- Email: [email protected]
Influence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle
Theriogenology 58 (2002) 1141±1151
In¯uence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle M. Lafria,b, C. Ponsarta, M. Nibarta, M. Durandc, A. Morela, N. Jeanguyota, F. Badinandb, K. De Marid, P. Humblota,* a
UNCEIA Services Techniques, 13 Rue JoueÈt B.P. 65, 94703 Maisons-Alfort, France b Ecole veÂteÂrinaire d'Alfort, 7 Av Gl de Gaulle, 94700 Maisons-Alfort, France c UCEAR, Les vesves, 38300 Chateauvillain, France d VIRBAC, 13eÁme rue, L.I.D., 06516 Carros, France Received 26 June 2000; accepted 16 November 2001
Abstract One of the major sources of success in embryo transfer is timing of AI relative to the LH surge and ovulation. The aim of this study was to compare the embryo production following superovulation during a PGF2a (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle). CIDR-B (CIDR-B ND, Virbac, Carros, France) was inserted on Day 11 of a previously synchronized cycle and left for 5 days. A total dose of 350 mg FSH was administered (eight injections i.m. for 4 days; ®rst on Day 13, decreasing doses) and PGF2a analog (750 mg i.m.; Uniandine ND, Schering-Plough, Levallois-Perret, France) injected at the time of third FSH injection. Arti®cial inseminations were performed 12 and 24 h after standing estrus (Day 0). Embryos were collected on Day 7. Luteinizing hormone was measured by EIA (Reprokit Sano®, Libourne, France) from blood samples collected every 3 h for 36 h, starting 24 h after PGF2a (control cycle) or 12 h after CIDR-B removal (CIDR-B cycle). The effects of treatment group and interval between the LH peak and AI (two classes, <10 and 10 h) on embryo production and quality were analyzed by ANOVA. No effect of treatment was observed on embryo production variables. The intervals between the end of treatment and onset of estrus and between end of treatment and LH surge were greater in heifers treated during a control than a CIDR-B cycle, respectively (45:5 1:4 versus 31:9 0:7; 42:0 1:6 versus 31:0 1:5; P < 0:05), but maximal LH and estradiol concentrations, at the preovulatory surge were similar in control and CIDR-B synchronized heifers. The numbers of viable and Grade 1 embryos were signi®cantly increased (P < 0:01) when animals had an interval from LH peak to ®rst AI 10 h (7:2 0:9 and 3:5 0:6) when compared to shorter intervals (4:2 1:1 and 2:0 0:7) whereas total number of embryos was unchanged (11:8 1:4 versus 10:3 1:8). It is concluded that late occurrence of LH peaks in relation to estrous behavior is * Corresponding author. Tel.: 33-1-43-53-51-00; fax: 33-1-43-53-51-01. E-mail address: [email protected] (P. Humblot).
0093-691X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 6 3 7 - 4
1142
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
associated with a lower embryo quality when ®rst AIs are performed systematically 12 h after standing estrus. Further studies are needed to know if results may be improved when making AI at a later time after standing estrus or if LH assays are useful to better monitor AI time. # 2002 Published by Elsevier Science Inc. Keywords: Cattle; Superovulation; FSH; LH; P4; Estradiol; CIDR
1. Introduction Success of embryo transfer is still limited by variable superovulatory response [1,2] and yield of viable embryos [3,4], although highly puri®ed FSH preparations are now available. This is mainly explained by large variations between donor cows which may be partly related to the highly variable interval between prostaglandin injection and the LH surge, averaging 42 h but ranging from 20 to 66 h [3,5±9]. This variability and especially the occurrence of late LH surges may contribute to lower mean results. Effectively, unfavorable effects of late LH peaks have been already reported on conception rates after AI [10] and on the number of transferable embryos following superovulation [6,11,12]. In order to reduce this variability, many authors have tested the use of treatments combining progestagens and FSH [9,10,13,14]. Indeed, superovulation treatments during a progestagen synchronized cycle have been reported to result in a better control of the LH surge, with intervals between the progestagen removal and the LH surge averaging 32 h and ranging from 18 to 51 h [9,15]. However, inconstant effects on embryo production results have been observed, since the use of progestagen has led to similar [7,9], decreased [16,17], or increased [14] embryo production results when compared to treatments performed during a PGF2a synchronized cycle. The discrepancies between those observations may be due to high individual variations within experimental groups. A part of this variability may result from differences in the interval between the LH peak and ovulation which was reported to be highly variable [3,18,19]. Laurincik et al. [18] have reported that the interval between the ®rst and the last ovulation lasted 8 h, with 75% of the ovulations occurring during the ®rst 4 h. Therefore, the control of the LH surge seems to be a major factor to determine timing of AI after superovulation. The present study aimed to compare the characteristics of hormonal patterns and embryo production following superovulation with FSH during a PGF2a or a CIDR synchronized cycle. This comparison was made using an experimental design allowing control of individual variability. The effect of the interval between the LH peak and ®rst AI and its relationships with embryo production results were most particularly investigated. 2. Materials and methods 2.1. Animals and treatment Twelve Charolais heifers, 19±24 months old and sexually mature were included in the study and randomly assigned to one group of treatment. They were kept in stalls and fed
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1143
Fig. 1. Experimental design for Group 1 cows (n 6) superovulated successively during a control cycle and then treated with CIDR-B (CIDR-B cycle). This sequence was inverted in Group 2 cows (n 6) which ®rst received FSH in CIDR-B cycle.
with hay ad libitum. All heifers were given Norgestomet (3 mg, i.m. and a 3 mg s.c. ear implant; Crestar ND Intervet, Angers, France) and estradiol valerate (5 mg, i.m.) and were observed for estrus. Thirteen days after the Norgestomet-synchronized estrus heifers were randomly assigned to treatment groups using a repeat-measure design: treatment Group 1 heifers (n 6) were ®rst subjected to a superovulation regimen during a cycle without insertion of a CIDR-B (control cycle), followed in 6 weeks by a regimen with CIDR-B (CIDR-B cycle). Treatment Group 2 heifers (n 6) were ®rst superovulated during a CIDR-B synchronized cycle, followed in 6 weeks during a cycle without CIDR-B (Fig. 1). Superovulation was induced in all heifers 13 days after the preceding estrus with an FSH preparation, containing 500 mg of pFSH and 100 mg of pLH (eight doses, i.m., every 12 h, decreasing from 62.5 to 25 mg, for a total of 350 mg; Stimufol ND, Merial, Lyon, France). Heifers on the control cycle were given PGF2a (750 mg, i.m.; Uniandine ND, ScheringPlough, Levallois-Perret, France) concurrently with FSH Dose 5 of the superovulation regimen. Heifers on the CIDR-B cycle had a CIDR-B (Controlled Internal Drug Release, Bovine; Virbac, Carros, France) inserted aseptically into the vagina on Day 11 after the preceding estrus; the device was removed 5 days later. PG was given concurrently with the FSH Dose 3. Estrous behavior was observed from 24 h after PG injection (control cycle) and from 12 h after CIDR-B removal (CIDR-B cycle). Arti®cial inseminations were performed 12 and 24 h after standing estrus (Day 0) with semen of the same ejaculate from a single bull. Thereafter, embryos were recovered on Day 7 by cervical ¯ushing. Embryos were classi®ed using the morphological IETS criteria of quality and viability [20]. 2.2. Blood samples Blood samples were collected by caudal venupuncture at ®rst standing estrus, ®rst FSH, estrus following superovulation and at ¯ushing and assayed for progesterone and estradiol 17b. Blood was sampled every 3 h for 36 h from 24 h after PG injection during the control
1144
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
cycle and 12 h after CIDR-B removal during the CIDR-B cycle for the measurement of LH and estradiol plasma concentrations. All blood samples were collected into heparinized tubes, centrifuged immediately after sampling at 3000 g for 10 min, and then stored at 20 8C until required for analysis. 2.3. Assays LH and progesterone were measured by enzyme immunoassay (Reprokit Sano® Sante animale, Libourne, France for LH assay and Ovucheck, 909263; Eurobio, Les Ulis, France for progesterone assay). Estradiol was measured by radioimmunoassay [21±23]. Intraassay coef®cients of variation were 17.7, 3, and 6.7% for progesterone, LH and estradiol assay, respectively. Corresponding inter-assay coef®cients of variation were 5.6, 10 and 8.8%. Assay sensitivities were less than 0.1, 0.1 ng/ml and 2 pg/ml, respectively. 2.4. Statistical analysis The LH surge was de®ned as LH concentrations, 50% higher than the basal level, calculated as the mean from three sequential LH measurements. The greatest LH concentration observed during the sampling period was considered as the LH peak value [5,6]. An estradiol surge was characterized [5,6] by a steady rise in plasma concentration above 20 pg/ml to a maximal concentration (estradiol peak) and followed by a decrease within 12±16 h. End of treatment was de®ned as the time of induction of luteolysis, which occurred after PG injection during control cycle and after CIDR-B removal during CIDR-B cycle. Pearson correlation coef®cients were calculated between embryo production, estradiol 17b, LH and progesterone concentrations and intervals between reproductive events (end of treatment, onset of estrus, time of AI) and LH peak. The effects of treatment, group, order of treatment, duration of interval between the LH peak and AI (two classes, <10 and l0 h), donor within group and their interaction on embryo production and quality were analyzed by ANOVA (Proc. GLM, SAS [24]). All results are presented as means S:E:M. 3. Results On average, 11:1 4:3 embryos per ¯ushing were recovered on Day 7, with a range of 3±20. No signi®cant effect of treatment was observed on the quality of embryos: the numbers of viable, Grade 1, degenerated embryos and unfertilized oocytes per ¯ushing were similar in heifers on the control and CIDR-B cycle and averaged 6:0 0:8, 2:9 0:5, 3:5 0:4 and 1:7 0:4, respectively (Fig. 2). 3.1. Characteristics of intervals between end of treatment and reproductive events The interval between the end of treatment and onset of estrus was signi®cantly longer in heifers on control than on CIDR-B cycle (45:5 1:4 versus 31:9 0:7 h, P < 0:01). These intervals were more variable during control than during CIDR-B cycle (P < 0:05) ranging
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1145
Fig. 2. Embryo production following a superovulation treatment during a PGF2a (control cycle, n 12) or a CIDR-B synchronized (CIDR-B cycle, n 12) cycle.
from 39 to 52 h and from 29 and 36 h, respectively (Table 1). In a similar way, heifers on control cycle presented greater time intervals between the end of treatment and the LH peak and between the end of treatment and AI than CIDR-B synchronized heifers. The interval between standing estrus and occurrence of the LH peak was highly variable for both treatments, and ranged from 8 to 15 h and from 10 to 7 h during control and CIDR-B cycles, respectively. The interval between the LH peak and ®rst AI was also highly variable, ranging from 0 to 25 h and averaged 12:1 1:3 h. However, the difference observed between control and CIDR-B cycles was not signi®cant (control: 13:9 2:0 versus CIDR-B: 10:3 1:6 h; P > 0:05) and the proportion of heifers inseminated at least 10 h after the LH peak was similar (7/12 in both groups). The interval from the LH peak to the ®rst AI was negatively correlated with the interval from the end of treatment to the LH peak in heifers on control cycle (r 0:66, P 0:02, n 12) and on CIDR-B cycle (r 0:78, P < 0:01, n 12). These events in¯uenced strongly embryo production and quality, as shown in Table 2. Moreover, the numbers of transferable and Grade 1 embryos were signi®cantly Table 1 Time intervals between end of treatment and reproductive events (h) in heifers superovulated during a PGF2a (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle) Interval (h)
End of treatment and estrus End of treatment and LH peak End of treatment and first AI
Control cycle
CIDR-B cycle
Mean S.E.M.
Range
Mean S.E.M.
Range
45.5 1.4a 42.0 1.6c 55.9 1.5a
39±52 36±54 48±63
31.9 0.7b 31.0 1.5d 41.0 0.9b
29±36 27±42 37±46
Values within a row with different superscripts differ: a vs. b: P < 0:01, c vs. d: P < 0:05.
1146
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
Table 2 Effects of treatment, group, interval between the LH peak and first AI and donor on quality of embryos (mean S:E:M:) Variables
Total
Transferable
Quality of Grade 1
Embryos degenerated
Unfertilized oocytes
Treatment Control CIDR-B P
11.0 1.1 11.0 1.4 NS
5.8 0.7 5.6 0.7 NS
3.0 0.5 2.6 0.5 NS
3.6 0.7 3.3 0.7 NS
1.5 0.7 2.1 0.7 NS
Group G1 G2 P
12.7 1.1 9.6 1.3 0.08
7.2 1.3 4.8 0.8 0.03
3.7 0.7 2.2 0.6 0.04
3.7 0.4 3.3 0.7 NS
1.8 0.5 1.6 0.8 NS
Interval from LH peak to first AI <10 h 10.3 1.8 >10 h 11.8 1.4 P 0.02
4.2 1.1 7.2 0.9 <0.01
2.0 0.7 3.5 0.6 <0.01
3.5 1.1 3.5 0.6 NS
2.6 1.1 1.1 0.9 NS
Donor within groupNS
0.10
0.09
NS
NS
higher in Group 1 heifers than in Group 2 heifers. However, no signi®cant effect of order of treatments and donor cow within group was observed. The number of total viable and Grade 1 embryos were signi®cantly decreased (P < 0:05), in heifers that presented an interval between the LH peak to the ®rst AI shorter than 10 h when compared to heifers with longer intervals. There was a negative correlation between the interval from the end of treatment to the LH peak and the number of total embryos in control heifers and CIDR-B synchronized heifers, respectively (r 0:53, P 0:07, n 12; and r 0:67, P 0:01, n 12). Moreover, the interval from LH peak to ®rst AI was positively correlated with the numbers of total, viable and Grade 1 embryos, respectively (r 0:45, P 0:03, n 24; r 0:50, P 0:01, n 24; and r 0:47, P 0:02, n 24). 3.2. Characteristics of hormonal patterns during and after superovulation The P4 plasma concentrations measured at time of the ®rst FSH injection were higher in heifers on CIDR-B cycle than on control cycle (14:8 0:7 versus 11:3 0:8 ng/ml; P < 0:05), whereas estradiol concentrations were similar in both treatment groups (control cycle: 5:9 0:8 versus CIDR-B cycle: 5:5 0:6 pg/ml; P > 0:05). The P4 and estradiol levels measured at time of the ®rst FSH injection were not correlated with embryo production variables. From the ®rst injection to time of ®rst AI, estradiol concentrations increased (control: 15:5 0:7 versus CIDR-B: 14:1 0:9 pg/ml; P > 0:05) whereas P4 concentrations decreased to a mean of 0:53 0:04 ng/ml (with all values lower than 1 ng/ml). No difference was observed between treatment groups. No effect of treatment (P > 0:05) was observed on basal LH levels, duration and the amplitude of the preovulatory LH surge which averaged 1:5 0:5 ng/ml, 8:6 0:6 h, and 12:0 1:4 ng/ml, respectively. The LH peak maximum value was similar in both treatment
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1147
Fig. 3. Pro®les of plasma concentrations of estradiol and LH in heifers superovulated during a PGF2a (control cycle, n 12, a) or a CIDR-B synchronized cycle (CIDR-B cycle heifers, n 12, b) measured for 36 h around the preovulatory surge (time of LH surge 0)
groups (14:2 2:2 versus 13:2 1:8 ng/ml; P > 0:05) (Fig. 3). The LH peak characteristics were not signi®cantly in¯uenced by the interval between the LH peak and the ®rst AI. However, the peak value of LH was negatively correlated to the interval between the PG injection and occurrence of the LH surge (r 0:65, P 0:02) in control heifers. Concomitantly, estradiol 17b concentrations increased after the end of treatment to a peak value occurring 40:5 2 h after PG injection in control heifers (ranging between 33 and 54 h) and 30:3 2 h after CIDR-B removal in CIDR-B synchronized heifers (ranging between 21 and 42 h). For both groups, estradiol 17b mean maximum value was observed
1148
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
(1 h) at time of the preovulatory LH maximum value. No differences between heifers on control and CIDR-B cycles were observed for those parameters. After the LH peak, estradiol 17b concentrations decreased steadily, reaching baseline values 54 h after PG and 42 h after CIDR removal in heifers on control and CIDR-B cycles, respectively, which corresponded to a delay of 18±24 h after the LH peak. The plasma concentrations of estradiol 17b measured at the time of the LH surge tended to be negatively correlated with the number of viable embryos (r 0:38, P 0:07, n 24). 4. Discussion The present study aimed to compare the embryo production after the control of luteolysis by a prostaglandin injection or a progesterone device. When using that experimental design allowing to control individual variations, no difference was observed between treatments for embryo production variables. This is in agreement with previous data [9,23,25] and these results con®rm that superovulation during a PGF2a or a progestagen synchronized cycle may lead to collection of similar numbers of transferable embryos. Concerning the hormonal patterns around the preovulatory LH surge, the maximal value of LH and estradiol 17b concentrations were not different between treatment groups and were in agreement with previous observations, which reported values of LH ranging from 16 to 24 ng/ml for LH peak values [7,11,26] and ranging from 30 to 80 pg/ml for estradiol 17b maximum values [7,9,27]. The LH peak occurred 42 h after the prostaglandin injection and 32 h after removal of the progesterone device. As previously reported, the intervals between the end of treatment and the peak of LH were less variable in heifers receiving the progesterone device treatment when compared to a control cycle [3,5±9]. This indicates that the control of luteolysis with a progestagen treatment leads to a better synchronization of the LH surge. Moreover, this was associated with a decreased variability of the interval between progestagen removal and onset of estrus. Surprisingly, better synchronization did not result in an improved embryo production and quality. First, this may be partly explained by the ®xed time AIs which were performed systematically 12 and 24 h after estrus detection. In agreement with previous observations [9,28], the interval between the LH surge and the beginning of estrus ranged from 8 to 15 h and from 10 to 7 h in control and CIDR-B synchronized heifers. Therefore, heifers were inseminated at different time intervals after the LH peak, independently of treatment groups and this may explain why embryo production was comparable between the two groups. Second, it has been reported that the synchronization of the LH peak is not directly related to the synchronization of ovulations [19,25]. Indeed, embryo production variables were mainly in¯uenced by the interval between the LH peak and the ®rst AI, as shown by the correlations between this interval and the numbers of viable and Grade 1 embryos. The numbers of transferable and Grade 1 embryos were signi®cantly decreased when the ®rst AI was performed within 10 h after the LH peak. This could be related to the longer interval between the end of treatment and the LH surge. Previous studies have reported that embryo production is decreased when the LH peak is delayed [6,11,12]. In the present experiment, this effect might be also illustrated by
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1149
the concomitant decrease of the maximal value and the amplitude of the preovulatory LH surge observed in control heifers. A negative effect of a short interval between the peak of LH and the ®rst AI on embryo production variables has been already reported as well [8,23]. A short interval between the peak of LH and the ®rst AI results in a long delay between ®rst AI and the beginning of ovulations, which can exceed 15 h considering that ovulations begin 24 h in average after the LH peak [3,18,25,26,29]. It may be hypothesized that this large delay between the ®rst insemination and ovulations could affect negatively the quality of semen and the fertilization process. This effect may be also related to the high estradiol 17b concentrations at AI measured in heifers which presented a delayed LH peak for which the storage and release of spermatozoa in the oviduct may be altered [30,31]. Furthermore, it is well known that superovulatory treatments are followed by a shorter time period from the induction of luteolysis to ovulations. Consequently, the process of the oocyte maturation may be modi®ed, and this may lead to ovulations of less mature oocytes which may have a reduced ability to undergo normal fertilization and a normal embryonic development [32±35]. We observed that the sequence of treatment may in¯uence the quality of embryos, since Group 1 heifers presented better embryo production results than Group 2 heifers. However, heifers were nested in each group. Since no effect of the order of treatments was observed, it may be hypothesized that this group effect is mainly due to differences between heifers constituting those groups. Moreover, there was no signi®cant effect of donors within group, which may indicate that heifers were quite similar within a group. In the present experiment, superovulation associated to a CIDR synchronized cycle resulted in similar embryo production to treatment during a PGF2a synchronized cycle but improved the LH peak synchronization. Despite this, time intervals between ®rst detection of estrus and the LH surge and between LH and ®rst AI were highly variable. The quality of embryos was decreased when the ®rst AI was performed too early after the LH peak. This may lead more reliable indicators of estrous behavior to test if results may be improved when making AI at a later time after standing estrus and to use more reliable indicators of estrus to better adjust time of AI to individual hormonal and behavioral responses to superovulation. References [1] Adams GP, Matteri RL, Kastelic JP, Ko JC, Ginther OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fertil 1992;94:177±88. [2] Guilbault LA, Grasso F, Lussier JC, Rouillier P, Maton P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fertil 1991;9:81±9. [3] Greve T, Callessen H, Hyttel R, Assey R. The effects of exogenous gonadotropins. Theriogenology 1995;43:41±50. [4] Saumande J. La production d'embryons chez les bovins: quelles voies de recherches pour augmenter l'ef®cacite des traitements de superovulation? INRA Prod Anim 1995;8(4):275±83. [5] Callesen H, Greve T, Hyttel P. Estrus characterization in superovulated cattle. Theriogenology 1993;40:1243±50. [6] Greve T, Callesen H, Hyttel P. Endocrine pro®le and egg quality in the superovulated cows. Nord Vet Med 1983;35:408±21.
1150
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
[7] Humblot P, Rodrigues JL, Nibart M, Silverstini, Tiezi FL, Jeanguyot N, et al. Effet du mode de synchronization des cycles sexuels sur la reÂponse hypophysaire et la fonction ovarienne apreÁs superovulation chez la vache. Elevage InseÂmination 1994;261:7±18. [8] Maurel MC, Thierry P, Menissier F, Astruc S, Bouguennec B, Coupet H, et al. LH monitoring in bovine embryo production. Application of an ELISA kit. AETE meeting. Lyon, 1994. p. 216. [9] Nibart M, Slimane N, Herrera R, Jeanguyot N, Mechkour F, Humblot P, et al. Variations des concentrations plasmatiques des hormones gonadotropes (FSH et LH) et des steroides (oestradiol-17b et progesterone) apreÁs diffeÂrents traitements de superovulation chez la vache. Elevage InseÂmination 1988;226:11±30. [10] Hansel W, Schechter RJ, Malven PV, Simmons KR, Black DL, Hackett AJ, et al. Plasma hormone levels in 6-methyl-17-acetoxyprogesterone and estradiol benzoate treated heifers. J Anim Sci 1975;40(4):671± 81. [11] Donaldson LE. LH and FSH pro®les at superovulation and embryo production in the cow. Theriogenology 1985;23(3):441±7. [12] Goff AK, Greve T, Bousquet D, King WA. Progesterone and luteinizing hormone pro®les in heifers used as oocyte donors. Theriogenology 1986;26:577±86. [13] Ellington JE, Elefson EE, McCall RM. Use of Norgestomet implant as an aid when superovulating low fertility dairy cattle. Theriogenology 1987;27:227. [14] Mapletoft RJ, Bo GA, Pierson RA. Recruitment of follicles for superovulation. Food Anim 1994;6(1):127±42. [15] Slimane W, Humblot P, Jeanguyot N, Thibier M. Detection par dosage enzymo-immunologique (EIA) au laboratoire et en ferme de la deÂcharge preÂovulatoire de LH chez la vache. Rec Med Vet 1994;170: 547±52. [16] Almeida AP. Superovulation in cattle: a combined treatment using Synchromate B with either PMSG or FSH. Theriogenology 1987;27(1):203. [17] Smith JF, Padgitt DD, Hayman DL. The effects of incorporating a Norgestomet ear implant into a superovulation regime. Theriogenology 1988;29(1):310. [18] Laurincik J, Oberfranc M, Hyttel P, Grafeneau M, Tomanek M, Pivko J, et al. Characterization of the periovulatory period in superovulated heifers. Theriogenology 1993;39:537±44. [19] Slimane N, Nibart M, Thibier M. DeÂlais d'apparition de l'oestrus et des ovulations apreÂs traitement de superovulation chez des femelles bovines de race laitieÁre. Elevage InseÂmination 1984;199:21±9. [20] Wright JM. Photographic illustrations of embryo developmental stage and quality codes. In: Stringfellow DA, Seidel SM, editors. Manual of the International Embryo Transfer Society. 3rd ed. Savoy, IL: IETS, 1998. p. 167±70. [21] Grimard B, Humblot P, Mialot JP, Sauvant D, Thibier M. Absence of response to estrus induction and synchronization treatment is related to lipid mobilization in suckled beef cows. Reprod Nutr Dev 1997;37:129±40. [22] Maurel MC. Development of an ELISA kit for the determination of LH, on farm. In: Proceedings of the 7th Reunion of AETE Meeting. Cambridge, 1991. p. 176. [23] Slimane W, Nibart M, Thuard JM, Keita JB, Humblot P. Effect of controlling the moment of AI with a ®eld LH on the number and quality of embryos collected from superovulated cows. In: Proceedings of the 11th AETE Meeting. Hannover, 1995. p. 240. [24] SAS, Statistical Analysis Systems Institute Inc. SAS/STST User's Guide, Release 6.03. Cary, NC: SAS Institute Inc., 1989. [25] Ka® M, McGowan MR. Factors associated with variation in the superovulatory response of cattle. Anim Reprod Sci 1997;48:137±57. [26] Yadav MC, Walton JS, Leslie KE. Plasma concentration of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotropin. Theriogenology 1986;26(4):523±40. [27] Maciel M, Edqvist LE, Gustafson H, Rodriguez-Martinez H. Preovulatory patterns of estradiol-17b in relation to the superovulatory response in cows and heifers. Reprod Dom Anim 1995;30:49±53. [28] Dieleman SJ, Bevers MM, Gielen T. Increase of the number of ovulations in PMSG/PG-treated cows by administration of monoclonal anti-PMSG shortly after the endogenous LH peak. Theriogenology 1987;27(1):222.
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1151
[29] Lacaze S, Coupet H, Blattes M, Perrin V, Hennequin M. In¯uences of arti®cial insemination number and bull on the percentage of transferable embryos in superovulated dairy cows. In: Proceedings of the 8th AETE Meeting. Lyon, 1992. p. 174. [30] Hunter RH, Wilmut I. Sperm transport in the cow: peri-ovulatory redistribution of viable cells within the oviduct. Reprod Nutr Dev 1984;24:597±608. [31] Nibart M. Le transfert embryonnaire et les biotechnologies appliqueÂes: bissection et sexage. Rec Med Vet 1991;167:261±90. [32] Assey RJ, Hyttel P, Roche JF, Boland M. Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers. Mol Reprod Dev 1994;39:8±16. [33] Callesen H, Greve T, Hyttel P. Premature ovulations in superovulated cattle. Theriogenology 1987;28:155±66. [34] Dominko T, First NL. Timing of meiotic progression in bovine oocytes and its effect on early embryo development. Mol Reprod Dev 1997;47:456±67. [35] Hyttel P, Callesen H, Greve T. Ultrastructure of in vivo fertilization in superovulated cattle. J Reprod Fertil 1988;82:1±13.
In¯uence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle M. Lafria,b, C. Ponsarta, M. Nibarta, M. Durandc, A. Morela, N. Jeanguyota, F. Badinandb, K. De Marid, P. Humblota,* a
UNCEIA Services Techniques, 13 Rue JoueÈt B.P. 65, 94703 Maisons-Alfort, France b Ecole veÂteÂrinaire d'Alfort, 7 Av Gl de Gaulle, 94700 Maisons-Alfort, France c UCEAR, Les vesves, 38300 Chateauvillain, France d VIRBAC, 13eÁme rue, L.I.D., 06516 Carros, France Received 26 June 2000; accepted 16 November 2001
Abstract One of the major sources of success in embryo transfer is timing of AI relative to the LH surge and ovulation. The aim of this study was to compare the embryo production following superovulation during a PGF2a (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle). CIDR-B (CIDR-B ND, Virbac, Carros, France) was inserted on Day 11 of a previously synchronized cycle and left for 5 days. A total dose of 350 mg FSH was administered (eight injections i.m. for 4 days; ®rst on Day 13, decreasing doses) and PGF2a analog (750 mg i.m.; Uniandine ND, Schering-Plough, Levallois-Perret, France) injected at the time of third FSH injection. Arti®cial inseminations were performed 12 and 24 h after standing estrus (Day 0). Embryos were collected on Day 7. Luteinizing hormone was measured by EIA (Reprokit Sano®, Libourne, France) from blood samples collected every 3 h for 36 h, starting 24 h after PGF2a (control cycle) or 12 h after CIDR-B removal (CIDR-B cycle). The effects of treatment group and interval between the LH peak and AI (two classes, <10 and 10 h) on embryo production and quality were analyzed by ANOVA. No effect of treatment was observed on embryo production variables. The intervals between the end of treatment and onset of estrus and between end of treatment and LH surge were greater in heifers treated during a control than a CIDR-B cycle, respectively (45:5 1:4 versus 31:9 0:7; 42:0 1:6 versus 31:0 1:5; P < 0:05), but maximal LH and estradiol concentrations, at the preovulatory surge were similar in control and CIDR-B synchronized heifers. The numbers of viable and Grade 1 embryos were signi®cantly increased (P < 0:01) when animals had an interval from LH peak to ®rst AI 10 h (7:2 0:9 and 3:5 0:6) when compared to shorter intervals (4:2 1:1 and 2:0 0:7) whereas total number of embryos was unchanged (11:8 1:4 versus 10:3 1:8). It is concluded that late occurrence of LH peaks in relation to estrous behavior is * Corresponding author. Tel.: 33-1-43-53-51-00; fax: 33-1-43-53-51-01. E-mail address: [email protected] (P. Humblot).
0093-691X/02/$ ± see front matter # 2002 Published by Elsevier Science Inc. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 6 3 7 - 4
1142
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
associated with a lower embryo quality when ®rst AIs are performed systematically 12 h after standing estrus. Further studies are needed to know if results may be improved when making AI at a later time after standing estrus or if LH assays are useful to better monitor AI time. # 2002 Published by Elsevier Science Inc. Keywords: Cattle; Superovulation; FSH; LH; P4; Estradiol; CIDR
1. Introduction Success of embryo transfer is still limited by variable superovulatory response [1,2] and yield of viable embryos [3,4], although highly puri®ed FSH preparations are now available. This is mainly explained by large variations between donor cows which may be partly related to the highly variable interval between prostaglandin injection and the LH surge, averaging 42 h but ranging from 20 to 66 h [3,5±9]. This variability and especially the occurrence of late LH surges may contribute to lower mean results. Effectively, unfavorable effects of late LH peaks have been already reported on conception rates after AI [10] and on the number of transferable embryos following superovulation [6,11,12]. In order to reduce this variability, many authors have tested the use of treatments combining progestagens and FSH [9,10,13,14]. Indeed, superovulation treatments during a progestagen synchronized cycle have been reported to result in a better control of the LH surge, with intervals between the progestagen removal and the LH surge averaging 32 h and ranging from 18 to 51 h [9,15]. However, inconstant effects on embryo production results have been observed, since the use of progestagen has led to similar [7,9], decreased [16,17], or increased [14] embryo production results when compared to treatments performed during a PGF2a synchronized cycle. The discrepancies between those observations may be due to high individual variations within experimental groups. A part of this variability may result from differences in the interval between the LH peak and ovulation which was reported to be highly variable [3,18,19]. Laurincik et al. [18] have reported that the interval between the ®rst and the last ovulation lasted 8 h, with 75% of the ovulations occurring during the ®rst 4 h. Therefore, the control of the LH surge seems to be a major factor to determine timing of AI after superovulation. The present study aimed to compare the characteristics of hormonal patterns and embryo production following superovulation with FSH during a PGF2a or a CIDR synchronized cycle. This comparison was made using an experimental design allowing control of individual variability. The effect of the interval between the LH peak and ®rst AI and its relationships with embryo production results were most particularly investigated. 2. Materials and methods 2.1. Animals and treatment Twelve Charolais heifers, 19±24 months old and sexually mature were included in the study and randomly assigned to one group of treatment. They were kept in stalls and fed
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1143
Fig. 1. Experimental design for Group 1 cows (n 6) superovulated successively during a control cycle and then treated with CIDR-B (CIDR-B cycle). This sequence was inverted in Group 2 cows (n 6) which ®rst received FSH in CIDR-B cycle.
with hay ad libitum. All heifers were given Norgestomet (3 mg, i.m. and a 3 mg s.c. ear implant; Crestar ND Intervet, Angers, France) and estradiol valerate (5 mg, i.m.) and were observed for estrus. Thirteen days after the Norgestomet-synchronized estrus heifers were randomly assigned to treatment groups using a repeat-measure design: treatment Group 1 heifers (n 6) were ®rst subjected to a superovulation regimen during a cycle without insertion of a CIDR-B (control cycle), followed in 6 weeks by a regimen with CIDR-B (CIDR-B cycle). Treatment Group 2 heifers (n 6) were ®rst superovulated during a CIDR-B synchronized cycle, followed in 6 weeks during a cycle without CIDR-B (Fig. 1). Superovulation was induced in all heifers 13 days after the preceding estrus with an FSH preparation, containing 500 mg of pFSH and 100 mg of pLH (eight doses, i.m., every 12 h, decreasing from 62.5 to 25 mg, for a total of 350 mg; Stimufol ND, Merial, Lyon, France). Heifers on the control cycle were given PGF2a (750 mg, i.m.; Uniandine ND, ScheringPlough, Levallois-Perret, France) concurrently with FSH Dose 5 of the superovulation regimen. Heifers on the CIDR-B cycle had a CIDR-B (Controlled Internal Drug Release, Bovine; Virbac, Carros, France) inserted aseptically into the vagina on Day 11 after the preceding estrus; the device was removed 5 days later. PG was given concurrently with the FSH Dose 3. Estrous behavior was observed from 24 h after PG injection (control cycle) and from 12 h after CIDR-B removal (CIDR-B cycle). Arti®cial inseminations were performed 12 and 24 h after standing estrus (Day 0) with semen of the same ejaculate from a single bull. Thereafter, embryos were recovered on Day 7 by cervical ¯ushing. Embryos were classi®ed using the morphological IETS criteria of quality and viability [20]. 2.2. Blood samples Blood samples were collected by caudal venupuncture at ®rst standing estrus, ®rst FSH, estrus following superovulation and at ¯ushing and assayed for progesterone and estradiol 17b. Blood was sampled every 3 h for 36 h from 24 h after PG injection during the control
1144
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
cycle and 12 h after CIDR-B removal during the CIDR-B cycle for the measurement of LH and estradiol plasma concentrations. All blood samples were collected into heparinized tubes, centrifuged immediately after sampling at 3000 g for 10 min, and then stored at 20 8C until required for analysis. 2.3. Assays LH and progesterone were measured by enzyme immunoassay (Reprokit Sano® Sante animale, Libourne, France for LH assay and Ovucheck, 909263; Eurobio, Les Ulis, France for progesterone assay). Estradiol was measured by radioimmunoassay [21±23]. Intraassay coef®cients of variation were 17.7, 3, and 6.7% for progesterone, LH and estradiol assay, respectively. Corresponding inter-assay coef®cients of variation were 5.6, 10 and 8.8%. Assay sensitivities were less than 0.1, 0.1 ng/ml and 2 pg/ml, respectively. 2.4. Statistical analysis The LH surge was de®ned as LH concentrations, 50% higher than the basal level, calculated as the mean from three sequential LH measurements. The greatest LH concentration observed during the sampling period was considered as the LH peak value [5,6]. An estradiol surge was characterized [5,6] by a steady rise in plasma concentration above 20 pg/ml to a maximal concentration (estradiol peak) and followed by a decrease within 12±16 h. End of treatment was de®ned as the time of induction of luteolysis, which occurred after PG injection during control cycle and after CIDR-B removal during CIDR-B cycle. Pearson correlation coef®cients were calculated between embryo production, estradiol 17b, LH and progesterone concentrations and intervals between reproductive events (end of treatment, onset of estrus, time of AI) and LH peak. The effects of treatment, group, order of treatment, duration of interval between the LH peak and AI (two classes, <10 and l0 h), donor within group and their interaction on embryo production and quality were analyzed by ANOVA (Proc. GLM, SAS [24]). All results are presented as means S:E:M. 3. Results On average, 11:1 4:3 embryos per ¯ushing were recovered on Day 7, with a range of 3±20. No signi®cant effect of treatment was observed on the quality of embryos: the numbers of viable, Grade 1, degenerated embryos and unfertilized oocytes per ¯ushing were similar in heifers on the control and CIDR-B cycle and averaged 6:0 0:8, 2:9 0:5, 3:5 0:4 and 1:7 0:4, respectively (Fig. 2). 3.1. Characteristics of intervals between end of treatment and reproductive events The interval between the end of treatment and onset of estrus was signi®cantly longer in heifers on control than on CIDR-B cycle (45:5 1:4 versus 31:9 0:7 h, P < 0:01). These intervals were more variable during control than during CIDR-B cycle (P < 0:05) ranging
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1145
Fig. 2. Embryo production following a superovulation treatment during a PGF2a (control cycle, n 12) or a CIDR-B synchronized (CIDR-B cycle, n 12) cycle.
from 39 to 52 h and from 29 and 36 h, respectively (Table 1). In a similar way, heifers on control cycle presented greater time intervals between the end of treatment and the LH peak and between the end of treatment and AI than CIDR-B synchronized heifers. The interval between standing estrus and occurrence of the LH peak was highly variable for both treatments, and ranged from 8 to 15 h and from 10 to 7 h during control and CIDR-B cycles, respectively. The interval between the LH peak and ®rst AI was also highly variable, ranging from 0 to 25 h and averaged 12:1 1:3 h. However, the difference observed between control and CIDR-B cycles was not signi®cant (control: 13:9 2:0 versus CIDR-B: 10:3 1:6 h; P > 0:05) and the proportion of heifers inseminated at least 10 h after the LH peak was similar (7/12 in both groups). The interval from the LH peak to the ®rst AI was negatively correlated with the interval from the end of treatment to the LH peak in heifers on control cycle (r 0:66, P 0:02, n 12) and on CIDR-B cycle (r 0:78, P < 0:01, n 12). These events in¯uenced strongly embryo production and quality, as shown in Table 2. Moreover, the numbers of transferable and Grade 1 embryos were signi®cantly Table 1 Time intervals between end of treatment and reproductive events (h) in heifers superovulated during a PGF2a (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle) Interval (h)
End of treatment and estrus End of treatment and LH peak End of treatment and first AI
Control cycle
CIDR-B cycle
Mean S.E.M.
Range
Mean S.E.M.
Range
45.5 1.4a 42.0 1.6c 55.9 1.5a
39±52 36±54 48±63
31.9 0.7b 31.0 1.5d 41.0 0.9b
29±36 27±42 37±46
Values within a row with different superscripts differ: a vs. b: P < 0:01, c vs. d: P < 0:05.
1146
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
Table 2 Effects of treatment, group, interval between the LH peak and first AI and donor on quality of embryos (mean S:E:M:) Variables
Total
Transferable
Quality of Grade 1
Embryos degenerated
Unfertilized oocytes
Treatment Control CIDR-B P
11.0 1.1 11.0 1.4 NS
5.8 0.7 5.6 0.7 NS
3.0 0.5 2.6 0.5 NS
3.6 0.7 3.3 0.7 NS
1.5 0.7 2.1 0.7 NS
Group G1 G2 P
12.7 1.1 9.6 1.3 0.08
7.2 1.3 4.8 0.8 0.03
3.7 0.7 2.2 0.6 0.04
3.7 0.4 3.3 0.7 NS
1.8 0.5 1.6 0.8 NS
Interval from LH peak to first AI <10 h 10.3 1.8 >10 h 11.8 1.4 P 0.02
4.2 1.1 7.2 0.9 <0.01
2.0 0.7 3.5 0.6 <0.01
3.5 1.1 3.5 0.6 NS
2.6 1.1 1.1 0.9 NS
Donor within groupNS
0.10
0.09
NS
NS
higher in Group 1 heifers than in Group 2 heifers. However, no signi®cant effect of order of treatments and donor cow within group was observed. The number of total viable and Grade 1 embryos were signi®cantly decreased (P < 0:05), in heifers that presented an interval between the LH peak to the ®rst AI shorter than 10 h when compared to heifers with longer intervals. There was a negative correlation between the interval from the end of treatment to the LH peak and the number of total embryos in control heifers and CIDR-B synchronized heifers, respectively (r 0:53, P 0:07, n 12; and r 0:67, P 0:01, n 12). Moreover, the interval from LH peak to ®rst AI was positively correlated with the numbers of total, viable and Grade 1 embryos, respectively (r 0:45, P 0:03, n 24; r 0:50, P 0:01, n 24; and r 0:47, P 0:02, n 24). 3.2. Characteristics of hormonal patterns during and after superovulation The P4 plasma concentrations measured at time of the ®rst FSH injection were higher in heifers on CIDR-B cycle than on control cycle (14:8 0:7 versus 11:3 0:8 ng/ml; P < 0:05), whereas estradiol concentrations were similar in both treatment groups (control cycle: 5:9 0:8 versus CIDR-B cycle: 5:5 0:6 pg/ml; P > 0:05). The P4 and estradiol levels measured at time of the ®rst FSH injection were not correlated with embryo production variables. From the ®rst injection to time of ®rst AI, estradiol concentrations increased (control: 15:5 0:7 versus CIDR-B: 14:1 0:9 pg/ml; P > 0:05) whereas P4 concentrations decreased to a mean of 0:53 0:04 ng/ml (with all values lower than 1 ng/ml). No difference was observed between treatment groups. No effect of treatment (P > 0:05) was observed on basal LH levels, duration and the amplitude of the preovulatory LH surge which averaged 1:5 0:5 ng/ml, 8:6 0:6 h, and 12:0 1:4 ng/ml, respectively. The LH peak maximum value was similar in both treatment
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1147
Fig. 3. Pro®les of plasma concentrations of estradiol and LH in heifers superovulated during a PGF2a (control cycle, n 12, a) or a CIDR-B synchronized cycle (CIDR-B cycle heifers, n 12, b) measured for 36 h around the preovulatory surge (time of LH surge 0)
groups (14:2 2:2 versus 13:2 1:8 ng/ml; P > 0:05) (Fig. 3). The LH peak characteristics were not signi®cantly in¯uenced by the interval between the LH peak and the ®rst AI. However, the peak value of LH was negatively correlated to the interval between the PG injection and occurrence of the LH surge (r 0:65, P 0:02) in control heifers. Concomitantly, estradiol 17b concentrations increased after the end of treatment to a peak value occurring 40:5 2 h after PG injection in control heifers (ranging between 33 and 54 h) and 30:3 2 h after CIDR-B removal in CIDR-B synchronized heifers (ranging between 21 and 42 h). For both groups, estradiol 17b mean maximum value was observed
1148
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
(1 h) at time of the preovulatory LH maximum value. No differences between heifers on control and CIDR-B cycles were observed for those parameters. After the LH peak, estradiol 17b concentrations decreased steadily, reaching baseline values 54 h after PG and 42 h after CIDR removal in heifers on control and CIDR-B cycles, respectively, which corresponded to a delay of 18±24 h after the LH peak. The plasma concentrations of estradiol 17b measured at the time of the LH surge tended to be negatively correlated with the number of viable embryos (r 0:38, P 0:07, n 24). 4. Discussion The present study aimed to compare the embryo production after the control of luteolysis by a prostaglandin injection or a progesterone device. When using that experimental design allowing to control individual variations, no difference was observed between treatments for embryo production variables. This is in agreement with previous data [9,23,25] and these results con®rm that superovulation during a PGF2a or a progestagen synchronized cycle may lead to collection of similar numbers of transferable embryos. Concerning the hormonal patterns around the preovulatory LH surge, the maximal value of LH and estradiol 17b concentrations were not different between treatment groups and were in agreement with previous observations, which reported values of LH ranging from 16 to 24 ng/ml for LH peak values [7,11,26] and ranging from 30 to 80 pg/ml for estradiol 17b maximum values [7,9,27]. The LH peak occurred 42 h after the prostaglandin injection and 32 h after removal of the progesterone device. As previously reported, the intervals between the end of treatment and the peak of LH were less variable in heifers receiving the progesterone device treatment when compared to a control cycle [3,5±9]. This indicates that the control of luteolysis with a progestagen treatment leads to a better synchronization of the LH surge. Moreover, this was associated with a decreased variability of the interval between progestagen removal and onset of estrus. Surprisingly, better synchronization did not result in an improved embryo production and quality. First, this may be partly explained by the ®xed time AIs which were performed systematically 12 and 24 h after estrus detection. In agreement with previous observations [9,28], the interval between the LH surge and the beginning of estrus ranged from 8 to 15 h and from 10 to 7 h in control and CIDR-B synchronized heifers. Therefore, heifers were inseminated at different time intervals after the LH peak, independently of treatment groups and this may explain why embryo production was comparable between the two groups. Second, it has been reported that the synchronization of the LH peak is not directly related to the synchronization of ovulations [19,25]. Indeed, embryo production variables were mainly in¯uenced by the interval between the LH peak and the ®rst AI, as shown by the correlations between this interval and the numbers of viable and Grade 1 embryos. The numbers of transferable and Grade 1 embryos were signi®cantly decreased when the ®rst AI was performed within 10 h after the LH peak. This could be related to the longer interval between the end of treatment and the LH surge. Previous studies have reported that embryo production is decreased when the LH peak is delayed [6,11,12]. In the present experiment, this effect might be also illustrated by
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1149
the concomitant decrease of the maximal value and the amplitude of the preovulatory LH surge observed in control heifers. A negative effect of a short interval between the peak of LH and the ®rst AI on embryo production variables has been already reported as well [8,23]. A short interval between the peak of LH and the ®rst AI results in a long delay between ®rst AI and the beginning of ovulations, which can exceed 15 h considering that ovulations begin 24 h in average after the LH peak [3,18,25,26,29]. It may be hypothesized that this large delay between the ®rst insemination and ovulations could affect negatively the quality of semen and the fertilization process. This effect may be also related to the high estradiol 17b concentrations at AI measured in heifers which presented a delayed LH peak for which the storage and release of spermatozoa in the oviduct may be altered [30,31]. Furthermore, it is well known that superovulatory treatments are followed by a shorter time period from the induction of luteolysis to ovulations. Consequently, the process of the oocyte maturation may be modi®ed, and this may lead to ovulations of less mature oocytes which may have a reduced ability to undergo normal fertilization and a normal embryonic development [32±35]. We observed that the sequence of treatment may in¯uence the quality of embryos, since Group 1 heifers presented better embryo production results than Group 2 heifers. However, heifers were nested in each group. Since no effect of the order of treatments was observed, it may be hypothesized that this group effect is mainly due to differences between heifers constituting those groups. Moreover, there was no signi®cant effect of donors within group, which may indicate that heifers were quite similar within a group. In the present experiment, superovulation associated to a CIDR synchronized cycle resulted in similar embryo production to treatment during a PGF2a synchronized cycle but improved the LH peak synchronization. Despite this, time intervals between ®rst detection of estrus and the LH surge and between LH and ®rst AI were highly variable. The quality of embryos was decreased when the ®rst AI was performed too early after the LH peak. This may lead more reliable indicators of estrous behavior to test if results may be improved when making AI at a later time after standing estrus and to use more reliable indicators of estrus to better adjust time of AI to individual hormonal and behavioral responses to superovulation. References [1] Adams GP, Matteri RL, Kastelic JP, Ko JC, Ginther OJ. Association between surges of follicle-stimulating hormone and the emergence of follicular waves in heifers. J Reprod Fertil 1992;94:177±88. [2] Guilbault LA, Grasso F, Lussier JC, Rouillier P, Maton P. Decreased superovulatory responses in heifers superovulated in the presence of a dominant follicle. J Reprod Fertil 1991;9:81±9. [3] Greve T, Callessen H, Hyttel R, Assey R. The effects of exogenous gonadotropins. Theriogenology 1995;43:41±50. [4] Saumande J. La production d'embryons chez les bovins: quelles voies de recherches pour augmenter l'ef®cacite des traitements de superovulation? INRA Prod Anim 1995;8(4):275±83. [5] Callesen H, Greve T, Hyttel P. Estrus characterization in superovulated cattle. Theriogenology 1993;40:1243±50. [6] Greve T, Callesen H, Hyttel P. Endocrine pro®le and egg quality in the superovulated cows. Nord Vet Med 1983;35:408±21.
1150
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
[7] Humblot P, Rodrigues JL, Nibart M, Silverstini, Tiezi FL, Jeanguyot N, et al. Effet du mode de synchronization des cycles sexuels sur la reÂponse hypophysaire et la fonction ovarienne apreÁs superovulation chez la vache. Elevage InseÂmination 1994;261:7±18. [8] Maurel MC, Thierry P, Menissier F, Astruc S, Bouguennec B, Coupet H, et al. LH monitoring in bovine embryo production. Application of an ELISA kit. AETE meeting. Lyon, 1994. p. 216. [9] Nibart M, Slimane N, Herrera R, Jeanguyot N, Mechkour F, Humblot P, et al. Variations des concentrations plasmatiques des hormones gonadotropes (FSH et LH) et des steroides (oestradiol-17b et progesterone) apreÁs diffeÂrents traitements de superovulation chez la vache. Elevage InseÂmination 1988;226:11±30. [10] Hansel W, Schechter RJ, Malven PV, Simmons KR, Black DL, Hackett AJ, et al. Plasma hormone levels in 6-methyl-17-acetoxyprogesterone and estradiol benzoate treated heifers. J Anim Sci 1975;40(4):671± 81. [11] Donaldson LE. LH and FSH pro®les at superovulation and embryo production in the cow. Theriogenology 1985;23(3):441±7. [12] Goff AK, Greve T, Bousquet D, King WA. Progesterone and luteinizing hormone pro®les in heifers used as oocyte donors. Theriogenology 1986;26:577±86. [13] Ellington JE, Elefson EE, McCall RM. Use of Norgestomet implant as an aid when superovulating low fertility dairy cattle. Theriogenology 1987;27:227. [14] Mapletoft RJ, Bo GA, Pierson RA. Recruitment of follicles for superovulation. Food Anim 1994;6(1):127±42. [15] Slimane W, Humblot P, Jeanguyot N, Thibier M. Detection par dosage enzymo-immunologique (EIA) au laboratoire et en ferme de la deÂcharge preÂovulatoire de LH chez la vache. Rec Med Vet 1994;170: 547±52. [16] Almeida AP. Superovulation in cattle: a combined treatment using Synchromate B with either PMSG or FSH. Theriogenology 1987;27(1):203. [17] Smith JF, Padgitt DD, Hayman DL. The effects of incorporating a Norgestomet ear implant into a superovulation regime. Theriogenology 1988;29(1):310. [18] Laurincik J, Oberfranc M, Hyttel P, Grafeneau M, Tomanek M, Pivko J, et al. Characterization of the periovulatory period in superovulated heifers. Theriogenology 1993;39:537±44. [19] Slimane N, Nibart M, Thibier M. DeÂlais d'apparition de l'oestrus et des ovulations apreÂs traitement de superovulation chez des femelles bovines de race laitieÁre. Elevage InseÂmination 1984;199:21±9. [20] Wright JM. Photographic illustrations of embryo developmental stage and quality codes. In: Stringfellow DA, Seidel SM, editors. Manual of the International Embryo Transfer Society. 3rd ed. Savoy, IL: IETS, 1998. p. 167±70. [21] Grimard B, Humblot P, Mialot JP, Sauvant D, Thibier M. Absence of response to estrus induction and synchronization treatment is related to lipid mobilization in suckled beef cows. Reprod Nutr Dev 1997;37:129±40. [22] Maurel MC. Development of an ELISA kit for the determination of LH, on farm. In: Proceedings of the 7th Reunion of AETE Meeting. Cambridge, 1991. p. 176. [23] Slimane W, Nibart M, Thuard JM, Keita JB, Humblot P. Effect of controlling the moment of AI with a ®eld LH on the number and quality of embryos collected from superovulated cows. In: Proceedings of the 11th AETE Meeting. Hannover, 1995. p. 240. [24] SAS, Statistical Analysis Systems Institute Inc. SAS/STST User's Guide, Release 6.03. Cary, NC: SAS Institute Inc., 1989. [25] Ka® M, McGowan MR. Factors associated with variation in the superovulatory response of cattle. Anim Reprod Sci 1997;48:137±57. [26] Yadav MC, Walton JS, Leslie KE. Plasma concentration of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotropin. Theriogenology 1986;26(4):523±40. [27] Maciel M, Edqvist LE, Gustafson H, Rodriguez-Martinez H. Preovulatory patterns of estradiol-17b in relation to the superovulatory response in cows and heifers. Reprod Dom Anim 1995;30:49±53. [28] Dieleman SJ, Bevers MM, Gielen T. Increase of the number of ovulations in PMSG/PG-treated cows by administration of monoclonal anti-PMSG shortly after the endogenous LH peak. Theriogenology 1987;27(1):222.
M. Lafri et al. / Theriogenology 58 (2002) 1141±1151
1151
[29] Lacaze S, Coupet H, Blattes M, Perrin V, Hennequin M. In¯uences of arti®cial insemination number and bull on the percentage of transferable embryos in superovulated dairy cows. In: Proceedings of the 8th AETE Meeting. Lyon, 1992. p. 174. [30] Hunter RH, Wilmut I. Sperm transport in the cow: peri-ovulatory redistribution of viable cells within the oviduct. Reprod Nutr Dev 1984;24:597±608. [31] Nibart M. Le transfert embryonnaire et les biotechnologies appliqueÂes: bissection et sexage. Rec Med Vet 1991;167:261±90. [32] Assey RJ, Hyttel P, Roche JF, Boland M. Oocyte structure and follicular steroid concentrations in superovulated versus unstimulated heifers. Mol Reprod Dev 1994;39:8±16. [33] Callesen H, Greve T, Hyttel P. Premature ovulations in superovulated cattle. Theriogenology 1987;28:155±66. [34] Dominko T, First NL. Timing of meiotic progression in bovine oocytes and its effect on early embryo development. Mol Reprod Dev 1997;47:456±67. [35] Hyttel P, Callesen H, Greve T. Ultrastructure of in vivo fertilization in superovulated cattle. J Reprod Fertil 1988;82:1±13.