eCG treatment for in vivo embryo production in sheep

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Theriogenology 75 (2011) 769 –776 www.theriojournal.com

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep F. Forcadaa,*, M. Ait Amer-Mezianeb, J.A. Abeciaa, M.C. Maurelc, J.A. Cebrián-Péreza, T. Muiño-Blancoa, B. Asenjod, M.I. Vázqueza, A. Casaoa a

Grupo de Biología y Fisiología de la Reproducción, Instituto de Investigación de Ciencias Ambientales de Aragón (IUCA), Universidad de Zaragoza, Spain b Centre National de l’Insemination Artificielle et de l’Amèlioration Génétique Alger, Algeria c Unité de Physiologie de la Reproduction des Mammifères Domestiques, INRA-Tours, France d Escuela Universitaria de Ingenierías Agrarias de Soria, Universidad de Valladolid, Spain Received 1 July 2010; received in revised form 17 October 2010; accepted 18 October 2010

Abstract This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n ⫽ 19) or simplified (n ⫽ 19) superovulatory treatment up to three times at intervals of ⱖ 50 d. The onset of estrus was 5 h earlier (P ⬍ 0.05) among ewes that received the eCG/FSH protocol (25.2 ⫾ 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ⫾ 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P ⬍ 0.05 to P ⬍ 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ⫾ 2.3 and 13.7 ⫾ 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P ⬍ 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies. © 2011 Elsevier Inc. All rights reserved. Keywords: Sheep; Superovulation; Embryo production; pFSH; eCG

1. Introduction

* Corresponding author: Tel.: ⫹34 976 761600; fax: ⫹34 976 761590. E-mail address: [email protected] (F. Forcada). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.10.019

In recent decades, the genetic diversity of livestock has declined and, in response, many developed countries have sought to preserve threatened breeds by offering financial incentives in the form of grants for the collection and cryopreservation of gametes and embryos. Embryo banks seem to be the most practical

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F. Forcada et al. / Theriogenology 75 (2011) 769 –776

means of maintaining genetic diversity and improving the genetic diversity of local endangered sheep genotypes that have few individuals, but are well adapted to specific breeding systems. Although somatic cell gene banks might be feasible for the conservation of species and breeds in the near future, the low success rates of the technique make embryo production more suitable for those purposes. Superovulation and the harvesting of embryos from donor females have been the most efficient means of obtaining embryos at a given stage of development; however, these techniques have limitations that make them difficult for practical use at the commercial level. In the last 30 years, several follicle stimulating hormones (FSH) have been used to induce superovulation in sheep using protocols that involve several administrations during the follicular phase, but the protocols are very labor intensive and rather stressful to the animals because of excessive handling. For those reasons, there is a demand for a simplification of superovulatory protocols. A single dose of FSH can be used to simplify the superovulation protocol, but premature luteal regression can occur frequently [1]. López Sebastián et al [2,3] identified the need for an oily vehicle that would reduce the absorption rate of the FSH when administered in a single dose, but this procedure has not been studied thoroughly [4 – 6]. Treatment with a single dose of eCG was also proposed due to its long half-life. The treatment is inexpensive but, at high doses, eCG has a detrimental effect on hormonal profiles, ovulation, fertilization, embryo recovery, and viability rates [7–9]. Ovarian stimulation by a single dose of FSH in saline and a small or moderate dose of eCG has been tested in some sheep breeds such as Merino [10,11], Sarda [5,12,13], Rasa Aragonesa [12] and Corriedale [14]. The best results have occurred when a commercial dose of eCG (500 – 600 IU) was combined with amounts of FSH that were 50 –75% lower than the total amounts used in treatments that used several doses [13,14]. When sheep embryos are collected surgically, exposure of the reproductive tract often leads to the formation of post-operative adhesions, which reduce the number of embryos recovered after repeated surgeries [15]; however, repeated superovulatory treatments and surgical embryo recovery can be a feasible way to optimize in vivo embryo production in females that have high genetic value or are of an endangered breed, but are at the end of their reproductive lives, when they are candidates for culling for non-reproductive reasons (e.g., age, teeth). The primary problem with repeated

embryo recoveries after superovulatory treatments containing eCG is the possibility of a humoral response to this gonadotropin because repeated eCG administrations can induce the production of anti-eCG antibodies [16,17], which might affect fertility. Ojalada Soriana is a Spanish breed of sheep of which there are 8,531 and 209 registered ewes and rams, respectively; therefore, the Spanish government considers the breed “in danger of extinction” and eligible to benefit from several programs for the production and cryoconservation of gametes and embryos. The objective of this study was to assess the efficacy of the repeated administration of a simplified superovulatory protocol (a single dose of FSH and eCG) for in vivo embryo production compared to the standard protocol of administering several incrementally decreasing doses of FSH; the in vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effect of concentrations of anti-eCG antibodies before each eCG/FSH treatment on embryo production. During the breeding season (fall-winter), Ojalada Soriana ewes that were near the end of their commercial reproductive lives were given the treatments as many as three times. 2. Materials and methods The study was conducted between September 2008 and March 2009 at the experimental farm of the University of Zaragoza, Spain (latitude 41 ° 40= N), and the procedures were approved by the Ethics Committee for Animal Experiments, University of Zaragoza. The care and use of the animals followed the Spanish Policy for Animal Protection RD1201/05, which meets the requirements of the European Union Directive 86/609 on the Protection of Animals used for Experimental and Other Scientific Purposes. 2.1. Animals The 38 mature Ojalada Soriana ewes (mean age ⫽ 7.9 ⫾ 0.3 yr; mean number of previous lambings ⫽ 5.8 ⫾ 0.3) were reared on the farm of the Spanish Association of Ojalada Soriana Breeders under a 3-lambings-in-2-years breeding system that included the use of progestagen and eCG treatments to induce and synchronize estrus. At the end of their commercial reproductive lives, the ewes were brought to the university experimental farm, where they were housed in communal yards that had uncovered areas and fed a concentrate ration, lucerne hay, and barley straw at rates

F. Forcada et al. / Theriogenology 75 (2011) 769 –776

designed to provide 1.2 times their maintenance requirements. Fresh, clean water was available at all times. 2.2. Experimental design Ewes underwent an ultrasound pregnancy examination when they arrived at the experimental farm. Estrus was synchronized using intravaginal sponges that contained 30 mg fluorogestone acetate (Sincropart®, Ceva Salud Animal S.A., Barcelona, Spain), which were inserted for 14 d. To induce superovulation, ewes received one of the following two treatments: ewes in the Simplified Group (S; n ⫽ 19) each received 6 mL (210 IU) of pFSH (Folltropin®, Bioniche Animal Health, Ireland) and 500 IU eCG (Sincropart®, Ceva Salud Animal S.A., Barcelona, Spain) in a single i.m. administration 48 h before the intravaginal sponge was removed. Ewes in the Decreasing Group (D; n ⫽ 19) received 8 mL (280 IU) of pFSH in six i.m administrations of decreasing dosages (2 mL, 1.5 mL, 1.25 mL ⫻ 2, and 1 mL ⫻ 2) at 12-h intervals, which began 48 h before the intravaginal sponge was removed. Rams of known fertility were placed with the ewes at the time of pessary withdrawal and ewes were examined for evidence of estrus every 8 h. Embryos were collected through a mid-ventral laparotomy 7 d after the onset of estrus. Ewes were anesthetized using an i.m. administration of 0.4 mL 2% xylazine and, 5 min later, an i.v. injection of 10 mL sodium thiopental (20 mg/mL) (Thiobarbital, Braun Medical, Jaen, Spain). The ovarian response was quantified using the number of functional corpora lutea that had a good morphological appearance in agreement with their age, consistent with an active luteal phase. Uterine horns were exposed and, using a Foley catheter, flushed with a pre-warmed phosphate-buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) and antibiotics (penicillin and streptomycin). To minimize the development of post-operative abdominal adhesions, before closure, reproductive tracts were flushed with a 2.5% heparin solution in saline. Ova and embryos were examined under a stereomicroscope (20 – 40⫻ magnification) and classified based on their stage of development and morphology [18]. Compacted morulae and early, expanded, and hatched blastocysts were considered viable embryos. Based on the day of development on which the embryos were collected and the high in vivo viability of vitrified ovine blastocysts and compacted morulae [19,20], only the embryos without imperfections and with a spherical/symmetrical shape [21] in these two stages of development (hatched blastocysts

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excepted) were deemed freezable. Throughout the experiment (September through March), the ewes were given the same superovulatory treatment as many as three times at intervals of at least 50 d. Thirteen ewes had to be eliminated from the study. Two ewes in each of the two groups died for non-pathological reasons and, in nine ewes, it was impossible to perform uterine flushing because of surgical adhesions that resulted from previous laparotomies. However, previous performances of recoveries one and/or two were included in the statistical analysis. 2.3. Vitrification and warming procedures Immediately after they were recovered, 38 blastocysts from Group S and 35 blastocysts from Group D were vitrified in a 3-step protocol [22]. The vitrification and warming solutions were prepared using PBS with antibiotics supplemented with 0.1% (wt/vol) PVA. Embryos were put into a 200-␮L drop of 1.4 M glycerol for 5 min, then into a 200-␮L drop of 1.4 M glycerol and 3.6 M ethylene glycol for 5 min. They were transferred into a 15-␮L column of 3.4 M glycerol and 4.6 M ethylene glycol and loaded into a 0.25-mL plastic insemination straw using a fine glass capillary pipette. In the straw, the embryos in the vitrification medium were separated by air bubbles from two columns (30 ␮L) of the vitrification medium and two columns (120 ␮L) of a 0.5 M sucrose solution. After the straws were sealed, they were immediately plunged into LN2 and kept there. For warming, the straws were transferred from the LN2 into a 35 °C water bath and kept there for 10 s. The contents of each straw were expelled into a Petri dish and, to facilitate the mixing of the two solutions, stirred gently for 15 s. Embryos were retrieved and, to remove intracellular cryoprotectants, transferred into a 200-␮L drop of a 37 °C sucrose solution (0.25 M) for 3 min. For rehydration and equilibration, the embryos were held for 10 min in PBS that contained 0.1% (wt/vol) PVA at 39 °C. After warming, the blastocysts were cultured in vitro in TCM-199 supplemented with 10% FCS in 5% CO2 in air at 39 °C and 100% humidity, and examined 12 h, 24 h, and 48 h later. Embryos that re-expanded and resume blastocoelic activity were considered viable. Hatching rate was evaluated 24 h and 48 h after warming. 2.4. Peroxide status evaluation High intracellular concentrations of hydrogen peroxide in blastocysts can contribute to cellular damage and apoptosis [23,24]. After 48 h in culture, the concentration of peroxides in each hatched embryo was

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quantified using dichlorodihydrofluorescein diacetate (DCHFDA) (Hashimoto et al [25]). Hatching and hatched blastocysts were transferred to a culture medium that contained 10 ␮M DCHFDA. After 20 min in culture, embryo nuclei were counterstained using HOECHST 33342 (10 ␮g/mL) for 10 min. Embryos were fixed using glutaraldehide 1.5% for 15 min, washed in PBS, and mounted on glass slides using CC/mount® mounting medium. The fluorescent emissions from the embryos were recorded under a fluorescent microscope equipped with FITC filters (excitation, 405– 435 nm; emission, 515 nm). To minimize subjective observation errors, the coded samples were evaluated by a blinded operator. A bright green fluorescence indicated that a cell was DCHFDA-positive, and the HOECHST stain facilitated the counting of cells. 2.5. Blood sampling and progesterone and anti-eCG antibody assays To evaluate previous cyclicity in each synchronization treatment using plasma progesterone concentrations, blood samples were collected from the ewes twice: 7 d and immediately before an intravaginal sponge was inserted. In addition, before each superovulatory treatment, the first blood samples from the ewes in the S Group were used to assay the plasma concentrations of anti-eCG antibodies. A direct, solid-phase RIA [“sensitivity ⫽ 0.02 ng/mL)”] (Count-A-Count TKPG; DPC) in a single assay measured plasma progesterone concentrations. At low (2 ng/mL), medium (10 ng/mL), and high (30 ng/mL) concentrations, the intra-assay CV was 7.9%, 2.7%, and 10.3%, respectively. A ewe was considered cyclic if the progesterone concentration was ⬎ 1 ng/mL in at least one of the two blood samples taken before the start of each superovulatory treatment. A specific quantitative ELISA (Roy et al [17,26]) measured the concentrations [“(␮g/mL)”] of anti-eCG antibodies in plasma. The samples were assayed as duplicate 100-␮L aliquots and antibody concentrations were expressed as ␮g/mL of plasma. Intraand inter-assay coefficients of variation were 7.1% and 11.5%, respectively. Before each superovulatory treatment, ewes were assigned to one of two groups based on whether their anti-eCG antibody concentrations were high (⬎ 3 ␮g/mL) or low (ⱕ 3 ␮g/mL). 2.6. Assessment of the response to superovulatory treatments The following information was recorded for each ewe: evidence of estrus or ovulation indicated by the presence of corpora lutea that had a good morpholog-

ical appearance in agreement with their age, the number of corpora lutea , the number of recovered oocytes and embryos, the number of fertilized embryos, the number of viable embryos (compacted morulae and early, expanded, or hatched blastocysts), and the number of freezable embryos (compacted morulae and blastocysts, except hatched blastocysts). We calculated rates of recovery (the number of ova and embryos recovered per the number of corpora lutea ), fertilization (the number of embryos recovered per the number of ova and embryos recovered), viability and freezability (the number of viable and freezable embryos per the number of ova and embryos recovered). 2.7. Statistical analysis The experiment was based on a 2 ⫻ 3 factorial design in which superovulatory treatment and the number of recovery were fixed effects, with the latter based on repeated measures; therefore, the model was without an interaction effect. The values expressed as percentages were compared between groups and among successive recoveries within each group using Chi-square or Fisher Exact tests, as appropriate. To integrate the percentages in the model to determine whether they were influenced by the considered effects, individual proportions were arcsine-transformed before being subjected to statistical analysis. Results were expressed as mean ⫾ SEM. The probability level for statistical significance was set to P ⬍ 0.05. 3. Results Plasma progesterone concentrations indicated that the 38 mature Ojalada Soriana ewes were cyclic when each of the superovulatory treatments was performed in the experiment. In five of the 94 superovulatory treatments, a ewe failed to exhibit estrus (two in Group D and three in Group S); however, in nine instances, ewes experienced premature luteal regression and, on three occasions, a ewe did not ovulate. After the first superovulation treatment, those events were significantly (P ⬍ 0.05) more frequent in Group D (33%) than they were in Group S (6%) (Table 1). The factorial analysis of variance revealed that the onset of estrus occurred significantly (P⬍0.05) earlier among the ewes that received the simplified superovulatory treatment (25.2 ⫾ 0.8 h) than among those that received the conventional treatment (30.1 ⫾ 1.0 h); otherwise, the treatments did not differ significantly in their effect on the parameters measured (Table 1). With the exception of the onset of estrus, the number of

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Table 1 The effect of the number of superovulatory treatment on the ovarian response, and embryo production in Ojalada ewes given 280 IU of pFSH administered in six incrementally decreasing doses (Group D), or 210 IU of pFSH and 500 IU of eCG administered together in a single dose (Group S). Number of treatments No. of ewes in estrus (%) Onset of estrus, h (mean ⫾ SEM) No. of ovulating ewes with functional corpora lutea (%) No. of corpora lutea (mean ⫾ SEM) No. of ova recovered (mean ⫾ SEM) Recovery rate (%) No. of embryos (mean ⫾ SEM) Fertilization rate (%) No. of viable embryos (mean ⫾ SEM) Viability rate (%) No. of freezable embryos (mean ⫾ SEM) Freezability rate (%)

Group D

Group S

1

2

3

1

2

3

18/19 (95) 29.3 ⫾ 1.6 12/18 (67)

15/16 (94) 29.3 ⫾ 1.5e 12/15 (80)

13/13 (100) 32.3 ⫾ 2.7 12/13 (92)

18/19 (95) 26.4 ⫾ 1.1a 17/18 (94)

13/15 (87) 21.2 ⫾ 1.5b,f 12/13 (92)

12/12 (100) 27.7 ⫾ 1.5a 12/12 (100)

15.9 ⫾ 2.0a 10.7 ⫾ 1.7a 128/191 (67) 9.2 ⫾ 1.9a 110/128 (86)a 7.8 ⫾ 1.7c,a 94/128 (73)a 6.4 ⫾ 1.5c,a 77/128 (60)a

13.2 ⫾ 1.6c 9.0 ⫾ 1.5a 108/158 (68) 4.3 ⫾ 1.4b 52/108 (48)b 3.8 ⫾ 1.4d 46/108 (43)b 3.1 ⫾ 1.0d 37/108 (34)b

8.8 ⫾ 1.5b,d 5.0 ⫾ 0.9b 61/106 (58) 2.6 ⫾ 0.9b 31/61 (51)b 2.5 ⫾ 0.9b 30/61 (49)b 2.5 ⫾ 0.9b 30/61 (49)

14.5 ⫾ 2.1a 11.3 ⫾ 1.8c,a 193/246 (78) 8.6 ⫾ 1.9a 147/193 (76)a 7.7 ⫾ 1.6a 131/193 (68)a 6.6 ⫾ 1.6c,a 113/193 (59)a

10.6 ⫾ 2.3 6.5 ⫾ 1.6d 78/127 (61) 3.4 ⫾ 1.4b 41/78 (53)b 3.1 ⫾ 1.3b 37/78 (47)b 2.6 ⫾ 1.1d 31/78 (40)b

7.5 ⫾ 1.5b 4.7 ⫾ 1.4b 56/90 (62) 3.3 ⫾ 1.4b 40/56 (71)a 2.9 ⫾ 1.4b 35/56 (63) 2.2 ⫾ 1.2b 26/56 (46)

Within a group, different superscripts in the same row indicate differences of P ⬍ 0.1 (c,d) and P ⬍ 0.05 (a,b). Between groups, different superscripts in the same row indicate differences of P ⬍ 0.05 (e,f).

superovulatory treatments administered to individual ewes had an effect on all of the parameters, which were significantly lower after the first treatment (P ⬍ 0.01 for all variables, and P ⬍ 0.05 for rates, except NS for recovery rate). The ovulation rates and numbers of ova recovered after the third treatment were significantly lower than they were after the first, but the number of embryos and rates decreased significantly after the first treatment, showing similar responses to the second and third treatments. The responses were similar in the two superovulatory treatment groups and the total number of viable embryos per ewe in the decreasing (14.1 ⫾ 2.3) and the simplified (13.7 ⫾ 2.5) treatments were similar after three superovulatory treatments. A subjective visual assessment of the embryos indicated that the stage of development of the embryos produced by the two superovulatory treatments were not noticeably different. Furthermore, the type of superovulatory treatment did not influence significantly the viability rates of vitrified embryos (60% and 53% of re-expansion for the decreasing and the simplified treatments), the mean number of cells (HOECHST staining) (228 ⫾ 50 and 157 ⫾ 33 cells, respectively), and the number of cells that were DCHFDA-positive (higher hydrogen peroxide concentrations) per embryo (34 ⫾ 8 and 33 ⫾ 8 cells, respectively). The proportion of ewes that exhibited high (⬎ 3 ␮g/mL) concentrations of antibodies before each superovulatory treatment increased after successive treatments (from 30% after the first to 50% after the third application) (Table 2). In addition, mean plasma anti-

body concentrations increased after each successive recovery. High antibody concentrations just before the superovulatory treatment with eCG were associated with significant (P ⬍ 0.05) reductions in the fertilization rate in each of the three successive recoveries, and in viability and freezability rates in the second and third recoveries (Table 2). 4. Discussion Our experiment demonstrated the efficacy of a simplified superovulation treatment for in vivo embryo production in mature Ojalada Soriana ewes, and the negative effect of high concentrations of anti-eCG antibodies before the simplified, eCG-containing superovulatory treatment on embryo viability. The results of the treatments using a single dose of eCG plus FSH and the protocol involving six incrementally decreasing doses of FSH did not differ significantly in the numbers of recovered or viable embryos, or with respect to the sensitivity of embryos to freezing temperatures. The two superovulation protocols differed only in the onset of estrus, which was earliest in the ewes that received eCG and FSH in a single dose. This effect has been previously observed in Indian crossbred [27] and Corriedale [14] ewes. A concomitant advance in the preovulatory LH surge when eCG is included in the treatment can occur [14]. Artificial insemination programs associated to MOET techniques should take into consideration those peculiarities in LH secretion and the

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Table 2 The effect of the plasma anti-eCG antibody concentrations (L: ⬍ 3 ␮g/mL; H: ⬎ 3 ␮g/mL) before each superovulatory treatment (one single administration of 210 IU of pFSH and 500 IU of eCG) on embryo production and viability of Ojalada ewes. Number of recovery

1

2

3

Anti-eCG antibody concentrations

L

H

L

H

L

H

No. of ewes Anti-eCG antibody concentrations (␮g/ mL) (mean ⫾ SEM) Onset of estrus, h (mean ⫾ SEM) No. of corpora lutea (mean ⫾ SEM) No. of ova recovered (mean ⫾ SEM) No. of embryos (mean ⫾ SEM) Fertilization rate (%) No. of viable embryos (mean ⫾ SEM) Viability rate (%) No. of freezable embryos (mean ⫾ SEM) Freezability rate (%)

12 0.83 ⫾ 0.47a

5 5.49 ⫾ 2.19b

7 1.16 ⫾ 0.63a

5 5.08 ⫾ 1.28b

6 1.44 ⫾ 0.33a

6 6.90 ⫾ 1.76b

26.15 ⫾ 1.25 16.92 ⫾ 2.50c 13.33 ⫾ 2.26c 10.67 ⫾ 2.36c 128/160 (80)a 9.33 ⫾ 2.03 112/160 (70) 7.83 ⫾ 2.01 94/160 (59)

27.20 ⫾ 2.33 8.60 ⫾ 3.17d 6.60 ⫾ 1.81d 3.80 ⫾ 1.93d 19/33 (58)b 3.80 ⫾ 1.93 19/33 (58) 3.80 ⫾ 1.93 19/33 (58)

22.29 ⫾ 1.71 8.86 ⫾ 2.61 6.14 ⫾ 1.91 5.00 ⫾ 2.02 35/43 (81)a 4.43 ⫾ 1.91 31/43 (72)a 3.57 ⫾ 1.76 25/43 (58)a

21.60 ⫾ 2.40 13.00 ⫾ 4.39 7.00 ⫾ 3.10 1.20 ⫾ 1.20 6/35 (17)b 1.20 ⫾ 1.20 6/35 (17)b 1.20 ⫾ 1.20 6/35 (17)b

29.33 ⫾ 2.23 7.33 ⫾ 2.43 4.50 ⫾ 2.45 4.50 ⫾ 2.45 27/27 (100)a 3.83 ⫾ 2.37 23/27 (85)a 3.67 ⫾ 2.38 22/27 (81)a

26.00 ⫾ 2.0 7.67 ⫾ 2.08 4.83 ⫾ 1.45 2.17 ⫾ 1.64 13/29 (45)b 2.00 ⫾ 1.63 12/29 (41)b 0.67 ⫾ 0.42 4/29 (14)b

Different superscripts indicate differences at P ⬍ 0.1 (c,d) and P ⬍ 0.05 (a,b).

timing of estrus associated with the simplified, superovulation protocol because ewes that receive eCG might have to be inseminated earlier than ewes that did not receive eCG. The optimal time for insemination should be identified for each breed because the protocols that involve multiple FSH administrations and those that involve a single dose of FSH plus eCG might not differ in their effect on the onset of estrus in all breeds. To improve the efficacy of the superovulatory treatments for embryo production in sheep, ultrasonography has been a very useful tool for the last 15 yr, and the characterization of follicle development within the ovine ovary has elucidated that the number and viability of recovered embryos are lower in the presence of a dominant follicle (see reviews by González-Bulnes et al [28] and Menchaca et al [29]); consequently, some have suggested starting the superovulation in the absence of a dominant follicle, shortly after ovulation. Our study did not find significant differences between the two superovulatory protocols in the number of ova and embryos recovered after flushing; however, most of the studies that examined the use of eCG in simplified superovulatory protocols reported recovery rates that were lower than those derived from standard superovulatory treatments that use several doses of FSH, even when moderately low doses of FSH and eCG (similar to that used in our study) were administered in a single dose [13,14]. After the first superovulatory treatment, the number of ewes that had regressed corpora lutea was higher in the group of ewes that were superovulated using several doses of FSH (6 of 18) than they were in the group that received the simplified protocol (1 of 18). Although the number of ewes in the decreasing protocol that failed to

exhibit functional corpora lutea after ovulation was lower in the second (3 of 15) and third (1 of 13) recoveries, the incidence of this behavior was not negligible given that the experiment was performed during the breeding season. The cause of the phenomenon is unclear, but undernutrition and the status of the follicular population at the start of the gonadotropin treatment probably were not the only factors involved. Indeed, premature luteal regression can occur in FSHsuperovulated ewes [30 –32] and in donor ewes that were treated with a single dose of FSH and a high (ⱖ 800 UI) [33] or low (400 UI) [34] dose of eCG. It is possible, however, that the breed or the individual nature of the ewes, coupled with sensitivity to the stress induced by the change of location or by the superovulatory treatment, played a significant role in the frequency of luteal regression. Indeed, the simplified superovulatory treatment evaluated in our study appeared to be well suited to the Ojalada breed because it produced good functionality of the induced corpora lutea. In our study, the efficiency of in vivo embryo production in mature Ojalada Soriana ewes decreased significantly after the first superovulatory treatment. Apparently, changes in the ovulatory response after repeated superovulation with FSH have not been reported [15,30], although Forcada et al [31] observed a significant decrease in ovulation rate after the third FSH treatment. The formation of post-operative adhesions might impair the number of embryos recovered or sperm transport, which might increase the number of unfertilized ova [15,30]. Although only 63% of the ewes received three successive gonadotropin treatments, the total number of viable embryos per ewe was similar for the two superovulatory protocols.

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In our study, the proportion of embryos that reexpanded after vitrification and warming was similar to those reported in studies that used PVA as a substitute for serum in the vitrification and warming solutions [13,22]. Although the differences were not statistically significant, the embryos from the simplified treatment had 31% fewer cells and a re-expansion rate after vitrification that was 7% lower than did the blastocysts from the standard protocol of successively decreasing doses. Leoni et al [13] reported that embryos derived from a FSH/eCG treatment had fewer cells and, therefore, a lower tolerance to cryopreservation than did those derived from a FSH alone; however, the technical and commercial viability of the FSH/eCG protocol requires further study. To our knowledge, this is the first report of results from three consecutive superovulatory treatments, each involving a single dose of FSH/eCG, and the protocol was as effective as the traditional treatment that involved several doses of FSH. Although the response can be highly variable among females, ewes treated previously with eCG responded to another exposure to the hormone by increasing anti-eCG antibodies from day 5–7, after which production decreased progressively for more than 2 mo [17]. In general, high concentrations of anti-eCG antibodies just before another exposure to the gonadotropin are associated with a lack of or delay in estrus and pre-ovulatory LH surge [17], which impairs fertility after AI. This effect is much less pronounced after natural mating. In our study, high concentrations of anti-eCG antibodies before the superovulatory treatment impaired fertilization rate and the effect increased with successive treatments. Although the eCG doses in commercial treatments and in the simplified superovulation protocol evaluated in our study were similar, the effect of the concentration of anti-eCG antibodies before a superovulation treatment involving FSH on fertilization can differ from the commercial treatment of induction and synchronization of estrus, without FSH, during anestrus or associated to AI. Therefore, studies are needed to characterize the response to repeated eCG-based superovulatory treatments in MOET programs, particularly in the context of embryo production following AI. In conclusion, our study demonstrated that the repeated administration of a simplified superovulation protocol -a single dose of FSH plus eCG-, in comparison with the standard FSH treatment, did not impair in vivo embryo production from Ojalada ewes at the end of their reproductive lives. However, the embryos derived from FSH treatment alone seemed to be more

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resistant to cryopreservation. Although the reductions in the responses to successive treatments were similar in the two protocols, in the simplified superovulatory treatment such reduction could be, at least partially, attributable to the high anti-eCG antibody concentrations exhibited by some of the ewes before they received the FSH and eCG treatment, which impaired fertilization rate. Therefore, further study is needed to confirm the latter effect and to better understand the effect of anti-eCG antibodies concentrations on embryo viability and embryo production after superovulation following AI.

Acknowledgments This study was supported by Grant RZ2008-0002 from INIA and Grant A26 from DGA (Spain). The authors are grateful to INRA-Tours (Unité de Physiologie de la Reproduction des Mammifères Domestiques) for the analysis of anti-eCG antibodies.

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