ELSEVIER

EFFECTS OF FSH COMMERCIAL PREPARATION AND FOLLICLJLAR STATUS ON FOLLICULAR GROWTH AND SUPEROVULATORY RESPONSE

IN SPANISH MERINO EWES

A. Gonzalez-Bulnes,a J. Santiago-Moreno, M.J. Cocero and A. Lopez-Sebastian

Dpto. de Reproduction Animal. SGIT/INIA (MAPA) Avda. Puerta de Hierro Km. 5,9 28040-Madrid (Spain)

Received for publication: November 16, 1999 Accepted: May 1, 2000

ABSTRACT

Ovarian follicular development was characterized in 24 Spanish Merino ewes to study effects of the follicular status and the FSH commercial product used on follicular growth and subsequent superovulatory response. Estrus was synchronized using 40 mg fluorogestone acetate sponges. The superovulatory treatment consisted in 2 daily im injections of FSH from 48 h before to 12 h after sponge removal. Sheep were assigned randomly to 2 groups treated with 6 decreasing doses (4, 4, 3, 3, 2, 2 mg) of FSH-PTM or with 6 doses of 1.25 mL of OVAGENTM. Growth and

regression of all follicles 22 mm were observed by transrectal ultrasonography, and recorded daily from Day 6 before sponge insertion to the first FSH injection, and then twice daily until estrus was detected with vasectomized rams. Differences were detected in follicular development from the first FSH injection to detection of estrus (-48 to 36 h from sponge removal) between groups. Administration of FSH-P increased the appearance of new follicles with respect to

OVAGEN (6.3 * 0.7 vs 4.8 t 0.4; P < 0.05). and the mean number of medium (4 to 5 mm) follicles

(8.9 f 1.2 vs 6.6 + 0.9; P < 0.05). However, the mean number of follicles that regressed in size after

sponge removal (5.9 .& 0.4 vs 3.3 f 0.4) and the number of preovulatory sized follicles that did not ovulate (60 vs 42.4%) were also higher in FSH-P treated ewes (P < 0.05). So, finally, there were no differences in ovulation rate, as determined by laparoscopy on Day 7 after sponge removal, between ewes treated with FSH-P or OVAGEN (6.3 + 1.9 vs 7.0 t 1.7 CL). In all the ewes, the ovulatory response was related (P < 0.05) both to the number of small follicles (2 to 3 mm in diameter) present in the ovaries at the start of treatment with exogenous FSH and to the number of

follicles that reached >4 mm in size at estrus, despite differences in the pattern of follicular development when using different commercial products. 0 2000 by Elsevw Science Inc.

Key words: follicular-growth, FSH/LH, sheep, superovulation.

Acknowledgments This work was supported in Spain by funds from CICYT (Project AGF 1460). A.G.B. and J.S.M. were supported by a research grant from the Ministry of Agriculture of Spain. Kind revision of the manuscript by Dr. E.K. Inskeep is gratefully acknowledged. a Correspondence and reprints requests: (E-mail: bulnesginia.es).

Theriogenology 54:1055-1064, 2000 0093-691X/00/$-see front matter 0 2000 Elsevier Science Inc. PII: SOO93-691X(00)00414-3

1056 Theriogenology

INTRODUCTION

Large variability in the superovulatory response to gonadotrophin treatments continues to be the main limiting factor in sheep embryo transfer programs (8). This high degree of variability has been associated with different factors such as the ovarian follicular status of the female or the gonadotrophin preparation used.

Studies of the influence of ovarian follicular status at the start of a superovulatory treatment in sheep showed that ovarian response to FSH is correlated positively with the number of small follicles on the first day of the superovulatory regimen, as described by Brebion and Cognie when using GnRH agonist pretreatment (4). The presence of a large follicle at the time of a superovulatory dose of eCG has been reported to decrease ovarian response (28) although a direct effect of large follicles on superovulatory response to eCG has not been observed when in vitro studies were performed (12).

Studies concerning the influence of gonadotrophin preparations have described a large variation in the ovarian response, which has been associated with the widely variable LH content in commercial FSH (17). High LH content was shown to decrease ovulatory response (7, 31), which led to use of highly purified ovine gonadotrophins. However, excessively low amounts of LH at the end of treatment also induced lower ovulation rates (7). Treatment with pure LH at sponge withdrawal during a superovulatory treatment with known FSH/LH ratio decreased variability in the ovulatory response without affecting mean ovulation rates (24). Both facts could be related to the role of LH in final follicular development. However, the influence of LH content on follicular growth during the FSH treatment and the influence of LH treatment on the intraovarian mechanism controlling follicular development in sheep are still not understood clearly.

We considered implications that could be applied in practice. This study was developed to elucidate possible effects of two different commercial products on follicular growth and subsequent ovulatory response. Possible interactions of FSH preparation with effects derived from the ovarian follicular status during the treatment also were assessed.

MATERIALS AND METHODS

Animals

The trial was developed during the beginning of the breeding season (June) and involved a total of 24 nonlactating Spanish Merino ewes, 4 to 6 years old. Ewes were maintained outdoors with access to indoor facilities at the experimental farm of the INIA in Madrid, Spain, latitude 40%.

Experimental Design

Estrus was synchronized with the insertion of intravaginal progestogen impregnated sponges (40 mg fluorogestone acetate, FGA, ChronogestR, Intervet International, Boxmeer, The Netherlands) for 14 days. The superovulatory treatment consisted of two daily intramuscular injections of FSH at 12-h intervals, starting 48 h before sponge removal and finishing 12 h after progestogen withdrawal. Sheep were assigned randomly to 1 of 2 similarly sized groups (Gl and

Theriogenology 1057

G2), but three of the ewes in G2 were taken off of the experiment during the FSH administration; so 12 ewes were in Gl and 9 were in G2. Gl was treated with FSH-PTM (Schering-Plough, Kenilworth, NY, USA) in 6 decreasing doses (4,4, 3, 3,2,2 mg) and G2 was injected with 6 equal doses of 1.25 mL of OVAGENTM (ICP, Auckland, N ew Zealand). Progestogen removal was coincident with the 5’ injection of FSH and estrus was detected with vasectomized rams between 36 and 48 h later.

Ultrasonic Equipment and Procedure

IJltrasonography was performed using an Aloka 500 SSD (Ecotron, Madrid, Spain), with a 7.5 MHz linear-array probe designed for examination of the human prostate. Observations were performed in the same manner as described by S&rick et al. (29) and validated for Merino ewes in

our laboratory by Gonzalez de Bulnes et al. (13). The largest diameter of each follicle 2 2 mm was measured and its position was recorded on a diagram of the ovaries to evaluate its development during successive observations. Ovarian ultrasonographies began on Day 6 before progestogen removal and finished when estrus was detected using vasectomized rams, with one observation per day until the first FSH injection and twice daily from this moment until estrous behavior.

Assessment of Ovarian Response

The number of corpora lutea (CL) was determined laparoscopically on Day 7 after estrus and was used as an index of superovulatory response.

Statistical Analysis

IJltrasonographic data showed patterns of follicular development, First, all follicles recorded by

ultrasonography were classified as large (26 mm), medium (4 to 5 mm) or small follicles (2 to 3 mm). After this, numbers of new follicles (not previously detected), and decreasing follicles (decreased in diameter from the previous day or disappeared) were determined. Finally, the size of the largest (LFl), the second largest (LF2) and the remaining follicles (RF) at each observation was recorded, without regard to the history and fate of these follicles, Data for each variable were examined by analysis of variance (ANOVA) for the effects of treatment (Gl and G2) and time within treatment. Several of these variables and ovulation rates were examined huther for effects of treatment and for relationships among variables after classification as to whether or not the animals showed a superovulatory ovarian response to the treatment. These relationships were assessed by Pearson correlation procedures. Comparisons of results expressed as percentages were performed by ANOVA of the transformed values for each individual percentage to the arcsine. Results were expressed as mean f S. E. M. and differences and correlations were accepted as statistically significant from P < 0.05.

RESIJLTS

The mean number of total (22 mm) follicles increased (P < 0.05) both in FSH-P and OVAGEN treatments, from the first FSH injection to detection of estrus (-48 to 36 h from sponge removal) due to a significant increase (P < 0.05j both in number of medium (4 to 5 mm) and large

(26 mm) follicles (Figure 1). The mean number of total follicles tended to be higher in ewes treated

1058 Theriogenology

with FSH-P (17.8 + 1.6 vs 14.5 k 0.9; P = 0.09). The mean number of medium follicles was higher

in FSH-P-treated than in OVAGEN-treated ewes (8.9 k 1.2 vs 6.6 + 0.9; P < 0.05), but the mean

number of large follicles (3.5 f 1 .O vs 3.0 + 0.9) did not differ.

Number of follicles

El Small II Medium W Large

25

20

I5

10

5

0

-48 -36 -24 -12 0 12 24 36 -48 -36 -24 -12 0 I2 24 36

B

Time (Hours) relative to sponge withdrawal

Figure 1. Mean number of small (2 to 3 mm), medium (4 to 5 mm), large (26 mm) and total

(22 mm) follicles in Spanish Merino ewes treated with FSH-P (A) or OVAGEN (B).

The mean number of newly-detected follicles tended to increase at 24 h after the first FSH injection (-24 h) and then remained constant until 24 h after progestogen removal in both groups

(Figure 2); however, there were more newly-detected follicles in FSH-P group (6.3 + 0.7 vs 4.8 + 0.4; P < 0.05). The mean number of follicles that regressed in diameter (Figure 3) increased from progestogen withdrawal to 24 h later in all the animals (P < 0.05); increase was greater (P < 0.05) in

FSH-P-treated (3.0 k 0.3 to 9.0 k 0.8) than in OVAGEN-treated ewes (2.5 :t 0.1 to 5.9 + 0.6).

Number of follicles -W-A-O-B Number of follicles

+A +B

10 -

8-

01 0-F -48 -36 -24 -12 0 12 24 36 -48 -36 -24 -12 0 12 24 36

Time (Hours) relative to sponge withdrawal Time (Hours) relative to sponge withdrawal

Figure 2. Number of newly-detected follicles Figure 3. Number of regressing follicles in

in Spanish Merino ewes treated Spanish Merino ewes treated with

with FSH-P (A) or OVAGEN (B). FSH-P (A) or OVAGEN (B).

Theriogenology 1059

The mean size of the largest follicle (LFl), the second largest (LF2) and the remaining follicles

t2 mm (RF) increased during the entire period of study in all the ewes (P < O.OS), but without differences in growth profiles between treatments (Figure 4).

Diameter (mm)

LFl - LF2 -_-mm RF-.-

I I I I

8- B

7-

5- - 4-

- _

--- e /

_--) 3- *

-_0

27 -48 -36 -24 -12 0 12 24 36 -_4g _36 _24 _12 o ,* 24 36

Time (Hours) relative to sponge withdrawal

Figure 4. Mean diameters of the largest (LFI), the second largest (LF2) and the remaining follicles (RF) in Spanish Merino ewes treated with FSH-P (A) or OVAGEN (B).

Estimation of number of corpora lutea (CL) showed that mean ovulation rates were similar in

ewes treated with FSH-P or OVAGEN (6.3 + 1.9 vs 7.0 ? 1.7 CL). While not a significant difference, four of 12 ewes in the FSH-P-treated group (33.3%) compared to 2 of 9 in the OVAGEN-treated group (22.2%) did not ovulate (nonresponding ewes). The remaining animals responded to the superovulatory treatment with a minimum of 4 CL (responding ewes); so no ewe had one, two or three ovulations. There was no difference in the ovulation rates of responding ewes

between treatments (mean 9.5 f 0.6 in FSH-P -treated ewes, 4 to 17 CL vs 9.0 ? 0.4 in ewes treated with OVAGEN, 4 to 13 CL).

Analysis of follicular status during the superovulatory treatment showed that the ovulation rate obtained was correlated with the number of small follicles at the first FSH injection, in both treatments (P < 0.05); the number of small follicles was higher in responding than in

nomesponding ewes (7.0 + 0.6 vs 1.2 f 0.3; P < 0.05) as highlighted in Table 1. On the other hand, no association was detected between ovulation rate and presence or persistence of a large growing follicle at start of treatment, Likewise there was no difference in the percentage of

females showing a large or persistent follicle between nonresponding (50%; 3 of 6) and responding ewes (46.6%; 7 of 15).

The number of CL obtained was correlated with the number of medium follicles at sponge

withdrawal (P < 0.05). However, the ovulation rate was related to the number of 24 mm (medium plus large) follicles, but not to the number of large follicles at detection of estrus (P <

0.05). This result indicates that a certain number of t4 mm follicles ovulated. There was no

difference in ovulation rate between groups despite the fact that there were more 24 mm follicles at estrus in FSH-P-treated than in OVAGEN-treated ewes (22.6 i 2.3 vs 15.0 * 2.2; P < 0.05).

1060 Theriogenology

Therefore, fewer of the >4 mm follicles ovulated in FSH-P-treated ewes (42.4% vs 60%; P < 0.05). Finally, it is important to note that the mean number of medium follicles at progestogen withdrawal was high in nonresponding ewes of both groups (mean number of 8.8 vs 10.8 in

responding ewes). The same was true for the mean number of 24 mm follicles at estrus (mean number of 14.6 vs 19.0 in responding ewes), although none of them ovulated.

Table 1. Overview of follicular status and ovulation rate at different times of treatment in FSH- P-treated and OVAGEN-treated ewes, responding (R) or not (NR) to the superovulatory protocol.

FSH-P OVAGEN

-

R NR R NR

Number of ewes 8 4 7 2

Number of 2 to 3 mm follicles at first FSH injection 7.4 + 0.5 1.1 * 0.2 6.7 f 0.7 1.5 * 0.5

Number of 4 to 5 mm follicles at sponge removal 12.9 f 1.4 10.5 + 0.8 8.4+ 1.9 5.5 f 0.5

Number of t4 mm follicles at estrus detection 22.6+2.3 15.2k4.0 15.0+22.2 13.5 f 3.5

Ovulation rate 9.5 k 0.6 0 9.0 * 0.4 0

DISCUSSION

Results from the current study confirm that ovulation rate in superovulated ewes is related to the number of small follicles at the start of FSH treatment, regardless of the commercial gonadotrophin used, as previously reported (4). However, in contrast to the study of Rubianes et al. (28) when using a single dose of eCG, no relationship was detected between the presence or persistence of a large follicle at the beginning of the treatment and the ovulation rate in response to several FSH doses. This discrepancy might be due to the different superovulatory protocols of the two studies. Ovulation rate in response to a single superovulatory injection of FSH combined

with prostaglandin Fza was affected by changes in diameter of the largest follicle just before the treatment and by the time of persistence of that follicle after the superovulatory treatment (18). This experiment is a good model to test for effects of the follicular dominance described in ewes after luteolysis (19, 26, 29). However, superovulatory regimens commonly used in sheep are based on the administration of several doses of exogenous FSH coincidentally with a

Theriogenology 1061

progestogen treatment. So, it would be possible to expect some variations in effects derived from large follicles when using progestogens. Moreover, the use of progestogen can change follicular growth patterns, increasing atresia of large follicles (16,23).

The CL number was related to the number of follicles 24 mm in size at the moment of estrus

detection but not to the more limited class of follicles 26 mm, which is the mean size established as ovulatory in the ewe (21) and particularly in Merino ewes (19). This fact, also found in superovulated ewes by Ktihholzer et al. (14) confirms that a certain number of medium size follicles are able to ovulate as a result of FSH administration, as previously described with PMSG treatments (12). Reduction in size of ovulatory follicles has been found not only with superovulatory treatments but during estrous cycles of prolific breeds such as Romanov (5) Booroola Merino (20) and Chios (2). Both follicles of polyovular ewes (10, 11) and follicles exposed to exogenous gonadotrophins (12) present an earlier end of maturation. In the first case, FSH administration has been associated with disturbances in the accumulation of antral fluids and with the proliferation of thecal and granulosa cells (33). However, all the ewes that had no CL at the end of this study had both medium and large follicles from the time of the sponge withdrawal to the termination of the experiment. Therefore, either the ovulatory stimulus or the competence of the follicles to ovulate, even though they had reached preovulatory size, seems to be deficient in these nonresponding animals.

In the current study, FSH-P and OVAGEN each stimulated the growth of a high number of follicles to preovulatory stages. However the former increased the waste of ovarian follicles, stimulating a higher number of them to grow without advantage, because they regressed during the treatment or were unable to ovulate. The main difference between the two commercial products seems to be related to LH contamination, considered as negligible by the manufacturing company. The very low level of LH in OVAGEN allowed the laboratory to recommend the injection of equal dosages instead of the decreasing dosage regimens previously used for FSH-P or other commercial products with presence of LH. Taking this into consideration, the higher number of growing follicles in FSH-P treated ewes could be an effect related to its LH content. Previous authors have described that the number of small growing follicles is increased in ewes when LH is added to FSH treatments (25). Studies done with cows showed that LH induced the growth of preantral follicles in an indirect way by regulating output of growth factors (I), because receptors for LH have not been detected in earlier stages of follicular development (6).

Conversely, it seems that administration of FSH-P negatively affected final follicular development. First, there was an increase in number of follicles that size-regressed in FSH-P-treated ewes with respect to the OVAGEN-treated ewes near es&us. This effect could be derived from the decreasing dosage of FSH at the end of the treatment with FSH-P. On the other hand, it has been described that increases in concentrations of LH in plasma increase the number of atretic follicles in sheep (32) without affecting the diameter of the largest follicle (15) as found in our current study. These variations in follicular development could be related to an alteration in the mechanism preventing atresia due to a change in the ovarian output of androgens and estrogens (22) or by a saturation of the LH receptors in cells of the theta, the granulosa or both (3). Second, ewes treated with FSH-P showed a lower proportion of preovulatory-size follicles that ovulated after sponge removal, with a higher percentage of ewes that didn’t ovulate in response to the treatment although they had follicles with an adequate size to ovulate. Both factors would predict a higher degree of ovulatory failure in this group. Although a minimum of LH is necessary for ovulation, high

1062 Theriogenology

concentrations of LH for prolonged periods before the LH surge in both heifers (30) and ewes (9) disturbed the ability of follicles to ovulate without changes in previous processes of recruitment and growth (27). One or both of these possibilities could be responsible for the ovulatory failure found in FSH-P-treated ewes, but it is not possible to elucidate which one because neither the LH peak nor follicular function were evaluated.

In conclusion, the ovulatory response to a superovulatory protocol is related to the number of small follicles present in the ovary at the beginning of the gonadotrophin treatment and their ability to grow until preovulatory stages, despite differences in follicular development induced by different commercial products.

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