Embryo production and endocrine response in ewes superovulated with PMSG, with or without monoclonal anti-PMSG administered at different times

EMBRYO PRODUCTION AND ENDOCRINE RESPONSE IN EWES SUPEROVULATED WITH PMSG, WITH OR WITHOUT MONOCLONAL ANTI-PMSG ADMINISTERED AT DIFFERENT TIMES G. Martemucci,

1 A. D’Alessandro, *a F. Toteda,’

A.M. Facciolongol

and

M. Gambacortal lDipartimento di Produzione Animale degli Studi di Bari, Via G. Amendola, 165/A 70126 Bari, Italia ZDipartimento di Scienze Animali, Vegetali e dell’Ambiente Universita de1 Molise, Via Cavour, 50 86100 Campobasso, Italia Universita

Received

for publication: Accepted:

May March

14,

1993 20,

1995

ABSTRACT Mature nonlactating Altamurana ewes (n=168) were synchronized in the seasonal anestrus period with FGA-impregnated intravaginal pessaries for I2 d. In Experiment 1, 48 ewes were divided into a 3 x 4 factorial design for anti-PMSG monoclonal antibody (AP) bioassay test. Concomitant injections of PMSG (1000, 1500, 2000 IU) and AP (0, I, 2, 3 ul/IU PMSG) were given, and ovarian response was evaluated by laparoscopy. In Experiment 2, 120 ewes were divid’ed into 8 experimental groups (n = 15 per group). The ewes treated with 1000 or 1500 IU PMSG at -24 h from sponge removal were given AP intravenously at 50 h after pessary withdrawal, 12 or 24 h after the onset of estrus, while the controls did not receive AP. Blood samples were collected from ewes (n = 6) treated with 1500 IU PMSG with or without antiPMSG. Ovarian response and embryo production were evaluated on Day 7 after sponge removal upon laparotomy. It was found that I ltl AP was effective in neutralizing 1 IU PMSG. No significant differences in serum concentrations of progesterone were observed among the groups of superovulated ewes, Estradiol- 17 B levels were reduced following AP treatment 12 h after the onset of estrus. At a lower dosage of superovulatory treatment (1000 IU PMSG), AP injected at 12 or 24 h after the onset of estrus significantly lowered large follicles (P
superovulation,

PMSG, monoclonal

antibody,

embryo production,

ewes

Acknowledgements Research was supported by the National Research Council of Italy, Special Project RAISA, Sub-project N. 3, Paper N. 1750. Authors thank Mr. L. Basso for technical assistance during surgical collection of embryos and Mr. L. Bongermino for the statistical processing analyses. aReprint requests. Present address: Dipartimento di Produzione Animale - Via G. Amendola, 65/A - 70126 Bari, Italia. Theriogenology 44:691-703, 1995 CJ 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0093-691 X/95/$1 0.00 SSDI 0093-691X(95)00249-9

Theriogenology

692 INTRODUCTION

Superovulation is a critical step in embryo transfer success. Several gonadotrophins have been utilized for superovulation in sheep, but PMSG has been one of the most widely used (1, 15, 18, 24), with the major advantage of PMSG being that it requires only one injection. However, it has been demonstrated that PMSG results in a highly variable response in sheep (13, 18, 24) and reduces both recovery rates and yield of transferable embryos (1, 15). This has been attributed to the long half-life of PMSG in the systemic circulation, which induces a second postovulatory wave of preantral and/or small antral follicle growth (4, 16) along with high levels of steroid production by follicles (17). It has been suggested that the adverse effect of PMSG on animal fertility could be minimized if estrogen concentrations were reduced (25). This could be achieved by neutralizing PMSG after sufficient follicular stimulation by injection of anti-PMSG as suggested by Bindon and Piper (3). In cows, administration of a monoclonal antibody to PMSG improves superovulatory response when administered at the onset of estrus or immediately after the LH peak (7, 8, 9), while other studies have shown inconclusive results (5, 28). The present study was designed to investigate 1) PMSG neutralization activity of monoclonal antibody anti-PMSG in superovulated ewes and 2) the ovulatory, endocrine and embryo quality response to time of anti-PMSG injection in ewes treated with 2 dosages of PMSG. MATERIALS AND METHODS Anti-PMSG Bioassay Mature nonlactating ewes (n=48) were treated in February to March with fluorogestone acetateimpregnated vaginal sponges (FGA, 30 mg; Intervet, Milan, Italy) for 12 d. The ewes were randomly assigned to 12 treatment groups in a 3 x 4 factorial design: 3 PMSG doses (1000, 1500 and 2000 IU) x 4 doses of monoclonal antibody against PMSG (0, 1, 2 and 3 pVIU PMSG). Saline was given to control ewes treated with 0 unit of anti-PMSG. The PMSG (Folligon, Intervet) was injected intramuscularly 24 h before pessary removal, and monoclonal anti-PMSG (Neutra-PMSG; Intervet) was administered intravenously immediately after PMSG injection. Ovarian response was determined at Day 7 after sponge removal by counting the number of corpora lutea by laparoscopic procedures previously described (12). The dosage of PMSG (IU) was plotted against the mean corpora lutea for each unitary dose of AP used (Figure 1). Superovulatory

Treatments and Sampling Procedures

Mature nonlactating Ahamurana ewes (n=120) were used in March, corresponding to the seasonal anestrus period (14). Estrus was synchronized with FGA-impregnated intravaginal pessaries left in situ for 12 d. The ewes were assigned at random to 1 of 8 treatment groups corresponding to the following factorial design. The factors were either PMSG (Folligon; Intervet) or PMSG plus monoclonal anti-PMSG (Neutra-PMSG; Intervet). The PMSG was administered at 2 dose levels (1000 or 1500 IU), and anti-PMSG was injected intravenously at 3 times (50 h after pessary removal or at 12 or 24 h after the onset of estrus of each ewe). Two groups received 1000 or 1500 IU PMSG alone and served as the controls. The PMSG was administered intramuscularly 24 h before pessary withdrawal. Since 1 pl anti-PMSG neutralized

693

Theriogenology

10 t?

9 --

$2

8-m 7--

-Control --o-. ljtI/IU PMSG 2ul/IU PMSG -+- 3ul/IU PMSG

6 ~~ 5 -4 -3 -2

**

/ *I\

1 v 0-c

0

** *xc-----t-- .---

**

.a ** ._____..-..~-

& I

_---_--____-__--+__&.~-___-_-___-_-T

1000

1500

2000

Dose of PMSG (IU) Figure 1. Inhibitory effect of anti-PMSG (Neutra-PMSG) on the ovulation superovulated with PMSG. ** Significantly different from the control (P
rate in ewes

the biological effect of 1 IU PMSG, the use of 1 and 1.5 ml anti-PMSG was considered sufftcient to neutralize 1000 and 1500 IU PMSG, respectively. Occurrence of estrus was monitored by teaser rams every 2 h starting from 16 h after sponge removal. All ewes detected in estrus were hand-mated every 6 to 8 h until they refused to mate. Hormonal profiles were performed on 6 ewes per group treated with 1500 IU PMSG with or without anti-PMSG. Blood samples were collected by jugular venipuncture at 12-h intervals from 24 h before pessary withdrawal until Day 3 after pessary removal and daily until Day 7 for estradiol-17 I3 assay. Progesterone concentrations were evaluated of the daily blood collection samples. The blood was drawn into lo-ml evacuated tubes (Becton - Dickinson Vacutainer Systems, Meylan Cedex, France). Serum was aliquoted after centrifugation and then stored at 20°C. Serum levels of estradiol-I7 I3 (Ez) and progesterone (P4) were determined by radioimmunoassay. Embryo Collection On Day 6 following estrus a ventral midline laparotomy was performed, the reproductive tract was exteriorized, and ovarian response (number of corpora lutea, number of large unruptured follicles > 4 mm) was assessed. Embryos were recovered by antegrade flushing of each uterine horn with modified PBS (26) supplemented with 10% fetal calf serum (FCS). The medium was delivered through a 20gauge intravenous catheter inserted in the tip of the horn and recovered by a glass catheter at the base of the uterus in sterile glass vessels (15). Ova were collected from the glass vessels under a stereomicroscope (x 40) after which the embryos were held in modified PBS (26) containing 20% FCS until evaluated. The collected ova were evaluated by phase contrast inverted microscopy (x 225) and classified as either nonfertilized (oocytes) or embryos.

694

Theriogenology

The embryos were classified for developmental stage as follows: 2 to 4 cells, 5 to 16 cells, monrla, early blastocyst, blastocyst, expanded blastocyst and hatched blastocyst. Embryo quality was evaluated under phase contrast inverted microscopy (x 225) and scored as excellent, good or poor according to morphological criteria previously described (13). Embryos at 2 to 4 cells were considered poor (13). Only excellent- and good-quality embryos were assessed for viability by their growth in vitro for 24 h. Embryos were placed in Whittingham’s modified PBS enriched with 20% FCS, refiltered before use (23) and held in an atmosphere of 5% CO,, 5% 02, 90% Nz (22) in an incubator (Labotect md 3015, Labor Technik, Gottingen, Germany) at 37 “C. Embryos collected at 5 to 16 cells, montla and early blastocyst stages were considered viable if after 24 h of incubation they developed into morulae, early blastocysts-blastocysts, expanded blastocysts and hatching blastocysts, respectively. Hormone Assay Serum concentrations of progesterone (PJ were estimated by a direct solid-phase tz51RIA method (Coat-A-Count TKPG; Diagnostic Products Corporation - DPC, Los Angeles, CA USA) in lOO+l samples in duplicate, according to the manufacturer, with the sensivity of 0.03 @ml. The inter-assay coefficients of variation were 13.3, 7.4 and 3.5% for the 3 commercial serum pools (1.5, 3.4 and 19.0 @ml, respectively; DPC, Los Angeles, CA USA); the intra-assay coefficients of variation were 6.6, 3.0 and 2.1%, respectively, for the same pools (n = 8). Estradiol-17 R (Ez) was assayed by the previously described RIA method (10). Briefly, serum samples of 5.0 ml were extracted by C2 columns (Bond Elut, Varian, Harbor City, CA USA) using 3.0 ml hexane:ethyl acetate 70:30 (v/v). After evaporation of the solvent, the residue was dissolved in 0.3 ml buffer (0.05 M - phosphate/ EDTA buffer, pH 7.4 + 0.1% BSA + 0.1% NaN,) to which 0.1 ml (- 30 pg/tube) 2, 4, 6, 7 - H3 - estradiol-17 l3 (Amersham, Milan, Italy) were added. Anti-estradiol-17 g-6 CMO-BSA antiserum (Analitycal Antibodies; Milan, Italy) was used at working dilution 1:lOOOO. The main cross-reactivities were 100, 1.6, 0.6 and 1.8% for estradiol, estrone, estriol and estradiol-17 CL,respectively; 4 mm, number of ova collected, number of ova fertilized, number of transferable embryos); and c), serum progesterone and estradiol measures were analyzed by least squares analysis of variance using the GLM procedure of SAS (20). The model utilized for the above was

Theriogenology

695

Yiik = p ’ ai + pj + "pij f

&ijk

where u = overall mean; CL= effect of PMSG dosage for a) (i = l-3) and b) (i = l-2); 0 = effect of anti-PMSG dosage for a) (j = l-4); effect of anti-PMSG treatment for b) (j = l-4); c$ = interaction; e = error. Data for c) were analyzed by following model for repeated measures Yijk

=

p

+

c(i +

hij

+

@k+ Apia +

Eijk

where p = overall mean; c1= effect of anti-PMSG treatment (i = l-4); h = animal within treatment (error for main effect of anti-PMSG treatments) (i = I-24); p = effect of sampling time for P, (k = l-9) and Ez (k = 1-12) ; $3 r interaction; E = error. Least squares means were compared by the predicted difference (PDIFF) option of GLM (20). Data of percentages of ova fertilized, number of embryos in relation to development stage and embryo viability rate were analyzed by the chi-square test (20). The F-test was utilized to compare synchronization degree of the onset of estrus (21). RESULTS Neutralization test results showed that anti-PMSG significantly (P 4 mm) was influenced (PcO.01) by doses of both PMSG and anti-PMSG, with a significant interaction of PcO.05. Use of 1000 rather

15

No. of ewes ovulating

1.5* 0.46

No. of follicles > 4 mm

5.1+ 0.84e O.l* 0.47%

0.4+ o.4d

24.3+ 1.19

14

15

15

24 hoursb

5.3+ 0.81e

22.7+ 1.19

15

15

15

12 hoursb

1000 IU

1.9+ 0.46c

3.1 l o.s1fg

23.35 1.19

15

15

15

Control

5.3 f 0.87 1.5* 0.49

1.8* 0.46

24.5+ 1.27

13

13

13

12 hoursb

6.5k 0.81

22.3* 1.19

15

15

15

50 hoursa

2.2k 0.46h

5.7k 0.81

23.1zt I.19

15

15

15

24 hoursb

1500 IU

;Hours after sponge removal. cIJours after the onset of estrus. Differences among anti-PMSG treatments (cd P
3.5* 0.81

No. of corpora lutea

22.0* 1.19

15

Onset of estrus (hours)

15

No. of ewes in estrus

50 hoursa

No. of ewes treated

Anti-PMSG administration

PMSG dose

2.7* 0.47

6.6+ 0.84h

25,9+ 1.19

1.5

15

15

Control

Table 1. Ovarian response following monoclonal anti-PMSG treatment at different times in superovulated ewes by 2 doses of PMSG (values are means k SEM).

Theriogenology

697

than 1500 IU PMSG induced fewer large follicles on the ovaries. Furthermore, in ewes receiving 1000 IU PMSG, the anti-PMSG significantly decreased (PPP
3.1 f o.49ce (82.l)e

1.7* o.49f

(67.5)g

(72 4)i (58.84

3.1 k o.51cei 1.3 + o.49d

a Hours after sponge removal. b Hours after the onset of estrus. cFf Differences among anti-PMSG treatments (cd P
Viable embryos

(1oo)cg

(96s)e

(82.2)d9

(86.8)d

2.2 f OBf

4.1 AZ0.60e

3.7 * 0.58

2.5 * 0.58f

Ova fertilized

2.5 f 0.63

4.1 f 0.65

3.9 f 0.63

3.0 f 0.63

Ova recovered

Control

24 hoursb

1000 IU 12 hoursb

50 hoursa

Anti-PMSG administration

PMSG dose

(z8.3)dm

0.9 f 0.49f

(82. I)f

3.5 f 0.58

4.1 * 0.63

50 hoursa

1.7 f 0.55 (62.2)c

1.8 f 0.55’ (5o.o)el

2.3 f 0.49e (73.4)C

(90.2)

2.9 i 0.65

3.2 f 0.71

Control

(78.6)dh

3.1 * 0.58

4.5 f 0.63

24 hoursb

(1oo)ce

3.1 + 0.65

3.4 f 0.71

12 hoursb

1500 IU

Table 2. Ova recovery and embryo production following monoclonal anti-PMSG treatment at different times in superovulated ewes by 2 doses of PMSG (values are means * SEM; percentages in brackets).

Phefiogenology

699

-----c.---

Control AP5OhpostFGA Ap 12 hpostestrus AP 24 h post estrus

20 18 16

-2

-1

0

1

2

3

4

5

6

Days from Estrus

Figure 2. Sequential patterns of mean (a) progesterone and (b) estradiol - 17p levels in ewes (n=6) superovulated with 1500 IU PMSG with or without monoclonal anti-PMSG (AP) administered at 50 hours after sponge removal (FGA) or at 12 or 24 hours after the onset of estrus (O=Day of estrus). Significant differences within each treatment: a vs b, a vs c, b vs c, P
Theriogenology

700

Levels of P, were influenced (PcO.05) by sampling time. The pattern of progesterone levels was regular, with values of P4 below 0.5 @ml until Day 1 after estrus followed by a very rapid increase on Days 2 to 6 post estrus (PP
Theriogenology

701

monoclonal anti-PMSG did not alter events leading to ovulation. Moreover, it could be possible to prevent PMSG-induced disorders during the final follicular maturation process that leads to an increase in the ovulation rate (8). Addition of anti-PMSG to the PMSG treatment did not reduce individual variability in the number of ovulations within donor ewes, In our study, treatment with anti-PMSG decreased the number of large follicles in ewes superovulated with 1000 II-l PMSG, especially if performed at 12 or 24 h after the onset of estrus. The high number of these large follicles in PMSG-superovulated ewes has been attributed to the long half-life of gonadotrophin and its continous stimulation of follicular recruitment and growth (27), thus suggesting that anti-PMSG injected at fixed times after signs of estrus effectively neutralizes the residual circulating PMSG, thereby preventing additional growth of small follicles into large follicles. Administration of monoclonal anti-PMSG at fixed times after the onset of estrus also significantly increased the mean number and rate of fertilized ova and viable embryo yields in ewes superovulated with 1000 IU PMSG, but the response was variable among the ewes. The positive effect of anti-PMSG on embryo production can be attributed to the absence of a highly estrogenic environment during early embryonic development. It has been reported that the presence of circulating PMSG during early embryonic development may adversely affect this development either directly or through stimulation of a second follicular wave after ovulation, producing high concentrations of estradiol (2, 4). When the higher dose of PMSG (1500 IU) was used, the anti-PMSG injection did not improve transferable embryo yield, perhaps because of the limited control of PMSG on the higher numbers of growing stimulated follicles, due to the inappropriate time of its administration. In conclusion, this study indicates that addition of anti-PMSG to the superovulatory treatment of 1000 IU PMSG improves ovarian response and transferable embryo yield when performed at fixed times following the onset of estrus, thus demonstrating that PMSG can serve embryo production if the duration of its biological activity is controlled by an anti-PMSG antibody. Further experiments on the optimal injection time of anti-PMSG with high dosages of PMSG may well serve to clarify the possibilities for superovulation control in sheep. REFERENCES 1. Armstrong DT, Evans G. Factors influencing success of embryo transfer in sheep and goats. Theriogenology 1983; 19: 31-42. 2. Bevers MM, Dieleman SJ. Superovulation with PMSG: Follicular growth and the variation in concentrations in the peripheral blood of progesterone, oestradiol, LH, cortisol, prolactin and PMSG. Anim Reprod Sci 1987; 15: 37-52. 3. Bindon BM, Piper LR. Induction of ovulation in sheep and cattle by injections of PMSG and ovine anti-PMSG immune serum. Theriogenology 1977; 8: 171 (Abstr.). 4. Bouters R, Moyaert I, Corijn M, Vandeplasche M. The use of a PMSG antiserum in superovulated cattle: endocrinological changes and effects on timing of ovulation. Zuchthygiene 1983; 18: 172-177. 5. Callesen H, Bak A, Greve T. Use of PMSG antiserum in superovulated cattle. Theriogenology 1992; 38: 959-968.

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Theriogenology

6. Cognie Y, Pelletier J. Preovulatory LH release and ovulation in dry and in lactating ewes after progestagen and PMSG treatment during the seasonal anoestrum. Ann Bioch anim Bioch Biophys 1976; 16: 529-536. 7. Dhont D, Bouters R, Spingemaille J, Coryn M, Vandeplassche M. The control of superovulation in the bovine with a PMSG-antiserum. Theriogenology 1978; 9: 529-534. 8. Dieleman SJ, Bevers MM, Gielen JTh. Increase of the number of ovulation in PMSG/PG treated cows by administration of monoclonal anti-PMSG shortly after the endogenous LH peak. Theriogenology 1987; 27: 222 (Abstr.). 9. Dieleman SJ, Bevers MM, Wurth YA, Gielen JTh, Willemse AH. Improved embryo yield condition of donor ovaries in cows after PMSG superovulation with monoclonal anti-PMSG administered shortly after the preovulatory LH peak. Theriogenology 1989; 3 1: 473-487. 10. Facciolongo AM, Toteda F, Bolelli G, D’Alessandro A, Gambacorta M, Martemucci G Influenza de1 trattamento con Gn-RH o hCG sulla risposta ovarica ed endocrina e sulla produzione di embrioni in pecore superovulate con PMSG. Zoo Nutr Anim 1994; 20: 119128. 11. Kummer V, Zraly Z, Holcak V, Veznick Z, Schlegelova J, Hruska K. Superovulation of cattle: Effect ofgoat anti-PMSG serum. Theriogenology 1980; 14: 383-390. 12. Martemucci G, Gambacorta M, Bellitti E, Manchisi A, Toteda F. Controllo dell’ovulazione con PMSG e GnRH in pecore trattate con progestageno. Zoo Nutr Anim 1984; 10: 1 l-22. 13. Martemucci G, Gambacorta M, Toteda F, Manchisi A, Bellitti E. Induzione della superovulazione nella pecora con PMSG, FSH-P, hMG, per il trapianto di embrioni. Zoo Nutr Anim 1988; 14: 379-386. 14. Martemucci G, Manchisi A, Rifino F, Melodia L. Studio dell’attivita sessuale delta pecora “Altamurana”. Ann Fat Agr Univ Bari 1979/80; 3 1: 20 1-2 16. 15. Martemucci G, Toteda F, Manchisi A, Lacalandra G, Gambacorta M. Risposta al trattamento di superovulazione con PMSG in pecore di razza Gentile di Puglia e Altamurana. Feconditl successiva a trapianto di embrioni. Zoo Nutr Anim 1988; 14: 165-l 72. 16. Monniaux D, Mariana JC, Gibson WR. Action of PMSG on follicular populations in the heifer. J Reprod Fert 1984; 70: 243-253. 17. Moor RM, Osborn JC, Crosby JM. Gonadotrophin-induced abnormalities in sheep oocytes after superovulation. J Reprod Fert 1985; 74: 167-172. 18. Mutiga ER, Baker AA. Superovulation response, ova recovery and fertility in Merino ewes superovulated either during the luteal phase of their estrus cycle or after intravaginal progestagen treatment. Theriogenology 1982; 17: 537-544. 19. Robertson HA, Rakha AM. The timing of the neural stimulus which leads to ovulation in the sheep. J Endocrin 1965; 32: 383-386. 20. SAS Institute Inc. SAXSTAT Guide for Personal Computers, version 6 Edition, Gary, NC: SAS Institute Inc., 1987. 21. Snedecor GW, Cochran WG. Statistical methods. 7th ed., The Iowa State University Press, Ames, USA, 1980. 22. Tervit HR, Whittingham DG, Rowson LEA. Successful culture in vitro of sheep and cattle ova. J Reprod Fert 1972; 30: 493-497. 23. Trounson AO, Willadsen SM, Rowson LEA. The influence of in-vitro culture and cooling on the survival and development of cow embryos. J Reprod Fert 1976; 47: 367-370. 24. Walker SK, Smith DH, Little DL, Warnes GM, Quinn P, Seamark RF. Artificial insemination and transfer of embryos by laparoscopy. In: DR Lindsay, DT Pearce (eds), Reproduction in Sheep. Canberra: Austral. Academy of Science, 1984; 306309.

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25. Walton EA, Armstrong DT. Ovarian function and early embryo development in immature rats given a superovulatory dose of PMSG later neutralized by antiserum. Biol Reprod 1981, 25: 272-280. 26. Whittingham DG. Survival of mouse embryos after freezing and thawing. Nature, London 1971; 233: 125-126. 27. Wise TH, Vernon MV, Maurer RR. Oxytocin, prostaglandin E and F, estradiol, progesterone, sodium and potassium in preovulatory bovine follicles either developed normally or stimulated by follicle stimulation hormone. Theriogenology 1986; 26: 757-778. 28. Zeitoun MM, Yassen AM, Hassan AA, Fathelbab AZ, Echternkamp SE, Wise TH, Maurer RR. Superovulation and embryo quality in beef cows using PMSG and monoclonal antiPMSG. Theriogenology 1991; 35: 653-667.