Superovulation of cattle with a recombinant-DNA bovine follicle stimulating hormone

Animal Reproduction Science, 33 ( 1993 ) 71-82 Elsevier Science Publishers B.V., Amsterdam

71

Superovulation of cattle with a recombinantD N A bovine follicle stimulating hormone J.M. Wilson*, A.L. Jones ~, K. Moore 2, C.R. Looney 3, K.R.

Bondioli 4

Granada BioSciences, Inc., Rt. I Box 201, Marquez, TX 77865, USA

ABSTRACT A recombinant DNA-derived bovine follicle stimulating hormone (rbFSH) was utilized as a superovulatory agent in mature bovine females. Several aspects of utilization of this product were tested; dose and duration of treatment, repeated usage and a supraoptimal dose. Also, the 'optimal' dose was tested by several different investigators. Doses from 0.5 to 12 mg twice daily (BID) were tested over a range of 3-5 days duration. All doses greater than 0.5 mg BID for 4 days produced more than two ova/embryos (defined as a superovulatory response). A dose of 24 mg given for 3 days was more efficacious than lower doses with equal duration, but larger doses and longer durations of treatment did not produce a greater response (P> 0.05 ). Moreover, when a supraoptimal dose was given, the response was not different (P> 0.05 ) from the 24 mg dose. Embryo production was not altered when individual cows were superovulated more than once with the selected dose nor was subsequent reproductive performance affected. The above studies also demonstrate that a recombinant DNA-derived glycoprotein hormone (i.e. pure follicle stimulatinghormone) can elicit a superovulatory response without exogenous luteinizing hormone (LH). The overall performance of rbFSH in these studies was similar to data previously reported for pituitary extracts.

INTRODUCTION

The major purpose of the administration of exogenous drugs with follicle stimulating hormone (FSH) activity is to induce a superovulatory response. Several compounds have been used. The two most widely used products are pregnant mare serum gonadotropin (PMSG) and follicle stimulating hormone-pituitary (FSH-P). The results obtained with these hormones have *Corresponding author: Reproductive Biology Laboratories, Shadyside Hospital, 5230 Centre Avenue, Pittsburgh, PA 15232, USA. ~Present address: Colorado Genetics, 7900 WCR 34, Platteville, CO 80651, USA. 2present address: Department of Anatomy, Texas A&M University, College Station, TX 77843, USA. 3Present address: TRANS-OVA, 2938 380th Street, Souix Centre, IA 51250, USA. 4present address: ABS, P.O. Box 459, DeForrest, WI 53532, USA.

© 1993 Elsevier Science Publishers B.V. All rights reserved 0378-4320/93/$06.00

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been variable. Refinements in superovulatory programs, to reduce the variability, increase predictability and embryo production, have had little effect. Superovulatory programs vary widely among practitioners in e.g. quantity of hormone, injection frequency, level or declining dose and the addition or deletion of other hormones. The hormonal differences, (i.e. FSH and luteinizing hormone (LH) activity) between superovulatory products have been indicated as reasons for differences in embryo quantity and quality in superovulated cows. Several studies have been conducted to identify factors which contribute to the variable results obtained with superovulatory products. Greve et al. (1984a,b) compared results from cows superovulated with both PMSG and FSH. In these studies the concentrations of LH and progesterone (P) were abnormal (i.e. LH, no surge, moderate, premature or delayed; P above 1 ng ml-~ at estrus or low, less than 1 ng ml-1, after superovulatory estrus). In another study, Yadav et al. (1986) found P concentration, prior to the LH surge, to be similar between PMSG-treated, FSH-treated and control animals. However, following the LH surge, P concentrations were greater in animals treated with PMSG than in those treated with FSH and both groups had concentrations greater than controls. The LH surge occurred earlier in the PMSG-treated cows and was higher in the FSH-treated cows. Various regimes have been utilized to reduce these problems. Kim et al. (1987) used polyclonal and monoclonal antibodies against PMSG following PMSG stimulation. In that study, PMSG: anti-PMSG treatment resulted in greater embryo production than did treatment with FSH-P. However, in a later study (Kim et al., 1988), FSH-P, a purified pituitary extract (Folltropin ®, Vetrepharm, Ont.) and a PMSG: bovine-PMSG antibody were compared. No differences were observed, for total or transferable embryos, between treatments. Several researchers (Alcivar et al., 1984; Murphy et al., 1984; McGowan et al., 1985 ) have compared FSH-P and human menopausal gonadotropin (hMG) and found no differences in embryo quantity or quality when these compounds were used for superovulation. Murphy et al. (1984) suggested that LH must be present in preparations used for superovulation with a ratio of approximately 5 : 1 (FSH: LH ). Addition of LH to commercial preparations of FSH-P reduced fertilization in those trials. Callesen et al. ( 1986, 1987) have shown that FSH-P and PMSG can cause premature ovulation, possibly as a result of LH contamination. Donaldson and Ward (1985) removed most of the LH from FSH-P preparations and increased the number of transferable embryos produced. Nine lots of FSH-P, used in a commercial embryo transfer program over a 6 year period, were analyzed for FSH and LH content by radioimmunoassay (Wilson, 1988 ). In that study, where FSH, LH and their ratio differed between lots, no differences were noted in the superovulatory response from more than 10 000 treatments. Mapletoft et al. ( 1988 ) compared Folltropin (a pituitary extract

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with 80% of LH removed) with FSH-P and found similar superovulatory resuits. However, Folltropin at supramaximal doses did not cause a reduction in the number or percentage of fertilized ova or transferable embryos. Superovulatory responses, using a recombinant DNA-derived bovine FSH (rbFSH), have given favorable results (Looney et al., 1988; Wilson et al., 1989 ). The rbFSH utilized in the studies presented here is the first recombinant hormone to be used in a domestic species for manipulation of the reproductive cycle. Additionally, rbFSH is the first cell line derived recombinant animal hormone, rather than a fermentation-derived bacterial product. The rbFSH is essentially an exact copy of naturally occurring hormone, free of contaminates, including LH. Therefore, rbFSH offers a unique tool for research and animal agriculture. MATERIAL

AND METHODS

A highly purified rbFSH preparation produced from a mammalian cell line co-transfected with C-DNAs encoding the alpha and beta sub-units of bovine FSH (bFSH) was used to induce superovulation in female bovines. Inference from superovulation studies utilizing a pituitary extract (FSH-P; Schering, Schering, Kenilworth, N J) was made to begin initial studies of dose and duration of treatment. Bovine FSH had elicited a biological response in the Steelman-Pohley in vivo ovarian weight assay and an immunological response, both parallel to an USDA FSH standard (Chappel et al., 1988 ). The milligram doses discussed in the present study cannot be compared with those of the bioassay, rather, these are milligram equivalents of radioimmunoactivity as compared with the USDA FSH standard. No direct comparisons with the pituitary extract (FSH-P) would be valid. Cows, adult, nulliparous and multiparous, lactating and non-lactating, used in these studies were randomly assigned to treatment groups. With the exception of clinical trials, where lactating dairy cows were included, animals were maintained in groups on pasture and given supplemental energy to maintain proper body condition. Animals were monitored twice daily by trained technicians, for estrus and health. During all studies each animal served as its own control, as cows normally ovulate only a single ovum. Following estrus and prior to treatment with rbFSH, cows were palpated per rectum to assure reproductive normalcy and the presence of a corpus luteum (CL) on at least one ovary. Some of the cows had been previously treated with other products. Also, some cows were treated more than once with rbFSH. Bovine FSH administration was initiated on Day 10 (estrus designated Day 0 ) of the estrous cycle. Cows were injected intramuscularly (i.m.) twice daily (at approximately 12 h intervals ). Prostaglandin a (PGF) (Lutalyse ®, 25 mg, Upjohn, Kalamazoo, MI) or the PGF analogue Estrumate ® (0.5 mg, Hayer, Shawnee, KS ) was administered concomitant with the fifth (3 and 4

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day regimen) or seventh (5 day regimen) injection of rbFSH. Estrus was expected approximately 48 h post PGF injection and cows were inseminated at 12 and 24 h following initiation of observed estrus or expected estrus. Nonsurgical embryo collections were performed at 6.5-7.5 days post-estrus. Embryos recovered were graded morphologically for quality and stage of development. The International Embryo Transfer Society (lETS) grading system was used (Grade 1, best quality; Grade 3, poorest quality). Breed, type, age, parity or previous embryo production were not considered in data analysis. Data were analyzed by analysis of variance with differences between means tested using Duncan's multiple range test. Specific study protocols are described below. Dose titration trials

One hundred and ninety-one, non-pregnant, non-lactating adult reproductively normal cows of various breeds were treated in two successive trials. Data from the two trials were pooled for analysis. Individual cows from a pool of available candidates were randomly assigned to treatment groups. Group and regimens are shown in Table 1. FieM trials

The 24 mg total dose (4 mg twice daily (BID) for 3 days) was selected as an optimal regimen. Embryo transfer practitioners were asked to use rbFSH to test the effectiveness of this regimen in a commercial clinical situation. TABLE1 Details o f dose and treatment duration trials No. cows

Total rbFSH dose admin. ( m g day - ~)

T r e a t m e n t duration ( days )

Total rbFSH dose admin. ( mg )

8 8 18 22 25 15 37 15 11 16 8 8

1.0 2.0 4.0 8.0 6.0 10.6 8.0 6.4 10.0 8.0 12.0 16.0

4 4 4 3 4 3 4 5 4 5 4 4

4 8 16 24 24 32 32 32 40 40 48 64

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Two hundred and three cows were selected and treated by these practitioners at various locations throughout the United States.

Stability of rbFSH This study was designed to test the effect of storage environment and the prostaglandin utilized on total ova and/or embryos recovered after superovulation with rbFSH (4 mg BID for 3 day regimen). Seventy-three adult, non-pregnant animals were used in a 3 × 2 factorial design. Cows were randomly assigned to one of six treatment groups to test: ( 1 ) the effect of different rbFSH storage environments; (2) luteolytic agent. Bovine FSH was stored and used as follows: ( 1 ) Group I: rbFSH was administered immediately after reconstitution. (2) Group II: rbFSH was stored refrigerated (approximately 4°C) after reconstitution for the duration of the injection period. (3) Group III: rbFSH was reconstituted, aliquoted into individual injection doses and frozen (approximately - 20 ° C ) until use. Each of the above groups were subdivided to receive either PGF or its analogue, cloprostenol.

Consecutive stimulation and breeding Each cow was subjected to three replicate treatments (superovulation) followed by artificial insemination for a natural calf, to test the effect of successive superovulations on subsequent fertility. To remain in the study each cow had to superovulate (i.e. produce more than two ova) at each stimulation. Thirty-four cows received the initial stimulation and 24 were stimulated three successive times. Normal stimulation intervals (approximately 60 days) were attempted. However, in some instances, with individual cows, this interval was extended. Immediately following each embryo collection, ovaries were observed via ultrasonography to quantify corpora lutea (CL) and follicular development. Following the third collection, PGF was administered and test animals were observed for estrous activity. As cows resumed normal estrous cyclicity (as determined by observed overt estrous activity or by palpation per rectum of ovarian structures) they were allowed one normal (approximately 21 day) estrous cycle prior to breeding by artificial insemination (AI). Pregnancy was determined by ultrasonography and palpation per rectum.

Supraoptimal dose of rbFSH To test the efficacy and safety of a supraoptimal dose on stimulation, embryo quality and subsequent reproductive performance, 18 adult, non-lactat-

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ing, multiparous cows were treated with 12 mg rbFSH BID for 4 consecutive days (96 mg total dose). This dose was four times greater than the dose selected in the dose titration trials. CL and follicular development were observed via ultrasonography following embryo collection. Post-collection, cows were treated with PGF for luteal regression and estrus induction. Following an observed estrus, cows were placed with bulls for rebreeding. Estrus was monitored twice daily until the conclusion of the study. Ultrasonography was used to monitor pregnancy status following breeding. RESULTS

Dose titration trials All doses greater than 0.5 mg BID for 4 days produced a mean superovulatory response of more than two ova (Table 2). However, a dose and duration of treatment effect was observed. A dose of 1 mg rbFSH BID for 4 days (8 mg total dose) produced less ( P < 0.05 ) total ova and viable embryos than higher doses tested. The total number of embryos/ova produced was greater ( P < 0.05) for the 24 mg total dose (either 3 mg BID for 4 days or 4 mg BID for 3 days). The 24 mg treatments were not different ( P > 0.05) in total embryos/ova produced than greater doses/duration. There were no differences between doses/durations with regard to the number of unfertilized ova TABLE2

The effect of dose and duration of treatment with rbFSH on the superovulatory response 1 ovum2

TRT ~ n

No. 53<4 1×4 2×4 3×4 4×4 5×4 6×4 8×4 4×3 5×3 3×5 4×5

8

1

8 6 18 17 25 25 37 35 11 10 8 7 8 8 22 20 15 13 15 14 16 15

Total

(£)

% 12.5 75.0 94.4 100 94.6 90.9 87.5 100 90.9 86.7 93.3 93.8

0.88 a 2.88 ab 6.83 b¢ 10.72 °a 1 3 . 0 0 ~d 12.82 ~a 11.63 ~a 12.25 ca 13.09 ca 11.40 ca 13.33 Cd 16.25 d

Viable embryos ()()

Degenerate embryos ( X )

Unfertilized ova ()()

No.

%

No.

No.

0.88 a 2.75 a 6.28 "b 9 . 2 4 b¢ 11.70 b¢ 10.55 b¢ 1 0 . 0 0 b¢ 10.38 b¢ 10.91 b¢ 10.07 b¢ 12.33 b¢ 12.81 c

100 95.5 91.9 86.2 90.0 82.3 86.0 84.7 83.3 88.3 92.5 78.8

0.13 a 0 . 3 3 ab 0 . 6 8 ab 0 . 8 9 ab 1.64 bc 1.13 ~b 1.25 ab 0.82 ab 0.87 ab 0.47 ab 2.69 c

%

0a

%

0 4.5 4.8 6.3 6.8 12.8 9.7 10.2 6.3 7.6 3.5 16.6

Milligram dose given twice daily × duration of treatment in days. 2Number of cows collected that produced more than one ovum. Means within columns with the same superscripts do not differ ( P > 0.05 ).

0 0.22 0.80 0.41 0.64 0.50 0.63 1.36 0.47 0.53 0.75

3.2 7.5 3.2 5.0 4.3 5.1 10.4 4.1 4.0 4.6

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(UFO); however, the number of degenerate embryos was higher ( P < 0.05 ) with the 4 mg BID 5 day regimen.

Field trials Two hundred and three cows, from 16 distinct breeds, were treated with rbFSH. One hundred and eighty-two (89.7%) produced at least one ovum. One hundred and seventy-two (84%) were superovulated (produced at least two ova). The mean ( _+SEM ) total numbers of ova, viable embryos, degenerate embryos and unfertilized ova were 10.51 + 0.58, 6.07 _+0.58, 1.72 +_0.20 and 2.74 + 0.34, respectively, for cows which superovulated (Table 3 ). There were differences ( P < 0.05 ) between cows, within investigator and between investigators, with regard to the numbers of total ova, viable embryos and degenerate embryos produced (individual data not shown). Neither geographical area nor season of the year had any effect ( P > 0.05 ).

Stability of rbFSH In this study, storage condition (rbFSH administered immediately following reconstitution or following refrigerated or frozen storage) and type of prostaglandin (PGF or an analogue) were tested. Sixty-seven (92%) of the 73 cows treated with rbFSH superovulated. There were no differences ( P > 0.05 ) observed between treatment groups for total ova, viable embryos, degenerate embryos or unfertilized ova (Table 4). However, there was a trend for greater productivity from cows treated with the PGF analogue.

Consecutive stimulations and rebreeding Thirty-one of the 34 cows treated with rbFSH during the first stimulation produced more than one ovum at collection. The mean total ova recovered TABLE 3 Embryo production ()( + SEM) in clinical trials using rbFSH for superovulation (4 mg rbFSH BID for 3 days, 24 mg total dose )

Cows treated ~ Cows >_ 12 Cows _>23

n

Total ova

Viable embryos

Degenerate embryos

Unfertilized ova

203 182 172

8.95 _+0.56 9.98__ 0.58 10.51_+0.58

5.16_+ 0.41 5.75 _+0.43 6.07_+0.58

1.46__ 0.17 1.63 _+0.19 1.72_+0.20

2.33 + 0.30 2.60 + 0.33 2.74_+0.34

tTotal number of cows treated. 2Cows producing at least one ovum/embryo. 3Cows producing two or more ova/embryos.

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TABLE 4 Total ova production (X + SEM) of cows treated with rbFSH (subjected to different storage conditions) and different prostaglandins Treatment I

n

Total

Viable embryos

Degenerate embryos

Unfertilized ova

NS 2

Group Group Group Group Group Group

12 12 12 12 13 12

9.17+ 1.31 10.50+ 1.92 8.33+1.52 11.33+1.95 6.83+ 1.64 10.17+2.15

8.25+ 1.25 7.83+ 1.16 6.75+1.34 10.75+1.96 5.77+ 1.70 7.42+1.62

0.42+0.19 2.50+0.74 0.83+0.27 0.33+0.14 0.31 +0.13 1.67+0.90

0.50+0.34 1.17+0.63 0.75+0.45 0.25+0.13 0.31 +0.13 1.08+0.79

0 1 1 0 2 2

I/A I/B II/A II/B III/A III/B

1Group I, test article administered immediately following reconstitution; Group II, test article reconstituted and stored at approximately 4 ° C during stimulation period; Group III, test article frozen (not less than 7 days) at approximately - 2 0 °C and thawed prior to use; A, prostaglandin treatment (Lutalyse ); B, prostaglandin treatment (Estrumate). 2Non-stimulators, total of less than two ova collected.

per collection was 14.52+ 1.40 ( X + S E M ) . The mean ( + S E M ) for viable embryos, unfertilized ova and degenerate embryos was 9.29 ___1.05, 1.77 + 0.65 and 3.45 +_.0.70, respectively. Twenty-seven of the 29 (two did not recycle post- collection ) cows remaining in this study, and treated with rbFSH during the second program produced more than one ovum. The mean ( + SEM) n u m b e r of days from the first to the second collection was 68.3 + 2.2. The mean total n u m b e r of ova recovered during the second stimulation was 12.15 + 1.44 (X + SEM). The mean ( _+SEM) numbers of viable embryos, unfertilized ova and degenerate embryos were 8.30 + 1.20, 0.85 + 0.29 and 3.0 + 0.95, respectively. Twenty-four of the 25 (two did not recycle post-collection) cows remaining following two consecutive stimulations and treated with rbFSH during the third program were successfully stimulated. The mean ( + SEM) number of days from the second to the third collection was 64.2 + 2.4 days. The mean total n u m b e r of ova collected during the third stimulation was 11.13 _+ 1.02 (X + SEM). The mean ( + SEM) numbers of viable embryos, degenerate embryos and unfertilized ova recovered were 7.5_+1.08, 2.54_+0.72 and 1.08 _+0.44, respectively (Table 5 ). Data from ultrasound observations of ovaries of superovulated cows immediately following embryo collections are shown in Table 6. These data show that total CL are almost identical to the total n u m b e r of ova recovered and that few follicles remain. Seventeen of the 24 (70.8%) cows which completed the three stimulation and collection programs conceived within three AI attempts. Ten cows conceived at the first AI breeding. The interval from the last collection to conception at first breeding was 48.2 ___6.1 days (.~ + SEM). Six cows conceived at

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TABLE 5 Embryo/ova (X -+ SEM) production during three stimulations (at approximately 60 day intervals) with rbFSH Stimulation No.

N~

Total ova

Viable embryos

Degenerate embryos

Unfertilized ova

First Second Third

31/34 27/29 24/25

14.52_+ 1.40 12.15_+ 1.44 11.13_+ 1.02

9.29_+ 1.05 8.30_+ 1.20 7.5+ 1.08

3.45_+0.70 3.0 _ + 0 . 9 5 2.54_+0.72

1.77_+0.65 0.85_+0.29 1.08-+0.44

1Number of cows successfully superovulated/number of cows treated. TABLE 6 Ovarian structures ()? + SEM) observed via ultrasonography during three stimulations with rbFSH Stimulation No.

Right ovary

First Second Third

Left ovary

CL

Follicles ( > 10mm)

CL

Follicles ( > 10mm)

8.61 +0.45 7.59 + 0.54 8.00 -+0.4

2.94+0.80 3.67 _+0.83 1.78 + 0.47

7.71 +0.45 6.85 _+0.45 7.30 _+0.60

3.03_+0.65 3.11 __%0.86 2.35 _+0.49

TABLE 7 Pregnancy rates following artificial inhsemination subsequent to three rbFSH stimulations Treatment

No.

Cows pregnant

Cows pregnant after first service

No.

%

No.

%

17 840

70.8 71.2

10 558

41.7 47.3

COWS

bFSH AI

241 11802

Services per pregnancy 1.47 1.70

~Animals superovulated three times with rbFSH. 2Data taken from 1986 AI season for the Granada purebred herd.

the second AI breeding. The average interval from the last collection to conception at the second breeding was 68.5 _+8.3 days. One cow conceived at the third AI breeding attempt which was 108 days from the last embryo collection date. These pregnancy rates are similar to those observed during the 1986 AI season for the Granada purebred herd from which the test animals for this study were obtained (Table 7 ). Of the seven cows determined not pregnant, five were bred by AI three times (maximum allowed by protocol) and two were bred twice. No estrous activity was observed for these two animals following PGF administration, although luteal tissue and follicles were observed.

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TABLE 8 Number ( X + SEM) of corpora lutea and follicles (over 10 m m in diameter) at embryo collection for cows given a supraoptimal ( 12 mg rbFSH BID for 4 days) dose of rbFSH Structures

Right ovary

Left ovary

Corpora lutea Follicles

7.5 + 0.72 4.8 _+0.98

7.6 + 0.45 5.0 + 0.89

Supraoptimal dose The mean n u m b e r of e m b r y o s / o v a recovered per collection from cows treated with a supraoptimal dose was 12.0+ 1.86 (.~ + S E M ) . The mean ( + SEM) numbers of viable embryos, degenerate embryos and unfertilized ova collected per cow were 8.56+1.07, 1.89+0.53, and 1.56+0.66, respectively. Ultrasonography data at the time of embryo collection are shown in Table 8. The n u m b e r of CL and follicles (approximately 10 m m or larger) were counted and recorded for each ovary. However, when ten or more CL were observed on an ovary only ten were recorded because of the difficulty of accurately distinguishing between more than ten CL. Cows were treated with P G F post-collection and observed for estrous activity. After estrus was observed cows were placed with bulls for rebreeding. Seventeen of 18 cows became pregnant post-collection. One cow was removed from the study because of chronic lameness. The mean ( _+SEM) n u m b e r of days from embryo collection to approximate breeding date (natural service) was 31.9 + 2.10 days (n = 17 ). The mean number of estrous cycles per conception for all cows determined to be pregnant was 1.24+0.10. DISCUSSION

These data demonstrate that a recominant DNA-derived bovine follicle stimulating hormone will induce superovulation in the cow. Dose titration studies indicate that a wide range of doses and durations of treatment are equally effective. These data also show that a threshold was reached (i.e. increases in dose a n d / o r duration did not increase the superovulatory response). As the dose and duration increased, the n u m b e r of embryos recovered or the n u m b e r of CL observed via ultrasonography did not increase correspondingly. Subsequent reproductive performance, as determined by rebreeding performance following multiple stimulation or stimulation with a supraoptimal dose, was not affected. Utilization of a prostaglandin analogue had no effect

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on embryo production. Although there was a trend toward an increased total number of ova/embryos using the analogue, this trend was not apparent in the number of viable embryos. Embryo production with rbFSH appears to be comparable with data in the literature for other superovulatory compounds. However, embryo quality, as determined by the percentage of embryos graded 1, 2 or 3 appeared to be increased using rbFSH when compared with data from superovulation with FSH-P (Wilson, 1988 ). Also, in contrast to previously published reports, exogenous LH was not necessary for follicular maturation or ovulation. The effect of adding LH to this FSH preparation has not been studied. CONCLUSION

Overall, these data demonstrate that this recombinant DNA-derived gonadotropin has biological activity similar to the biological activity of the product extracted from pituitaries. Moreover, rbFSH will induce successful superovulation without the inclusion of other gonadotropins. These data also show that several dose and duration regimens will yield similar results. However, there was no advantage to extending the duration of treatment beyond 3 days. The availability of pure gonadotropins should provide researchers with the tools necessary to begin to understand better follicular development, maturation, ovulation and superovulation.

REFERENCES Alcivar, A.A., Maurer, R.R. and Anderson, L.L., 1984. Superovulatory responses in FSH- or Pergonal®-treated heifers. Theriogenology, 22: 635-642. Callesen, H., Greve, T. and Hyttel, P., 1986. Preovulatory endocrinology and oocyte maturation in superovulated cattle. Theriogenology, 25:71-86. Callesen, H., Greve, T. and Hyttel, P., 1987. Premature ovulations in superovulated cattle. Thereiogenology, 28:155-166. Chappel, S., Looney, C.R. and Bondioli, K.R., 1988. Bovine FSH produced by recombinant DNA technology. Theriogenology, 29: 235. Donaldson, L.E. and Ward, D.N., 1985. Superovulation in cattle: Dose response to FSH-W with and without LH contamination. Theriogenology, 23: 189. Greve, T., Callesen, H. and Hyttel, P., 1984a. A characterization of plasma LH-profiles in superovulated dairy cows. Theriogenology, 21: 237. Greve, T., Callesen, H. and Hyttel, P., 1984b. Plasma progesterone profiles and embryo quality in superovulated dairy cows. Theriogenology, 21: 238. Kim, H.N., Rorie, R.W., Young, C.R., White, K.L. and Godke, R.A., 1987. The use of antiPMSG antibodies with PMSG for superovulating beef cattle. Theriogenology, 27: 243. Kim, H.N., Roussell, J.D., Pool, S.H. and Godke, R.A., 1988. The effect of a commerciallyavailable purified FSH and bovine anti-PMSG serum on the superovulation of dairy heifers. Theriogenology, 29: 267.

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Looney, C.R., Bondioli, K.R., Hill, K.G. and Massey, J.M., 1988. Superovulation of donor cows with bovine follicle stimulating hormone (bFSH) produced by recombinant DNA technology. Theriogenology, 29:271. Mapletoft, R.J., Gonzalez, A., Lussier, J.G., Murphy, B.D. and Carruthus, T.D., 1988. Superovulation of beef heifers with Folltropin and FSH-P. Theriogenology, 29: 274. McGowan, M.R., Braithwaite, M., Jochle, W. and Mapletoft, R.J., 1985. Superovulation of beef heifer with pergonal (HMG): A dose response trial. Theriogenology, 24:173-184. Murphy, D.B., Mapletoft, R.J., Manns, J. and Humphrey, W.D., 1984. Variability in gonadotropin preparations as a factor in the superovulatory response. Theriogenology, 21: 17-125. Wilson, J.M., 1988. Superovulation: FSH update. Proc. 7th Annual Meeting of the American Embryo Transfer Association, Reno, NV, pp. 31-43. Wilson, J.M., Moore, K., Jones, A.L. and Looney, C.R., 1989. Recombinant bovine follicle stimulating hormone: Dose and duration regimens for superovulation of embryo donors. Theriogenoiogy, 31: 273. Yadav, M.C., Walton, J.S. and Leslie, K.E., 1986. Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotropins. Theriogenology, 26: 523-540.