Radioimmunoassay and enzymeimmunoassay of plasma progesterone as monitors of progesterone sponge treatment in ewes

THERIOGENOLOGY

RADIOIMMUNOASSAY AND ENZYMEIMMUNOASSAY OF PLASMA PROGESTERONEAS MONITORSOF PROGESTERONE SPONGETREATMENT IN EWES H.F. Mac Donnell! S. Mullins ‘and A.C. Gordon2 Department of Agriculture, University College Dublin, 1 Lyons, Newcastle, P.O., Co. Dublin and 2 Belfield, Dublin 4, Ireland Received

for

publication: Accepted:

July June

8, 6,

1987 1988

ABSTRACT The daily plasma progesterone (P) concentrations achieved during insertion of P (750 mg) sponges into two groups of ewes were examined. Group I received prostaglandin (PG) treatment, which was required to suppress the P production (to levels of < 0.3 ng hormone/ml plasma) from the corpora lutea (CL) of a previous superovulation treatment, following which these Group I ewes and the anestrous Group II ewes were sponge treated. Radioimmunoassay (RIA) and enzymeimmunoassay (EIA) were used to measure the P levels in both groups. Progesterone (750 mg) sponges with and without citric acid impregnation were inserted into all the ewes for 12 days (d). Citric acid lowered the P levels reaching the plasma from the sponges, but it did not mask the characteristic profile (during the treatments) determined by the states of the ewes (single PG and double PG injected. Group I or in the anestrous Group II). The plasma P levels in Group I and II ewes rose to at least 7.0 ng/ml at intervals during treatment. The duration and magnitude of the P concentrations in the plasma were higher in the single PG compared with the double PG ewes during sponge insertion in Group I. The anestrous Group II ewes showed two major peaks (Day 1, P 2.0 ng/ml was maintained over the entire treatment in the single PG and in the anestrous hormone-treated ewes, and was of shorter duration (7 d) in the double PG-treated animals. These endogenous patterns in P profiles of the ewes indicate that the hormone level during sponge insertion varies in magnitude and duration, parameters determined by the phys:iological/endocrinological state of the ewes at the start of the treatment. The EIA correlated significantly (PiO.001) with the RIA for concentration, when analyzed daily on an individual animal basis. Key words:

ewe, progesterone,

prostaglandin,

the

measurement of

P

radioimmunoassay. enzymeimmunoassay

Acknowledgments: The antiserum used in this study was donated by Professor Oystein Andresen Professor Ian Gordon (University College, (Norges Veterinaerhdgskole, Oslo). Dublin) provided the research facilities for this study, where Hugh McGovern and Mary Gallagher carried out the field work. The guidance of the UCD Computer Centre in implementing programmes is much appreciated.

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THERIOGENOLOGY

INTRODUCTION Fertility can be achieved in sheep following various treatment regimes in the Increasing the level of exogenous P administered either by nonbreeding season (l-3). implant (4) or impregnated sponge (5) increased the conception rate in ewes when these treatments were accompanied by gonadotropic stimulation. As reviewed (3), attempts to control estrus and ovulation in ewes, whether during the breeding season or in anestrus, are based on attempts to simulate the activity of the CL. Different modes of progesterone/progestagen administration have been applied, for various lengths of time to both cyclic and anestrous ewes by daily injections for 14 d, feeding of orally-active and subcutaneous insertion of silastic impregnated with implants progestagens In out-of-season progesterone/progestagen applications, it progesterone/progestagens. is of prime importance to administer a gonadotropic substance such as pregnant mare serum gonadotropin (PMSG) to stimulate follicular maturation and ovulation comparable to that which occurs during the natural breeding season (1.3). A high dose of progestagen for a fixed time, followed by its rapid withdrawal, is required for good estrus induction/synchronization. The degree of absorption of the progestagen from the intravaginal sponge is related to both dose and procedure used for impregnation of the sponge (1). Despite some adverse effects on conception, intravaginal administration of natural progesterone and its synthetic derivatives has been used universally. Currently, due to the advent of freezing/storage facilities for viable embryos, there is a requirement to examine the feasibility of treating these donor ewes (previously superovulated) with prostaglandin/progesterone sponges to reinduce cyclicity in these animals. Normal luteal function (levels >1.5 ng progesterone/ml plasma over 9 d) and acceptable fertility (73% conception rate) were achieved in anestrous ewes when silastic implants (3 X 375 mg) were used as a progesterone pretreatment for gonadotropin releasing hormone (GnRH; 2). Increased sensitivity to exogenous P was observed in anestrus (5) when compared to cyclic ewes on similar doses (500 mg and 800 mg) of the hormone (6). While luteal phase levels of plasma progesterone were achieved and/or maintained during sponge insertion in the cyclic ewe, this phenomenon did not ensure high conception rates (6,7), suggesting that the hormone profiles and times of occurrence of peak levels are the critical factors to be examined before such sponge treatments achieve full fertility. The percentage lambing rates increased (PcO.05) linearly with increasing doses of P (500 mg to 1000 mg) in anestrus (5). It was of interest, therefore, to investigate the peripheral plasma P levels achieved using 750 mg (mid-point on this dose response curve) P sponges in ewes in the nonbreeding season to examine the effects of the intravaginal route of administration of the hormone on the magnitude and duration of P levels Citric acid has been reported to enhance reached endogenously during sponge insertion. the absorption of the peptide. Leuprolide, intravaginally in rats (8). In our study of P-impregnated sponge treatment of ewes, we decided to examine whether this acid has a similar effect on the absorption of P. The aims of the experiments were to examine 1) the efficacy of single and double PG injections in inducing regression of CLs (from an earlier superovulation treatment) and the influence of PG on the subsequent plasma hormone profile during sponge treatment; 2)

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the plasma progesterone concentration achieved during intravaginal insertion of 750 mg of citric acid to the P sponges into anestrous sheep; 3) whether the addition hormone-impregnated sponges (750 mg) affects the plasma P concentrations reached: and 4) the plasma P levels measured by EIA and RIA in order to examine the degree of correlation between the P profiles obtained by both assay techniques.

MATERIALSAND METHODS Animals The sheep used in these experiments were of mixed breeds Greyface) and were located on the east coast of Ireland.

(Galway, Suffolk

and

Grouo I. Eight ewes were selected for the PG-P sponge study. These ewes had been previously subjected to superovulation with PMSG in the nonbreeding season, but the details are not considered relevant to this study of Group I, apart from the possibility of residual PMSG effects (1,500 IU administered at least 12 d before the first PG injection). These eight ewes were injected intramuscularly (i.m.) with PG (125 ug Cloprostenol. ICI) and the plasma P (RIA and EIA) was measured at 2- or 3- d intervals for evidence of cessation of P production. Four of the eight ewes were given a second PG injection (125 ug) after 12 d because of a persistance in these animals to secrete P (Figure 2). The progesterone sponge treatment commenced in 4 of the 8 ewes 14 days after the first PG, and in the remainder (n = 4) two days after the second PG injection (Figures 1 and 2). The animals were then segregated on the basis of PG injection (single PG. n = 4; double PC, n = 4) and subjected to 1) blank sponges (n = 2) 2) 750 mg P (n = 2), 3) 750 mg P + 5% citric acid (n = 2), and 4) 750 mg P + 10% citric acid (n = 2) impregnated sponge treatments for 12 d in the nonbreeding season (March, 1985). Grout’ II. Sixteen anestrous ewes were examined (June. 1985) in Group II. Twelve ewes were treated for 12 d with 1) blank sponges (n = 4), 2) 750 mg P (n = 4), and 3) 750 mg P + 5% citric acid impregnated sponges (n = 4) while the remaining four ewes were left as untreated (no sponges) controls. Blood Sampling Blood samples for P determination were collected from all ewes by jugular venipuncture, on a daily (10 h) basis during sponge insertion (Groups I and II) and additiona.lly, before treatment with sponges in Group I (see above for assessment of PG Plasma was separated and stored at -2O’C until assayed. efficacy). Sponge Preparation Polyurethane sponges were prepared as described (5.9) and impregnated with 750 mg P using a method reported earlier (10). The citric acid was impregnated onto the 750-mg P sponges Iby dissolving 5.0 g and 10.0 g of the substance in 100 ml of the stock P solution (250 mg progesterone/5.0 ml methanol). Three applications (5.0 ml) resulted in 750-mg sponges containing the required percentage of citric acid.

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I’HERIOGENOLOGY

Plasma Progesterone

Measurement

Radioactive progesterone (1,2,6,7,16.17- 3 H-progesterone, Extraction procedure. Amersham Internatl.. U.K.) was added to each sample of plasma before extraction of the hormone with hexane for analysis. Each plasma P concentration obtained in both assays was corrected to 100% recovery of the hormone in the result interpolation. Following were dissolved in 0.1% gelatin-phosphate evaporation of the hexane, the extracts buffered saline (0.01 M, pH 7.0) and allocated in parallel to the EIA and RIA. The antiserum (used in the assays at 1:5,000 dilution) was raised against !&J. ll-cl-hemisuccinate P linked to bovine serum albumin and its cross-reactivity was checked. Cross reactions with other steroids were 6.7% with deoxycorticosterone, 0.9% with corticosterone and 17 cx-OH-progesterone, and less than 0.5% with cholesterol, 17 cr-OH-pregnenolone, oestrone. hydrocortisone. dihydrotestosterone, and testosterone (11). The RADASS computer programme (12) was used for interpolation of results and was designed to reject assays if the coefficient of determination was less than 0.98. The RIA as used in this work has already been applied in a superovulated heifer study (13). The sensitivity of the RIA was 0.10 ng/tube. and it ranged from 0.3 ng to 111.0 ng P per milliliter plasma. In our study, the coefficient of variation within assays was 8.7% (n = 17. mean 3.45 ng/ml), and between assays was 11.8% (n = 29, mean 4.86 ng/ml). EIA. The extracted samples were used in a commercial EIA system (Noctech Ltd., Dublin, Ireland). This EIA is based on that published (14) using solid phase microtitre plate immunoassay with horse radish peroxidase-labelled progesterone. This antiserum was raised against the Ilo -hemisuccinate P linked to bovine serum albumin and showed a cross reactivity of 4.6% with 17c1 -OH-progesterone and less than 0.2% with pregnenolone This system contains a software and data reduction package for result (15). interpolation. The sensitivity of the EIA was 2.5 pg/well and ranged from 0.08 ng to 8.0 ng per milliliter plasma. The between-assay coefficient of variation was 8.0% (n = 11, mean 5.62 ng/ml) in this study, and statistics relating to microtitre plate reproducibility are available (Noctech, Ireland, Ltd.). Statistical

Analyses

Group I ewes (Table 1) were examined for differences in plasma P levels observed during sponge Treatments 2, 3 and 4 following the single and double PG injection regimens. Due to the small number (n = 2) of ewes per sponge treatment (Group I) the results were not analyzed statistically, but manifested trends in the plasma P profiles are discussed in relation to treatments used. In Group II, the synchronization of major peaks (Day 1 and Days 11 to 12) observed in mean plasma P levels, and the day-to-day fluctuations occurring in the individual ewes treated across the 12-d treatment period (Treatments 2 and 3) led to the analysis of the data using two, paired t-tests (a and b) as follows a) Group II ewes (Treatments 2 and 3) were analyzed using the paired t-test (two-tailed) on the differences observed per ewe per day in the plasma P concentrations across the 12-d treatment period. This paired t-test was also applied to Treatments 2 and 3 separately per ewe per day to examine for differences between treatments on the timing of increases or decreases observed in plasma hormone levels during the periods of sponge insertion. b) A paired

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THERIOGENOLOGY

t-test (two-tailed) was applied to mean levels of P observed in ewes daily on both Treatments 2 and 3 across the treatment period to detect any overall difference in magnitude of hormone concentration reached which might be attributed to the method of hormone administration. The IStudent’s t-test was also applied to Group II (anestrous ewes) to compare the mean plasma levels of P observed on a daily basis in ewes, and on the mean of the differences observed in the plasma progesterone levels per day during citric acid (5%) supplementation (n = 3, Treatment 3) with those achieved using the 750-mg P sponges alone (n = 4, Treatment 2). To correlate the progesterone values (Table 3) obtained by both RIA and EIA techniques, the statistical analysis was applied within the assay limits of 0.3 ng and 8.0 ng progesterone per milliliter plasma, the lower limit of the RIA and the upper limit of Therefore, in the determination of correlation coefficients the EIA, respectively. between both assays, levels above 8.0 ng/ml were included in the analysis as “8.0 ng” values.

RESULTS Group I The RIA plasma P profiles (Figures 1 and 2) following the first PG injection in the eight ewes indicate that four of the animals subsequently manifested P levels of, at the very least, luteal magnitude (ranging from 3.02 to > 16.0 ng/ml by the ninth day after the first injection). Therefore, these four ewes (Figure 2) were given a second injection of PG, administered 2 d before insertion of the progesterone sponges. The peripheral plasma P levels of Group I after insertion of the sponges for the animals i.n Treatments 2, 3 and 4 showed a definite pattern in the duration, timing and magnitude of peak values of the hormone in the plasma, depending on whether the ewes were subjected to a single (Figure 1) or double (Figure 2) PG injection. This luteal pattern !was also manifested, although to a minor extent, even in Treatment 1 animals, when blank sponges were inserted. These results suggest that the control ewes in Group I were secreting progesterone (2.29 ng/ml) on the 5th day during (single PG. Figure l), and (2.68 ng/ml) on the 2nd day after (double PG, Figure 2). blank sponge removal during These effects were also reflected in the hormone profiles of the nonbreeding season. the ewes in Treatments 2. 3 and 4. Table 1 illustrates the endocrinological response of the animals to impregnated sponge treatments on the basis of whether the ewes received Similar timing of peak hormone levels occurred during sponge one or two PG injections. treatment of ewes following the single PG. These peak magnitudes decreased in a descending order, depending on the treatment applied, 2, 3 or 4 (Figure 1). Similarly, a sequence of peaks at fixed times were observed in the plasma P during sponge treatment following double PG treatment, but these peaks were at different stages compared to those ob:served following the single PG treatment (Figure 2). Higher plasma progesterone levels were observed in the ewes treated with hormone impregnated sponges Treatments 2, 3 and 4 following the single PG when compared to those following the double PG - where trends suggest that plasma progesterone declined more abruptly in ewes on all treatments (Table 1. Figure 2).

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N

3

a

Figure

(

1.

i

PC’

Plasma progesterone treatment measured

ng / ml

progesterone

Plasma

16

18

.

l ,

Before Before

levels in ewes (Group by RIA (Table 1).

sponge

I)

following

single

insertion

mg progesterone

mg progesterone

sponge

0750

(3750

PC injection

+ 10% citric

sponge

sponge

and during

acid

acid

insertion

+ 5% citric

sponge

insertion

0 ‘(50 mg progesterorte

A blank

Days of

during

, during

, during

m

Before

, during

A

Before

impregnated

insertion

insertion

sponge

-10

3

-14

PC 1'

1

12

-6

t Before

1

I

during

mg progesterone+

mg progesterone+

mg progesterone

double PG injection

and during impregnated

sponge

10% citric acid sponge insertion

5% citric acid sponge insertion

sponge insertion

sponge insertion

insertion

0750

Day of sponge

1

i:

P -,

+,

0750 0750

during during

ablank

, during

Figure 2. Plasma progesterone levels in ewes (Group I) following treatment measured by RIA (Table 1).

( ng / ml

progesterone

Plasma

16

la-l

of

sponge insertion

Progesterone (750 “9

2

1 Blank sponge

)

sp0”ges

plasma

Treatments

1.

Days before

Table

r\ 0.30

1 PC

insertion of the

(t) and sponges.

removal

(0.

4

2.33

2.61

2.96

6.42

3.86

7.10

either

1.22

14

1.17

2.17

4.06

I .96

1.67

1.20

3.93

0.30

0.30

4.41

adminisLered

3.58

3.08

3.02

6.54

0.30

0

ewes

2.04

4.86

7

0.30

my?,

PG treated)

2.49

2.68

3.61

3.44

7.25

3.36

5.97

10.05 5.75

0.30

0.30

6

0.30

2.29

5

(superovulated

1.89

0.74

I

Prostaglandin

4.88

3.52

1.03 0.96

1.53

5.60

2.84

1.64

1.42

0.69

A

1 PG

2 PC

5.87

5.00

8.37

0.85

1.74

3

Group

2.55

3.57

I 1.49

of

1.88

3.75

0.30

A

10.40

I PG

0.30

2 PG

2 PG

I -

0.30

0. 30

7.23

0.30

0.30

0.53

AO.30

2

1

IPC

0

concentrations

2 PG

progesterone

d

(1

1.95

4.21

1.41

1.44

0.98

3.02

0.37

0.30

9

PGI

during

or,

1.11

3.34

I.39

0.62

0.81

3.32

0.56

0.30

10

14

11

d

1.26

1.84

2.57

0.44

0.30

I.18

1.89

0.54

treatment

12

and

2 d

~1.69

I 3.13

~2.46

I 1.93

v.72 I

(2

0.30

PGI

0.35

0.30

0.30

0.30

0.30

0.30 0.30

0.30

0.30

2.68

0.30

+2

0.30

0.30

2.50

0.30

+1

progesterone

1.83

V1.82

I 1.44

with

THERIOGENOLOGY

The addition of citric acid to the P in the sponges lowered the amount of P reaching the plasma in all ewes treated (Figures 1 and 2). This decrease did not mask the higher P levels detected in ewes following the single PG while P sponges were in situ, when compared to the double PG treated ewes (Table 1). Group II Two ewes in Group II (n = 16) lost sponges during included in the analysis of the anestrous treated animals.

the treatments

and were not

Table 2 shows the plasma P levels, (mean * SEM, ng/ml) throughout the blood sampling period (flay 0 --> 12, to Day +2) for the 14 remaining ewes in Group II. Progesterone levels remained low throughout the sampling period, with mean hormone concentrations of approximately 0.30 ng/ml in both the blank sponge treated ewes and the four untreated control animals. These results confirm the anestrous state of the ewes in Group II because the P concentrations (Table 2) are at the lower limits of the RIA (sensitivity limit 0.3 ng/ml plasma). Treatment 2 with 750 mg progesterone impregnated sponges of Group II animals over a 12-d period shows that the progesterone levels (mean + SEM, n = 4) rose sharply (P~0.05) .in the four ewes by the first day of sponge treatment from the < 0.30 ng/ml observed on Day 0 to approximately 7.0 ng/ml on Day 1 (Table 2, Figure 3). The plasma progesterone profiles of these ewes presented a synchronized response, but a plateau period olccurred between two major peaks, Day 1 and Days 11 to 12, respectively. The concentration of P during the sponge treatment period maintained plasma progesterone levels > 2.0 ng/ml over the interval between both peaks. The second major (Pt0.10) plasma progesterone peak (Days 11 to 12) was manifested before the time of sponge removal (Figure 3). The progesterone concentration in the plasma fell significantly (P~0.05) to very low levels following withdrawal of the sponges and remained low for 2 d. The plasma P concentrations (mean i SEM, n = 3) obtained for the ewes inserted with 750 mg ? plus 5% citric acid impregnated sponges (Treatment 3) over a 12-d period significantly lowered (PcO.01) the overall concentrations of peripheral plasma P (when the mean plasma P levels of Treatment 2 and 3 from Day 1 to 12 of the treatment period were compared) but the two major peaks on Day 1 and Days 11 to 12 (PCO.01 and P
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Plasma

Mean

0.02

SEM

0.09

1.57

6.84

0.00

0.30

1.01

3.50

0.00

0.30

0.00

0.30

2

0.09

2.01

0.15

3.09

-

0.00

3.18

-

-

0.00

0.23

0.34

2.99

0.00

0.30

0.00

0.30

6

7

0.57

3.03

0.00

0.30

0.12

8

0.55

3.27

0.00

0.30

0.56

2.71

0.00

0.30

0.04

0.34

9

treatment

0.07

0.37

during

0.42

ewes

0.21

1.47

0.00

0.30

0.14

0.44

10

with

1.76

6.46

0.00

0.30

0.00

0.30

II

12

V1.31

5.43

I Vo.00

0.30

vo.00

0.31

progesterone

0.14

2.29""

0.08

2.69'

0.38

2.60

0.15

2.13

0.16

1.82

0.40

2.53

0.11

1.77

0.25

3.00

vo.37

4.46

0.30

0.60

0.03

0.33

0.00

0.30

0.00

0.30

+I

sponges

2.5-6.3,3.5-4.4,2.1-3.8,1.9-4.5,l.7-3.9,1,7-4.2,1.0-2.0,3.5-l1.0,3.0-P.1,0.3-0.4

0.23

3.95"

0.01

0.31

0.00

0.30

5

Canrstrous)

4.22":'

0.00

0.30

0.00

0.30

4

II

3.3-4.7,1.8-2.1,2.9-3.4,2.0-2.5,2~6-2.8,2.0-3.3,~.9-2.4,~.5-2.~,2.1-3.3.l.6-2.0,2.7-3.5,3,9-5.1.0.3-1.2

0.40

4.01

3

Group

0.30

of

Days of sponge insertion (t) and removal (1). "P
-

Range

0.30

0.00

A

0.3-0.7,3.5-10.0,1.9-6.4

SEM

"=a

Hean

Range

(750 mg)

i+zleanAo.39

SEM

A

0.32

n=3

Fka"

0.00

0.30

0.37

0.04

A

SEM

n=4

1

concentrations

0

progesterone

n=4

2

treated

2.

Prcqesterone

Not

Table

-

0.00

0.30

-

0.00

0.30

0.00

0.30

0.00

0.30

+2

THERIOGENOLOGY

Plasma 6 progesterone

I ng / ml ) 4

0

2

4

6

8

10

12

+2

Day of sponge insertion Figure 3. Mean plasma progesteronelevels (* SEM, Group II) beforeA during a, blank sponge (n = 3); before n and during q , 750 mg progesteronesponge in : 4); and before l and during 0, 750 mg progesterone+ 5% citric acid sponge (n = 3) insertion as measured by RIA.

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I-HERIOGENOLOGY

between-animal uniformity in the mean plasma P levels period (Table 2. Figure 3).

per day throughout

Comparison of RIA and EIA as Monitors of Plasma Progesterone

the treatment

Profiles

Throughout this entire study the hormone concentrations of Groups I and II obtained by RIA and EIA were paired as measured per day for each ewe treated. The plasma progesterone profiles measured in all ewes of both groups were reflected similarly by both RIA and EIA. Figure 4 illustrates the paired RIA and EIA progesterone profiles obtained in a ewe that required the second PG injection and treatment with a 750 mg progesterone sponge for 12 d. Figure 5 shows the correlation of plasma progesterone concentrations by RIA and EIA in one of the anestrous ewes treated with a 750 mg The correlations shown (Table 3) between RIA and EIA were progesterone sponge. significant (PtO.OO1 to Pt0.05) for each ewe with the exception of one animal in Group I (Pt0.20). In Group II, the >2.0 ng progesterone/ml (RIA) maintained by sponge Treatments 2 and 3 between the two major peaks equated to >l.O ng/ml (EIA) using the equation - RIA = 1.45EIA + 0.64, obtained by linear regression (P
DISCUSSION Throughout this study of plasma progesterone levels that arise from hormone impregnated sponge treatment of sheep, the techniques of EIA and RIA have been compared. The EIA and RIA of progesterone in the extracted plasma resulted in significantly (P 14.0 ng/ml) in these animals. The second PG (12 d later) suppressed luteal function in these ewes (n = 2) and in the remaining 2 ewes that had shown basal progesterone following the first PG. The latter four ewes manifested a renewed secretion of progesterone during this 12-d interval before sponge insertion (Figure 2).

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16

Plasma progesterone

t ng / ml )

PG

Figure 4.

f

PG

/

Day of sponge insertion

Comparison of plasma progesteronelevels in Ewe 216 measured by RIA and EIAM (treatedwith 750-mg progesterone sponge). Correlation coefficient (r) : 0.875.

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315

THERIOGENOLOGY

Sponge out

Plasma progesterone ( ng / ml

)

k

0 Day of

Figure

316

5.

sponge

. lb

. l-2

-

+i

insertion

Comparison of plasma progesterone level s in Ewe 8 measured and EIA W (treated with 75C+mg progesterone sponge). Correlation coefficient (t-j = 0.816.

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l

E

5;

2

:

>

Group II

Group I

Progesterone 750 mg + 5% Ltr1c acJ

Progesterone (750 mg)

2

Progesterone 00 & ZJ

Progesterone (750 mg)

2

Blank

1

Treatments

21 20

13 14

0.843 0.665 0.609

12

0.576 0.215

1 PG 2 PG

20 21

0.792

0.832 0.935

1 PG 2 PG

19 21

14 13 13

0.858 0.875

1 PG

2 PG

(df) 21 20

Degrees of freedom

0.816 0.866 0.796

0.695 0.785

ir)

Correlation coefficients

PG 2 PG

1

Prostaglandin injection

EIA

F

s

0

8

E

0.01 0.05

and RIA

0.001

0.001

0.001 0.001 0.001

0.01 0.20

0.001 0.001

0.001 0.001

(P-==) 0.001 0.001

Levels of significance

Table 3. Correlation coefficientsbetween plasma progesteronelevels in treated ewes measured by

THERIOGENOLOGY

Following both single and double PG treatments, the ewes subsequently treated with blank sponges showed small increases ((3.0 ng/ml) in P for 2 to 4 d (Figures 1 and 2). The single PG treated ewes showed a sustained level of progesterone during hormone administration when compared with ewes sponge treated after the double PG regimen in Group I. Citric acid (5% and 10%) impregnation of the 750-mg progesterone sponges reduced the amount of P (Table 1) measured in the plasma whether this type of sponge treatment followed the single or double PG injection. However, the plasma hormone profiles observed in the ewes characteristic of sponge treatments following one or two PG were mirrored during citric acid supplementation of the treatments at these lower plasma P levels. The Group I study requires further investigation on a larger number of ewes to clarify whether the longer duration of high plasma progesterone levels observed in sponge-treated ewes after a single PG injection (both in blank sponge and hormone treated animals) is evidence of residual PMSGactivity, persistent large follicles and/or The CLs in a nonresponsive state (17,18) synergistic or otherwise with PG effects. endogenous patterns in P profiles of the ewes indicate that the hormone level during These latter two parameters sponge insertion varies in magnitude and duration. determined by the physiological/endocrinological state of the ewes at the start of the treatment are factors that require further investigation. The absence of plasma P in the controls confirmed the anestrous state of Group II ewes. The anestrous ewes that received the progesterone (750 mg with and without citric acid) sponges showed synchronization of the initial (Day 1) and final (Days 11 to 12) peaks. It is also apparent that even when P was assayed on a daily basis, hormone peaks could be missed in the plasma (Figure 3). resulting in a truncated peak at Days 11 to 12 in Group II. Between the two major peaks during the hormone treatment of anestrous ewes, the mean peripheral hormone profile exhibited a plateau-like (about 2.0 ng/ml) The major peaks remained synchronized despite the between-ewe variation interval. observed in the mean hormone levels in Treatment 2, where P was administered alone. This variation was much reduced with citric acid impregnation (Treatment 3) in Group II. However, as in Group I ewes, this supplementation with citric acid did not alter the characteristic peak:trough plasma progesterone profiles observed in any of the sheep treated, regardless of their physiological/endocrinological state at the commencement of sponge treatment (Groups I and II). In the natural estrous cycle of the ewe, the normal life span of the CL shows a peak:trough-like pattern in circulating P (19). which varies depending on breed of ewe being studied (20). Two major peaks in plasma P levels were also manifested in the anestrous ewe at the beginning and end of a progesterone implant GnRH treatment (2). The anestrous ewe may have a controlled feed-back mechanism operating, susceptible at fixed time intervals to alterations in the endocrinological environment (progesterone in the present study), which relates to the waves of follicular growth reported to occur in out-of-season ewes (21). A more recent report in cyclic ewes suggests that progesterone secretion may have its pattern of luteal activity predetermined by both gonadotropic feed-back from the pituitary and follicular luteinization in the ovine ovary (22). Pituitary control may occur in anestrus.

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THERIOGENOLOGY

Similarities observed between the dose responses by cyclic (6) and anestrous (5) ewes, although the latter were more sensitive to the exogenous hormone, suggest that ewes in both physiological/endocrinological states have a rhythmic or predetermined way (at fixed time intervals) of reacting to this hormone when administered from an exogenous source. This has been demonstrated recently (23) using controlled internal drug releasing (CIDR) devices in cyclic ewes. The 69% conception rate following 1000 mg progesterone sponge treatment (5) and 73% conception rate following 1125 mg progesterone silastic implant GnRH treatment (2) may be indicative of the predictability of anestrous ewes to exogenous progesterone administration (both treatments accompanied by gonadotropic stimulation), these ewes being in similar endocrinological/physiological states. Our study (using 750 mg P) confirms that the timing, magnitude and duration of progesterone concentration in ovine plasma retains a uniform profile when the hormone is administered during anestrus (Group II). Plasma P profiles during sponge treatment of ewes that had been subjected to a PMSG/‘PGpretreatment prior to progesterone sponge treatment are different from those profiles observed in sponge-treated anestrous ewes. The reasons for this variability of response to progesterone sponge treatment between Groups I and II requires further study to resolve which factors are dominant in producing the altered plasma P profiles, residual PMSG,number and timing of PG injections or other factors. It has been reported that a luteal phase prior to estrus is required in sheep to ensure survival of embryos (24). The variable fertility reported to follow treatment of flocks of sheep (cyclic and anestrous) with progestagen and progesterone (1,3) sponges might be resolved, and a uniform optimum fertility ensured if the patterns of induced luteal function could be equated to their subsequent fertility potential. This direct correlation of luteal function (in terms of plasma progesterone profiles), prior to conception, to the subsequent lambing rates of individual ewes should eliminate incorsistent fertility.

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McLeod, B.J.. Haresign, W. and Lamming, G.E. Response of seasonally anoestrous ewes to small-dose multiple injections of GnRH with and without progesterone J. Reprod. Fertil. pretreatment. -65:223-230 (1962).

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Meinecke-Tillmann, S., Evers, P. and Meincke, B. PMSG-plasma concentrations and ovarian response after in Merino ewes. Theriogenology: 27:259 (1987).

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Moore, N.W. and Miller. B.G. The use of the ovariectomised ewe in studies on survival of embryos during early pregnancy : A role for progesterone preceding conception. m: Lindsay, D.R. and Pearce, D.T. (eds.). Reproduction in Sheep. Cambridge University Press, Cambridge, 1984, pp. 112-114.

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