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Repeated superovulation of high-prolificacy Rasa Aragonesa
ewes before culling as an inexpensive way to obtain
high-quality embryos
*
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F. Forcada , J.A. Abecia, J.M. Lozano, O. Zuniga
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Departamento de Produccion Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet, 177, 50013 Zaragoza,
Spain
Received 10 June 1999; received in revised form 4 October 1999; accepted 19 January 2000
Abstract
The purpose of this study was to assess the ovulatory response and embryo production after repeated superovulation of selected high-prolificacy Rasa Aragonesa ewes at the end of their reproductive life. A total of 211 superovulatory treatments were performed during the breeding seasons of 3 consecutive years. Ewes were given the same gonadotrophin treatment up to three times at intervals of at least 50 days. They were synchronized with intravaginal progestagen sponges and treated with ovine follicle stimulating hormone (oFSH) equivalent to a total dose of 176 NIH-FSH-S1 units in eight decreasing doses administered at 12-h intervals from 72 h before sponge removal. Embryos were recovered by laparotomy 7 days after the onset of oestrus. There was no effect of year on the superovulatory response as measured by the percentages of ewes in oestrus (80%) or ovulating (67%). An average of four freezable embryos (compacted morulae and blastocysts) were recovered at each treatment from the 141 ovulating ewes, with no significant differences amongst years, although the process seemed to be less efficient after the third treatment than after the first or second ones (2.4 vs. 4.1 (P,0.10) or 4.5 (P,0.05)
freezable embryos, respectively). This was primarily attributable to a lower ovulation rate (three fewer corpora lutea;
P,0.10). Results indicate that the methodology used in the present study could be an efficient and inexpensive way to
obtain high-quality embryos from selected high-prolificacy animals before culling. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Sheep; Superovulation; Embryo production; oFSH
1. Introduction factors which limit genetic improvement in sheep. The techniques of multiple ovulation and embryo Limited prolificacy and a relatively long inter- transfer (MOET) have been developed in this species generational interval are two of the most important to accelerate genetic improvement by increasing the number of offspring produced by each superior female.
*Corresponding author. Tel.:134-976-761-600; fax:1
34-976-Several follicle stimulating hormone (FSH) prepa-761-612.
E-mail address: [email protected] (F. Forcada). rations have been used in the last 15 years to induce
superovulation in sheep using protocols involving 2. Materials and methods several injections during the follicular phase
(Arm-strong and Evans, 1983; Rexroad and Powell, 1991; The experiment was conducted at the experimental D’Alessandro et al., 1996). The effects of single FSH farm of the University of Zaragoza, Spain (latitude
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injections also have been tested (Lopez-Sebastian et 418409 N), which meets the requirements of the al., 1993; Dattena et al., 1994). At present, only European Community Commission (1986).
purified ovine FSH (oFSH) (OvagenE, Immuno- The Rasa Aragonesa breed is a local Spanish Chemical Products Ltd., New Zealand) and porcine genotype that shows a short seasonal anoestrous
FSH (pFSH) (Folltropin -V, Vetrepharm, Canada; period between May and July (Forcada et al., 1992)
Pluset , Serono Veterinary, Rome) are commercially and a reduced prolificacy. Only mature Rasa Aragon-available. Although the ovulatory response of the esa ewes older than 8 years, with more than six oFSH can vary with batches, the commercial use of lambings in their productive life and selected for this pituitary preparation is recommended for induc- prolificacy (mean litter size of at least 1.4 lambs per tion of superovulation in donors and repeated treat- lambing), were used in the study. They were main-ments are not associated with a decreased ovarian tained on farms registered by ANGRA (Spanish response (Baril et al., 1993). Association of Rasa Aragonesa Breeders), where the Surgical embryo collection procedures have not mean litter size of these selected animals under an changed in the last few decades. Exteriorization of accelerated lambing management (three lambings in
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Spain) were administered by i.v. injection. Both cedure of the Statistical Analysis System (SAS uterine horns were exposed and flushed with pre- Institute Inc., 1987).
warmed phosphate buffered saline (PBS) sup-plemented with 1% bovine serum albumin (BSA;
Sigma, St. Louis, MO, USA) and antibiotics (penicil- 3. Results lin and streptomycin). The reproductive tract was
flushed with a 2.5% heparin solution in saline before Of the 211 ewes treated, only 141 (67%) ovulated closure in order to minimize the post-operative with functional corpora lutea (Table 1) and were development of abdominal adhesions. The embryos included in the analysis to test the effect of the were evaluated under a stereo microscope at a number of the gonadotrophin treatment on embryo magnification of 20–403, and classified by their production variables. Despite exhaustive veterinary stage of development and morphological appearance examination of the ewes on arrival to the experimen-according to Winterberger-Torres and Sevellec tal farm to establish that they were not pregnant, (1987). Given the excellent viability in vivo of 20% of them did not show oestrus after the vitrified ovine blastocysts (Szell and Windsor, 1994; superovulatory protocol; moreover, the percentage of Naitana et al., 1995, 1997), and even compacted ewes in oestrus had a tendency to be influenced by
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morulae (Martınez and Matkovic, 1998), only select- year of the treatment (P50.09). A high percentage ed embryos in these two stages of development, of premature luteal regression (12.4%), which was without any imperfections and with spherical / not dependent on the year of breeding (Table 1), also symmetrical shape (Linder and Wright, 1983), were was observed. An average of 4.0 freezable embryos deemed freezable in this experiment. (compacted morulae and blastocysts) were recovered
Table 1
The effect of the year in which the superovulatory treatment with oFSH was performed on the ovulatory response and embryo production of Rasa Aragonesa ewes at the end of their reproductive lives
Year (autumn–winter) 1996–1997 1997–1998 1998–1999 Total
No. of treated ewes 109 44 58 211
No. of ewes in oestrus (%) 87 (79.8) 31 (70.4) 51 (87.9) 169 (80.1)
No. of non-ovulating ewes (%) 5 (5.7) 1 (3.2) 1 (2.0) 7 (4.1)
No. of ewes with regressed 9 (10.3) 3 (9.7) 9 (17.6) 21 (12.4)
corpora lutea (%)
No. of ovulating ewes with 73 (83.9) 27 (87.1) 41 (80.4) 141 (83.4)
functional corpora lutea (%)
Early and compacted morulae, and early, expanded and hatched blastocysts.
b
Compacted morulae and blastocysts (except hatched blastocysts). There were no significant differences between years in any of the parameters studied (P.0.05).
Table 2
The effect of the number of the superovulatory treatment with oFSH on the ovarian response and embryo production of Rasa Aragonesa ewes at the end of their reproductive lives
Number of treatment 1 2 3
No. of treated ewes 113 73 25
No. of ewes in oestrus (%) 84 (74.3) 63 (86.3) 22 (88.0)
No. of non-ovulating ewes (%) 2 (2.4) 4 (6.3) 1 (4.5)
No. of ewes with regressed 12 (14.3) 7 (11.1) 2 (9.1)
corpora lutea (%)
No. of ovulating ewes with 70 (83.3) 52 (82.5) 19 (86.4)
functional corpora lutea (%)
c c d
Ovulation rate (no. of corpora lutea) 11.460.8 11.660.9 8.561.5 (mean6S.E.M.)
Early and compacted morulae, and early, expanded and hatched blastocysts.
b
4. Discussion 1996). However, the high ovulation rate obtained in our study (average of 11 corpora lutea from the 141 Since the cost-effectiveness of superovulatory ovulating ewes) indicates a good response to the treatments is a critical factor for the MOET tech- gonadotrophin treatment in the selected Rasa niques in small ruminants, the genetic value of the Aragonesa ewes when compared with others of the produced embryos should more than cover the same breed or with other Mediterranean genotypes
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expense of the superovulation, recovery and cryo- (Folch et al., 1991; Lopez-Sebastian et al., 1993; preservation procedures. The repeated use of selected D’Alessandro et al., 1996). This was achieved in ewes as donors at the end of their reproductive life, spite of their relatively high average age.
when they are candidates for culling, could be a The ovulatory response after repeated superovula-useful tool to reduce the unit cost of high-quality tion tended to decrease at the third oFSH treatment
embryos. compared with the first or second treatments
Cognie, Y., 1999. State of the art in sheep-goat embryo transfer. number of freezable embryos recovered was 2.7 and
Theriogenology 51, 105–116. 4.0, respectively. Although the individual nature of
Cognie, Y., Benoit, F., Poulin, N., Khatir, H., Driancourt, M.A., the ewes used in this experiment would have im- 1998. Effect of follicle size and of the FecB Booroola gene on paired the real return on investment, the response oocyte function in sheep. J. Reprod. Fertil. 112, 379–386.
D’Alessandro, A., Martemucci, G., Toteda, F., Gambacorta, M., obtained per ovulating ewe can be considered good.
Manchisi, A., 1996. Superovulation and embryo production in However, additional research on synchronization of
ewes using a commercial p-FSH. Small Ruminant Res. 19, the response induced by an oFSH treatment is 255–261.
needed in order to improve the percentages of Dattena, M., Vespignani, S., Branca, A., Gallus, M., Ledda, S., Naitana, S., Cappai, P., 1994. Superovulatory response and recovered ova and fertilized embryos as well as the
quality of embryos recovered from anestrous ewes after a homogeneity of the embryo development stage
ob-single injection of porcine FSH dissolved in PVP. Theriogenol-tained. In fact, 0.4 and 0.7 additional fully hatched ogy 42, 235–239.
blastocysts per superovulatory treatment and ovulat- European Community Commission, 1986. Scientific Procedure ing ewe were also recovered. Using excellent and and Breeding of Animals for use in Scientific Procedure
Establishments, Directive 86 / 609 / EEC. good compacted morulae and blastocysts, we can
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Folch, J., Cocero, M.J., Ramon, J.P., Fernandez-Arias, A., Alabart, expect an in vivo viability of 50% after transfer of
J.L., 1991. Embryo recovery in the oviduct improves the vitrified-thawed sheep embryos to recipients (Szell efficiency of superovulation in the ewe. In: 7th Scientific and Windsor, 1994; Naitana et al., 1995, 1997; Meeting of the European Embryo Transfer Association,
Cam-´
Martınez and Matkovic, 1998). bridge, Vol. 1, p. 144, Abstract.
Forcada, F., Abecia, J.A., Sierra, I., 1992. Seasonal changes in In conclusion, the results of the present study
oestrous activity and ovulation rate in Rasa Aragonesa ewes indicate that the use of prolific Rasa Aragonesa ewes
maintained at two different body condition levels. Small at the end of their productive life as embryo donors Ruminant Res. 8, 313–324.
seems to result in a lower percentage of ovulating Ishwar, A.K., Memon, M.A., 1996. Embryo transfer in sheep and ewes and fewer recovered ova and fertilized embryos goats: a review. Small Ruminant Res. 19, 35–43.
Linder, G.M., Wright, W., 1983. Bovine embryo morphology and than typically seen in younger animals. However, the
evaluation. Theriogenology 20, 407–416. good ovulatory response to the superovulatory
treat-Lindsay, D.R., 1996. Environment and reproductive behaviour. ment of the ovulating ewes in response to three Anim. Reprod. Sci. 42, 1–12.
successive gonadotrophin treatments, indicates that Lopez-Sebastian, A., Gomez-Brunet, A., Lishman, A.W., Johnson,´ ´ ´ the methodology used in the present study is an S.K., Inskeep, E.K., 1993. Modification by propylene glycol of ovulation rate in ewes in response to a single injection of FSH. efficient and inexpensive method for the collection of
J. Reprod. Fertil. 99, 437–442. high-quality embryos from high-prolificacy ewes
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Marıa, G.A., Ascaso, M.S., 1999. Litter size, lambing interval and selected before culling. lamb mortality of Salz, Rasa Aragonesa, Romanov and F ewes
1
on accelerated lambing management. Small Ruminant Res. 32, 167–172.
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Martınez, A.G., Matkovic, M., 1998. Cryopreservation of ovine Acknowledgements embryos: slow freezing and vitrification. Theriogenology 49,
1039–1049.
This study was supported by Grant 95-0032-OP Naitana, S., Dattena, M., Gallus, M., Loi, P., Branca, A., Ledda, S., Cappai, P., 1995. Recipient synchronization affects viability from C.I.C.Y.T. (Spain). The authors thank ANGRA
of vitrified ovine blastocysts. Theriogenology 43, 1371–1378. for providing the Rasa Aragonesa ewes used in the
Naitana, S., Ledda, S., Loi, P., Leoni, G., Bogliolo, L., Dattena, present study. M., Cappai, P., 1997. Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryop-reserve ovine embryos at different stages of development. Anim. Reprod. Sci. 48, 247–256.
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