Factors affecting superovulation in heifers treated with PMSG

ELSEVIER

FACTORS AFFECTING SUPEROVULATION IN HEIFERS TREATED WITH PMSG D. Gouldingi

D.H. Williams,2 J.F. Rochei andM.P. Boland3

Faculties of iVeterinary Medicine, Qcience and 3Agriculture University College Dublin, Belfield, Dublin 4, Ireland Received for publication : MUMIO, 1995 Accepted: September 26,

1995

ABSTRACT In this study we determined 1) if the immunoneutralization of PMSG affected the ovulatory response, the number of large follicles and embryo yield compared with that of PMSG alone or pFSH, and 2) whether the stage of the estrous cycle at which PMSG was injected affected the ovulatory response and yield of embryos in superovulated heifers. Estrus was synchronized in 99 (Experiment 1) and 71 (Experiment 2) heifers using prostaglandin F2a (PG) analogue, cloprostenol, given 11 d apart in replicate experiments over 2 yr. In Experiments 1 and 2, heifers were randomly allocated to 1 of 3 treatments (initiated at mid-cycle): Treatment 1 - 24 mg of pFSH (Folhropin) given twice daily for 4 d; Treatment 2 - a single injection of 2000 IU PMSG; Treatment 3 -2000 IU PMSG followed by 2000 IU of Neutra-PMSG at the time of first insemination. In Experiment 3, 116 heifers were given 2000 IU PMSG on Day 2 (n=28). Day 3 (n = 27), Day 10 (n = 41) or Day 16 (n = 20) of the estrous cycle. The PG was given at 48 h (500 pg cloprostenol) and 60 h (250 pg cloprostenol) after the first gonadotropin treatment. Heifers were inseminated twice during e&us. and embryos were recovered on Day 7, following slaughter and graded for quality. The numbers of ovulations and large follicles (210 mm) were also counted. There was no effect of treatment on ovulation rate in Experiment 1, but in Experiment 2 it was greater (P < 0.002) in heifers given PMSG (14.7rt1.5) than pFSH (7.91.4) or PMSG-neutra-PMSG (8.7k1.5). The number of large follicles was higher following PMSG than pFSH treatment in Experiment 1, and it was higher (P < 0.004) in heifers given PMSG (5.5~tO.8) than pFSH (1.12kO.7) or PMSG-neutra-PMSG (2.7N.8) in Experiment 2. The use of Neutra-PMSG did not affect the numbers of embryos recovered or numbers of Grade 1 or 2 embryos, but it did decrease the number of Grade 3 embryos in both experiments. In Experiment 3, the ovulation rate decreased (PC 0.004) when PMSG was given on Day 3 (5.7*1.46) of the cycle rather than on Day 2 (12.3*1X%), Day 10 (13.4*1.45) or Day 16 (12.91.87). There was no effect of day of treatment on the numbers of large follicles. The mean numbers of embryos recovered were lower (PC 0.01) in heifers treated on Day 3 (2.1a.67) than on Day 2 (6.8*1.0), Day 10 (6.4ti.86) or Day 16 (7.8k1.87). It is concluded that Neutra-PMSG given to heifers treated with PMSG did not improve embryo yield or quality and that treatment with PMSG early in the cycle can result in acceptable embryo yields provided sufficient time elapses between treatment and luteolysis. Key words: superovulation,

heifers, embryos, FSH, PMSG

Acknowledgments We thank Vetrepharm, Canada, for Folltropin; Intervet, Holland for PMSG and Neutra-PMSG ; Goodman International, Ireland, for the use of the animals and facilities; and BioResearch, Ireland, for financial support.

Theriogenology 45:765-773, 1996 0 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0093-691 X/96/$15.00 PI I 50093-691 X( 96)00006-6

Theriogenology INTRODUCTION The lack of a predictable and reliable supply of embryos is a major limiting factor in the widespread use of embryo transfer (11). Thus, it is a high priority to understand the factors that affect the yield of transferable embryos following superovulation (31). Maximum response to superovulation in cattle requires the administration of a gonadotropin at preselected stages of the estrous cycle followed by control of luteolysis, synchronous ovulation, high fertilization and early embryonic developmental rates. The gonadotropins commonly used are PMSG or FSH of porcine or ovine origin (2,lO). Because of its availability, relatively low cost, effectiveness and ease of use, with only a single injection needed, PMSG has been used extensively to superovulate cattle. The half-life of PMSG greatly exceeds that of FSH in cattle, and it involves rapid (t 0.5 40 to 50 h) and slow (t 0.5 118 to 123 h) clearance components (30). This long half-life of PMSG often induces extra follicular growth during the first follicular wave post ovulation with a resultant increased estradiol concentration in the blood, which may have a deleterious effect on early embryonic development (1,326). Immunoneutralization of circulating PMSG by injecting antibodies against the hormone will decrease the PMSG concentration by about 85% within 1 h of administration (5), and the use of anti-PMSG to improve the embryo recovery rate following superovulation with PMSG has recently been reviewed (l), indicating considerable debate or contradictions in the literature. Some researchers have reported increased numbers of transferable embryos following the use of anti-PMSG compared with that of FSH or PMSG (7), while others have failed to substantiate these claims (33). Early research indicated that PMSG antiserum, after PMSG injection, decreased subsequent excessive follicular development and resulted in higher numbers of normal embryos recovered from superovulated animals (7). Recent data suggest that the time of administration of the PMSG antibodies in relation to the time of occurrence of the LH surge is of curucial importance (32). Neutralization of PMSG shortly (6 to 8 h) after the preovulatory LH surge results in an increase in the ovulation rate compared with earlier neutralization (5 7,32). There is a large individual variation in the superovulatory response of heifers following a standard PMSG or pFSH treatment regimen, which means that sound experimental design requires large numbers of similarly managed animals per treatment in order to draw meaningful conclusions (13). For example, the equivocal reports in the literature on the merits of neutralization of PMSG at or around the time of insemination could be related to the small number of animals per treatment used in several of the earlier studies. Accordingly, 2 experiments were designed in separate years, using large numbers of heifers per treatment, to determine if neutralization of PMSG in superovulated cattle would increase the number of transferable embryos recovered and decrease the number of large follicles present at the time of embryo recovery. Another factor that appears to affect the ovulation rate and embryo yield is the stage of the cycle at which gonadotropin is administered (12). The presence of a dominant follicle at the time of superovulation treatment reduces the ovulatory response in some studies (4,15), though not consistently (34). Therefore, an experiment was designed to determine if administration of PMSG early in the cycle, before the selection of the dominant follicle, would affect the superovulatory response obtained compared with that of treatment at mid- or late-luteal stages of the estrous cycle.

767

Thetiogenology MATERIALS AND MElHODS Animals and Treatments

Simmental and Charolais crossbred beef heifers, 18 to 24 mo old, were maintained in a slatted floor house with access to water ad libitum. They were fed a complete diet of silage, brewers grains and corn gluten, which was calculated to give a daily liveweight gain of at least 0.7 kg. The reproductive tracts of heifers were palpated per rectum prior to the study to confirm that they were normal and that heifers were cyclic. E&us (Day 0) was synchronized using 1 injection of 500 sg cloprostenol (prostaglandin F2a analogue {PG}, Estrumate, Coopers, Bray, Ireland). Experiments 1 and 2 Heifers were randomly assigned to 1 of 3 superovulatory treatments initiated between Days 9 and 14 of the estrous cycle: 1) a series of 8 im injections of porcine FSH (pFSH, Folltropin, Vetrepharm, London Canada; n=33 in Experiment 1 and n=26 in Experiment 2) administered twice daily for 4 d at 4.5,3.5, 2.5 and 1.5 mg per injection 12 h apart; 2) a single im injection of 2000 IU PMSG (Intervet,Boxmeer Holland; n=33 in Experiment 1 and n=22 in Experiment 2); and 3) a single im injection of 2000 IU PMSG and 2000 IU Neutra-PMSG (Intervet; n=33 in Experiment 1 and n=23 in Experiment 2) at the time of the first insemination. Luteolysis was induced in all heifers using 2 injections of PG, first at 48 h (500 rg) and then at 60 h (250 rg) after initiation of gonadotropin treatment. Observation for estrus commenced 24 h after the second PG injection, and the heifers were observed 4 times daily for 25 min at 0700. 1200, 1700 and 2100 h. All heifers were inseminated twice by the same technician using frozen-thawed semen from the same bull. The first insemination was at 8 to 12 h after the onset of the detected estrus and the second approximately 12 h later. Experiment 3 Estrus was synchronized in heifers (n = 116) using 2 injections of PG 11 d apart and were allocated to receive 2000 IU PMSG in a single injection on Day 2 (n= 28), Day 3 ( n = 27), Day 10 ( n = 41) or Day 16 (n = 20) of the estrous cycle. Heifers treated on Day 2 received 500 ug cloprostenol at 72 h and 250 pg at 84 h after PMSG. Heifers in all other groups received similar doses of PG at 48 and 60 h after PMSG, and heifers were inseminated twice. Embryo Recovery and Classification The heifers were slaughtered 7 d after estrus, and the reproductive tracts were recovered within 20 min. The tracts were then severed at the uterotubal junction, and each uterine horn was flushed with 20 ml of PBS containing 3% heat inactivated steer serum (12). The numbers of corpora lutea and large follicles (~10 mm) on each ovary were recorded. Embryos were recovered, counted, assessed for fertilization and graded on a scale 1 to 5 by the same operator, as described previously (3). Statistical Analysis Data on numbers of ovulations, large follicles and embryos (recovered, freezable, transferable and degenerated and unfertilized oocytes or ova) were analyzed using a t-test and a Wilcoxon rank sum test to compare means. Each variable was expressed as a proportion of the number of ovulations and of embryos recovered for the combined data and for each treatment sepamtely; significance was tested with a t-test (13).

Theriogenology RESULTS Estrous Response There was no difference (P > 0.1) in the estrous response (97%, 94% and 94%) or in the interval from PGF2a to estrus (49.2kl.3, 49.6A.7 and 48A1.7 h ) for heifers treated with pFSH, PMSG or PMSG-Neutra-PMSG respectively. Charian Response Experiments 1 and 2. There was no effect of treatment on the number of corpora lutea (CL) in Experiment 1 (Table 1); however, in Experiment 2, heifers given PMSG had a higher (P=O.OOl) ovulation rate. The significant difference in the ovulation rate in Experiment 2 was due to the high number of PMSG-treated heifers (10122) with 215 ovulations and the low number with 0 to 4 ovulations (1122) compared with heifers treated with Neutra-PMSG (2123 and 8123; P=O.Ol) or pFSH (3126 and 10126; FkO.011). The use of PMSG and Neutra-PMSG compared with PMSG alone did not significantly affect the number of large follicles obtained on Day 7 of the cycle after superovulation in Experiment 1; however it decreased (P=O.W04) the number of large follicles in Experiment 2 compared with that of PMSG-treated heifers but did not differ in the number of follicles compared with pFSH treatment. There were fewer (P
Theriogenology Table 1.

769

Ovulatory response and embryo production rates (mean&EM) for heifers superovulated with pFSH, PMSG or PMSG plus Neutra-PMSG at the midluteal phase of the estrous cycle in 2 experiments over 2 years in association with induced luteolysis following injection of a PGF2u analogue.

Treatment

pFSH

PMSG

Neutra-PMSG

Experiment 1 No. of heifers 33 corpora lutea 18.2*1.9 large follicles (r10 mm) 2.2iO.8a embryos recovered 11.3i1.4a freezable embryos (Grade I/ 2) 2.6iO.5 transferable embryos (Grade 3) 2.lrt0.3a degenerate embryos (Grade 415) 6.4*1.3 unfertilized ova 2.2il.lab

33 13.5zk1.9 4.6kO.Sb 7.2*1.4b 2.1kO.5 1.3ko.3ab 3.71t1.3 1.3*1.1a

33 16.8zt1.9 3.8*0.8ab 9.8*1.4ab 2.1*0.5 0.8*0.3b 6.8zk1.3 4.8*1. lb

Experiment 2 No. of heifers corpora lutea large follicles (210 mm) embryos recovered freezable embryos(Gmde I/ 2) transferable embryos (Grade 3) degenerated embryos (Grade 45) unfertilized ova

22 14.7rtl.Sb 5.%0.8b 9.4+o.9b 3.8ti.6 1.2*0.3a 2.2*0.4b 2.0*0.4b

23 8.7*1.5a 2.7k0.8a 4.7*0.9a 2.7a.7 0.4*0.3b 0.9*0.4a o.ai0.4a

26 7.5*1.4a 1. kO.7a 5.7ztzO.8a 2.4iO.6 1.2k0.3a 1.6+00.4ab 0.6*0.3a

P value

0.21 0.10 0.11 0.66 0.01 0.21 0.09

0.002 0.0004 0.001 0.25 0.04 0.08 0.01

a,bFigures within rows with different superscripts are different (PC 0.05 at least). Table 2.

Ovulatory response and embryo production rates (mean&EM) for heifers superovulated with 2000 IU PMSG given on Day 2,3, 10 or 16 in association with PG to induce estrus in Experiment 3. Day of cycle that PMSG was given

No. of heifers corpora lutea large follicles (rlOmm) embryos recovered freezable embryos transferable embryos degenerated embryos unfertilized ova

ab P
Day 2

Day 3

Day 10

Day 16

28 12.3*1.6a

27 5.7*1.5b 6.3il.2 2.1*0.7d 0.6*0.2d 0.3io. 1 l.lrtO.4 0.8*0.3

41 13.4*1.5a 5.2*0.9 6.4+0.9c

20 12.5*1.9a 5.3rt1.9 7.8*1.4c 1.8rt0.8c 1.9zto.7 1.8*0.4 2.5ti.8

3.6rt0.8 6.8*1.OC 2.8&.7c 0.4io.3 2.3kO.8 2.2kO.8

%%8” 1.8*0:5 0.9*0.3

770

Theriogenology

Exneriments 1 and 2. There was no effect (m.10) of treatment on the number of freezable embryos recovered in either experiment. The frequency distribution of the number of freezable embryos recovered from heifers assigned to the different treatments was significantly different (P=O.OS) in Experiment 1 but not in Experiment 2 (P=O.6). This was due to the high number (17) of pFSH-treated heifers with more than 3 freezable embryos compared with the PMSG- (10) or Neutra-PMSG- (11) treated heifers in Experiment 1. There were significant differences between treatments in the number of transferable embryos in Experiments 1 (P=O.Ol) and 2 (kO.04). The Neutra-PMSG heifers had the lowest number of transferable embryos in both years, and differed from the pFSH-treated heifers (P=O.O03) in Experiment 1 and the pFSH-treated (P=O.O4) and PMSG-treated heifers (P=O.O2) in Experiment 2. The frequency distribution of the number of transferable embryos recovered per treatment was significantly different in Experiment 1 (P=O.OOS)but not in Experiment 2 (P=O.24). The difference was due to the high number (24) of Neutra-PMSG heifers with no transferable embryos and the high number (12) of pFSH-treated heifers with ~53transferable embryos. The Neutra-PMSG-treated heifers had the highest number of unfertilized ova and differed significantly from PMSG-treated heifers (P=O.O4) in Experiment 1. The PMSG treated heifers had the highest number of unfertilized ova and differed significantly from the pFSH- (P=O.O06) and the Neutra-PMSG-treated heifers (P=O.O2) in Experiment 2. Exneriment 3. In this experiment, the number of freezable embryos recovered, but not the number of transferable embryos was affected by treatment. Heifers given PMSG on Day 3 had lower (PC 0.01) numbers of freezable embryos than heifers in the other treatments. More heifers (14120) treated on Day 16 produced 2 1 ova than those given PMSG on Day 2,3 or 10 (zz40% of these heifers produced single-cell ova). Overall, half (49%) of the treated heifers yielded no freezable embryos across all treatments, and there was a higher percentage of heifers in this category following treatment on Day 3 (67%) and Day 16 (70%) than on Day 2 (35%) or Day 10 (37%). DISCUSSION In view of the known variation that exists in superovulatory responses, adequate numbers of heifers (170 in Experiments 1 and 2 and 116 in Experiment 3) kept under similar management conditions and on a similar level of nutrition were used to obtain statistically reliable data. These heifers were subsequently slaughtered to achieve an accurate assessment of the ovulatory response and to eliminate the variabilities associated with data obtained by nonsurgical recovery of embryos. As has been reported in another study using Neutra-PMSG (8), the results of its use were not consistent between the 2 experiments on similar animals on the same feedlot over 2 yr. Consequently, meaningful interpretation of data is difficult, and caution is needed in drawing conclusions from many previous experiments in the literature where lower numbers of animals per treatment group were used. These experiments confirm the superiority of pFSH over PMSG as a superovulatory agent (13) and the failure of Net&a-PMSG to enhance the yield of Grade 1 to 3 embryos. The number of large follicles present 7 d after insemination was consistently lower following pFSH treatment; neutra-PMSG reduced the number of large follicles in Experiment 2 but not in Experiment 1. The explanation for the major differences in the ovulation rates of heifers assigned to the superovulatory treatments in Experiments 1 and 2 is not clear. Both the pFSH and Net&a-PMSG treatments had a higher proportion of heifers with lower ovulation rates than the PMSG regimen. Similar results were reported previously (13) where 20 to 30% of the animals had ovulation rates between 0 and 9 following similar PMSG or pFSH

771

Theriogenology

treatments . In addition, immunization of heifers against an 01 l-26 inhibin fragment conjugated to human serum albumin resulted in, at best, 50% of heifers responding with multiple ovulations (28). Thus it may be necessary to determine the innate sensitivity of each animal to exogenous gonadotropin so that different doses or treatment regimens may be used for optimal response from animals. Endogenous FSH concentrations and antral follicle numbers vary quite considerably between individual heifers in the same experimental treatment (29). further suggesting that individual responses to the same dose of an FSH-like hormone will vary. These results confirm that PMSG induces more large follicles than pFSH treatment and that the Neutra-PMSG treatment had an equivocal effect on reducing the number of large follicles. This is in agreement with other studies on the contrary effects of NeutraPMSG on the incidence of ovarian overstimulation (8,14,23,26,33). The difference in embryo recovery rates between experiments can be explained by the differences in respective ovulation rates. It has been shown (13) that a positive correlation exists between the number of ovulations and the number of embryos recovered. Neutra-PMSG did not increase embryo recovery, which is similar to the finding of one study (26) and in contrast to that of others (7,17). The Neutra-PMSG treatment in each experiment resulted in the highest number of heifers yielding no transferable embryos . This was reflected in the lowest mean number recovered in both years and differed from both the pFSH-treated heifers over the 2 yr and from the PMSG-heifers in Experiment 2. Thus, it may be deduced that the incorporation of Neutra-PMSG into a superovulatory regimen led to a deterioration rather than improvement in embryo quality (26,33) or had no effect (35), as previously reported. The proportion of unfertilized ova, in Experiment 2 was much higher than normally reported from our laboratory (13). The results from Experiment 3 indicate that it is possible to induce a high superovulatory response and yield of freezable embryos by treating heifers with PMSG on Day 2 of the cycle provided that PG is not given until Day 5, giving follicles a longer period of growth before ovulation. This is in contrast with our earlier data, in which pFSH was the gonadotropin used (12). However, in that earlier experiment (12) pFSH was omitted on Day 3, and this may have contributed to the lower ovulatory response and embryo yield recorded, due to an early onset of atresia of the initial FSH stimulated follicles. There was no difference in the response between heifers treated on Day 10 or 16, and it is anticipated that the heifers would have been superovulated during the period of atresia of the first and second dominant follicles. Recent data (4) indicate a severe negative effect of the dominant follicle on superovulation and embryo yield in cows. In conclusion, pFSH treatment resulted in fewer unovulated follicles than PMSG; Net&a-PMSG treatment in Experiment 1 decreased the numbers of unovulated follicles but had no effect in Experiment 2. Considerable variability existed not only between but also within years in terms of embryo yield and quality. This suggests that it is not only the treatment but also the fundamental physiological processes that regulate the number of follicles that develop and ovulate, which contributes to the variability in embryo production following superovulation. REFERENCES 1. Alfuraiji MM, Atkinson T, Broadbent PJ, Hutchinson JSM. Superovulation in cattle using PMSG followed by PMSG-monoclonal antibodies. Anim Reprod Sci 1993; 3399-109.

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