- Email: [email protected]
Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep
Theriogenology 66 (2006) 896–905 www.journals.elsevierhealth.com/periodicals/the
Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep I. Ammoun a, T. Encinas a, A. Veiga-Lopez b, J.M. Ros a, I. Contreras b, P. Gonzalez-An˜over a, M.J. Cocero b, A.S. McNeilly c, A. Gonzalez-Bulnes a,b,* a
Departamento de Toxicologia y Farmacologı´a, Facultad de Veterinaria, UCM, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain b Departamento de Reproduccion Animal, INIA. Avda. Puerta de Hierro s/n, 28040 Madrid, Spain c MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Little France Crescent, Edinburgh EH16 4SB, UK Received 20 October 2005; received in revised form 10 February 2006; accepted 11 February 2006
Abstract Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4 h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4 h post-administration, maximum plasma FSH concentration, Cmax, and both the area under the plasma concentration-time curve extrapolated to the infinite, AUCinf, and to the last moment of sampling, AUClast) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 1.32 mg, 2 1.10 mg, and 3 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7 0.6 versus 10.0 0.4 in N and 9.8 0.6 in M, P < 0.05 for both) and embryos (7.9 0.8 versus 4.3 0.4 in N, P < 0.05, and 6.7 0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54 0.08 ng/ml for R, 0.45 0.05 ng/ml for N and 0.35 0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4 2.2), M (18.9 1.5, n.s.) and N sheep (14.1 1.4; P < 0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment. # 2006 Elsevier Inc. All rights reserved. Keywords: Breed; FSH kinetics; Follicular dynamics; Sheep; Superovulation
1. Introduction
* Corresponding author. Tel.: +34 91 347 4022; fax: +34 91 347 4014. E-mail address: [email protected] (A. Gonzalez-Bulnes). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.02.024
Protocols for superovulation and embryo transfer are widely used to increase progeny produced in sheep, like in other ruminants, thereby providing an alternative tool both for spread of selected animal genetics
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
and for ex situ preservation of endangered breeds. However, despite the improvements of the last few years, the ovarian response to exogenous gonadotrophin treatments remains highly variable. Factors causing such high variability are the major constraint for multiple ovulation and embryo transfer (MOET) protocols [1,2], being classified either as extrinsic (depending on the protocol of treatment used for multiple ovulation) or intrinsic factors (derived from species, breed, age, nutrition, reproductive and lactational status of donors). Breed is widely recognized as a major factor of variation [3]. In a very exhaustive study using over 9000 superovulated sheep, the breed factor accounted for approximately 30% of the variability in the embryo yields obtained in response to FSH treatments [4]. Early studies established that most of the differences in superovulatory response were related to the different prolificacy of the breeds used in MOET [5], with highly prolific breeds having a greater response to exogenous stimulation [6–8]. These differences were also found when comparing nonprolific breeds, where an interaction between the type of gonadotrophin used and the breed was also described [9]. Thus, due to the variation in superovulatory responses derived from genetic factors [10], it has become important to establish a reliable method of superovulation for each individual breed as the first step toward the establishment of reliable least variable embryo transfer programmes, particularly when this involves the potential conservation of endangered species. Rubia del Molar and Negra de Colmenar are two native non-prolific Spanish breeds listed as endangered species (FAO DAD-IS; http://dad.fao.org/en/Home.htm) and currently included in conservation programmes by freezing of semen and embryos. Most of the work on superovulation of sheep has been carried out in highlyproducing breeds, while only limited information concerning the superovulatory response of other breeds is currently available [11]. The first aim of the present study was to determine the pharmacokinetic behaviour of ovine FSH (oFSH) administered in single and multiple doses in Rubia del Molar and Negra de Colmenar sheep. The second objective was to analyze in practice the pharmacodynamics, in terms of effects on follicular growth, ovulation rate and embryo yields. The second objective would also help to verify a previous hypothesis suggesting that differences in rates of follicular growth and function [12,13] may be the underlying reason for the different responses to exogenous hormone observed between breeds.
897
2. Material and methods 2.1. Animals This study included 2 consecutive experiments, conducted during the breeding season (November 2004 and February 2005), which involved a total of 30 ewes, distributed in 3 groups according to their breed (10 Manchega, 10 Negra de Colmenar and 10 Rubia del Molar sheep), without differences in age distribution (range: 4–7 years old). Manchega sheep were used as control, being a breed in which the follicular response to the FSH treatment is well known [14]. Animals were in good body condition, and were part of the herd, maintained outdoors with access to indoor facilities, of the experimental farm of the Centro Nacional de Seleccion y Reproduccion Animal (CENSYRA, Madrid, Spain, latitude 408N). The farm meets the requirements of the European Union for Scientific Procedure Establishments and the experiment was performed under Project Licence from the INIA Scientific Ethic Committee. None of the sheep had been previously used in superovulatory protocols. 2.2. Experiment 1 Breed differences in the pharmacokinetics of FSH after the administration of a single intramuscular dose of oFSH. 2.2.1. Experimental procedure A total of 21 sheep were used, 7 ewes from each breed. Animals were treated with an intravaginal progestagen impregnated sponge (40 mg fluorogestone acetate, FGA, Chronogest1, Intervet International, Boxmeer, The Netherlands) for 14 days in a similar way as for our routine embryo production protocol [14]. In order to reduce the endogenous secretion of FSH to its nadir levels [15], a subcutaneous dose of 1.5 mg of the GnRH antagonist (GnRHa) teverelix (Antarelix1, Zentaris, AG, Frankfurt, Germany), was administered 4 days after sponge insertion. On the day after the GnRHa administration (considered Day 0 of the experimental design), all the animals were treated with a single dose of 1.32 mg of oFSH (OvagenTM, equivalent to 17.6 mg NIADDK/20 ml, ICP, Auckland, NZ); administered deeply in the semitendinous muscle, like all the other i.m. treatments used in current study. This dose corresponds to the one used in first injection of oFSH in multiple decreasing dose regimes in superovulatory protocols used in our laboratory. Serial sampling was carried out for determination of FSH kinetics at 24,
898
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
12, 0, 0.33, 0.66, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24 and 32 h from oFSH administration. Jugular blood samples (5 ml) were obtained with heparinized vacuum blood evacuation tubes (Vacutainer1 Systems Europe, Becton Dickinson, Meylan Cedex, France). Blood samples were immediately centrifuged at 2000 g for 15 min. Just after that, the plasma was stored at 20 8C until assayed for FSH plasma concentration levels. 2.2.2. Pharmacokinetic analysis Plasma FSH was measured in duplicate using previously described double-antibody radioimmunoassay [16]. The sensitivity of the assay was 0.1 ng/ml, and all samples were measured in a single assay with an intraassay coefficient of variation of 8.9%. After correction of the plasma concentrations by subtraction of the basal level of FSH before injection, experimental FSH concentration versus time data after intramuscular injection were analyzed for each individual animal by a least-squares regression algorithm using the PKCALC1 program [17] and were curve-fitted according to bi-exponential equations (Cp = A ekat + B ebt). Pharmacokinetic parameters were calculated for each individual animal but reported as means of seven animals. The maximum serum FSH concentration (Cmax) and time (tmax) were read directly from the concentration versus time data. The area under the plasma concentration-time curve (AUClast) was calculated from 0 to last sampling time (Clast) or estimated by trapezoidal rule with extrapolation to infinity using Clast/b (AUC01). Mean residence time (MRT) was calculated by dividing AUMC by AUC.
was assessed by ultrasonography at first FSH dose. For prevention of the presence of a possible large dominant follicle and enhancement of the population of gonadotrophin-responsive follicles at first FSH dose, sheep were injected with a single dose of 1.5 mg of the GnRH antagonist teverelix (Antarelix1, Zentaris AG, Frankfurt, Germany) on Day 9 [19]. To avoid the persistence of a corpus luteum beyond the sponge withdrawal, ewes were also treated with a single dose of 125 mg i.m. cloprostenol (Estrumate1, Mallinckrodt Vet GmbH, Friesoythe, Germany), coincidentally with the first FSH injection on Day 12. Superovulatory treatment consisted of eight i.m. decreasing doses of oFSH (OVAGENTM, ICP, Auckland, New Zealand, 3 1.32 mg, 2 1.10 mg, and 3 0,88 mg) administered twice daily starting from the morning of Day 12–24 h after progestagen withdrawal (which was coincident with the 6th FSH injection at 60 h). Serial sampling for determination of FSH kinetics was carried out, with vacuum blood evacuation tubes, as described above. Blood samples were taken just before administration of each oFSH dose, and at 1, 2, 4, 6, 16 and 24 h after the last oFSH dose. Follicular population was assessed by transrectal ultrasonography, twice daily from first oFSH injection to sponge withdrawal. Oestrus detection was performed at 24, 36 and 48 h after sponge withdrawal, using trained adult rams at a rate of one ewe/one male. Mating was allowed and repeated 12 h later. Thereafter, sheep remained with rams at a rate of one ram/six ewes for two additional days following the second mating. Ovarian response was assessed 7 days after sponge withdrawal by counting corpora lutea, when embryos were obtained by laparotomy.
2.3. Experiment 2 Breed differences in the pharmacokinetic and pharmacodynamic behaviour of FSH after the administration of a multiple-dose superovulatory protocol. 2.3.1. Experimental procedure Oestrus was synchronized in all the animals (n = 30) with the insertion of an intravaginal progestagen impregnated sponge (40 mg fluorogestone acetate, FGA, Chronogest1, Intervet International, Boxmeer, The Netherlands) on Day 0, replaced by a new one that was maintained from Days 9 to 14. Previously, all the sheep had been treated with two i.m. injections of 125 mg of cloprostenol (Estrumate1, Mallinckrodt Vet GmbH, Friesoythe, Germany), given 10 days apart, to assure the presence of a corpus luteum at progestagen insertion which has been found to improve superovulatory yields [18]. Permanence of corpus luteum
2.3.2. Pharmacokinetic analysis Blood samples were centrifuged and stored and plasma FSH concentrations were measured as described above. Pharmacokinetic parameters were calculate as described for the first experiment and the following were estimated: the minimal concentration during the steady state (CSSmin), the area under the serum concentration curve (AUC080), the maximum concentration (Cmax), the time of maximum concentration (tmax) and the elimination half-life (t1/2b) for the last oFSH dose. 2.3.3. Pharmacodynamic analysis Assessment of follicular population. The number of follicles 2 mm (follicles able to grow in response to the oFSH treatment; 20), follicles 3 mm (differentiated follicles able to grow and give rise to a viable embryo in response to FSH treatment; 21) and follicles 4 mm (size of preovulatory follicles in superovulated
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
sheep; 20) were determined by transrectal ultrasonography. The number of follicles with 2 and 3 mm in size were obtained by subtracting the number of follicles 2 and 3 mm from the number of follicles 3 and 4 mm. Sizes, and difference in size, between the largest and second largest follicles (LF1 and LF2, respectively) were also determined. Ultrasonographies were performed using an Aloka SSD500 (Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5 MHz lineararray transducer, as previously described by Schrick et al. [22] and validated in our laboratory [23]. Assessment of ovulation and embryo recovery. Ovarian response was assessed by determining the number of corpora lutea by laparoscopy on Day 7 after progestagen removal. Laparoscopic observation also avoided the laparotomy for embryo recovery in nonresponding females and possible haemorrhage caused by exteriorization of ovaries in responding ewes. Embryo recoveries were performed by midventral laparotomy and by flushing both uterine horns as previously described [18]. The following information was recorded for each ewe: number of corpora lutea, total number of recovered embryos and rate of recovery (obtained by dividing, in every sheep, the total number of oocytes/ embryos by the total number of corpora lutea). 2.4. Statistical analysis Effect of breed on FSH concentrations and pharmacokinetic and pharmacodynamic parameters were evaluated by serial analysis of variance (split-plot ANOVA). The comparison between means and effects of breed on percentage of animals showing oestrus, timing of oestrus detection, rate of ovulation and rate of embryo recovery were tested by ANOVA followed by a Kruskal–Wallis test when Levene’s test showed nonhomogeneous variances; individual percentages were previously transformed to the arcsine. Pearson and Spearman correlation analysis were carried out to assess possible correlations between the number of follicles of various size categories, timing of oestrus detection, ovulation rate and embryo recovery. All results were expressed as mean S.E.M. and the statistical significance was accepted from P < 0.05.
899
Fig. 1. Mean plasma concentration S.E.M. versus time curves for FSH after single intramuscular injection of oFSH (1.32 mg) in Manchega (filled circles), Rubia del Molar (open circles), and Negra de Colmenar breeds (filled squares). The asterisk indicates significant differences (P < 0.05).
breeds and samples, ranging in mean values of 0.66 0.07 ng/ml for Manchega group (M), 0.64 0.19 ng/ml for Negra de Colmenar (N) and 0.55 0.09 ng/ml for Rubia del Molar sheep (R). The intramuscular single dose of oFSH induced a significant increase of plasma FSH in all the animals (P < 0.05). The highest level was reached between 1 and 8 h postadministration (Fig. 1), with a mean time to peak concentration (tmax) of 2.7 h (Table 1), in all the breeds. The plasma FSH levels after administration were similar between breeds; however, Negra de Colmenar ewes showed a higher FSH concentration at 4 h postadministration (1.84 0.27 ng/ml versus 1.03 0.11 in R and 0.96 0.20 ng/ml in M; P < 0.05 for both). After reaching the maximum value, plasma FSH concentrations followed a mono-exponential decreasing curve, without statistical differences between groups; basal levels were reached between 16 and 24 h. The values of absorption half-life were similar between groups; however, there was a trend for a slower plasma elimination, and a longer mean residence time (MRT), in Rubia del Molar sheep. Overall, the maximum concentrations (Cmax) values of FSH were higher in Negra de Colmenar than in Manchega and Rubia del Molar sheep, as detailed in Table 1. The values of AUC extrapolated to the infinite (AUCinf) and to the last time of sampling (AUClast) were also higher in Negra de Colmenar than in Manchega and Rubia del Molar sheep (P < 0.05).
3. Results 3.1. Effects of the breed on pharmacokinetics after a single dose of oFSH Plasma FSH concentrations from 24 to 0 h of exogenous oFSH administration were similar between
3.2. Effects of the breed on pharmacokinetics of multiple doses of oFSH The intramuscular administration of the exogenous oFSH in eight step-down doses increased plasma FSH levels in all the animals. The mean FSH plasma
900
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Table 1 Pharmacokinetic parameters (mean S.E.M.) of FSH in Manchega, Rubia del Molar and Negra de Colmenar sheep after single (1.32 mg) and multiple (3 1.32 mg; 2 1.1 mg; 3 0.88 mg) i.m. administrations of oFSH Breed Manchega Single dose Cmax (ng/ml) tmax (h) AUClast (ng h/ml) AUCinf (ng h/ml) t1/2abs (h) t1/2b (h) MRT (h) Multiple dose Cmax (ng/ml) tmax (h) CSSmin (ng/ml) AUC0–80 (ng h/l)
2.10 0.21 a 2.71 0.47 9.28 0.95 a 9.54 1.26 a 0.92 0.25 3.40 0.48 6.30 0.79 1.31 0.27 3.57 0.43 0.32 0.05 25.70 3.69
Rubia del molar
Negra de colmenar
1.97 0.31 a 2.67 0.98 9.68 2.16 a 10.43 2.01 a 0.97 0.19 6.16 2.92 10.34 4.32
2.98 0.40 b 2.71 0.75 15.79 2.45 b 15.75 1.99 b 1.13 0.19 3.69 0.52 6.93 0.89
1.80 0.23 3.71 0.52 0.54 0.04 40.47 2.74
1.50 0.36 3.57 0.61 0.44 0.10 32.88 3.19
Cmax, maximum plasmatic concentration; tmax, time of peak; CSSmin, minimum plasma concentration at steady state; AUClast, AUCinf, AUC0–80, area under the curve from time 0 to the last time of sampling, to infinity or to 80 h, respectively; t1/2abs and t1/2b, absorption and elimination half-lives; MRT, mean residence time. Mean S.E.M. within rows with different letters differ (P < 0.05).
concentrations were similar between breeds (0.35 0.05 ng/ml for M, 0.45 0.05 ng/ml for N and 0.54 0.08 ng/ml for R), as depicted in Fig. 2. The mean values of pharmacokinetic parameters after oFSH administration were similar between the three breeds (Table 1). However, it is important to note that, although statistical differences were not found, the minimum and maximum concentration values (CSSmin and Cmax) in steady state and both the time to maximum concentration (tmax) and hormone availability from first injection until the end of treatment (AUC0–80) tended to be higher in Rubia del Molar than in the other breeds.
Fig. 2. Mean plasma concentration of FSH S.E.M. versus time curves during the intramuscular administration of multiple doses of oFSH (3 1.32 mg; 2 1.1 mg; 3 0.88 mg), in a twice daily regimen, in Manchega (filled circles), Rubia del Molar (open circles) and Negra de Colmenar breeds (filled squares).
The analysis of pharmacokinetics after the last oFSH dose showed that the plasma FSH peak was reached between 1 and 3 h post-administration in all animals, with similar values between breeds (1.45 0.42 for M, 1.50 0.48 for N and 1.59 1.53 for R). Thereafter, plasma FSH concentration showed a mono-exponential decreasing curve until the lowest levels, being reached at 24 h post-administration (Fig. 2). 3.3. Effects of the breed on pharmacodynamics of multiple doses of oFSH. Dynamics of follicular development Two ewes of the Negra de Colmenar breed and one Manchega sheep were excluded from subsequent analysis due to pregnancy detection or uterine adhesion. Thus, the results are available from 9 Manchega (M), 10 Rubia del Molar (R) and 8 Negra de Colmenar (N) sheep. The number of total follicles (2 mm) at start of the FSH treatment (0 h) was similar between breeds (16.7 1.4 for M, 15.4 1.1 for R, and 13.2 0.8 for N) and the mean number of follicles 3 and 4 mm were also similar (Fig. 3). The administration of oFSH induced a significant increase in the mean number of follicles in each category in all the sheep. However, this increase was higher in 4 mm follicles. The relative percentage of follicles 4/2 mm increased during the treatment from around 35% at 0 h to 70% at 60 h, without significant differences between breeds. The size of the
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Fig. 3. Mean number of follicles with 2 mm (dark grey area), 3 mm (medium grey area), 4 mm (light grey area) and 2 mm in size (total area) versus time curves during the intramuscular administration of multiple doses of oFSH (3 1.32 mg; 2 1.1 mg; 3 0.88 mg), in a twice daily regimen, in Manchega (A), Rubia del Molar (B) and Negra de Colmenar sheep (C).
901
largest (LF1) and second largest follicle (LF2), similar between breeds at start of FSH treatment (Fig. 4), were also affected by the superovulatory treatment. The oFSH treatment induced an increase in both LF1 and LF2 diameters in all the sheep (P < 0.05), except for LF1 in Negra de Colmenar ewes, which remained around 5.7 mm. The population of follicles in each category increased in response to the FSH treatment in all the sheep, but the mean number of follicles were affected by the breed. The mean number of preovulatory sized follicles (4 mm) growing during the treatment was similar in Manchega and Rubia del Molar ewes (12.4 0.8 and 13.6 1.0, respectively) and lower in Negra de Colmenar sheep (8.6 0.8, P < 0.005 for M and P < 0.001 for R). Finally, at sponge withdrawal (60 h), the number of 4 mm follicles was higher in Rubia del Molar (24.4 2.2) than in Manchega (18.9 1.5, n.s.) and Negra de Colmenar sheep (14.1 1.4; P < 0.01). Size distribution of the population of follicles was also affected by the breed. During the entire FSH treatment, Manchega sheep showed the highest mean values of LF1 when compared to Rubia del Molar and Negra de Colmenar (6.3 0.1 mm versus 5.8 0.1, P < 0.001 and 5.7 0.8, P < 0.0001, respectively). The same was found for LF2 (5.2 0.1 in M versus 4.7 0.1 in R and 4.4 0.1 mm in N, P < 0.005). At sponge withdrawal (60 h), LF1 and LF2 were larger in Manchega than in Rubia del Molar and Negra de Colmenar sheep (LF1: 7.0 in M versus 6.4 0.6 in R, P < 0.05, and 6.0 0.2 mm in N, P < 0.05; LF2: 5.9 0.1 in M versus 5.3 0.2 in R, n.s., and 5.0 0.2 mm in N, P < 0.05).
Fig. 4. Mean sizes of the largest follicle (LF1; left hand) and the second largest follicle (LF2; right hand) versus time curves during the intramuscular administration of multiple doses of oFSH (3 1.32 mg; 2 1.1 mg; 3 0.88 mg), in a twice daily regimen, in Manchega (filled circles), Rubia del Molar (open circles) and Negra de Colmenar (filled squares) sheep. S.E.M. have been omitted for clarity of the figures.
902
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Table 2 Differences in superovulatory yields in Manchega, Rubia del Molar and Negra de Colmenar sheep (k = 3; n = 8, 9 and 10, respectively) after multiple (3 1.32 mg; 2 1.1 mg; 3 0.88 mg) i.m. administration of oFSH Breed
Animals (n) Corpora lutea (n) Recovered embryos (n) Recovery rate (%)
Manchega
Rubia del molar
Negra de colmenar
8 9.8 0.1 b 6.7 0.5 70.7 2.7 a
9 13.7 0.6 a 7.9 0.8 a 53.0 3.4
7 10.0 0.4 b 4.3 0.4 b 40.0 3.3 b
Mean S.E.M. within rows with different letters differ (P < 0.05).
3.4. Effects of the breed on pharmacodynamics of multiple doses of oFSH. Ovulatory response and embryo yields All the Manchega sheep, 90.0% of Rubia del Molar and 87.5% of Negra de Colmenar sheep showed oestrus signs after sponge withdrawal; no statistical differences were found in the timing of oestrus behaviour between breeds. Thereafter, 88.9% of Manchega, 90.0% of Rubia del Molar and 87.5% of Negra de Colmenar sheep ovulated in response to the superovulatory treatment. All these animals superovulated with at least four corpora lutea (CL). The number of CL was higher in Rubia del Molar sheep (P < 0.05), whilst Negra de Colmenar females showed the lower recovery rate and, hence, number of recovered embryos (Table 2). The follicular population at starting the FSH treatment affected superovulatory responses. The number of CL obtained in response to the treatment was related, in all the sheep, to the mean number of follicles with 2–3 mm in diameter at first FSH dose (r = 0.284, P < 0.05). However, the number of recovered embryos was related to the number of follicles 3 mm (r = 0.369, P < 0.005). In Manchega ewes, a higher number of 4 mm follicles at 0 h was associated to a decrease in the ovulation rate (r = 0.740, P < 0.05). Statistical analysis showed that in all the groups, the number of both corpora lutea and recovered embryos were influenced by the follicular population at 12 h. Again, ovulation rate was correlated with the number of 2–3 mm follicles (r = 0.284, P < 0.05) in all the sheep, but a step-down analysis showed that the number of follicles of 3 mm exerted the strongest effect (r = 0.621, P < 0.005). The size distribution of the follicles at 0 h also affected the superovulatory yields in all the breeds; ewes with a smaller LF2 diameter showed lower recovery rates (r = 0.464, P < 0.05). In Manchega sheep, a higher difference between LF1 and
LF2 was also related to a lower recovery rate (r = 0.801, P < 0.05). The follicular population growing in response to the FSH treatment also affected superovulatory responses. In all the sheep, both the number of CL and embryos were mainly determined by the number of preovulatory follicles, with 4 mm in size at progestagen withdrawal, in response to the FSH treatment (r = 0.783, P < 0.0005 and r = 0.573, P < 0.005, respectively). The effect of the number of 4 mm follicles on the ovulation rate was also found when breeds were analyzed separately; in Rubia del Molar sheep, the CL number was also higher with a higher size of LF1 and LF2 (r = 0.690 and r = 0.580, respectively; P < 0.05). However, unlike the relationship between follicles and ovulation rate, no effect of the number of 4 mm follicles on the number of recovered embryos was found when breeds were considered separately, suggesting alterations in follicular function. 4. Discussion The evaluation of the kinetics of the exogenous ovine FSH (oFSH) and the ovarian response in superovulated sheep in the current study confirms the existence of a breed-related variability and provides evidence supporting the role of a differential response of ovarian follicles to oFSH between breeds rather than a differential kinetics of the exogenous gonadotrophin. The present results showed significant differences between the three breeds studied in terms of ovarian response to a step-down oFSH treatment and in terms of number of corpora lutea and oocytes/embryos obtained after the treatment. The superovulatory response of the Manchega group used in the current study as a control was similar to previous reports using the same treatment in the same breed [19,24]. The results showed a better ovulatory response in the Rubia del Molar breed than in Manchega and Negra de Colmenar sheep. Rubia del
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Molar ewes also showed a higher number of recovered oocytes/embryos, although differences with the Manchega breed did not reach statistical significance. These effects of genetic factors on superovulatory yields have been previously reported either in breeds differing in prolificacy (Chios versus Friesian, 11) or in non-prolific breeds (Welsh Mountain versus Scottish Blackface, 25; Manchega versus Churra versus Merino, 9). Thus, the study of Picazo et al. [9] indicated an interaction between breed and treatment. Differences in superovulatory responses between breeds may be related either to a differential kinetic behaviour of the exogenous gonadotrophin or to a differential follicular dynamics and function in response to the hormone. The pharmacokinetic study developed in the first trial indicates that the administration of a single dose of 1.32 mg of oFSH provokes a significant increase in the plasma FSH in all the animals, without statistical differences in absorption and elimination of the hormone between breeds. Data obtained were similar to previous reports [26,27], although the elimination process seems to be slower in our experiment than in those studies. Differences may be caused either by the FSH preparation used, or by the route of administration or by the high inherent variability of FSH elimination, as described in the bovine [28,29]. In our study, we noted that mean plasma FSH concentrations and areas under the plasma concentration-time curve were higher in Negra de Colmenar than in Rubia del Molar and Manchega breed. These differences may be due to the fact that we administered the same FSH dose in all the females, corresponding to the first dose in multiple decreasing dose regimes used in our laboratory for Manchega sheep. The dose was not adjusted to individual body weight and Negra de Colmenar ewes, with a lower weight (around 35 kg versus 45 kg in Rubia del Molar and 70 kg in Manchega), received in fact a higher dose. This hypothesis is reinforced by the fact that the mean residence time (MRT) was similar between breeds, discarding possible differences between breeds in the mechanisms of distribution and elimination of the gonadotrophin. In the second experiment, without differences between animals and breeds, plasma FSH concentrations were maintained in an equilibrium state in spite of the fact that oFSH was administered in an step-down regime. Although Rubia del Molar showed a trend for higher FSH concentrations both in equilibrium and after the last administration than Manchega and Negra de Colmenar sheep, differences were not significant. These findings support a minor role of differences in oFSH
903
kinetics on breed differences in the superovulatory response and confirm a previous hypothesis [30,31] that a minimum plasma FSH threshold level is necessary to achieve a superovulatory effect [32]. However, the lack of a correlation between plasma FSH concentration during treatment and the superovulatory response obtained indicates that final yields would depend on intrinsic factors. Most of the studies on embryo production in sheep and other species indicate that, relative to intrinsic factors, ovarian status and follicular dynamics are the main factors responsible for the variability of superovulatory responses (for review see [1,14,33]). In the second trial of the current study, the superovulatory response was related to the distribution of the follicular population present in the ovaries at the first FSH dose; which conforms with previous results with different commercial gonadotrophin preparations and administration protocols [20,21,24,34]. In brief, a higher ovulatory response was associated to a higher number of small (2–3 mm in size) gonadotrophin-responsive follicles, able to grow to ovulatory sizes in response to the administration of exogenous oFSH. However, the number and viability of embryos seems to be more related to the number of follicles of 3 mm in diameter [21], i.e. those that had reached a differentiated stage, as indicated by the secretion of inhibin A [35]. Thereafter, in the current experiment, the FSH treatment induced a progressive increase in the number and size of antral follicles in all the females. The patterns of follicular development in Manchega sheep were similar to those reported in a previous study with the same breed and superovulatory protocol [36]. The current results indicate a higher recruitment and growth rate of follicles in Rubia del Molar sheep, leading to a higher number of 4 mm follicles at sponge withdrawal; which was related to the number of preovulatory follicles at oestrus [20]. These differences between breeds in the patterns of follicular development in response to the oFSH treatment coincide with a recent study of our group showing differences in the patterns of follicular development in non-stimulated sheep of the three breeds (Gonzalez-An˜over, Unpublished study). In conclusion, the combined analysis of pharmacokinetic and pharmacodynamic parameters in the current study indicate that breed differences in superovulatory response would be mainly explained by breed differences in follicular dynamics in response to exogenous oFSH rather than to any differences in the dynamics of FSH absorption and clearance. A high individual variability within breeds was also found. Such differences might be related to a higher
904
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
expression, or a higher sensitivity, of FSH receptors in the ovary [37–39]. However, the elucidation of the mechanisms involved was not possible in the current experiment, thus further studies are required to determine the causes for individual variability and, from a practical point of view, for the selection of highresponsive individuals within each breed. Acknowledgments We gratefully acknowledge C. Martinez, J. Dochao and J. Cuevas for the management of the animals and their help during the development of the experimental work and I. Swanston and G. Johnstone for their technical assistance in the FSH assays. This work was supported by funds from INIA (project RZ 00-009) and Comunidad de Madrid (Convenio de Colaboracion INIA-DGADR-ITDA). IA was supported by a CIHEAM-IAMZ Grant, AVL and IC were supported by PhD fellowships from INIA and UCV, respectively, and AGB by the Programme Ramon y Cajal. References [1] Cognie Y. State of the art in sheep–goat embryo transfer. Theriogenology 1999;51:105–16. [2] Driancourt MA. Regulation of ovarian follicular dynamics in farm animals: implications for manipulation of reproduction. Theriogenology 2001;55:1211–39. [3] Torres S, Cognie Y, Colas G. Transfer of superovulated sheep embryos obtained with different FSH-P. Theriogenology 1987;27:407–19. [4] Vivanco HM, Greaney KB, Varela H. Explaining the variability in superovulatory responses and yield of transferable embryos in sheep embryo transfer. Theriogenology 1994;41:329 (abstract). [5] Cahill LP, Dufour J. Follicular population in the ewe under different gonadotrophin levels. Ann Biol Anim Biochem Biophys 1979;19:1475–81. [6] Bindon BM, Chang TS, Turner HN. Ovarian response to gonadotrophin by Merino ewes selected for fecundity. Austr J Agric Res 1971;22:809–20. [7] Piper LR, Bindon BM, Curtis YM, Cheers MA, Nethery RD. Response to PMSG in Merino and Booroola Merino crosses. Proc Austr Soc Reprod Biol 1982;14:82–9. [8] Smith JF. Selection for fertility and response to PMSG in Romney ewes. Proc NZ Soc Anim Prod 1976;36:247–51. [9] Picazo RA, Cocero MJ, Barragan ML, Lopez-Sebastian A. Effects of LH administration at the end of an FSH superovulatory regimen on ovulation rate and embryo production in three breeds of sheep. Theriogenology 1996;45:1065–73. [10] Bondurant RH. Embryo transfer in sheep and goats. In: Morrow DA, editor. Current therapy in theriogenology. Philadelphia: WB Saunders Company; 1986. p. 63–6. [11] Boscos C, Vainas E, Kouskoura Th, Vafiadis D, Dellis S. Superovulatory response of Chios and Friesian ewes to two FSH-P dose levels. Reprod Dom Anim 1997;32:195–8.
´ Shae T. [12] Bindon BM, Piper LR, Cahill LP, Driancourt MA, O Genetic and hormonal factors affecting superovulation. Theriogenology 1986;25:53–70. [13] Bartlewski PM, Beard AP, Cook SJ, Chandolia RK, Honaramooz A, Rawlings NC. Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oestrous cycle in breeds of sheep differing in prolificacy. J Reprod Fertil 1999;115:111–24. [14] Gonzalez-Bulnes A, Baird DT, Campbell K, Cocero MJ, GarciaGarcia RM, Inskeep EK, et al. Multiple factors affecting multiple ovulation and embryo transfer in sheep and goats. Reprod Fertil Develop 2004;16:421–35. [15] Lopez-Alonso A, Encinas T, Garcia-Garcia RM, Veiga-Lopez A, Ros JM, McNeilly AS, et al. Administration of single short-acting doses of GnRH antagonist modifies pituitary and follicular function in sheep. Dom Anim Endocrinol 2005;29: 476–87. [16] Campbell BK, McNeilly AS, Picton HM, Baird DT. The effect of a potent gonadotrophin-releasing hormone antagonist on ovarian secretion of oestradiol, inhibin and androstenedione and the concentration of LH and FSH during the follicular phase of the sheep oestrous cycle. J Endocrinol 1990;126:377–84. [17] Shumaker RC. PKCALC: a BASIC interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metabol Rev 1986;17:331–48. [18] Gonzalez-Bulnes A, Berlinguer F, Cocero MJ, Garcia-Garcia RM, Leoni G, Naitana S, et al. Induction of the presence of corpus luteum during superovulatory treatments enhances in vivo and in vitro blastocysts output in sheep. Theriogenology 2005;64:1392–403. [19] Lopez-Alonso C, Encinas T, Veiga-Lopez A, Garcia-Garcia RM, Cocero MJ, Ros JM, et al. Follicular growth, endocrine response and embryo yields in sheep superovulated with FSH after pretreatment with a single short-acting dose of GnRH antagonist. Theriogenology 2005;64:1833–43. [20] Gonzalez-Bulnes A, Santiago-Moreno J, Cocero MJ, LopezSebastian A. Effects of FSH commercial preparation and follicular status on follicular growth and superovulatory response in Spanish merino ewes. Theriogenology 2000;54:1055–64. [21] Veiga-Lopez A, Gonzalez-Bulnes A, Garcia-Garcia RM, Dominguez V, Cocero MJ. The effect of previous ovarian status on ovulation rate and early embryo development in response to superovulatory FSH treatments in sheep. Theriogenology 2005;63:1973–83. [22] Schrick FN, Surface RA, Pritchard JY, Dailey RA, Townsend EC. Inskeep EK Ovarian structures during the estrous cycle and early pregnancy in ewes. Biol Reprod 1993;49:1133–40. [23] Gonzalez-Bulnes A, Santiago-Moreno J, Garcia-Lopez M, Gomez-Brunet A, Lopez-Sebastian A. Observacio´n del ovario en la oveja y eficacia en la deteccio´n de foliculos y cuerpos luteos mediante ecografı´a transrectal. Inv Agrar 1994;10:319–29. [24] Gonzalez-Bulnes A, Santiago-Moreno J, Cocero MJ, Souza CJH, Groome NP, Garcia-Garcia RM, et al. Measurement of inhibin A and follicular status predicts the response of ewes to superovulatory FSH treatments. Theriogenology 2002;57: 1263–72. [25] Bari F, Khalid M, Haresign W, Murray A, Merrell B, Richards R. An evaluation of the success of MOET in two breeds of hill sheep maintained under normal systems of hill flock management. J Anim Sci 1999;69:367–76. [26] McNeilly AS. Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
[27]
[28]
[29]
[30]
[31]
[32]
subsequent ovulation rate and corpus luteum function in the ewe. J Reprod Fertil 1985;74:661–8. Fry R, Cahill L, Cummins J, Bindon BM, Piper LR, Clarke I. The half-life of follicle stimulating hormone in ovary intact and ovariectomized Booroola and control Merino ewes. J Reprod Fertil 1987;81:611–5. Demoustier MM, Beckers JF, Van der Zwalmen P, Closset J, Gillared JL. Ectors Fr. Determination of porcine plasma folltropin levels during superovulation treatment in cow. Theriogenology 1988;30:379–86. Prakash N, Narayana K, Honne-Gowda P, Ramachandra SG. Disposition kinetics of bufflalo pituitary FSH following single intramuscular administration in female buffalo calves (Bubalus bubalis). Buffalo Bull 1999;18:86–91. Ben-Rafael Z, Levy T, Schoemaker J. Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril 1995;63:689–700. Briant C, Toutain PL, Ottogalli M, Magallon T, Guillaume D. Kinetic studies and production rate of equine (e) FSH in ovariectomized pony mares. Application to the determination of a dosage regimen for eFSH in a superovulation treatment. J Endocrinol 2004;182:43–54. Picton HM, McNeilly AS. Administration of FSH to ewes during chronic treatment with GnRH agonist: evidence to support an FSH-threshold theory for follicle selection. J Reprod Fertil 1991;93:43–51.
905
[33] Cognie Y, Baril G, Poulin N, Mermillod P. Current status of embryo technologies in sheep and goats. Theriogenology 2003;59:171–88. [34] Brebion P, Belloc JP, Briois M. Elite Lacaune ewes pretreated with a GnRH antagonist yield more useable embryos following pFSH. In: Proceedings of the Sixth Meeting Europ Assoc Embryo Transfer; 1990 (Abstract). [35] Gonzalez-Bulnes A, Souza CJH, Campbell BK, Baird DT. Systemic and intraovarian effects of dominant follicles on ovine follicular growth. Anim Reprod Sci 2004;84:107–19. [36] Gonzalez-Bulnes A, Garcia-Garcia RM, Santiago-Moreno J, Lopez-Sebastian A, Cocero MJ, Baird DT. Patterns of follicular growth in superovulated sheep and influence on endocrine and ovarian response. Reprod Dom Anim 2002;37:357–61. [37] Driancourt MA, Fry RC, Clarke IJ, Cahill LP. Variations in patterns of follicle development in prolific breeds of sheep. J Reprod Fertil 1986;78:565–75. [38] Abdennebi L, Monget P, Pisselte C, Remy JJ, Salesse R, Monniaux D. Comparative expression of luteinizing hormone and follicle-stimulating hormone receptors in ovarian follicles from high and low prolific sheep breeds. Biol Reprod 1999;60:845–54. [39] Dufour J, Cognie Y, Mermillod P, Mariana J, Romain RF. Effects of the Booroola Fec gene on ovarian follicular populations in superovulated Romanov ewes pretreated with a GnRH antagonist. J Reprod Fertil 2000;118:84–95.
Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep I. Ammoun a, T. Encinas a, A. Veiga-Lopez b, J.M. Ros a, I. Contreras b, P. Gonzalez-An˜over a, M.J. Cocero b, A.S. McNeilly c, A. Gonzalez-Bulnes a,b,* a
Departamento de Toxicologia y Farmacologı´a, Facultad de Veterinaria, UCM, Avda. Puerta de Hierro s/n, 28040 Madrid, Spain b Departamento de Reproduccion Animal, INIA. Avda. Puerta de Hierro s/n, 28040 Madrid, Spain c MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, University of Edinburgh, Little France Crescent, Edinburgh EH16 4SB, UK Received 20 October 2005; received in revised form 10 February 2006; accepted 11 February 2006
Abstract Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4 h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4 h post-administration, maximum plasma FSH concentration, Cmax, and both the area under the plasma concentration-time curve extrapolated to the infinite, AUCinf, and to the last moment of sampling, AUClast) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 1.32 mg, 2 1.10 mg, and 3 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7 0.6 versus 10.0 0.4 in N and 9.8 0.6 in M, P < 0.05 for both) and embryos (7.9 0.8 versus 4.3 0.4 in N, P < 0.05, and 6.7 0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54 0.08 ng/ml for R, 0.45 0.05 ng/ml for N and 0.35 0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4 2.2), M (18.9 1.5, n.s.) and N sheep (14.1 1.4; P < 0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment. # 2006 Elsevier Inc. All rights reserved. Keywords: Breed; FSH kinetics; Follicular dynamics; Sheep; Superovulation
1. Introduction
* Corresponding author. Tel.: +34 91 347 4022; fax: +34 91 347 4014. E-mail address: [email protected] (A. Gonzalez-Bulnes). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.02.024
Protocols for superovulation and embryo transfer are widely used to increase progeny produced in sheep, like in other ruminants, thereby providing an alternative tool both for spread of selected animal genetics
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
and for ex situ preservation of endangered breeds. However, despite the improvements of the last few years, the ovarian response to exogenous gonadotrophin treatments remains highly variable. Factors causing such high variability are the major constraint for multiple ovulation and embryo transfer (MOET) protocols [1,2], being classified either as extrinsic (depending on the protocol of treatment used for multiple ovulation) or intrinsic factors (derived from species, breed, age, nutrition, reproductive and lactational status of donors). Breed is widely recognized as a major factor of variation [3]. In a very exhaustive study using over 9000 superovulated sheep, the breed factor accounted for approximately 30% of the variability in the embryo yields obtained in response to FSH treatments [4]. Early studies established that most of the differences in superovulatory response were related to the different prolificacy of the breeds used in MOET [5], with highly prolific breeds having a greater response to exogenous stimulation [6–8]. These differences were also found when comparing nonprolific breeds, where an interaction between the type of gonadotrophin used and the breed was also described [9]. Thus, due to the variation in superovulatory responses derived from genetic factors [10], it has become important to establish a reliable method of superovulation for each individual breed as the first step toward the establishment of reliable least variable embryo transfer programmes, particularly when this involves the potential conservation of endangered species. Rubia del Molar and Negra de Colmenar are two native non-prolific Spanish breeds listed as endangered species (FAO DAD-IS; http://dad.fao.org/en/Home.htm) and currently included in conservation programmes by freezing of semen and embryos. Most of the work on superovulation of sheep has been carried out in highlyproducing breeds, while only limited information concerning the superovulatory response of other breeds is currently available [11]. The first aim of the present study was to determine the pharmacokinetic behaviour of ovine FSH (oFSH) administered in single and multiple doses in Rubia del Molar and Negra de Colmenar sheep. The second objective was to analyze in practice the pharmacodynamics, in terms of effects on follicular growth, ovulation rate and embryo yields. The second objective would also help to verify a previous hypothesis suggesting that differences in rates of follicular growth and function [12,13] may be the underlying reason for the different responses to exogenous hormone observed between breeds.
897
2. Material and methods 2.1. Animals This study included 2 consecutive experiments, conducted during the breeding season (November 2004 and February 2005), which involved a total of 30 ewes, distributed in 3 groups according to their breed (10 Manchega, 10 Negra de Colmenar and 10 Rubia del Molar sheep), without differences in age distribution (range: 4–7 years old). Manchega sheep were used as control, being a breed in which the follicular response to the FSH treatment is well known [14]. Animals were in good body condition, and were part of the herd, maintained outdoors with access to indoor facilities, of the experimental farm of the Centro Nacional de Seleccion y Reproduccion Animal (CENSYRA, Madrid, Spain, latitude 408N). The farm meets the requirements of the European Union for Scientific Procedure Establishments and the experiment was performed under Project Licence from the INIA Scientific Ethic Committee. None of the sheep had been previously used in superovulatory protocols. 2.2. Experiment 1 Breed differences in the pharmacokinetics of FSH after the administration of a single intramuscular dose of oFSH. 2.2.1. Experimental procedure A total of 21 sheep were used, 7 ewes from each breed. Animals were treated with an intravaginal progestagen impregnated sponge (40 mg fluorogestone acetate, FGA, Chronogest1, Intervet International, Boxmeer, The Netherlands) for 14 days in a similar way as for our routine embryo production protocol [14]. In order to reduce the endogenous secretion of FSH to its nadir levels [15], a subcutaneous dose of 1.5 mg of the GnRH antagonist (GnRHa) teverelix (Antarelix1, Zentaris, AG, Frankfurt, Germany), was administered 4 days after sponge insertion. On the day after the GnRHa administration (considered Day 0 of the experimental design), all the animals were treated with a single dose of 1.32 mg of oFSH (OvagenTM, equivalent to 17.6 mg NIADDK/20 ml, ICP, Auckland, NZ); administered deeply in the semitendinous muscle, like all the other i.m. treatments used in current study. This dose corresponds to the one used in first injection of oFSH in multiple decreasing dose regimes in superovulatory protocols used in our laboratory. Serial sampling was carried out for determination of FSH kinetics at 24,
898
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
12, 0, 0.33, 0.66, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24 and 32 h from oFSH administration. Jugular blood samples (5 ml) were obtained with heparinized vacuum blood evacuation tubes (Vacutainer1 Systems Europe, Becton Dickinson, Meylan Cedex, France). Blood samples were immediately centrifuged at 2000 g for 15 min. Just after that, the plasma was stored at 20 8C until assayed for FSH plasma concentration levels. 2.2.2. Pharmacokinetic analysis Plasma FSH was measured in duplicate using previously described double-antibody radioimmunoassay [16]. The sensitivity of the assay was 0.1 ng/ml, and all samples were measured in a single assay with an intraassay coefficient of variation of 8.9%. After correction of the plasma concentrations by subtraction of the basal level of FSH before injection, experimental FSH concentration versus time data after intramuscular injection were analyzed for each individual animal by a least-squares regression algorithm using the PKCALC1 program [17] and were curve-fitted according to bi-exponential equations (Cp = A ekat + B ebt). Pharmacokinetic parameters were calculated for each individual animal but reported as means of seven animals. The maximum serum FSH concentration (Cmax) and time (tmax) were read directly from the concentration versus time data. The area under the plasma concentration-time curve (AUClast) was calculated from 0 to last sampling time (Clast) or estimated by trapezoidal rule with extrapolation to infinity using Clast/b (AUC01). Mean residence time (MRT) was calculated by dividing AUMC by AUC.
was assessed by ultrasonography at first FSH dose. For prevention of the presence of a possible large dominant follicle and enhancement of the population of gonadotrophin-responsive follicles at first FSH dose, sheep were injected with a single dose of 1.5 mg of the GnRH antagonist teverelix (Antarelix1, Zentaris AG, Frankfurt, Germany) on Day 9 [19]. To avoid the persistence of a corpus luteum beyond the sponge withdrawal, ewes were also treated with a single dose of 125 mg i.m. cloprostenol (Estrumate1, Mallinckrodt Vet GmbH, Friesoythe, Germany), coincidentally with the first FSH injection on Day 12. Superovulatory treatment consisted of eight i.m. decreasing doses of oFSH (OVAGENTM, ICP, Auckland, New Zealand, 3 1.32 mg, 2 1.10 mg, and 3 0,88 mg) administered twice daily starting from the morning of Day 12–24 h after progestagen withdrawal (which was coincident with the 6th FSH injection at 60 h). Serial sampling for determination of FSH kinetics was carried out, with vacuum blood evacuation tubes, as described above. Blood samples were taken just before administration of each oFSH dose, and at 1, 2, 4, 6, 16 and 24 h after the last oFSH dose. Follicular population was assessed by transrectal ultrasonography, twice daily from first oFSH injection to sponge withdrawal. Oestrus detection was performed at 24, 36 and 48 h after sponge withdrawal, using trained adult rams at a rate of one ewe/one male. Mating was allowed and repeated 12 h later. Thereafter, sheep remained with rams at a rate of one ram/six ewes for two additional days following the second mating. Ovarian response was assessed 7 days after sponge withdrawal by counting corpora lutea, when embryos were obtained by laparotomy.
2.3. Experiment 2 Breed differences in the pharmacokinetic and pharmacodynamic behaviour of FSH after the administration of a multiple-dose superovulatory protocol. 2.3.1. Experimental procedure Oestrus was synchronized in all the animals (n = 30) with the insertion of an intravaginal progestagen impregnated sponge (40 mg fluorogestone acetate, FGA, Chronogest1, Intervet International, Boxmeer, The Netherlands) on Day 0, replaced by a new one that was maintained from Days 9 to 14. Previously, all the sheep had been treated with two i.m. injections of 125 mg of cloprostenol (Estrumate1, Mallinckrodt Vet GmbH, Friesoythe, Germany), given 10 days apart, to assure the presence of a corpus luteum at progestagen insertion which has been found to improve superovulatory yields [18]. Permanence of corpus luteum
2.3.2. Pharmacokinetic analysis Blood samples were centrifuged and stored and plasma FSH concentrations were measured as described above. Pharmacokinetic parameters were calculate as described for the first experiment and the following were estimated: the minimal concentration during the steady state (CSSmin), the area under the serum concentration curve (AUC080), the maximum concentration (Cmax), the time of maximum concentration (tmax) and the elimination half-life (t1/2b) for the last oFSH dose. 2.3.3. Pharmacodynamic analysis Assessment of follicular population. The number of follicles 2 mm (follicles able to grow in response to the oFSH treatment; 20), follicles 3 mm (differentiated follicles able to grow and give rise to a viable embryo in response to FSH treatment; 21) and follicles 4 mm (size of preovulatory follicles in superovulated
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
sheep; 20) were determined by transrectal ultrasonography. The number of follicles with 2 and 3 mm in size were obtained by subtracting the number of follicles 2 and 3 mm from the number of follicles 3 and 4 mm. Sizes, and difference in size, between the largest and second largest follicles (LF1 and LF2, respectively) were also determined. Ultrasonographies were performed using an Aloka SSD500 (Aloka Co. Ltd., Tokyo, Japan) equipped with a 7.5 MHz lineararray transducer, as previously described by Schrick et al. [22] and validated in our laboratory [23]. Assessment of ovulation and embryo recovery. Ovarian response was assessed by determining the number of corpora lutea by laparoscopy on Day 7 after progestagen removal. Laparoscopic observation also avoided the laparotomy for embryo recovery in nonresponding females and possible haemorrhage caused by exteriorization of ovaries in responding ewes. Embryo recoveries were performed by midventral laparotomy and by flushing both uterine horns as previously described [18]. The following information was recorded for each ewe: number of corpora lutea, total number of recovered embryos and rate of recovery (obtained by dividing, in every sheep, the total number of oocytes/ embryos by the total number of corpora lutea). 2.4. Statistical analysis Effect of breed on FSH concentrations and pharmacokinetic and pharmacodynamic parameters were evaluated by serial analysis of variance (split-plot ANOVA). The comparison between means and effects of breed on percentage of animals showing oestrus, timing of oestrus detection, rate of ovulation and rate of embryo recovery were tested by ANOVA followed by a Kruskal–Wallis test when Levene’s test showed nonhomogeneous variances; individual percentages were previously transformed to the arcsine. Pearson and Spearman correlation analysis were carried out to assess possible correlations between the number of follicles of various size categories, timing of oestrus detection, ovulation rate and embryo recovery. All results were expressed as mean S.E.M. and the statistical significance was accepted from P < 0.05.
899
Fig. 1. Mean plasma concentration S.E.M. versus time curves for FSH after single intramuscular injection of oFSH (1.32 mg) in Manchega (filled circles), Rubia del Molar (open circles), and Negra de Colmenar breeds (filled squares). The asterisk indicates significant differences (P < 0.05).
breeds and samples, ranging in mean values of 0.66 0.07 ng/ml for Manchega group (M), 0.64 0.19 ng/ml for Negra de Colmenar (N) and 0.55 0.09 ng/ml for Rubia del Molar sheep (R). The intramuscular single dose of oFSH induced a significant increase of plasma FSH in all the animals (P < 0.05). The highest level was reached between 1 and 8 h postadministration (Fig. 1), with a mean time to peak concentration (tmax) of 2.7 h (Table 1), in all the breeds. The plasma FSH levels after administration were similar between breeds; however, Negra de Colmenar ewes showed a higher FSH concentration at 4 h postadministration (1.84 0.27 ng/ml versus 1.03 0.11 in R and 0.96 0.20 ng/ml in M; P < 0.05 for both). After reaching the maximum value, plasma FSH concentrations followed a mono-exponential decreasing curve, without statistical differences between groups; basal levels were reached between 16 and 24 h. The values of absorption half-life were similar between groups; however, there was a trend for a slower plasma elimination, and a longer mean residence time (MRT), in Rubia del Molar sheep. Overall, the maximum concentrations (Cmax) values of FSH were higher in Negra de Colmenar than in Manchega and Rubia del Molar sheep, as detailed in Table 1. The values of AUC extrapolated to the infinite (AUCinf) and to the last time of sampling (AUClast) were also higher in Negra de Colmenar than in Manchega and Rubia del Molar sheep (P < 0.05).
3. Results 3.1. Effects of the breed on pharmacokinetics after a single dose of oFSH Plasma FSH concentrations from 24 to 0 h of exogenous oFSH administration were similar between
3.2. Effects of the breed on pharmacokinetics of multiple doses of oFSH The intramuscular administration of the exogenous oFSH in eight step-down doses increased plasma FSH levels in all the animals. The mean FSH plasma
900
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Table 1 Pharmacokinetic parameters (mean S.E.M.) of FSH in Manchega, Rubia del Molar and Negra de Colmenar sheep after single (1.32 mg) and multiple (3 1.32 mg; 2 1.1 mg; 3 0.88 mg) i.m. administrations of oFSH Breed Manchega Single dose Cmax (ng/ml) tmax (h) AUClast (ng h/ml) AUCinf (ng h/ml) t1/2abs (h) t1/2b (h) MRT (h) Multiple dose Cmax (ng/ml) tmax (h) CSSmin (ng/ml) AUC0–80 (ng h/l)
2.10 0.21 a 2.71 0.47 9.28 0.95 a 9.54 1.26 a 0.92 0.25 3.40 0.48 6.30 0.79 1.31 0.27 3.57 0.43 0.32 0.05 25.70 3.69
Rubia del molar
Negra de colmenar
1.97 0.31 a 2.67 0.98 9.68 2.16 a 10.43 2.01 a 0.97 0.19 6.16 2.92 10.34 4.32
2.98 0.40 b 2.71 0.75 15.79 2.45 b 15.75 1.99 b 1.13 0.19 3.69 0.52 6.93 0.89
1.80 0.23 3.71 0.52 0.54 0.04 40.47 2.74
1.50 0.36 3.57 0.61 0.44 0.10 32.88 3.19
Cmax, maximum plasmatic concentration; tmax, time of peak; CSSmin, minimum plasma concentration at steady state; AUClast, AUCinf, AUC0–80, area under the curve from time 0 to the last time of sampling, to infinity or to 80 h, respectively; t1/2abs and t1/2b, absorption and elimination half-lives; MRT, mean residence time. Mean S.E.M. within rows with different letters differ (P < 0.05).
concentrations were similar between breeds (0.35 0.05 ng/ml for M, 0.45 0.05 ng/ml for N and 0.54 0.08 ng/ml for R), as depicted in Fig. 2. The mean values of pharmacokinetic parameters after oFSH administration were similar between the three breeds (Table 1). However, it is important to note that, although statistical differences were not found, the minimum and maximum concentration values (CSSmin and Cmax) in steady state and both the time to maximum concentration (tmax) and hormone availability from first injection until the end of treatment (AUC0–80) tended to be higher in Rubia del Molar than in the other breeds.
Fig. 2. Mean plasma concentration of FSH S.E.M. versus time curves during the intramuscular administration of multiple doses of oFSH (3 1.32 mg; 2 1.1 mg; 3 0.88 mg), in a twice daily regimen, in Manchega (filled circles), Rubia del Molar (open circles) and Negra de Colmenar breeds (filled squares).
The analysis of pharmacokinetics after the last oFSH dose showed that the plasma FSH peak was reached between 1 and 3 h post-administration in all animals, with similar values between breeds (1.45 0.42 for M, 1.50 0.48 for N and 1.59 1.53 for R). Thereafter, plasma FSH concentration showed a mono-exponential decreasing curve until the lowest levels, being reached at 24 h post-administration (Fig. 2). 3.3. Effects of the breed on pharmacodynamics of multiple doses of oFSH. Dynamics of follicular development Two ewes of the Negra de Colmenar breed and one Manchega sheep were excluded from subsequent analysis due to pregnancy detection or uterine adhesion. Thus, the results are available from 9 Manchega (M), 10 Rubia del Molar (R) and 8 Negra de Colmenar (N) sheep. The number of total follicles (2 mm) at start of the FSH treatment (0 h) was similar between breeds (16.7 1.4 for M, 15.4 1.1 for R, and 13.2 0.8 for N) and the mean number of follicles 3 and 4 mm were also similar (Fig. 3). The administration of oFSH induced a significant increase in the mean number of follicles in each category in all the sheep. However, this increase was higher in 4 mm follicles. The relative percentage of follicles 4/2 mm increased during the treatment from around 35% at 0 h to 70% at 60 h, without significant differences between breeds. The size of the
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Fig. 3. Mean number of follicles with 2 mm (dark grey area), 3 mm (medium grey area), 4 mm (light grey area) and 2 mm in size (total area) versus time curves during the intramuscular administration of multiple doses of oFSH (3 1.32 mg; 2 1.1 mg; 3 0.88 mg), in a twice daily regimen, in Manchega (A), Rubia del Molar (B) and Negra de Colmenar sheep (C).
901
largest (LF1) and second largest follicle (LF2), similar between breeds at start of FSH treatment (Fig. 4), were also affected by the superovulatory treatment. The oFSH treatment induced an increase in both LF1 and LF2 diameters in all the sheep (P < 0.05), except for LF1 in Negra de Colmenar ewes, which remained around 5.7 mm. The population of follicles in each category increased in response to the FSH treatment in all the sheep, but the mean number of follicles were affected by the breed. The mean number of preovulatory sized follicles (4 mm) growing during the treatment was similar in Manchega and Rubia del Molar ewes (12.4 0.8 and 13.6 1.0, respectively) and lower in Negra de Colmenar sheep (8.6 0.8, P < 0.005 for M and P < 0.001 for R). Finally, at sponge withdrawal (60 h), the number of 4 mm follicles was higher in Rubia del Molar (24.4 2.2) than in Manchega (18.9 1.5, n.s.) and Negra de Colmenar sheep (14.1 1.4; P < 0.01). Size distribution of the population of follicles was also affected by the breed. During the entire FSH treatment, Manchega sheep showed the highest mean values of LF1 when compared to Rubia del Molar and Negra de Colmenar (6.3 0.1 mm versus 5.8 0.1, P < 0.001 and 5.7 0.8, P < 0.0001, respectively). The same was found for LF2 (5.2 0.1 in M versus 4.7 0.1 in R and 4.4 0.1 mm in N, P < 0.005). At sponge withdrawal (60 h), LF1 and LF2 were larger in Manchega than in Rubia del Molar and Negra de Colmenar sheep (LF1: 7.0 in M versus 6.4 0.6 in R, P < 0.05, and 6.0 0.2 mm in N, P < 0.05; LF2: 5.9 0.1 in M versus 5.3 0.2 in R, n.s., and 5.0 0.2 mm in N, P < 0.05).
Fig. 4. Mean sizes of the largest follicle (LF1; left hand) and the second largest follicle (LF2; right hand) versus time curves during the intramuscular administration of multiple doses of oFSH (3 1.32 mg; 2 1.1 mg; 3 0.88 mg), in a twice daily regimen, in Manchega (filled circles), Rubia del Molar (open circles) and Negra de Colmenar (filled squares) sheep. S.E.M. have been omitted for clarity of the figures.
902
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Table 2 Differences in superovulatory yields in Manchega, Rubia del Molar and Negra de Colmenar sheep (k = 3; n = 8, 9 and 10, respectively) after multiple (3 1.32 mg; 2 1.1 mg; 3 0.88 mg) i.m. administration of oFSH Breed
Animals (n) Corpora lutea (n) Recovered embryos (n) Recovery rate (%)
Manchega
Rubia del molar
Negra de colmenar
8 9.8 0.1 b 6.7 0.5 70.7 2.7 a
9 13.7 0.6 a 7.9 0.8 a 53.0 3.4
7 10.0 0.4 b 4.3 0.4 b 40.0 3.3 b
Mean S.E.M. within rows with different letters differ (P < 0.05).
3.4. Effects of the breed on pharmacodynamics of multiple doses of oFSH. Ovulatory response and embryo yields All the Manchega sheep, 90.0% of Rubia del Molar and 87.5% of Negra de Colmenar sheep showed oestrus signs after sponge withdrawal; no statistical differences were found in the timing of oestrus behaviour between breeds. Thereafter, 88.9% of Manchega, 90.0% of Rubia del Molar and 87.5% of Negra de Colmenar sheep ovulated in response to the superovulatory treatment. All these animals superovulated with at least four corpora lutea (CL). The number of CL was higher in Rubia del Molar sheep (P < 0.05), whilst Negra de Colmenar females showed the lower recovery rate and, hence, number of recovered embryos (Table 2). The follicular population at starting the FSH treatment affected superovulatory responses. The number of CL obtained in response to the treatment was related, in all the sheep, to the mean number of follicles with 2–3 mm in diameter at first FSH dose (r = 0.284, P < 0.05). However, the number of recovered embryos was related to the number of follicles 3 mm (r = 0.369, P < 0.005). In Manchega ewes, a higher number of 4 mm follicles at 0 h was associated to a decrease in the ovulation rate (r = 0.740, P < 0.05). Statistical analysis showed that in all the groups, the number of both corpora lutea and recovered embryos were influenced by the follicular population at 12 h. Again, ovulation rate was correlated with the number of 2–3 mm follicles (r = 0.284, P < 0.05) in all the sheep, but a step-down analysis showed that the number of follicles of 3 mm exerted the strongest effect (r = 0.621, P < 0.005). The size distribution of the follicles at 0 h also affected the superovulatory yields in all the breeds; ewes with a smaller LF2 diameter showed lower recovery rates (r = 0.464, P < 0.05). In Manchega sheep, a higher difference between LF1 and
LF2 was also related to a lower recovery rate (r = 0.801, P < 0.05). The follicular population growing in response to the FSH treatment also affected superovulatory responses. In all the sheep, both the number of CL and embryos were mainly determined by the number of preovulatory follicles, with 4 mm in size at progestagen withdrawal, in response to the FSH treatment (r = 0.783, P < 0.0005 and r = 0.573, P < 0.005, respectively). The effect of the number of 4 mm follicles on the ovulation rate was also found when breeds were analyzed separately; in Rubia del Molar sheep, the CL number was also higher with a higher size of LF1 and LF2 (r = 0.690 and r = 0.580, respectively; P < 0.05). However, unlike the relationship between follicles and ovulation rate, no effect of the number of 4 mm follicles on the number of recovered embryos was found when breeds were considered separately, suggesting alterations in follicular function. 4. Discussion The evaluation of the kinetics of the exogenous ovine FSH (oFSH) and the ovarian response in superovulated sheep in the current study confirms the existence of a breed-related variability and provides evidence supporting the role of a differential response of ovarian follicles to oFSH between breeds rather than a differential kinetics of the exogenous gonadotrophin. The present results showed significant differences between the three breeds studied in terms of ovarian response to a step-down oFSH treatment and in terms of number of corpora lutea and oocytes/embryos obtained after the treatment. The superovulatory response of the Manchega group used in the current study as a control was similar to previous reports using the same treatment in the same breed [19,24]. The results showed a better ovulatory response in the Rubia del Molar breed than in Manchega and Negra de Colmenar sheep. Rubia del
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
Molar ewes also showed a higher number of recovered oocytes/embryos, although differences with the Manchega breed did not reach statistical significance. These effects of genetic factors on superovulatory yields have been previously reported either in breeds differing in prolificacy (Chios versus Friesian, 11) or in non-prolific breeds (Welsh Mountain versus Scottish Blackface, 25; Manchega versus Churra versus Merino, 9). Thus, the study of Picazo et al. [9] indicated an interaction between breed and treatment. Differences in superovulatory responses between breeds may be related either to a differential kinetic behaviour of the exogenous gonadotrophin or to a differential follicular dynamics and function in response to the hormone. The pharmacokinetic study developed in the first trial indicates that the administration of a single dose of 1.32 mg of oFSH provokes a significant increase in the plasma FSH in all the animals, without statistical differences in absorption and elimination of the hormone between breeds. Data obtained were similar to previous reports [26,27], although the elimination process seems to be slower in our experiment than in those studies. Differences may be caused either by the FSH preparation used, or by the route of administration or by the high inherent variability of FSH elimination, as described in the bovine [28,29]. In our study, we noted that mean plasma FSH concentrations and areas under the plasma concentration-time curve were higher in Negra de Colmenar than in Rubia del Molar and Manchega breed. These differences may be due to the fact that we administered the same FSH dose in all the females, corresponding to the first dose in multiple decreasing dose regimes used in our laboratory for Manchega sheep. The dose was not adjusted to individual body weight and Negra de Colmenar ewes, with a lower weight (around 35 kg versus 45 kg in Rubia del Molar and 70 kg in Manchega), received in fact a higher dose. This hypothesis is reinforced by the fact that the mean residence time (MRT) was similar between breeds, discarding possible differences between breeds in the mechanisms of distribution and elimination of the gonadotrophin. In the second experiment, without differences between animals and breeds, plasma FSH concentrations were maintained in an equilibrium state in spite of the fact that oFSH was administered in an step-down regime. Although Rubia del Molar showed a trend for higher FSH concentrations both in equilibrium and after the last administration than Manchega and Negra de Colmenar sheep, differences were not significant. These findings support a minor role of differences in oFSH
903
kinetics on breed differences in the superovulatory response and confirm a previous hypothesis [30,31] that a minimum plasma FSH threshold level is necessary to achieve a superovulatory effect [32]. However, the lack of a correlation between plasma FSH concentration during treatment and the superovulatory response obtained indicates that final yields would depend on intrinsic factors. Most of the studies on embryo production in sheep and other species indicate that, relative to intrinsic factors, ovarian status and follicular dynamics are the main factors responsible for the variability of superovulatory responses (for review see [1,14,33]). In the second trial of the current study, the superovulatory response was related to the distribution of the follicular population present in the ovaries at the first FSH dose; which conforms with previous results with different commercial gonadotrophin preparations and administration protocols [20,21,24,34]. In brief, a higher ovulatory response was associated to a higher number of small (2–3 mm in size) gonadotrophin-responsive follicles, able to grow to ovulatory sizes in response to the administration of exogenous oFSH. However, the number and viability of embryos seems to be more related to the number of follicles of 3 mm in diameter [21], i.e. those that had reached a differentiated stage, as indicated by the secretion of inhibin A [35]. Thereafter, in the current experiment, the FSH treatment induced a progressive increase in the number and size of antral follicles in all the females. The patterns of follicular development in Manchega sheep were similar to those reported in a previous study with the same breed and superovulatory protocol [36]. The current results indicate a higher recruitment and growth rate of follicles in Rubia del Molar sheep, leading to a higher number of 4 mm follicles at sponge withdrawal; which was related to the number of preovulatory follicles at oestrus [20]. These differences between breeds in the patterns of follicular development in response to the oFSH treatment coincide with a recent study of our group showing differences in the patterns of follicular development in non-stimulated sheep of the three breeds (Gonzalez-An˜over, Unpublished study). In conclusion, the combined analysis of pharmacokinetic and pharmacodynamic parameters in the current study indicate that breed differences in superovulatory response would be mainly explained by breed differences in follicular dynamics in response to exogenous oFSH rather than to any differences in the dynamics of FSH absorption and clearance. A high individual variability within breeds was also found. Such differences might be related to a higher
904
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
expression, or a higher sensitivity, of FSH receptors in the ovary [37–39]. However, the elucidation of the mechanisms involved was not possible in the current experiment, thus further studies are required to determine the causes for individual variability and, from a practical point of view, for the selection of highresponsive individuals within each breed. Acknowledgments We gratefully acknowledge C. Martinez, J. Dochao and J. Cuevas for the management of the animals and their help during the development of the experimental work and I. Swanston and G. Johnstone for their technical assistance in the FSH assays. This work was supported by funds from INIA (project RZ 00-009) and Comunidad de Madrid (Convenio de Colaboracion INIA-DGADR-ITDA). IA was supported by a CIHEAM-IAMZ Grant, AVL and IC were supported by PhD fellowships from INIA and UCV, respectively, and AGB by the Programme Ramon y Cajal. References [1] Cognie Y. State of the art in sheep–goat embryo transfer. Theriogenology 1999;51:105–16. [2] Driancourt MA. Regulation of ovarian follicular dynamics in farm animals: implications for manipulation of reproduction. Theriogenology 2001;55:1211–39. [3] Torres S, Cognie Y, Colas G. Transfer of superovulated sheep embryos obtained with different FSH-P. Theriogenology 1987;27:407–19. [4] Vivanco HM, Greaney KB, Varela H. Explaining the variability in superovulatory responses and yield of transferable embryos in sheep embryo transfer. Theriogenology 1994;41:329 (abstract). [5] Cahill LP, Dufour J. Follicular population in the ewe under different gonadotrophin levels. Ann Biol Anim Biochem Biophys 1979;19:1475–81. [6] Bindon BM, Chang TS, Turner HN. Ovarian response to gonadotrophin by Merino ewes selected for fecundity. Austr J Agric Res 1971;22:809–20. [7] Piper LR, Bindon BM, Curtis YM, Cheers MA, Nethery RD. Response to PMSG in Merino and Booroola Merino crosses. Proc Austr Soc Reprod Biol 1982;14:82–9. [8] Smith JF. Selection for fertility and response to PMSG in Romney ewes. Proc NZ Soc Anim Prod 1976;36:247–51. [9] Picazo RA, Cocero MJ, Barragan ML, Lopez-Sebastian A. Effects of LH administration at the end of an FSH superovulatory regimen on ovulation rate and embryo production in three breeds of sheep. Theriogenology 1996;45:1065–73. [10] Bondurant RH. Embryo transfer in sheep and goats. In: Morrow DA, editor. Current therapy in theriogenology. Philadelphia: WB Saunders Company; 1986. p. 63–6. [11] Boscos C, Vainas E, Kouskoura Th, Vafiadis D, Dellis S. Superovulatory response of Chios and Friesian ewes to two FSH-P dose levels. Reprod Dom Anim 1997;32:195–8.
´ Shae T. [12] Bindon BM, Piper LR, Cahill LP, Driancourt MA, O Genetic and hormonal factors affecting superovulation. Theriogenology 1986;25:53–70. [13] Bartlewski PM, Beard AP, Cook SJ, Chandolia RK, Honaramooz A, Rawlings NC. Ovarian antral follicular dynamics and their relationships with endocrine variables throughout the oestrous cycle in breeds of sheep differing in prolificacy. J Reprod Fertil 1999;115:111–24. [14] Gonzalez-Bulnes A, Baird DT, Campbell K, Cocero MJ, GarciaGarcia RM, Inskeep EK, et al. Multiple factors affecting multiple ovulation and embryo transfer in sheep and goats. Reprod Fertil Develop 2004;16:421–35. [15] Lopez-Alonso A, Encinas T, Garcia-Garcia RM, Veiga-Lopez A, Ros JM, McNeilly AS, et al. Administration of single short-acting doses of GnRH antagonist modifies pituitary and follicular function in sheep. Dom Anim Endocrinol 2005;29: 476–87. [16] Campbell BK, McNeilly AS, Picton HM, Baird DT. The effect of a potent gonadotrophin-releasing hormone antagonist on ovarian secretion of oestradiol, inhibin and androstenedione and the concentration of LH and FSH during the follicular phase of the sheep oestrous cycle. J Endocrinol 1990;126:377–84. [17] Shumaker RC. PKCALC: a BASIC interactive computer program for statistical and pharmacokinetic analysis of data. Drug Metabol Rev 1986;17:331–48. [18] Gonzalez-Bulnes A, Berlinguer F, Cocero MJ, Garcia-Garcia RM, Leoni G, Naitana S, et al. Induction of the presence of corpus luteum during superovulatory treatments enhances in vivo and in vitro blastocysts output in sheep. Theriogenology 2005;64:1392–403. [19] Lopez-Alonso C, Encinas T, Veiga-Lopez A, Garcia-Garcia RM, Cocero MJ, Ros JM, et al. Follicular growth, endocrine response and embryo yields in sheep superovulated with FSH after pretreatment with a single short-acting dose of GnRH antagonist. Theriogenology 2005;64:1833–43. [20] Gonzalez-Bulnes A, Santiago-Moreno J, Cocero MJ, LopezSebastian A. Effects of FSH commercial preparation and follicular status on follicular growth and superovulatory response in Spanish merino ewes. Theriogenology 2000;54:1055–64. [21] Veiga-Lopez A, Gonzalez-Bulnes A, Garcia-Garcia RM, Dominguez V, Cocero MJ. The effect of previous ovarian status on ovulation rate and early embryo development in response to superovulatory FSH treatments in sheep. Theriogenology 2005;63:1973–83. [22] Schrick FN, Surface RA, Pritchard JY, Dailey RA, Townsend EC. Inskeep EK Ovarian structures during the estrous cycle and early pregnancy in ewes. Biol Reprod 1993;49:1133–40. [23] Gonzalez-Bulnes A, Santiago-Moreno J, Garcia-Lopez M, Gomez-Brunet A, Lopez-Sebastian A. Observacio´n del ovario en la oveja y eficacia en la deteccio´n de foliculos y cuerpos luteos mediante ecografı´a transrectal. Inv Agrar 1994;10:319–29. [24] Gonzalez-Bulnes A, Santiago-Moreno J, Cocero MJ, Souza CJH, Groome NP, Garcia-Garcia RM, et al. Measurement of inhibin A and follicular status predicts the response of ewes to superovulatory FSH treatments. Theriogenology 2002;57: 1263–72. [25] Bari F, Khalid M, Haresign W, Murray A, Merrell B, Richards R. An evaluation of the success of MOET in two breeds of hill sheep maintained under normal systems of hill flock management. J Anim Sci 1999;69:367–76. [26] McNeilly AS. Effect of changes in FSH induced by bovine follicular fluid and FSH infusion in the preovulatory phase on
I. Ammoun et al. / Theriogenology 66 (2006) 896–905
[27]
[28]
[29]
[30]
[31]
[32]
subsequent ovulation rate and corpus luteum function in the ewe. J Reprod Fertil 1985;74:661–8. Fry R, Cahill L, Cummins J, Bindon BM, Piper LR, Clarke I. The half-life of follicle stimulating hormone in ovary intact and ovariectomized Booroola and control Merino ewes. J Reprod Fertil 1987;81:611–5. Demoustier MM, Beckers JF, Van der Zwalmen P, Closset J, Gillared JL. Ectors Fr. Determination of porcine plasma folltropin levels during superovulation treatment in cow. Theriogenology 1988;30:379–86. Prakash N, Narayana K, Honne-Gowda P, Ramachandra SG. Disposition kinetics of bufflalo pituitary FSH following single intramuscular administration in female buffalo calves (Bubalus bubalis). Buffalo Bull 1999;18:86–91. Ben-Rafael Z, Levy T, Schoemaker J. Pharmacokinetics of follicle-stimulating hormone: clinical significance. Fertil Steril 1995;63:689–700. Briant C, Toutain PL, Ottogalli M, Magallon T, Guillaume D. Kinetic studies and production rate of equine (e) FSH in ovariectomized pony mares. Application to the determination of a dosage regimen for eFSH in a superovulation treatment. J Endocrinol 2004;182:43–54. Picton HM, McNeilly AS. Administration of FSH to ewes during chronic treatment with GnRH agonist: evidence to support an FSH-threshold theory for follicle selection. J Reprod Fertil 1991;93:43–51.
905
[33] Cognie Y, Baril G, Poulin N, Mermillod P. Current status of embryo technologies in sheep and goats. Theriogenology 2003;59:171–88. [34] Brebion P, Belloc JP, Briois M. Elite Lacaune ewes pretreated with a GnRH antagonist yield more useable embryos following pFSH. In: Proceedings of the Sixth Meeting Europ Assoc Embryo Transfer; 1990 (Abstract). [35] Gonzalez-Bulnes A, Souza CJH, Campbell BK, Baird DT. Systemic and intraovarian effects of dominant follicles on ovine follicular growth. Anim Reprod Sci 2004;84:107–19. [36] Gonzalez-Bulnes A, Garcia-Garcia RM, Santiago-Moreno J, Lopez-Sebastian A, Cocero MJ, Baird DT. Patterns of follicular growth in superovulated sheep and influence on endocrine and ovarian response. Reprod Dom Anim 2002;37:357–61. [37] Driancourt MA, Fry RC, Clarke IJ, Cahill LP. Variations in patterns of follicle development in prolific breeds of sheep. J Reprod Fertil 1986;78:565–75. [38] Abdennebi L, Monget P, Pisselte C, Remy JJ, Salesse R, Monniaux D. Comparative expression of luteinizing hormone and follicle-stimulating hormone receptors in ovarian follicles from high and low prolific sheep breeds. Biol Reprod 1999;60:845–54. [39] Dufour J, Cognie Y, Mermillod P, Mariana J, Romain RF. Effects of the Booroola Fec gene on ovarian follicular populations in superovulated Romanov ewes pretreated with a GnRH antagonist. J Reprod Fertil 2000;118:84–95.