frozen semen frtxebulls belonging to the &ate A.I. station. 56 to 72 how titer the onset of oestrus the heifers were killad at the alaughterhouse and their reproductive organs taken to the laborAfter inspectionof ovaries the oriducts ax& %%e horns were flushed with salbe containiag-u&n. The ova were first evaluated urder the stereorricreocope. Phase-contrastmicroscopy was wed for detailed exaaimtian of ova fixed in acetalcoholand stained with acetic OXWin.
108
MARCH 1975 VOL. 3 NO. 3
THERIOGENOLOGY
MARCH
1975
VOL. 3 NO. 3
109
THJZRIOGENOLOGY
Cleaved ova with nuclei in all blastomeresand distinct polar bodies were evaluatedas fertilized. The presence of pronuclei,the occurrenceof sperm tails in the cytoplasm, and extrusion of the seco_ndpolar body, were criteria for fertilizationof uncleaved ova. RESULTS The onset of oestrus was observed in all experimental heifers within 56 to 96 hours. In most animals the first symptoms of oestrusappeared on the third day, regardless of whether a single or two injections of FGF &were given. In Groups 1 and 2 abnormally long and intensvveoestrus was observed. In some heifers uterine cervix was open for more than 48 hours. Ovulation occurred in all heifers, with two exceptions in Group 1 (Table I>. Even in these cases, well developed preovulatoryfollicleswere found on the ovaries. Treatment with P?&SGat-ted the developmentof a higher number of follicles in all animals but individual response was variable. The mean number o# follicles cer heifer was 15, of which 11.8 per heifer ovulated. The number of ova obtained from Group 1 (2 x 0.5mg FGF d was relatively low, since from 'Iovulated follicles onlf4 ova were found. With reference to the abnormally long oestrus, it seems likely that at least in some animals the ova escaped into the uterine horns at a time when they were expected to be in the oviducts. The proportion of ova obtained from PBG-stimulated heifers was also low (66.9%). Here again, some ova were flushed from the uterine horns, although according to the onset of oestrus and ~~h$ime of slaughteringthey should have been in the ovi.
The fertilizationrate of ova flushed from heifers injected with HCG was relatively high ('75%).On the other hand, fertilization in FHSGtreated heifers m low - 43% These unsatisfactoryresults were caused mainly by complete failure of fertilizationin some aniPPelswith relktively high ovulationrate. DISCUSSION The results concerningoestrus synchronizationare comparable with those referred by other authors l&2,3,4, 5). The main aim of our study was to follow the ovulation and fertilizationrate in the first synchronizedoestrus. Ovulation failed to occur onl in two heifers, both treated with/2 x 0.5 mg HGF2a? The reason for this failure
MARCH
1975 VOL. 3 NO. 3
THERIOGENOLOGY
is difficult to explain, especially as the oestrus behavior was completely norms1 in both animals. Except for the &%-treated group, the fertilization rate attained a satisfactorylevel higher than after synchronization with tilengestrolaceiate (MGA)(8). In superovulated heifers, on the other hand, the fertilizationrate was relatively low and did not reach the values described by other authors (9). This may have been due to wrong choice of the inseminationtime, the quality of the spermatozoa or unfavorable conditionsfor transport and capacitation of the spermatozoacaused by the abnormal hormonal conditions. This view would be also in agreement with the absence of spermatozoa on the zona pellucida of unfertilized ova. The relative high proportionof the ova flushed from the uterine horns in some animals would support this opinion. The purpose of HCG administrationin the last group was to fix the time of ovulation. In the normal oestrous cycle a transient Ui surge occurs soon after the onset of oestrus (10). In W-synchronized heifer8 ovulation occurred 32 to 40 hours after IiCGwection (11). Conception after inseminationcarried out with respect to this time interval was far below expectation (12). On the contrary, in our experiments the proportionof fertilized ova was relatively high after BCG treatment. Cur knowledge of the level of pre- and postimplantationlosses in relation to fertility in the given treatment, is likewise altogether inadequate. We can conclude, however, from preliminary,unpublished results, that using ;PGF2+for synchronizationand HCG for induction of ovulationgive conception rates which are comparable with figures obtained in normal oestrus (57.2% pre nant in normal oestrus and 68.6% pregnant after FGF2dand fliCG treatment). ~BZIKXS 1. Bowson, L.E.A., Tervit, R., and &a&, A. Synchronization of oeetrus in cattle by means of prostaglandin F d Froc. VII. Int. Congr. Anim. Reprod. and A.1. M&hen. vol. IIt 866469 (19'72). 2.
Louis, T.Y. Bafs, H.D. and Morrow, D.A. Estrus_and ovulation after uterine &z 2din cows* J. Anim. Scl. 35: 247-248 (1972).
3. Louis, T.Y., Bafs, H.D., and Se&n, B.E. Progesterone, LH eetrus and ovulationafter pros landin F din hehers. Proc. Sot. Exp. Biol. 152-266 (1973).
MARCH
1975 VOL. 3 NO. 3
111
THERIOGENOLOGY
4.
Louis, T.M., Hafs, H.D., and Morrow, D.A. Intrauterine administrationof prostaglandinF &in cows: progesterestrogen,LH estrus and ovuPation. J. Anim. Sci. g '347.353 (39743
5.
Philipsen H., and Rasbech, N.O. The use of prostalandin l&F aC>for heat synchronizationin heifers. L sberetn. ?nst. f. Sterilitetsforskn.,Kobenhavn: 157-162 (1973).
6.
Inskeep, ES. Potentialuses of prostaglandinsin control of reproductivecycles of domestic animals. J. Anim. Sci. 36: 1149-ll59 (1973).
7.
Lauderdale, J.W., Seguin, B.E., Stellflug,J.N., Chenault, JR., Thatcher, W.W., Vincent, C.K., and Loyancano,A.F. Fertility of cattle following PGF2d injection.J. Anim. Sci. g_t 964-967 (1974).
8.
Hill, JR., Jr., Lamond, D.R., Henricks, D.M., Dickey, J.F., and Niswender,G.D. The effect of melengestrol acetate (laca)on ovarianfunction and fertilization in beef heifers. Biol. Repcod. St 16-22 (197l)
9.
gleden, B.P., Lewis, Susan, Gumming, I&., and Lawson, R.A.S. Superovulationin the cow following treatment with PMSG and prostaglandinF2d J. Reprod. Fert. 36: 435-456 (1974).
D., and Karg, H. RadioimmunologischeLH10. SC-, Bestin Blutserumvom Rind unter besondererBerllcbichtigungde8 Brunstzyklus.Acta endocr., Copenh. 6& 96-102 (1969). 11.
Roche, J.F., and Crowley, J.P. Control of oestrus and owulation in heifers. Proc. VII. Int. Congr. Bnim. Repod. and A.I. N&chen, Vol. II: 860-863 (1972).
12. Roche, J.F., and Crowley, J.P. The fertility of heifers 3memlnated at predeterminedintervalsfollowing treatment with m and HCG to control ovulation. J. Reprod. Fert. -358 211-216 (1973).
112
MARCH
1975 VOL. 3 NO. 3