Superovulatory responses of cattle

THERIOGENOLOGY

SUPEROVULATORY RESPONSES OF CATTLE

Danielle MONNIAUX, D. CHUPIN and J. SAUMANDE I.N.R.A. Station de Physiologie de la Reproduction 37380 Nouzilly FRANCE

ABSTRACT

Non-histological examination of superovulated ovaries of cows does not allow one to distinguish between corpora lutea and luteinized follicles. A better estimation of ovulation rate could, therefore, be made from the number of embryos recovered or from the levels of E2-17/3 in the plasma 60 hours after PMSG. For comparison of different treatments, it is necessary to characterize activities of the stimulatory agents used. Administration of anFSH - LH preparation twice a day at decreasing doses gives the best mean responses, but no treatment has been found which can clearly decrease the large variation between individuals in their responses. Numerical, kinetic and endocrine ovarian factors can partly explain the variability of ovarian responses to PMSG in the heifer. Individual differences in follicular populations at the time of treatment, or in E -17/3levels after stimulation, could be related to differences in resionses in ovulation rate. Normal follicles >1.7 mm diameter before treatment would usually ovulate following PMSG injection, whereas early atretic follicles of the same size mostly luteinize.

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1983 VOL. 19 NO. 1

THERIOGENOLOGY

INTRODUCTION

Despite many improvements in each step of the .methodology of embryo production and transfer, the main drawback is still the impossibility to be sure that a potential donor will provide a suitable number of good embryos at a given time. The major component of this problem is the large variation between individuals in their ovulatory response to treatments. As suggested by previous investigations (1, 2, 3, 4), individual variation in ovulatory responses could be attributed mainly to the choice of superovulatory treatments applied to the animals and the different responsiveness of animals to a given treatment. According to SAKMANDE et al. (5), ovarian responsiveness seems to be related to ovarian status at the time of treatment. Moreover, part of the variability is also due to the method of estimation of ovarian response (6); therefore we need to have a true determination of ovulation yield or at least to know the accuracy and limits of techniques of estimation before drawing any conclusion on the effects of a particular treatment. Thus in this paper we will focus our attention on three main points : i

: determination and estimation of ovarian response to

ii

: improvement of superovulatory treatments in order to

treatment, recover more viable embryos, iii : importance of the ovary in the variability of responsiveness to stimulation.

I -

DETERMINATION AND ESTIMATION OF OVARIAN RESPONSE TO TREATMENT

Two types of estimators can be used to measure ovarian response: the number of luteal or follicular structures observed on the superovulated ovaries and the levels of a hormone, measured in plasma or milk before, during or after treatment, which are known to be well correlated with the number of ovulations. The first type of estimator is currently measured by rectal palpation, endoscopy or direct observation of ovaries. The second is very simple and convenient, before as well as after ovulation. Studies of progesterone in milk before stimulation (7), E2-17p in plasma between stimulation and ovulation (8, 9, 10) and progesterone in plasma after ovulation (10) have yielded promising results. Estimation of ovarian response by counting luteal and follicular structures Generally, people working on superovulation estimate ovarian response by counting by rectal palpation the number of corpora lutea and large unovulated follicles on days 7 or 8 of the cycle. Less frequently, endoscopy or direct observation of ovaries has been used.

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TI-IERIOGENOLOGY

- The first question we asked was whether rectal palpation, endoscopy or direct observation are suitable methods for counting the number of corpora lutea. A comparison of the three techniques (11, 6) showed large discrepancies in the results, especially those obtained by rectal palpation. Moreover, the precision of the estimation by rectal palpation decreases when ovulation rate increases and is totally inadequate when more than 10 corpora lutea are present on the ovaries. However when less than 10 corpora lutea are present, 76% of the estimations by rectal palpation fall in the right class of ovulatory responses (Table 1).

Table 1: Comparison of the estimation of ovulation observation of ovaries and by rectal palpation. Estimation by direct observation Classes

of ovulatior

of

Comparison

to

estimation

rate by direct

by rectal

palpation

ovaries

No.

animals per class

No. animals with exact coincidence

No. animals ir:which the 2 estimations

between the timatior,s

fall class

2 es-

icto

the

o-4

5

2

4

5-10

12

2

9

13

0

8

>lO

same

- The second question we asked was which "luteal structure "has or has not effectively ovulated 7 or 8 days after oestrus. After dissection, many of the luteal structures had the appearance of pseudo-corpora lutea, the stigma of ovulation had often disappeared and a cavity was sometimes present (photo 1). It was often impossible to distinguish the true corpora lutea from the luteinized follicles. Finally, differences between the 2 types of sructures were defined histologically in ovaries of 10 stimulated heifers slaughtered 48 hours after preovulatory discharge of LH. Luteal structures were then very young and could be easily identified. In a young corpus luteum (photo 2) the granulosa had been disrupted and antral fluid was partly ejected but in some cases the oocyte was not ejected. Luteinized follicles (photo 3) showed an oocyte enclosed with cumulus cells dispersed and first signs of luteinisation in the granulosa (photo 4), similar to the granulosa of a young corpus luteum.

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THERIOGENOLOGY

Photo 1 : LUTEAL STRUCTURES ON SUPEROVULATED OVARIES OF A FRIESIAN COW AT D_

Photos 2-3-4 : LUTEAL STRUCTURES ON SUPEROVULATED OVARIES OF FRIESIAN HEIFERS 48 HOURS AFTER.LH PEAK 2 : Corpus luteum 3 : Luteinized follicle 4 : Wall of a luteinized follicle

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THERIOGENOLOGY

Table 2 gives results of the numbers of CL counted 48 hours after preovulatory discharge of LH by direct observation and histology in the 10 heifers studied. The relationship between the 2 techiques is very close (rs* = 0.94) and the concordance is significant (p < 0*05). But if we consider the total number of luteal structures (CL plus luteinized follicles), which is the number effectively taken into account at days 7 or 8 of the cycle, the relationship between this number and the actual number of ovulations is not as strong (rs = 0.77) and the concordance is not significant; the relationship between the number of embryos recovered and the actual number of ovulations is higher than with the total number of luteal structures (rs = 0.92 with a significant concordance vs rs = 0.83 with a non-significant concordance). The true number of CL is the number obtained by histology; accuracy of the technique of estimation by direct observation was defined as follow: a-_

AC

.s/tL-h.i 2-k;

‘1

x A00

ti = true number of CL obtained by histology ni = number of CL estimated by direct observation. Accuracy was here 85 X; it was = 42.5 % for the estimation by counting the number of total luteal structures and 65 % for the estimation by counting the number of embryos recovered. with:

Table 2: Number ofluteal structures or:superovulated ovaries of 10 Friesiac heifers, 48 hours after LH peak: results of icdividual ar.imals Number of CL by histology

Number of CL by direct observatioc

0 2 2 1

0 2 1 1

3 6 6 a 2

3 6 6 a 3

10

6

Number of luteal structures (CL + luteicized follicles)

Number of embryos recovered

1 2

0 1

3 5 5 8'

0 0 1 6

a 9

z 2

15

7

* rs = Spearman Rank Correlation Coefficient JANUARY

1983 VOL. 19 NO. 1

59

THERIOGENOLOGY In conclusion, the number of embryos recovered may be in some cases a better estimation of the ovulatory response to treatment than the number of luteal structures observed, even by a direct observation, 7 or 8 days after oestrus. On the other hand, adjustment of the technique of embryo recovery is possible only if the actual number of ovulations is known and this requires histological studies.

- The third question was whether the large u.novulated follicles seen on the ovaries of animals stimulated with PMSG on days 7 or 8 must be taken into account as part of the ovarian response to treatment. These follicles explain a lowering in the quality of embryos recovered (12). We postulated 00) that such follicles grew after ovulation stimulated by PMSG still present in the blood of treated animals and provided the following arguments for this hypothesis: - The long half life of PMSG ensures that the concentration at time of oestrus is more than half that recorded just after injection (13).

- Such large follicles are not observed after superovulation with FSH (14) or HMG (15). - The injection of anti-PMSG at oestrus in heifers treated with 5000 IU PMSG decreases the number of large follicles observed and E2- 17 k concentrations after ovulation, improves ovulation rate and quality of embryos (16). Anti-PMSG prevents the formation of the many large follicles normally observed after an injection of 7500 Iu of PMSG (SAUMANDE, 1982 unpublished results).

so, if these follicles mostly grow after ovulation, they cannot be considered as follicles which have grown at the same time as those that ovulate so are not relevant in the calculation of ovulation yield. . Estimation of ovarian response by different hormonal assays The prediction of ovulation rate by plasma levels of progesterone after ovulation has been tried: a few hours after ovulation, there was a good relationship between the concentration of progesterone and the number of corpora lutea, but it was not sufficiently strong to accurately predict the number of corpora lutea (10). On day 8 or later a similar relationship was also .found (17, 8, 18, 19) but with the same conclusion. However, it is difficult to draw final conclusions from these results because ovulation rate was estimated by counting luteal structures at day 8 or later and nothing is known about the ability of luteinized follicles to secrete progesterone in similar amounts. Prediction of ovulation rate before ovulation has also been et a1.(7) reported that there was a attempted. Recently, SCHILLING -relationship between progesterone levels in milk at the time of the

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THERIOGENOLOGY treatment and ovulation rate estimated by rectal palpation that would explain 16 % of the total variance in ovulation rate. This is not sufficient to be a good estimator of ovulation rate. Levels of E2-17p in plasma of heifers following injection of PMSG were also found to be well correlated with luteal structures after ovulation and the correlation coefficient was very high (r = 0.90 between ovulation rate and the preovulatory peak of E2-17p) (10). Recent results of studies on the heifer (see III, endocrine factors) indicate that ovulation rate might be estimated from the levels of E2-17pin plasma between 60 hours and 66 hours after PMSG injection. A new method of prediction of ovulation rate after PMSG, proposed by CHUPIN and PROCUREUR (ZO), is the echography of large follicles at the beginning of oestrus.

II IMPROVEMENT OF SUPEROVULATORY TREATMENTS Treatments using different stimulatory products (PMSG, FSH - LH from different hormonal preparations) were tested in order to try to increase the number of good embryos recovered. Comparison of results obtained can be made only if the characteristics of the stimulatory agents are well defined.

. Characterization of the stimulatory agents A qualitatively and quantitatively better definition of the stimulatory agents is needed. The affinity of PMSG for ovarian receptors varies with the species, so bovine corpora lutea were used to develop a standardized radioreceptor assay for PMSG which allows the measurement of the amount of PMSG present in different preparations (fig. 1). Radioreceptor assays were also developed for FSH and LH with receptors prepared from total calf ovaries to allow the comparison of FSH and LH activity of different hormonal preparations that may be used to induce superovulation in cattle (fig. 2) Unfortunately, when the curves are linearized using the logit/log transformation, the lines obtained are not parallel, so a strict comparison of activity is not possible, due to the heterogeneity of the products used. Nevertheless, the following differences have to be observed: 1 unit of SIGMA (St Louis Missouri USA, lot F 8001) or BURNS BIOTEC(B.B.; Omaha, Nebraska, USA, lot 540881) pituitary preparations have the same FSH activity, far greater than 1 unit of U.C.B (Bruxelles, lot I 107) product. In the LH system, 0.5 IU SIGMA has the same activity as 2ng of pure bovine LH and BB preparation. These data have to be compared with those obtained in porcine radioreceptor assay; the ratio LH/FSH activity was 5 for PMSG and varied between 2 and 5 for SIGMA and BB products; in these preparations the amount of LH is around 5 fold greater than the amount of FSH, measured by RIA (Y. COMBARNOUS 1982, personal communication).

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THERIOGENOLOGY

I.

I wa

0.01

0.1 I

0.31

0.m

1.a

2.

‘

*o

10

”

l

,,*

PISO 2s

Fig.

1:

bovine radioreceptor a: Purified PMSG b: Gestyl (Organon) c: PNSG (Intervet)

Fig.

2:

5

Comparison of different

-a.-

10

batches assay

of PMSG

Comparison of different stimulatory Preparations in an FSH radioreceptor

in a

assay

Amounts are expressed in m IU/tube, except for b FSH (ng/tube)

62

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1983 VOL. 19 NO. 1

THERIOGENOLOGY

So it will be more appropriate to call these products "FSH - LH" preparations. These observations also question the necessity of adding LH to superovulatory treatments using these preparations. . Comparison of treatments with PMSG and FSH - LH preparations Adult cows of Charolais breed were injected with PMSG (2500 IV) or a FSH - LH (BB) preparation (50 mg). Doses were chosen in order to have in each case the maximal ovulatory response. PMSG was injected at day 13 + 3 after natural oestrus. The FSH-LH preparation was injected twice daily over a 4-day period beginning Ll + 3 days after natural oestrus. Results (Table 3) showed that with t%e FSH-LH preparation, mean ovulation rate, estimated by rectal palpation at days 7 or 8 of the cycle, was slightly higher than with 2500 IU PMSG; as a consequence, the percentage of cows with more than 3 good embryos recovered by a non-surgical technique was higher with FSH. Effect of PMSG (2500 Ill)or FSH-LH (50 mg Sigma) preparation Table 3: on ovarian stimulation and embryo production in Charolais COWS

No. cows FNSG

28

FSH_J_H 30

*

Ovulation rate (CL) g cows with Mean 2 SD O-l 2-5 >5

Embryos recovered

Good embryos $ cows with Mean 2 SD 0 1-3 >3

8.1 + 7.4

25

25

50

6.0 z 6.4

3.3 2 4.6

44

28

28

11.1 + 6.7"

10

13

77** 8.6 z 6.7

4.4 2 4.3

37

13

509'

P
These results agree those of Eldsen -et ale(21) but others (22, 23, 24) found no differences between FSH and PMSG. However, there were differences between these studies in the dose of hormone injected per treatment, the graduation of doses within injection regime and the source of hormone preparation. These factors needed to be more precisely studied.

. Effects of different schedules of treatments with FSH-LH preparations CHUPIN and PROCUREUR (25) found no difference between the results of the injection of 32 mg and 50 mg of BB preparation; they also found that Sigma preparation was as efficient as BB preparation. However, the frequency of injections was important: Friesian cows received an FSH - LH preparation over a 4-day period beginning 11 + 3 days after natural estrus in 1, 2, or 3 injections per day. Tbe total dose (32 or 50 mS) injected was divided in equal doses given at each injection. Results (Table 4) showed that 2 or 3 injections increased ovulation rate slightly , and, more clearly, the mean number of good embryos recovered.

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1983 VOL. 19 NO. 1

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THERIOGENOLOGY

Effect of Number of FSH-LH injections per day in Friesian cows

Table 4. No .

injec- No. tions cows --

Good embryos $ cows with Mean 2 SD 0 l-3 >3

Ovulation rate (CL) Embryos $ cows with Mear.2 SD O-f 2-5 >5 recovered

3

24

10.0 2 5.5

4

13

83

6.9 + 6.2

4.1 f 5.0

38

20

42

2

24

10.2 f. 5.0

4

21

75

6.7 f. 6.3

5.1 f 5.4

25

25

50

1

23

7.4 + 5.6a 22

22

57

3.6 f 3.8b

1.8 f 2.8'

52

22

26

a p
(From Chupin and Procureur, 1982)

Moreover, the graduation of doses within injection regime was important: in a second experiment, Friesian cows received 2 injections per day of the FSH - LH preparation over a 4 day period beginning 11 + 3 days after natural oestrus. The total dose (32 or 50 mg) injected was divided in equal or decreasing doses. Results (Table 5) showed that a decreasing dosage schedule increased the ovulation rate and the number of good embryos recovered. Effect Table 5: Friesian cows

No. cows

of dosage

of FSH-LH preparation

Ovulation rate (CL) s cows with Mean + SD O-1 2-5 >5

per injection

in

Good embryos $ cows with Mean 2 SD 0 l-3 >3

Embryos recovered

--- Constact dosage/injection --28

9.3 +

7.6

14

21

64

5.8 f

6.4

2.4 2

3.6 50

25

25

--- Decreasing dosage/injection ---

26

15.9 + 15.0b 8

19

73a 12.2 +

12.3'

5.3 +6.2b35

19

46a

a P<0.10; b p
64

JANUARY 1983VOL. 19 NO. 1

THERIOGENOEOGY III INPORTAXCE OF THE OVARY IN THE VARIABILITY OF RESPOhTSIVENNESSTO PMSG The question of whether the variability of ovulatorv responses of cows to PMSG could be related to the variability of ovarian status at the time of treatment was posed by SAUMANDE et al (5); in the following experiments we tried to answer this queszon?hrough several different approaches and our conclusion was that ovarian status at the time of treatment was a large determinant of the ovulatory response to PMSG in cattle. 1) Some evidence for the role of ovarian factors : preliminary studies In the calf _-_____-___ On 36 prepuberal Friesian calves, ovarian status was determined by endoscopy the day before PMSG injection (600, 800, 1050 or 1400 In). E ,178 (26) was assayed on plasma collected every 2 hours after PMSG kjection. Animals were slaughtered 10 days after PMSG and ovaries were studied. PMSG induced follicles greater than 10 mm diameter on the ovaries. Moreover, for animals receiving 800, 1050 or 1400 IU of PMSG: - There were significant correlations (p < 0.05) between the total number of follicles before treatment and the number of follicles, ovulated or not, greater than 10 mm diameter after treatment (fig 3 a). - There were significant correlations (p < 0.05) between the total number of follicles before treatment and oestradiol levels in plasma after treatment (fig 3 b).

Fig.

3:

Relationships between and ovarian response Regression lines

JANUARY

1983 VOL. 19 NO. I

ovarian status before treatment to PXSG in the 3 month-old calf:

65

THERIOGENOLOGY

so, in the calf, the number of follicles which can be detected by endoscopy at the time of treatment would be a determinant of the follicular and endocrine response to PMSG. . _______-----_ In the heifer the

In 1.6 heifers, aged 15 months, stimulation was modified by two ways:

ovarian

status

before

- Prestimulation with PMSG (1500 IU) at DO (12 hours after oestrus) - Electra-cauterization of all the follicles greater than 8 mm diam. at Di6 using endoscopy (27). Animals were assigned to 4 groups according to prestimulation or not and (or) cauterization or not. In all the animals, an endoscopy was performed at D 16 (1st endoscopy) and PMSG (2000 Ill)was injected at the same time. A 2nd endoscopy was performed between D4 and D8 of the new cycle. Prestimulation with PMSG increases (p < 0.05) the number of follicles greater than 8 mm diam. on the ovaries at D , but decreases (p < 0.05) the number of follicles that ovulate a h6 ter stimulation. Cauterization of follicles greater than 8 mmh;; ~&~,,;,"r;~;;; (non-significantly) the number of follicles stimulation, but cauterization is ineffective in prestimulated animals (Table 6). Table 6: Effect of prestimulatinr. with PMSG (1500 IIJ)and cauterization of follicles larger than 8 mm diameter on ovarian response to PMSG (~000 IU) in the heifer

kar

2 SD

CL

No prestimulation Prestimulatior 1st endoscopy 2nd endoscopy 1st endoscopy 2nd endoscopy --- co cauterization --1.5 + 0.6 9.8 2 6.6 1.3 5 0.5 2.5 2 1.7

Foil. >8mm

1.8 2 1.3

Foil. <8mm

0.3 + 0.5

6.3 + 6.5

---

6.0 2 5.0

9.5 + 8.5

6.3 + 6.5

2.3 t 2.1

0

--1.3 2 0.5

3.5 f 3.8

CL

1.8 2 0.5

cauterization 5.5 -c 4.4

Foll.>&nm

2.5 2 1.7

5.0 + 1.4

6.0 - 5.2

9.5 + 5.3

Foll.
2.0 2 1.0

0

1.5 2 2.4

0

PMSG given at the beginning of the cycle would recruit follicles which become atretic before D so the second injection would be ineffective. Cauterization of foLA icles at D decreases the number of follicles which could be stimulated, so ovu116 ation rate after PMSG is reduced. This reduction in mean ovulation rate after cauterization has been shown previously (28). It means that follicles greater than 8 mm

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THERIOGENOLOGY

at the time of treatment will ovulate after PMSG. If animals are prestimulated, cauterization would mostly act upon atretic follicles so ovulation rate is unchanged compared to ovulation rate of non-prestimulated animals.

so, in the heifer, changes in the number or quality of large follicles at the time of PMSG injection can apparently affect the ovulatory response to stimulation. In order to verify this point, a more complete study was undertaken in the heifer. 2) Main ovarian factors liable to act upon ovulatory response to PMSG in the heifer A positive relationship between the number of growing follicles with more than 2 layers of granulosa cells and natural ovulation rate has been found in the sheep (29) by comparison of 2 breeds with different ovulation rates. In the Merinos, animals with 2 ovulations have more normal follicles greater than 2 mm diam. than animals with 1 ovulation (30). In sheep, breeds with the highest natural ovulation rates are the most sensitive to PMSG (31, 32, 33). In the hamster or in the rat, relationships between follicular populations and ovulation rate after PMSG have been fonnc~~(~34;35). In the stimulated cow, however, no precise data is available. The aim of this work was to study the relationships in the heifer between follicular populations at the time of PMSG injection, and ovulatory responses to stimulation. The questions we asked were: are there differences between follicular populations of heifers with different responsiveness to PMSG?; on which types of follicles (size, quality) is PMSG the most effective, and what sort of changes (numerical, kinetic, endocrine) are observed after PMSG?; By which mechanism are the follicles that ovulate selected ? To attempt to answer to these questions, eighteen cyclic Friesian heifers were randomly assigned to 2 groups: one experimental group (10 animals) and one control group (8 animals). The experiment (Table 7) consisted of hemicastration immediately followed, by the stimulation of the remaining ovary in the 10 animals of the experimental group (PMSG, 2000 IV). Control animals did not receive PMSG. Animals were slaughtered 48 hours after the time of the LH preovulatory discharge, known by a rapid LH radioimmunoassay, and the second ovary was removed. Both ovaries were studied by classic histological techniques. A. the follicles larger than 70 urn diameter(follicles with more than 2 layers of granulosa cells) were counted, measured, and checked for atresia according to previous studies (37, 38). Mitotic indexwas estimated on normal follicles with the beginningofanantrum (between 115 pm and 280 urn diameter) and follicles larger than 0,5 mm diameter E2-178(26) and LH (39) were assayed in on the plasma.

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THERIOGENOLOGY

Table 7:

Experimental design

* Norgesotmet:

SG 21009, Searle

N.B. The stimulatory treatment is the "cocktail treatment" of Saumande et al. (36) Results of this study showed that three kinds of factors liable to act upon ovulatory response to PMSG were found : numerical factors, kinetic factors and endocrine factors. factors * Numerical -------_-________ - In the 5 control animals which had ovulated, hemicastration without PMSG had no effect on the mean number of total follicles (372 before hemicastration vs 356 after hemicastration), of total atretic follicles (59 before hemicastration vs 52 after hemicastration), of normal follicles greater than 1.7 mm diam. (9 before hemicastration vs 10 after hemicastration) or of atretic follicles greater than 1.7 mm diam. (9 before hemicastration vs 10 after hemicastration). So no short-term effect of hemicastration on follicular populations could be found and any difference between ovaries of stimulated animals could thus be attributed to PMSG. - In the 10 experimental animals, the number of ovulations on the "stimulated ovaries" (removed 48 hours after LH peak) varied between 0 and 10 and the number of luteinized follicles between 0 and 7. The total number of normal follicles on the ovaries before and after PMSG was quite variable between animals: it varied between 53 and 755 on the "control" ovaries (ovaries removed before PMSG injection), and between 73 and 881 on the "stimulated" ovaries. Variability between animals was quite large, but variability between the 2 ovaries within each animal was small: there was a highly significant correlation between the number of normal follicles on the control and on the stimulated ovary. The same result was found for atretic follicles (fig 4). Moreover, it was found for all size classes of follicles. So, the "control ovary" is a suitable control of the "stimulated" ovary in this study.

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The large variability of folilcltParpopulatlioas observed between animals in the bovine has been often noticed in both non-st%mulated and Sti¶dL~t@d aaiimls (37, 4c, 41, 42$ 43). fn t?& studyy stimulation by PMSG has not overthrown foHlicular populations and in each animal the number of grcniing fallicles is steiM_Iy*even after stimulation; this number is probably an indivi.duaT charaetehist~e of the animal, and it suggests that regulation of follisulaf growth is different between heifers.

There was a close reEationship between the total number of grow%ng fc4licBes In the ovary before treatment and the nsrmber of young corpora lutea pius Puteinized follicles on the stimulated ovary CfBg, 5) Crs = 0,gg ; pto.oor>. This correlation was observed w%th different kinds of foPlicles (norm& or atretic; greantral. astral follicles, or foPlfePes greater than 0.5 mm diameter) (Table 8). However, the number of ovulations was not correlated signiffcantly with any kind of felfieular variable measured before treatment. 8x2 the other hand, the number of lutefnized foS1icPes was sfgnificantly correlated with the num3er of atretd,c follicles graater than 0.5 mm before treatment CptC,O§). SD it is likely that numerical factors can explain a large part of the total ovulatory and Puteal response to PHSG, but they are insufficient to explain the ovulatcrp reslponse atresia anui Ka’everthePess, between alone. the relationship Buteinization is noticeable.

49

THERIOGENOLOGY

Fis. 5 : RELATIONSYIP mn:Eix

FOLLICLLAR POPULATIONS 3EFORE TRrATMEP:T AXD OVhRIAX RESPONSE TO PMSG IN THE UEIFERS

Table 8. Relationships between follicular populations before treatment and ovarian response to PMSG in the heifer: Rank correlation coefficients No. follicles on control ovary Stimulated Preantral follicle Antral follicle Follicles )0.5 mm ovary Normal Atretic Normal Atretic Normal Atretic ----_I_Ovulations

0.54

0.43

0.62

0.49

0.53

0.41

Luteinized follicles

0.62

0.54

0.32

0.60

0.61

0.70"

Ovulations & luteinized follicles

O-87***

0.81""

0.72,

0.87"s

0.92***

0.83'"

* P<0.05; ** p
70

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1983 VOL. 19 NO. 1

THERIOGENOLOGY

significantly while the number of atretic preantral follicles was unchanged. In antral follicles, the number of normal follicles remained unchanged whereas the number of atretic follicles decreased significantly. So, the, short-term effects of PMSG would be to stimulate the growth of normal preantral follicles and to rescue some atretic antral follicles, as has been suggested in previous studies in vivo and -in vitro (44, 45, 46). -These results suggest that atretic follicles rescued by PMSG would fail to ovulate and only luteinize following preovulatory discharge.

ONORMLFOLLICLES CONTROL OATRElIC

OVARY

FOLLICLES

NORMAL FOLLICLES STltWATED

PREANTRAL

Fiq. 6

:

FOLLICLES

AHTRAL

OVARY

FOLLICLES

EFFECT OF PMSG ON FOLLICULAR POPULATIONS IN THE HEIFER

Kinetic factors --_--_--------The mitotic index was estimated on normal follicles forming an antrum and on normal and early atretic follicles greater than 0,5 mm diameter, and was quite variable between animals for each type of follicle and each type of ovary. Mean mitotic index varied with the size of follicle and the type of ovary (fig. 7). In control ovaries, mitotic index of normal follicles increased as the follicles move from forming antrum to

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1983 VOL. 19 NO. 1

71

Xi Si MIi t

= = = =

nu&er of fcll%cles in class i an the cmtrcl ovaq mean foliicuiar awa of class i mean mitntic index of fslPicles ia ciass i on the esntra: ovilry tine from FMSG injection to Xl peak

This index was civ~sen to provide a simplified parameter which took info account the different numerical end kinetic features of rerminal growth. It suggests that the Ni follicles in class i grow, after PNSG, from the size Si at a rate proportional to KIi s.nd during the time t. Growth index may be calculated for one of the ten defined size classes, or for two or m.oreof these classes.

Results showed that: the "'growth index'" of normal follicles greeter than I.9 m diameter on control ovaries was well correlated with nhe number of ovulations observed on the stimulated ovary (r-s= C186; p < D,DI)‘ The value of the rank correlation coefficient between the nnmber of normal follicles greater than 1,7 mm diameter on the control ovaries and the number of ovulations after stimulation is a little lower frs = O-79; p
Fig. 8:

Relationships between large follicles before treatment and ovarian response to treatment

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1983 VOL. 19 NO. 1

N. Follicles = Pjormal follicles 5 1.7 mm diameter on control ovary E.A.Follicles 4 Early atretic fol icles a 1.7 mm on control ovary

73

THERIOGENOLOGY

These results might indicate that normal follicles greater than 1.7 mm diameter present on the control ovary at the time of the stimulation would have their growth rate stimulated proportional to their initial growth rate and would mostly ovulate; early atretic follicles of the same sizes would be rescued by PMSG, grow at a rate proportional to their initial growth rate and would mostly luteinize. Endocrine factors ------------_--_Fluctuations in plasma E -178 1evels after hemicastration and PMSG injection were quite varla *?Ile between animals: peak levels varied between 14 and 106 pg/ml, and were reached between 76 hours and 150 hours after PMSG. The concentration of E2-17 p at peak was correlated with the number of ovulations + luteinized follicles on the stimultited ovary (rs = 0,69), which is in agreement with previous results (8.?,1$>. Different types of endocrine responses were observed (fig. 9):

Fig. 9:

74

Different types of rises of (E2 17 beta) in plasma after implant removal (values have been corrected for E 2 injected at the beginning of treatment) a: Biphasic rise b: Rise "type 1" c: Rise "type 2"

JANUARY

1983 VOL. 19 NO. 1

THERIOGENOLOGY

- After implant removal (48 hours after PMSG), 6 animals had a biphasic rise: a rise "type l", slow and regular, was followed by a sharp rise "type 2" whose duration was always less than 28 hours. Animals with this endocrine response had ovulations and luteinized follicles on their stimulated ovaries. - 3 animals had only a "type 1" rise. These animals always ovulated and had never more than 1 luteinized follicle. - 1 animal had only a "type 2" rise. This animal did not ovulate, and 1 luteinized follicle was found on its stimulated ovary. The number of ovulations after stimulation was correlated with the characteristics rise [initial concentration: rs = 0,66 rs of = othe 68 1st (
JANUARY 1983VOL. 19 NO. 1

75

P CO,aol __----_

-----____-

P co,01 A_-______-_-__

_------

Fig. -_

P(O.as ____-__-_

IO : a : VALUES OF RANt'CORREL.ATIONCOEFFICIENT BETWEEN E LEVELS AFTER IMPLANT REXWAL AND THE 2 TYPES OF 2 OVARIAIiRESPFXSE TO STIXJLATIGN _b

: VALUES OF R&X CCRRELATIOX COEFFICIENT BETbJEENE, <.EVELSAFTXR Zf.:?L.X;T RE::i?VAL AND THE GROWTH INDEX (‘F _I 2 TYPES OF FOLLiCLES BEFORE STIMLKATION.

76

JANUARY

1983 VOL. 19 NO. 1

Results

variation stimulatioc

of this work pointed out the complexity rjf indivihual cows in their cvulatory between

thP

problem response

of to

_

two days after Exact histological determination of r esponses oestrus showed that two types of luteal structures could be At this distinguished: the corpora lutea and the luteinized follicles. time, direct observatioc of ovaries in slaughtered animals or, to a permit determination of the lesser extent D ecdoscopy of living animals, When t’ne observations number of ovulated follicles with good accuracy. the two structures cannot be easily are made some days later, estimation of ovulation rate may be incorrect no identified; thus, matter which technique is used (direct observation, endoscopy, or rectal Rectal palpation performed seven days after oestrus is palpation). particuiariy inadequate to estimate ovulation rate because, when the accuracy of the technique number of luteal structures increases, decreases. At this time, the number of embryos recovered may be a Elsewhere, endocrine criteria of better estimation of ovulation rate. ovulation rate, such as oestradiol levels in plasma 60 hours after PMSG, need further invest,igation before they could be of practical use. One of the main problems in superovulation is the existence of Recovery of 0 poorly responding animals, whatever the treatment used. or 1 embryo after treatment suggests that the ovulation rate is less From our results, there are two types of poorly responding than 3. animals: -- The first one includes animais with less than 200 normal, growing follicles per ovary at the time of stimulation. These animals always respond poorly. Attempts to increase responsiveness of these animals might be achieved by long-term pretreatment to stimulate folliculogenesis, perhaps applied before puberty. tie wever s the efficiency of such pretreatment is very uncertain because the follicular structure and sensitivity of these animals probably have genetic origins. -- The second one includes animals with a large number of normal, growing follicles, but with a high rate of atresia in follicles that have a large diameter at the time of stimulation. From our results, early atretic follicles greater than ‘I.7 mm in diameter before PMSG injection cannot ovulate after PMSG, but only luteinize, Perhaps for these follicles, PMSG was given at the wrong time. If so, a short-term pretreatment could be considered to increase the number of large, normal follicles and/or rescue large, early atretic follicles before stimulation. For this purpose, pretreatment with an FSH-like agent or immunization against an ovarian steroid before stimulation could be attempted to suppress or reduce the rate of atresia at the time of stimulation, Other strategies than modification of ovarian status at the time of ovulation could be developed: -- One is to pick out of an unselected herd the best responding animals just before or just after the stimulatory treatment. With this objective, hormonal estimators are the most useful. The decision to stimulate or to inseminate an animal would be made according to the results of this estimation.

JAIWJAKY 1983 WQL. 19 NO. 1

77

THERIOGENOLOGY -- Another is to attempt genetic selection of animals that respond well, which, according to our results,are animals withmore than 500 It will be necessary for this normal, growing follicles per ovary. genetic approach to have good estimators of ovarian response available. The endocrine estimators are very attractive and practical in this This selection could be made before puberty, if ovarian regard. responsiveness to PMSG before and after puberty are well correlated (5). LITERATURE CITED (I)

Gordon, I., Williams, G., and Edwards, J. 1962. 'he use of serum gonadotrophin (PMS) in the induction of twin pregnancy in the cow. J. Agric. Sci., -59, 143-198.

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Saumande, J., Chupin, D., Mariana, J.C., Ortavant, R.and Mauleon, Factors affecting the variability of ovulation rates P. 1978. after PMSG stimulation. In: "Control of reproduction in the cow." Ed. J. M. Sreenan. The Hague, M. Nijhoff. 195-224.

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Guay, P. and Bedoya, M. 1981. A study of the equivalence between rectal palpation, laparoscopy, laparotomy and ovarian dissection for evaluation of the ovarian response of PMSG-superovulated cows. Can. Vet. J. 22, 353-355.

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Schilling, E., Haupt, P., Smidt, D., Sacher, B., Elsaesser. F., and von Schutzbar, W. Die Variabilittit des 1981. Superovulationserfolges bei KUhen und deren mwlichen Ursachen. 2. Tierzehtg. Ztihtungsbiol. 98, 88-99.

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Testart, J., Kann, G., Saumande, J. and Thibier, M. 1977. Oestradiol-17 beta, progesterone, FSH and LH in superovulated prepuberal calves. J. Reprod. Fert. -51, 329-336.

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Boland. M.P., Crosby, T.F., and Gordon, I. 1978. Morphological normality of cattle embryos following superovulation using PMSG. Theriogenology lo, 175-180.

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Lamond, D.R.and Gaddy, R.G. 1972. Plasma progesterone in cows with multiple ovulations. J. Reprod. Fert. 3, 307-311.

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Sreenan, J.M., Beehan, D., and Gosling, J.P. 1978. Ovarian responses in relation to endocrine status following PMSG stimulation in the cow. In: "Control of reproduction in the cow?' Ed. J.M. Sreenan. The Hague, M. Nijhoff. 144-155.

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Chupin. D. and Procureur, R. Prediction of ovarian 1983. response to PMSGby ultrasonic echography. Theriogenology 19, 119. Elsden, R.P., Nelson, L.D., and Seidel, G.E., Jr. 1978 Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotropin. lheriogenology 2, 17-26.

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:24!

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198Oa

of past-treatment

estraus

cycle

a

Camm,

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E.R.,

transferabie

Ginther, o,‘I, s affec’sirig embryos and length

and

factor

Theriogenoiogy 2,

397-406_

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Greenwald, G.S. 1974. Role of follicle stimulating hormone and luteinizing hormone in follicular development and ovulation. Handbook of Physiology, Sect. 7, 4, 293-323.

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Mariana, J.C, 1979. PMSG responsiveness during adult life after partial oogonia destruction with misulban in the rat embryo. Ann. Biol. anim. Bioeh. Biophys. -I 19 1469-1474.

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Hey,

H.F*,

Regezeratlcc Fert, zp

N0 0 r R,M., Crar., of atretin ova-ian !95-207: 9

D.G,, fcllieLes

and --in

Dot:, vitra,

R,M, 6.

1979. Repsod.