Superovulation of dairy cows with purified FSH supplemented with defined amounts of LH

THERIOGENOLOGY SUPEROVUtATlON OF DAIRY COWS WITH PURIFIED FSH SUPPLEMENTED WITH DEFINED AMOUNTS OF LH A. Herrler,’

F. Elsaesser,

N. Parvizi

and H. Niemann2

lnstitut fur Tierzucht und Tierverhalten 3057 Neustadt 1, Mariensee, Federal Republic Received

for publication:

August

6,

Accepted: November

(FAL) of Germany

1990 20, 1990

ABSTRACT This study investigated the effects of a purified follicle stimulating hormone (FSH) preparation supplemented with three different amounts of bovine luteinizing hormone (bLH) and a commercially available FSH with a high LH contamination on superovulatory response, plasma LH and milk progesterone levels in dairy cows. A total of 112 lactating Holstein-Friesian crossbred dairy cows were used for these experiments; the cows were randomly assigned to treatment groups consisting of purified porcine FSH (pFSH) supplemented with bLH. Group 1 was given 0.052 IU LH140 mg armour units (AU) FSH (n = 6); Group 2 was given 0.069 IU LH (n = 32); Group 3 received 0.423 IU LH (n = 34); while Group 4 cows (n = 36) were superovulated with a commercially available FSH-P@. This compound appeared to contain 8.5 IU LH/40 mg AU FSH according to bioassay measurement. All animals received a total of 40 mg AU FSH at a constant dose twice daily over a 4-d period. Levels of milk progesterone and plasma LH were determined during the course of superovulatory treatment. The Group 1 treatment did not reveal multiple follicular growth, and no embryos were obtained. Superovulation of Group 3 cows resulted in significantly (PcO.05) more corpora lutea (CL; 12.6k1.1) and fertilized ova (5.1r1.3) compared with Groups 2 and 4 (10.1+0.9 and 2.6kO.6, 9.OkO.9 and 2.7tO.5, respectively). Due to a high percentage of degenerated embryos (33%) Group 3 yielded only one more transferable embryo than Groups 2 and 4. Among groups, LH levels differed in the period prior to induction of luteolysis and were similar thereafter. The progesterone pattern following FSH/LH administration reflected the amount of LH supplementation. Milk progesterone levels on the day prior to embryo collection were correlated to the number of CLs and recovered embryos. It is concluded that under the conditions of our experiment superovulation with 0.423 IU LH/40 mg AU FSH may yield a significantly improved superovulatory response in dairy cows. It is further suggested that LH supplementation exerts its effects mainly on follicular and oocyte maturation during the period prior to luteolysis. Key words:

superovulation,

dairy cows,

embryo

yield,

FSH, LH,

progesterone

A portion of this study has been presented as an abstract at the XIV Annual meeting of \he Embryo Transfer Society in Fort Collins, CO, USA in January 1988. Present address: Lehrstuhl f. Anatomie u. Reproduktionsbiologie, Klinikum der RWTH, 5100 Aachen, FRG. 2Correspondence and reprint requests.

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THERIOGENOLOGY

INTRODUCTION The primary factor limiting embryo production in dairy cattle is the variability of the ovarian response following induction of superovulation with commercially available FSH preparations. One causative factor for this could be highly variable FSH/LH ratios, ranging from 4.4 : 1 to 0.2 : 1 in the available superovulatory drugs(l-3). This prevents systematic investigation for improving the superovulatory response, since it adds further variability to an already highly variable system. Several investigators have demonstrated that FSH preparations with a high LH content resulted in poor ovarian responses (2, 4, 5). It was suggested that high amounts of LH are harmful to folliculogenesis and lead to premature ovulation (6, 7), disturbances in cytoplasmatic and/or nucleic maturation of the oocytes (6, 8, 9), luteinization of granulosa cells (10, 11) and a deviating LH peak (12-14). On the other hand, it has also been shown that a minimal LH amount in the FSH preparation is required to obtain multiple ovulations. Highly purified FSH preparations yielded significantly lower ovulation rates than LH-supplemented FSH preparations (4, 5, 15). Recent studies in the rat have shown that purified FSH administered in high dosages induced high ovarian responses (16). Infusion of subthreshold FSH dosages supplemented with LH led to similar ovulation rates. Increasing the LH content decreased ovulation rates (10, 16), which confirms the findings obtained in the bovine (5, 15). The purpose of our present study was to evaluate the superovulatory response of dairy cows following treatment with purified FSH preparations supplemented with different amounts of LH and to compare it to a commercially available FSH in which a high LH contamination had been determined. In addition, plasma LH and milk progesterone (P4) levels were measured during the course of the respective treatments.

MATERIALS AND METHODS A total of 112 Holstein-Friesian crossbred dairy cows from the lnstitut fur Tierzucht und Tierverhalten experimental herds were used for these experiments. The cows were 4.8kO.2 (x*SEM) years of age and yielded an average of 25+0.7 kg/d milk. The animals were superovulated 6 to 12 wk post partum. The gonadotropin treatment started in midcycle (9 to 13 d post estrus, estrus = Day 0). Prior to treatment, the cows were checked for the presence of a functional midcycle CL by palpation per rectum. Superovulation was induced with a purified FSH preparation supplemented with different amounts of LH: purified pFSH, 40 mg AU supplemented with 0.052 IU bLH (Group 1, n = 6), 0.069 IU bLH (Group 2, n = 32), or 0.423 IU bLH ( Group 3, n = 36). These preparations were obtained from Drs. J. F. Beckers and J. Viviers IRSIA Research unit, 1070, Brussels, Donnay (Faculte de Medicine Veterinare, Belgium). The effects of superovulation with purified FSH preparations were compared with those obtained following treatment with a commercially available FSH-P@ (Burns Biotec, Omaha, NE 68103, USA, batch No. 534M85; Group 4, n = 34). This preparation was highly contaminated with LH (40 mg AU containing 8.5 IU LH). The biological LH-activity in all preparations was determined by Prof. H. 0. Hoppen

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(Veterinary University, Hannover, to van Damme et al. (17).

FRG)

using

a modified

in vitro

bioassay

according

All cows received a total of 40 AU mg FSH at 5mg AU FSH eve 12 h over a 41 d period. A prostaglandin analogue (650 pg Cloprostenol, Estrumate , ICI, UK) was administered on Day 3 of the FSH treatment. All cows were inseminated 12 and 24 h after the last FSH injection. Seven days later, the ovaries were examined for the number of CLs and follicles by palpation per rectum by an experienced veterinarian, and the embryos were recovered nonsurgically on the same day. To determine LH levels during the course of superovulation treatment, a silicon catheter was inserted into the Vena jugularis externa of 16 cows 12 h prior to the first FSH injection. In addition to the four animals from Groups 2, 3 and 4, blood samples were also collected from four additional cows which received an equivalent amount of physiological NaCl solution (sham treatment). The first blood sample was collected immediately prior to the first FSH injection, and sampling was maintained for 96 h (i.e., 12 h after the last FSH injection). Blood samples were collected from two of four animals per group every 20 min over the first 12 h, while the remaining two animals were sampled every 3 h. This sampling schedule was changed every 12 h. Blood samples were stored in heparinized tubes at 7°C for a maximum of 2 h before being centrifuged at 1000 g for 10 min. The plasma was stored at -18°C. Bovine LH was determined by RIA according to Pomerantz et al. (18) and Ponzilius et al. (19), with minor modifications. Purified bLH and a specific antibody to LH were purchased from UCB (Brussels, Belgium). The lowest level of LH detection was 0.2 nglml plasma. The intra- and inter-assay coefficients of variation were 4 and 7.5%, respectively. The crossreactions with bFSH, bTSH and pLH were ~0.1, 0.2 and 30%, respectively. Data were pooled over 6 h from animals of each group. In addition, milk samples were collected from Groups 2, 3 and 4 every 24 h (whole evening milk) from the day prior to the first gonadotropin injection until embryo collection. After addition of 20 ul 7.5% thiomersal solution milk samples containing 5 ml were frozen and stored at -18°C for a maximum of 3 mo. Milk progesterone levels were determined by an enzymeimmuno assay (EIA) according to Van de Wiel and Koops (20). The lowest level of progesterone detection in this dilution The intraand inter-assay coefficients of (1:200) was 1 ng/ml progesterone. variation were 12.2 and 21.4%, respectively. The crossreactions with 17,hydroxyprogesterone, estradiol and cortisol were 0.005, co.002 and O.l%, respectively.

t-test.

Data were evaluated by analysis Data are expressed as x+SEM.

of variance

and were tested

for significance

by

RESULTS Gonadotropin

Treatments

Superovulation was terminated after

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With Defined

FSH/LH

Preparations

with purified FSH supplemented with 0.052 IU LH (Group 1) six animals had been treated, since it did not result in multiple

35 NO. 3

THERIOGENOLOGY Table 1. Ovarian response and embryo yield following gonadotropin different FSH/LH ratios Group n (I.U. LH)

No. of CLS

treatment with

No. of follicles

No. of ova + embryos

No. of fertilized ova

No. of unfertilized ova

No. of transferable embryos

2 (0.069)

32 10.la LO.9

1.4a LO.3

5.5 LO.9

2.6a iO.6

3.0 ~0.8

2.2 LO.5

3 (0.423)

36 1 2.6aVb 2.3a +l . 1 LO.4

8.4a +1.5

5.1 a,b 51.3

3.4 LO.7

3.4 ~0.6

4 (8.5)

34 9.0b LO.9

4.8a LO.9

2.7’ LO.5

2.1 LO.7

2.2 LO.5

1 .6 LO.4

Results in the same column with the same superscript are different: PcO.05

follicular growth (0 to 4 CL, 0 to 2 follicles, only one unfertilized ovum). Treatment with FSH containing 0.423 IU LH per 40 mg AU (Group 3) yielded significantly more CLs and fertilized ova (PcO.05) compared with Group 2 (Tablel). However, the percentage of degenerated and retarded embryos was considerably higher in Group 3 than in Group 2 (33 and 13%, respectively), and resulted in only one additional transferable embryo in this group (3.450.6 versus 2.2kO.5, respectively). The LH levels in the various treatment groups differed in pattern prior to PGF20 injection (Figure l), and they were similar thereafter (Figure 2). The LH levels in Group 2 fluctuated around the levels of the sham-treated cows in a 12-h rhythm. In Group 3, LH levels were elevated for the first 24 h compared with the sham treated group. Progesterone levels remained constant in Group 2 until the PGF2, injection, while they increased significantly in Group 3 (Figure 3). Cows (two from each of the treatment groups) with elevated progesterone levels (~2 ng/ml milk) at the time of estrus yielded an abnormally high number of unfertilized ova (73%). Progesterone levels steadily increased after artificial insemination and were significantly different between Groups 2 and 3 12 h prior to embryo collection (38.3k3.9 ng/ml and 51.0f5.9 nglml, respectively; PcO.05). Progesterone levels on Day 6 were correlated with the number of CLs (r=0.57; n=66; P-zO.01) and the number of recovered ova and embryos (r=0.36; n=66; PcO.01).

Superovulation

With a Commercially

Available

FSH Preparation

In Group 4 the mean number of CLs, ova and embryos and fertilized ova was significantly lower than in Group 3 (PqO.05) and was similar to that of Group 2 (Table 1). The LH levels were higher in Group 4 than in Group 3 (Figure 1). As with Groups 2

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FSH

1.l

FSH

FSH

FSH

PGF2n FSH

I

I

I

I

I

Time

PGF2a FSH

FSH

FSH

FSH

FSH

(hours)

1 .l 1 “E 1 0.9

-z

-is ~ 0.8 5 9 0.7 z 0.6 0.5

6

0

Figure

1.

18

24

Time

(hours)

30

36

42

48

Mean LH levels prior to induction of luteolysis during gonadotropin treatment with different FSHlLH ratios in dairy cattle. Group 4 received a pLH contaminated FSH preparation which showed a 30% crossreaction with bLH in RIA (see Materials and Methods). For reasons of clarity, SEM are not shown. (x

MARCH

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Group

2,

l

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Group

3,oGroup

4,O sham).

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THERIOGENOLOGY

PGF2a FSH

I

FSH

FSH

FSH

I

I

I

3 2.75 2.5 1 2.25 z $2 E 2 1.75

0.5 1

I

I

I

I

I

I

t

48

54

60

66

72

78

84

90

Time

PGF2a FSH

FSH

I

3

(hours)

FSH

I

FSH

I

I

-

2.75. 2.5 .

0.5

48

I

I

I

I

I

I

54

60

66

72

78

84

Time

Figure

(hours)

2. Mean LH levels after induction of luteolysis during gonadotropin treatment with different FSH/LH ratios in dairy cattle. Group 4 received a pLH contaminated FSH preparation which showed a 30% crossreaction with bLH in RIA (see Materials and Methods). For reasons of clarity, SEM are not shown. (x

638

’

Group 2,aGroup 3,OGroup 4,0shem).

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THERIOGENOLOGY

FSH

PGF2a

4

4

Embryo recovery

A I JJ.

6

55 504540. 35. z

30.

2 z

25. 20. 15105.

-4

-2

2

0

4

6

8

Days Figure

3. Milk progesterone (x

Group

2,

levels

following

A Group 3,0

superovulation

Group

with

3

4)

and 3, LH levels of Group 4 were different prior to luteolysis (Figure l), and were similar to those of the controls thereafter (Figure 2). In Group 4, progesterone levels increased during the first 24 h after the initial FSH/LH injection (Figure 3) and were significantly higher than in Groups 2 and 3 (A=5.5 ng/ml progesterone, A=0 and A=l.& respectively; PcO.05). Subsequently, progesterone reached the same levels as in Groups 2 and 3. Progesterone levels on Day 6 were correlated with the number of CLs (r=0.40; n=32; P
DISCUSSION Results of our experiments characterize, for the first time, hormonal parameters, ovarian response and embryo yields in dairy cows following superovulatory treatment using gonadotropin preparations with defined FSH/LH ratios. These results emphasize the significance of a proper FSH/LH ratio for achieving improved ovarian response in dairy cows. Similar patterns of ovarian response

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following treatment of cows with FSH preparations containing comparable FSH/LH ratios were obtained by other investigators (4, 5, 15). Although the superovulatory results following treatment with 0.423 IU LH were considerably better than previous ones in our herd (21). the number of embryos recovered was still lower than that of other studies (4, 5). This might have been due to factors such as breed, environment and/or to the decreasing dosages of FSH administration in those studies, which had previously been shown to be more effective than using one constant FSH dosage, as we did (22). Since the variability of ovarian response could not be reduced in our study, the effect of the individual donor and the surrounding environment needs further study. Determination of LH levels revealed some insights into the effects of treatments with gonadotropin preparations containing defined FSHlLH ratios and did not reflect LH contaminations of the respective FSH preparations. Cows in the various treatment groups exhibited deviations in the LH pattern during the period prior to induction of luteolysis, when follicles are recruited and undergo final growth and maturation. The LH level for the first 24 h of treatment in Group 3 was similar to that of the sham treated group during the first 12 h after induction of luteolysis. In Group 4, LH levels for the first 18 h of treatment were similar to levels of the sham-treated cows 12 to 24 h after induction of luteolysis. Speculatively, these deviations may have interfered with follicular development and oocyte maturation (6, 23, 24). In particular, the high levels of LH could have resulted in a luteinization of follicles (7, li), a desensitization of the LH receptors (25) or premature ovulations (7, 26, 11). Low LH levels could have caused deviating patterns in oocyte cytoplasma and/or nucleus maturation (23). All these deviations may have led to the release of nonfertilizable oocytes or to early embryonic degeneration, which could explain the high percentage of unfertilized ova and degenerated embryos. Since major differences in the LH levels were mainly found prior to the induction of luteolysis, it is suggested that this is the period in which LH mainly exerts a beneficial effect on follicular and oocyte maturation. After induction of luteolysis exogenous LH may become less important, as endogenous LH levels increase. Milk progesterone levels prior to artificial insemination increased following FSH/LH application and reflected the different LH contents of the respective gonadotropin preparations, thus reaching the highest levels in animals treated with the most LH contaminated gonadotropin preparation. Following Al, progesterone levels steadily increased and reflected the number of CLs on Day 6 of treatment. The significant correlation found between progesterone levels and the number of CLs or recovered ova and embryos allows progesterone levels to be used to some extent for the prediction of superovulatory responses (27, 28), as we have shown for progesterone levels prior to gonadotropin treatment (29). The use of gonadotropin preparations with defined FSHlLH ratios can eliminate one important source of variability. Application of FSH containing 0.423 IU LH/40 mg AU resulted in slightly increased LH levels and in significantly more CLs, ova and embryos, and fertilized ova. As recombinant FSH becomes available (30), optimal FSH/LH ratios can be composed according to the ovarian status of each donor cow. This may improve embryo yields under commercial embryo conditions as well as facilitate further basic investigations to study the mechanisms of ovarian variability following superovulatory treatment.

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