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Effect of LH-RH on meiotic maturation of preovulatory oocytes in superovulated heifers
I)
.4nimul Reproduction Science, 25 ( I99 I89- I97 Etsevier Science Publishers B.V., Amsterdam
Effect of LH-RH on meiotic maturation of preovulatory oocytes in superovulated heifers J. LaurinEik”,J. Pichab, D. PichovP and J. Pivko” ‘Research lnstiruteof,4ninrai Praducrion. Hlchovskd 2. 94992 Nirra. Czechoslovakia bRes~arch Institute of Feed Suppletnears and Veterinary Drugs. 254 49Jilovk u Prahy. Cteehoslovakia (Accepted I I January 199
1)
LaorinMk, J., Picha, J.. Pichov& D. and Pivko, J., 1991. Efleceel of LH-RH on meiotic maturation of preovulatoryoocytes in superovulated heifers. Anim. Reprod. Sci., 25: 189-197. Eleven heifers (Group 1) were stimulated for 4 days by 20 mg of PSH-P administered in declining doses. Cloprostenol(O.75 mg) was administered 48 h after the first PSH-Pdose. Eight heifers (Group 2) were stimulated according to the same patterns, but 40 h after the injection of clopmrtenol they were treated with 50 w of LH-RH analog. The onset of the LH peak was observed 35.9f :.26 and 40.2?0.03 h afier the administration ofcloprostenol in Groups I and 2 respectively (PcO.01). The
concentration
of
LH reached a maximum aAer 40.IJ? 1.44h in Group1and42.0+0.17 h in Group 2 (P~-0.05).The EH concentration was 11.1 f3.49 ngml-’ in Group I and 58.5+5.05 ngml-’ in Group 2 (P~0.01). The oocytes were recovered in both groups 56h after the cloproslenol was administered. In Groups I and 2 respectively, 19.2% and 5.5% of oocy~eswith compact cumulus were recovered (PcO.01). and 78.3% and 94.3% of oocytes with expanded cumulus were recovered ). M these oocyies. Z0.M and 8.6% lacked the perivitelline space (P
(P
INTRODUCTION
Major problems with superovulation in cattle includehigh variability in response (measured as palpable corpora lutea or number of recovered embryos), treated cows not expressing overt estrus, and cows ovulating over a wide interval of time. In addition, superovulatory results vary owing to age, breed, season, nutrition, level of milk production, frequency of breeding and the type and batch of superovulatory compound used (Gordon, 1975; Brand et al., 1978; Newcomb, 1980). Some investigators have injected gonadotropin releasing hormone or analogs at various times near the onset of estrus in an attempt to control the time of LH surge and therefore ovulation (Prado
190
1.IAUlUNCfKETAL
Delago et al., 1984; Takahashi and Kanagawa, 1984; Wubishet et al., 1986; Savage et al., 1987). Treatment Muses a rapid rise in LH when administered before the endogenous LH peak, but this does not appear to significantly improve oocyte maturation or ovulation rate. A decrease of embryo recovery and increase of unfertilized oocytes (Walton and Stubbings, 1986) took place in superovulated cows after LH-RH application. Treatment with LH-RH of nonstimulated ‘cows at insemination improved conception rates of repeatbreeding dairy cows (Stevenson et al., 1988). An increase in goodquaiity embryos after supplemental LH-RH treatment was reported in two studies (Takahashi and Kanwwa, 1984; Wubishet et al., 1986). For these reasons, the main aim of this work was to determine the influence of LH-RH on oocyte maturation in superovulated heifers. MATERIALSAND METHODS
Nineteen heifers form 13 to 18 months of age, 280-350 kg weight, with regular sexual cycles were divided into two groups differing in hormonal treatment. Eleven heifers (Group I ) with corpus luteurn were synchronized with 0.75 mg doprostenol (Oestrophan inj. a.u. v., Spofa, Praha). Starting on Day 13, they were stimulated with follicle-stimulating hormone isolated from the pituitary gland of pigs (Folicotropin inj., Spofa, Praha), with a total dose of 20 mg. The FSH treatment was done at 12-h intervals for 4 days in the following doses: I x 6, 1 x 4,4 x2,2 x 1mg respectively.The luteolytic compound was given again, 48 h after the beginning of the FSH stimulation. Eight heifers (Group 2) were synchronized and stimulated on tbt?same scheme as animals of Group I, but 40 h after the administration ofthe se\?nn.d cloprostenol, 50 gg of superanalog (DTle’) LH-RH (Supcrgestran inj., Spofa, Praha ) was injected. Blood samples were removed from the jugular vein using an indwelling cannula, inserti: 2 days before the first sampling. Sampling began 18 h aRer *he _---___‘______~_______ **rn*dina-+i-n nf ._ c!oprostend in heifers of Group 1, Bloodwasremoved at 2-h intervals for 36 h. In Group 2 (IV= 8) blood sampling began 18 h after the secosd cloprosteuol injection. The first 12 samples were taken every 2 hours. After the twelfth sample, LHRH was applied and subsequently blood was removed at I5-min intervals for 4 h, then twice more at l-h intervals. Blood was colleccaedinto heparinized test tubes and centrifuged immediately for 15 min at 2740 I, Plasma was stored at - 20°C. Chzmges in LM concentration of peripheral blood were compared in both groups of heifers during the preovulaLtoryperiod. Luteinizing hormone was determined using a double antibody assay f Stupnicki and Madej, 1976). Immunogene NIH-LH-B 9 was used to prepare the antibodies, and for USDAbLH-I- I iodination. Antibodies with titer I: 150(100showed a cross-reaction
LH-RHAND MHOTIC h4ATURATlON
191
with b-FSH and b-STH of O-O!% and 0.01% respectively, and b-TSH 6.9% with bovine prolactin. Sensitivity of the method was 50 pg bL.H. Intra- and interassay coefficients were calculated as 8.6% and 12.4% respectively. Ovaries were observed by laparoscopy in the midventral route (Holland et al., 198 1) in both groups 65 h after the application of cloprostenol (Braclcett, 1982 ). The inner diameter of aspiration needles was 1.Omm (Lambert et al., 1983; LaurinEik et al., 1988). Oocytes were recovered from clear follicles (Grimes and Ireland, 1986) with an outside diameter2 8 mm (CaIlesen et al., 1986). The appearance of cumulus cells was compared in recovered ow cytes and was differentiated into compact, expanded and partial. The absence of presence of the perivitelline space (without or with WS), or presence of the extruded first polar body (PI31), wasevaluated in these oocytes (Greve et al., 1984a). Preovulatory oocytes with PVS from both groups were cultured in 150@ of Parker’s medium (M 199; Sevac, Praha) supplemented with 2.92 mM Ca-lactate, 2 mM Na-pyruvate, 33.9 m&tNa-bicarbonate, 4.43 LUMN-( 2hydroxyethyl)piperaxine-N’-(2-ethanesulfonic) acid (Hepes) (Serva, Heidelberg), 50 III ml-’ penicillin, 50 pg ml-’ streptomycin sulphate and 20% bovine serum under pa&o oil in an atmosphere of 5% CC&, 10% O2 and 85% Nr and at a temperature of 385°C (Pavlok et al., 1988). Gocytes were cultured experimentally for 8 h. Oocytes which excluded PBI after this interval were noted. The rest of the oocytes were fixed (ethanol: acetic= 3: 1) and stained with 1% acetic orceine. The degree of nuclear maturation after this short-term culture was noted. The log-logit system was used to calculate the LH concentration in samples of blood plasma, The area under the curve was determined according to the method of Jensen et al. ( 1983 )_The X*-test was used to compare the appearance of the cumulus cells and ooeytcs, as well as the degree of nuclear maturation of oocytes, between groups. When the results of the $test were signiticant, further comparisons were made using a U-test. RESULT’S
Figure 1 depicts the LH concentration from 16 to 50 h after the cloprostenol administration in Group 1 [stimulation with FSH-P) and Group 2 (stimulation with FSH-P and LH-RH 1. Table 1 presents results for the beginning of preovulatory LH peak, the period of maximum LH concentration after the cloprostenol administration, and the period of maximum LH concentration after the onset of the LH peak. Concentrations of LH did not differ statistically (Fig 1) in the individual samples from 16 to 24 h after cloprostenol administration. In Group 1, an increase in LH was observed in one heifer 28 h, and in two heifers 34 h, after cloprostenol injection. Non-significant differences during this period are caused by the increase of luteinizing hormone variability as a result of the preovulatorypeak. The preovulatory peak occurred in another four heifers of the first group 36 h after
I. lAURINi%
192
16
5t
26
hours
ET AL.
!3rter
cloprosiend
Fig, t , LH concentrations in stimulated heifers. ? n~mals of Soup I (We) were stimulated over 4 days by 20 mgofFSH-P in doses. AnimalsofGroup 2 (. l . ) were stimulated according to the same patterns, but 40 h after cloprostenol they were treated with 50 j$ of LH-
decreasing
RH analog. TABLE I LH xcretion
in heifers stimulated with FSH-P or FSH-P and LH-RH
Croup no. and treatment
I. FSH-P 2. FSH.P+ LH-BH
No. of
LH onset’
heifers
(h+SD)
II 8
35.4 + 1.26 40.2 i: 0.03 .*-\
Prcovulatory LH !xak Period of manimal LH concenlfidiun’ (h+_SD)
LH concmm-a!!3C (n~ml-‘+SP)
Maximum LH cancen1ratio ancr onset of LH peak (h*SD)
Area under Ihe curve
40.14? 1.44 42.00f3.17 n.s.’
11.1+3.49 58.5f 5.0s l *
4.33+ 1.25 2.lOiO.18 l .
119.: 144.6’
I
‘Atier c~oprosrenolapplication. ‘6 hours afkr spplicalion of (ISTIe*) LH-RH. 62.5% of heifen had LH concentrationshigher than in the pc~iod b&xv application. ‘Statistical s@ticancr: n.s.. not stgnificaot (f>O.OS); . a. $rcwps differ significantly (P .c 0.01).
administration, and the average LH concentration rose signifi1.45ng ml- I. A maximum mean LH concentraml- ’ ) wasobserved 40 h after cloprostenol administration in Group 1. This significant increase was statistically confirmed up ta 46 huurs. A different ~att~~~ of LH concentration with time was observed in Group 2 (Fig. I 1. The preovulatory LH peak was not observed in any heifer up to 40 h after cloprostenol administration. Mean concentrations of the luteinking hormone in individual samptes after luteolysis were within the permitted var-
cloprosbwl
cantly(P6O.O.S)to 4.32 ?
2
10.75+0.96 8.84kO.63 n.5.’
19.2 5.7
94 66 l *
no.
23 4 112
Expanded
%
no.
0’
“0.
Partial ---
from preovulatory
78.3 94.3
%
cells
Compact
Character of cumulus
2.5 0
96
24 6 l *
no.
- PVS
+ PVS
65 36 n.s.
no.
54.1 51.4
%
31 28 *I
“0.
PBI
treatments
perivilellinespace.
20.0 8.6
91,
Characlerofoocytes’
follicles after different stimulauon
‘SYtt~bok: - PVS. without perivitellinc space; +PVS, with perivite!he space; PB I, first polar body presentin the 2Statistic81 significance:ns. not significant(P> 0.05); l*,groupsdiffersipificantly (~<0.01).
120 70
I. FSH-P 2. FSH-P+LH-RH
Mean ? SD
complexes recovered by laparozxcopic aspiration
No. of ooeytes
Gmup no. and trcatmenr
Character of cumulus-oocyte
TABLE
25.9 40.0
%
194
1. LALWNtk
ITAL.
TAILE
3
Nuclear
maturation ofprcovulatory oocytes’ with perivitellinespace experimentally cultured in vitro
Group no. and treatment
I. FSH-P 2. FSH-P+LH-RH
No. of cultivated oocytes
With PB 1 no.’ %’
65 36
27 31 l*I
41.5 86.1
Without PB I Degenerated
AI-T1 -
MI no.z
%”
no.z
%<
no.z
%4
7 0
10.7 0
23 3 **
35.4 8.3
8 2 n.s.’
12.4 5.6
‘Abbreviations: PB I, first polar body; M I, metaphasc I; A I,
anaphaseI: T
I, telophase 1.
2Numberof noqtes. ‘Oocytes with PB 1, as a percentage of the number ofcultivated oocytes. ‘Oocytes in individual stages.as a percentage of the number ofoocytes without PB I. ‘Statistical significance: n.s., not significant; t l differ ~igniiicantly (PC 0.01).
,groups
iability span. Administration ol”LH-RH increased the LH concentration to 8.322.76 ng ml-! in the course of 30 minutes. It was significantly higher (P
LH-RH :.8D n(ElOTIC MATURATION
195
tith .l released first polar body reached 86.1% in the group treated with LHKH. When oocytes cultivated for 8 h were stained, it was observed that all cocytes from Group 2 were in Anaphase I or Interphase I. Groups Pid not differ in the percentage of degenerated oocytes. DlSCUSSION
The onset of a preor &tory peak in heiftz was observed 40.9 18.4 h (Grew et al., 1984b), and even in 36 h (Yadav et al., 1983) after prostaglandin F2 alpha administration in heifers stimulated with pregnant mar: serum gonadotropin (PMSG) The preovul3tory LH peak occurred 42.0 5 8.4 h (Greve et al., 1984b) or 49.7 h (Yadav e? al., 1983) after FSH-P administration. Comparison of these data with resu!ts obtained in Group 1 showed that the preovulatory onset 3f an early LH peak in the present experiment is similar to the results of superovulation after PMSG treatment. However, plotting the data for Group 2 based on these results suggcts that both the LH rise and the pituitary gland response to LH-RH treatment were not influenced by previous LH secretion (Voss et al., 1986; Savage et aL, 1987; Lamming and McLeod, i988) and an LH increase occurred in all heifers after LH-RH administration, in contrast to other studies (Walton and Stubbings, 1986; Savage et al., 1987). Luteinizing hormone is a physiological agent which induces meiotic maturation of mammalian oQcytes in vivo. It predominantly affects follicular cells. After the preovulatory LH increase, the disintegration fif the cumulus oophorus structure proceeds from acculaulation of giycosaminog!ycans in the iutercellular space (r)ekel and Phillips, 1979) and a decrease of metabolic cooperation between the oocyte and cumulus takes pIa=. In this experiment the effect of exogenous LH-RH resulted in a significantly lower number of oocytes with expanded cumulus, compared with the untreated group and with previous data (Sirard and Lambert, 1985). Unilateral enlargement of the perivitelline space occurs with gradual maturation of the oocytes, the indication of which is visible 2 h after the preovulatory LH peak (Hyttel et al., 1986). Enlrrgement ofthe perivitelline space is related to subsequent release of the first polar body in the preovulatory period (Greve et al., 1984a). Both the expansion of the cumulus oophorus and meiotic maturation were improved in the group treated with LH-RH, compared with the untreated group and with the results of other authors (Greve et al., 1984aJ. The stimulating effect of LH-RH was confirmed partly by a lower percentage of oocytes without a perivitelline space, partly by a higher percentage of oocytes with a released polar body, and partly by a high percentage of oocytec with a released polar body after short-term cultivation. Although it was observed in the past that the LH peak is not a prerequisite for meiotic maturation in heifers (Goff et al., 19861, the results of this exper-
196
J. UURINt?K
ET AL.
iment point out the important synchronizing influence of LH-RH upon meiotic maturation. Positive results were achieved owing to the optimal timing of LH-RH administration. Results of short-term culture also confirmed that it is appropriate to divide the oocytes, after their aspiration from preovulatory foiiicles, into oocytes with or without perivitelline space, and those with a released polar body. In conclusion, the present study indicates that the utilization of LH-RH increases the number of recovered oocytes that are suitable for the study of problems associated with in vitro fertilization. At the same time, it enables the recovery of embryos at the same stage of development in embryo-transfer programs. ACKNOWLEDGEMENTS
The authors would like to thank NlQDK National Hormone and Pituitary Program, Baltimore, Maryland, for kindly supplying the bovine LH preparations used for the assay and antibody production, and Garett Rarden of University College Cork for his help with English.
REFERENCES Brackett. B.G., Bosquet. D., Roice, M.L., Donawick, W.J.. Evans, J.F. and Dressel, M.A., 1982. Normal development following in vitro fertilization in the cow. Biol. Reprod., 27: 147-158. Brand. A., Trounson, A.O., A&II-IS, M.H., Dmst, M. and Zaayer. D., 1978. Superovulalion and non-surgical embryo recovery in the lactating dairy cow. Anim. Prod., 26: 55-60. Callesen, H.. Greve, T. and Hyttel, P., 1986. Preovulatory endocrinology and oocyte maturation in superovulaled cnlrle. Theriogenology, 25: 71-86. Dekel, N. a&IdPhillips, D.M., 1979. Maturation of the rm cumulus oophorus: a scanning electron microscopic study. Biol. Reprod., 2 I: 9-18. Gold A.K.. Greve. T., Bousquet, D and King, W.A., 1986. Pmestemne and lureinizing hormone profiles in heifers used as ooeyte donors. Therioaenology. 26: 577-586. Gordon. 1.. 1975. Problems and prospects in cattle e transfer. Ir. Vet. J., 3: 21-30. Grew T., Bousquet, D., King. W.A. and Berteridge. K.J.. 1984~1. In viwo fertilization and cleavage of in vivo molured bovine oocytes. Theriogenology, ZZ: 15 I - 165. Greve. T., Callescn, N. and Plyrtel, P., 1984b. Plasma progesterone profiles and embryo quality in superovulated dairy cow. Theriogenology, 21: 238. Grimes, R.W. and Ireland. J.J.. 1986. Rehtionship of macroscopicappearance ofthe surface of bovine ovarian follicles. concentrations of stemids in follicular fluid, and maturation of ow cytes in vitro. Biol. Reprod., 35: 725-732. Nollnnd. E.J.. Bindoo, KM., Piper, L.R., Thimonier, J.. Cornish. K.A. and Radford, H.M.. I98 I. Endoscopy io cartle: techniques for ovarian examination by the panlumbar and midvenrral romes. Anim. Rep&. Sci., 4: 127-l 35,
LH-RH AN3 MEIOTIC biATURATION
197
Hyttel, P., Callesen, H. and Greve, T., 1986. Ultr&ructural features of preovufatoyr oocyte maturation in superovulated cat;le. J. Reprod. Fertil., 76: 645~65.:. Jensen, A.M., Pedersen, KM.. Agger, J.F. and Madej, A., 1983. Plasma luteinizing hormone response to increasing doses Xc:‘orh+c g~--~ ..aYotrophin-releasing hormone in heifers. Acta Vet. Stand., 24: 221-224. Lambert. R.D., Bernard, C.. Rioux. J.E., B&land, R.. D’Amours, D. and Montreuil, A., 1983. Endoscopy in cattle by the pamlumbar route: technique for ovarian examination and follicular aspiration. Tberiogenology, 20: i 49-161. Lamming, E.E. and M&cod, B.J., 1988. Continuous infusion of GnRH reduces the LH response to an intravenous GnRH injection but dces net inhibit endogenous LH secretion in COW.J. Repmd. Fertil., 82: 237-246. Lautincik. J.. Pivko, J., Grafenau, P. and Oberfranc. M., 1988. Oocyte recovery by aspiration of the preovulatory follicles of heifers. Vet. Med. (Prague), 33: ‘27-734. Newcomb, R., 1980. Investigation of factors affecting superovulation and non-surgical embryo recovery from lactaling British Friesian cows. Vet. Rcc , 106: 48-52. Pavlok, A., Tamer, H., hkl!l~, J., Fulka, J.. K&fold, P. and Duschinski, U., 1988. Fertilizetion of bovine oocytes in vitro: effect of ditferent sources of gametes on fertilization rate and frequency of fertilization anomalies. Anim. Reprod. Sci., 16: 207-213. Prado Delago. A.R., El&n, R.P. and Seidel. Jr., G.F , 1984. Effects of GnRH on response to superovulation in cattle. Theriogenology. 21: 254. Savage, NC., Howell, W. and Mapletofl. R.J., 1987. Superovulation in the cow using estradiol I78 or CaRH in conjunction with FSH-P. Thetiogenoiogy. 27: 383-394. Shard. M.A. and Lambert, R.D., 1%. In vitro fertilization of bovine follicular oocytes obtained by lapmpy. Biol. Repmd., 33: 487-494. Stevenson. J.S.. Fran% K.D. and Call, E.P., 1988. Conception rates in repeat-hrccders and dany cattle with unnbaervcd CstNuSafier prostaglandin Fz alpha and gonadotropin-releasing hormone. Theriogenology, 29: 45 I-460. Stupnicki, R. and Madej, A., 1976. Radioimmunoassay of bovine LH in blood p1ass.a. Endocrinologie, 68: 6- 13. Takahashi, Y. and Kanagawa, H.. 1984. Effects of LH-RH snalogue on the ovulation rate and embryo quality in heifers superovulated with PMSG and PGFs alpha. Jap. J. Vet. Res., 32: 183-189. Voss, H.J., Allen, S., Aqua1 3, P. and Foote. R.H., 1986. Incorporation of a GnRH analog into a superovulatory regin 1 f- dry holstein cows. Theriogenology, 25: 2 IO. Walton, J.S. and dubbings, R.J., 1986. Factors affecting the yield of variable embryos by superovulated Holstein-Friesian cows. Theriogenology, 26: 167-177. Wubishet. A.. Gnves, C.N.. Spahr. S.L.. Kesler, D.J. and Favero, R.J., 1986. EffectsofCnRH treatr .ents on superovulatory response of daity cows. Theriugenology, 25: 423-427. Yadav, MC.. Leslie, K.E. and Walton, J.S., 1983. The timing of the supemvulatory surge of LH and embryo production in superovulated COWS. Theriogenology, 19: 150.
.4nimul Reproduction Science, 25 ( I99 I89- I97 Etsevier Science Publishers B.V., Amsterdam
Effect of LH-RH on meiotic maturation of preovulatory oocytes in superovulated heifers J. LaurinEik”,J. Pichab, D. PichovP and J. Pivko” ‘Research lnstiruteof,4ninrai Praducrion. Hlchovskd 2. 94992 Nirra. Czechoslovakia bRes~arch Institute of Feed Suppletnears and Veterinary Drugs. 254 49Jilovk u Prahy. Cteehoslovakia (Accepted I I January 199
1)
LaorinMk, J., Picha, J.. Pichov& D. and Pivko, J., 1991. Efleceel of LH-RH on meiotic maturation of preovulatoryoocytes in superovulated heifers. Anim. Reprod. Sci., 25: 189-197. Eleven heifers (Group 1) were stimulated for 4 days by 20 mg of PSH-P administered in declining doses. Cloprostenol(O.75 mg) was administered 48 h after the first PSH-Pdose. Eight heifers (Group 2) were stimulated according to the same patterns, but 40 h after the injection of clopmrtenol they were treated with 50 w of LH-RH analog. The onset of the LH peak was observed 35.9f :.26 and 40.2?0.03 h afier the administration ofcloprostenol in Groups I and 2 respectively (PcO.01). The
concentration
of
LH reached a maximum aAer 40.IJ? 1.44h in Group1and42.0+0.17 h in Group 2 (P~-0.05).The EH concentration was 11.1 f3.49 ngml-’ in Group I and 58.5+5.05 ngml-’ in Group 2 (P~0.01). The oocytes were recovered in both groups 56h after the cloproslenol was administered. In Groups I and 2 respectively, 19.2% and 5.5% of oocy~eswith compact cumulus were recovered (PcO.01). and 78.3% and 94.3% of oocytes with expanded cumulus were recovered ). M these oocyies. Z0.M and 8.6% lacked the perivitelline space (P
(P
INTRODUCTION
Major problems with superovulation in cattle includehigh variability in response (measured as palpable corpora lutea or number of recovered embryos), treated cows not expressing overt estrus, and cows ovulating over a wide interval of time. In addition, superovulatory results vary owing to age, breed, season, nutrition, level of milk production, frequency of breeding and the type and batch of superovulatory compound used (Gordon, 1975; Brand et al., 1978; Newcomb, 1980). Some investigators have injected gonadotropin releasing hormone or analogs at various times near the onset of estrus in an attempt to control the time of LH surge and therefore ovulation (Prado
190
1.IAUlUNCfKETAL
Delago et al., 1984; Takahashi and Kanagawa, 1984; Wubishet et al., 1986; Savage et al., 1987). Treatment Muses a rapid rise in LH when administered before the endogenous LH peak, but this does not appear to significantly improve oocyte maturation or ovulation rate. A decrease of embryo recovery and increase of unfertilized oocytes (Walton and Stubbings, 1986) took place in superovulated cows after LH-RH application. Treatment with LH-RH of nonstimulated ‘cows at insemination improved conception rates of repeatbreeding dairy cows (Stevenson et al., 1988). An increase in goodquaiity embryos after supplemental LH-RH treatment was reported in two studies (Takahashi and Kanwwa, 1984; Wubishet et al., 1986). For these reasons, the main aim of this work was to determine the influence of LH-RH on oocyte maturation in superovulated heifers. MATERIALSAND METHODS
Nineteen heifers form 13 to 18 months of age, 280-350 kg weight, with regular sexual cycles were divided into two groups differing in hormonal treatment. Eleven heifers (Group I ) with corpus luteurn were synchronized with 0.75 mg doprostenol (Oestrophan inj. a.u. v., Spofa, Praha). Starting on Day 13, they were stimulated with follicle-stimulating hormone isolated from the pituitary gland of pigs (Folicotropin inj., Spofa, Praha), with a total dose of 20 mg. The FSH treatment was done at 12-h intervals for 4 days in the following doses: I x 6, 1 x 4,4 x2,2 x 1mg respectively.The luteolytic compound was given again, 48 h after the beginning of the FSH stimulation. Eight heifers (Group 2) were synchronized and stimulated on tbt?same scheme as animals of Group I, but 40 h after the administration ofthe se\?nn.d cloprostenol, 50 gg of superanalog (DTle’) LH-RH (Supcrgestran inj., Spofa, Praha ) was injected. Blood samples were removed from the jugular vein using an indwelling cannula, inserti: 2 days before the first sampling. Sampling began 18 h aRer *he _---___‘______~_______ **rn*dina-+i-n nf ._ c!oprostend in heifers of Group 1, Bloodwasremoved at 2-h intervals for 36 h. In Group 2 (IV= 8) blood sampling began 18 h after the secosd cloprosteuol injection. The first 12 samples were taken every 2 hours. After the twelfth sample, LHRH was applied and subsequently blood was removed at I5-min intervals for 4 h, then twice more at l-h intervals. Blood was colleccaedinto heparinized test tubes and centrifuged immediately for 15 min at 2740 I, Plasma was stored at - 20°C. Chzmges in LM concentration of peripheral blood were compared in both groups of heifers during the preovulaLtoryperiod. Luteinizing hormone was determined using a double antibody assay f Stupnicki and Madej, 1976). Immunogene NIH-LH-B 9 was used to prepare the antibodies, and for USDAbLH-I- I iodination. Antibodies with titer I: 150(100showed a cross-reaction
LH-RHAND MHOTIC h4ATURATlON
191
with b-FSH and b-STH of O-O!% and 0.01% respectively, and b-TSH 6.9% with bovine prolactin. Sensitivity of the method was 50 pg bL.H. Intra- and interassay coefficients were calculated as 8.6% and 12.4% respectively. Ovaries were observed by laparoscopy in the midventral route (Holland et al., 198 1) in both groups 65 h after the application of cloprostenol (Braclcett, 1982 ). The inner diameter of aspiration needles was 1.Omm (Lambert et al., 1983; LaurinEik et al., 1988). Oocytes were recovered from clear follicles (Grimes and Ireland, 1986) with an outside diameter2 8 mm (CaIlesen et al., 1986). The appearance of cumulus cells was compared in recovered ow cytes and was differentiated into compact, expanded and partial. The absence of presence of the perivitelline space (without or with WS), or presence of the extruded first polar body (PI31), wasevaluated in these oocytes (Greve et al., 1984a). Preovulatory oocytes with PVS from both groups were cultured in 150@ of Parker’s medium (M 199; Sevac, Praha) supplemented with 2.92 mM Ca-lactate, 2 mM Na-pyruvate, 33.9 m&tNa-bicarbonate, 4.43 LUMN-( 2hydroxyethyl)piperaxine-N’-(2-ethanesulfonic) acid (Hepes) (Serva, Heidelberg), 50 III ml-’ penicillin, 50 pg ml-’ streptomycin sulphate and 20% bovine serum under pa&o oil in an atmosphere of 5% CC&, 10% O2 and 85% Nr and at a temperature of 385°C (Pavlok et al., 1988). Gocytes were cultured experimentally for 8 h. Oocytes which excluded PBI after this interval were noted. The rest of the oocytes were fixed (ethanol: acetic= 3: 1) and stained with 1% acetic orceine. The degree of nuclear maturation after this short-term culture was noted. The log-logit system was used to calculate the LH concentration in samples of blood plasma, The area under the curve was determined according to the method of Jensen et al. ( 1983 )_The X*-test was used to compare the appearance of the cumulus cells and ooeytcs, as well as the degree of nuclear maturation of oocytes, between groups. When the results of the $test were signiticant, further comparisons were made using a U-test. RESULT’S
Figure 1 depicts the LH concentration from 16 to 50 h after the cloprostenol administration in Group 1 [stimulation with FSH-P) and Group 2 (stimulation with FSH-P and LH-RH 1. Table 1 presents results for the beginning of preovulatory LH peak, the period of maximum LH concentration after the cloprostenol administration, and the period of maximum LH concentration after the onset of the LH peak. Concentrations of LH did not differ statistically (Fig 1) in the individual samples from 16 to 24 h after cloprostenol administration. In Group 1, an increase in LH was observed in one heifer 28 h, and in two heifers 34 h, after cloprostenol injection. Non-significant differences during this period are caused by the increase of luteinizing hormone variability as a result of the preovulatorypeak. The preovulatory peak occurred in another four heifers of the first group 36 h after
I. lAURINi%
192
16
5t
26
hours
ET AL.
!3rter
cloprosiend
Fig, t , LH concentrations in stimulated heifers. ? n~mals of Soup I (We) were stimulated over 4 days by 20 mgofFSH-P in doses. AnimalsofGroup 2 (. l . ) were stimulated according to the same patterns, but 40 h after cloprostenol they were treated with 50 j$ of LH-
decreasing
RH analog. TABLE I LH xcretion
in heifers stimulated with FSH-P or FSH-P and LH-RH
Croup no. and treatment
I. FSH-P 2. FSH.P+ LH-BH
No. of
LH onset’
heifers
(h+SD)
II 8
35.4 + 1.26 40.2 i: 0.03 .*-\
Prcovulatory LH !xak Period of manimal LH concenlfidiun’ (h+_SD)
LH concmm-a!!3C (n~ml-‘+SP)
Maximum LH cancen1ratio ancr onset of LH peak (h*SD)
Area under Ihe curve
40.14? 1.44 42.00f3.17 n.s.’
11.1+3.49 58.5f 5.0s l *
4.33+ 1.25 2.lOiO.18 l .
119.: 144.6’
I
‘Atier c~oprosrenolapplication. ‘6 hours afkr spplicalion of (ISTIe*) LH-RH. 62.5% of heifen had LH concentrationshigher than in the pc~iod b&xv application. ‘Statistical s@ticancr: n.s.. not stgnificaot (f>O.OS); . a. $rcwps differ significantly (P .c 0.01).
administration, and the average LH concentration rose signifi1.45ng ml- I. A maximum mean LH concentraml- ’ ) wasobserved 40 h after cloprostenol administration in Group 1. This significant increase was statistically confirmed up ta 46 huurs. A different ~att~~~ of LH concentration with time was observed in Group 2 (Fig. I 1. The preovulatory LH peak was not observed in any heifer up to 40 h after cloprostenol administration. Mean concentrations of the luteinking hormone in individual samptes after luteolysis were within the permitted var-
cloprosbwl
cantly(P6O.O.S)to 4.32 ?
2
10.75+0.96 8.84kO.63 n.5.’
19.2 5.7
94 66 l *
no.
23 4 112
Expanded
%
no.
0’
“0.
Partial ---
from preovulatory
78.3 94.3
%
cells
Compact
Character of cumulus
2.5 0
96
24 6 l *
no.
- PVS
+ PVS
65 36 n.s.
no.
54.1 51.4
%
31 28 *I
“0.
PBI
treatments
perivilellinespace.
20.0 8.6
91,
Characlerofoocytes’
follicles after different stimulauon
‘SYtt~bok: - PVS. without perivitellinc space; +PVS, with perivite!he space; PB I, first polar body presentin the 2Statistic81 significance:ns. not significant(P> 0.05); l*,groupsdiffersipificantly (~<0.01).
120 70
I. FSH-P 2. FSH-P+LH-RH
Mean ? SD
complexes recovered by laparozxcopic aspiration
No. of ooeytes
Gmup no. and trcatmenr
Character of cumulus-oocyte
TABLE
25.9 40.0
%
194
1. LALWNtk
ITAL.
TAILE
3
Nuclear
maturation ofprcovulatory oocytes’ with perivitellinespace experimentally cultured in vitro
Group no. and treatment
I. FSH-P 2. FSH-P+LH-RH
No. of cultivated oocytes
With PB 1 no.’ %’
65 36
27 31 l*I
41.5 86.1
Without PB I Degenerated
AI-T1 -
MI no.z
%”
no.z
%<
no.z
%4
7 0
10.7 0
23 3 **
35.4 8.3
8 2 n.s.’
12.4 5.6
‘Abbreviations: PB I, first polar body; M I, metaphasc I; A I,
anaphaseI: T
I, telophase 1.
2Numberof noqtes. ‘Oocytes with PB 1, as a percentage of the number ofcultivated oocytes. ‘Oocytes in individual stages.as a percentage of the number ofoocytes without PB I. ‘Statistical significance: n.s., not significant; t l differ ~igniiicantly (PC 0.01).
,groups
iability span. Administration ol”LH-RH increased the LH concentration to 8.322.76 ng ml-! in the course of 30 minutes. It was significantly higher (P
LH-RH :.8D n(ElOTIC MATURATION
195
tith .l released first polar body reached 86.1% in the group treated with LHKH. When oocytes cultivated for 8 h were stained, it was observed that all cocytes from Group 2 were in Anaphase I or Interphase I. Groups Pid not differ in the percentage of degenerated oocytes. DlSCUSSION
The onset of a preor &tory peak in heiftz was observed 40.9 18.4 h (Grew et al., 1984b), and even in 36 h (Yadav et al., 1983) after prostaglandin F2 alpha administration in heifers stimulated with pregnant mar: serum gonadotropin (PMSG) The preovul3tory LH peak occurred 42.0 5 8.4 h (Greve et al., 1984b) or 49.7 h (Yadav e? al., 1983) after FSH-P administration. Comparison of these data with resu!ts obtained in Group 1 showed that the preovulatory onset 3f an early LH peak in the present experiment is similar to the results of superovulation after PMSG treatment. However, plotting the data for Group 2 based on these results suggcts that both the LH rise and the pituitary gland response to LH-RH treatment were not influenced by previous LH secretion (Voss et al., 1986; Savage et aL, 1987; Lamming and McLeod, i988) and an LH increase occurred in all heifers after LH-RH administration, in contrast to other studies (Walton and Stubbings, 1986; Savage et al., 1987). Luteinizing hormone is a physiological agent which induces meiotic maturation of mammalian oQcytes in vivo. It predominantly affects follicular cells. After the preovulatory LH increase, the disintegration fif the cumulus oophorus structure proceeds from acculaulation of giycosaminog!ycans in the iutercellular space (r)ekel and Phillips, 1979) and a decrease of metabolic cooperation between the oocyte and cumulus takes pIa=. In this experiment the effect of exogenous LH-RH resulted in a significantly lower number of oocytes with expanded cumulus, compared with the untreated group and with previous data (Sirard and Lambert, 1985). Unilateral enlargement of the perivitelline space occurs with gradual maturation of the oocytes, the indication of which is visible 2 h after the preovulatory LH peak (Hyttel et al., 1986). Enlrrgement ofthe perivitelline space is related to subsequent release of the first polar body in the preovulatory period (Greve et al., 1984a). Both the expansion of the cumulus oophorus and meiotic maturation were improved in the group treated with LH-RH, compared with the untreated group and with the results of other authors (Greve et al., 1984aJ. The stimulating effect of LH-RH was confirmed partly by a lower percentage of oocytes without a perivitelline space, partly by a higher percentage of oocytes with a released polar body, and partly by a high percentage of oocytec with a released polar body after short-term cultivation. Although it was observed in the past that the LH peak is not a prerequisite for meiotic maturation in heifers (Goff et al., 19861, the results of this exper-
196
J. UURINt?K
ET AL.
iment point out the important synchronizing influence of LH-RH upon meiotic maturation. Positive results were achieved owing to the optimal timing of LH-RH administration. Results of short-term culture also confirmed that it is appropriate to divide the oocytes, after their aspiration from preovulatory foiiicles, into oocytes with or without perivitelline space, and those with a released polar body. In conclusion, the present study indicates that the utilization of LH-RH increases the number of recovered oocytes that are suitable for the study of problems associated with in vitro fertilization. At the same time, it enables the recovery of embryos at the same stage of development in embryo-transfer programs. ACKNOWLEDGEMENTS
The authors would like to thank NlQDK National Hormone and Pituitary Program, Baltimore, Maryland, for kindly supplying the bovine LH preparations used for the assay and antibody production, and Garett Rarden of University College Cork for his help with English.
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