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Repeated superovulation of high-prolificacy Rasa Aragonesa ewes before culling as an inexpensive way to obtain high-quality embryos
Livestock Production Science 66 (2000) 263–269 www.elsevier.com / locate / livprodsci
Repeated superovulation of high-prolificacy Rasa Aragonesa ewes before culling as an inexpensive way to obtain high-quality embryos ´˜ F. Forcada*, J.A. Abecia, J.M. Lozano, O. Zuniga ´ Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet, 177, 50013 Zaragoza, Departamento de Produccion 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 Limited prolificacy and a relatively long intergenerational interval are two of the most important *Corresponding author. Tel.: 134-976-761-600; fax: 134-976761-612. E-mail address: [email protected] (F. Forcada).
factors which limit genetic improvement in sheep. The techniques of multiple ovulation and embryo transfer (MOET) have been developed in this species to accelerate genetic improvement by increasing the number of offspring produced by each superior female. Several follicle stimulating hormone (FSH) preparations have been used in the last 15 years to induce
0301-6226 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00168-8
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F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269
superovulation in sheep using protocols involving several injections during the follicular phase (Armstrong and Evans, 1983; Rexroad and Powell, 1991; D’Alessandro et al., 1996). The effects of single FSH ´ et injections also have been tested (Lopez-Sebastian al., 1993; Dattena et al., 1994). At present, only purified ovine FSH (oFSH) (OvagenE, ImmunoChemical Products Ltd., New Zealand) and porcine FSH (pFSH) (Folltropin -V, Vetrepharm, Canada; Pluset , Serono Veterinary, Rome) are commercially available. Although the ovulatory response of the oFSH can vary with batches, the commercial use of this pituitary preparation is recommended for induction of superovulation in donors and repeated treatments are not associated with a decreased ovarian response (Baril et al., 1993). Surgical embryo collection procedures have not changed in the last few decades. Exteriorization of the reproductive tract often leads to the formation of post-operative adhesions in the uterus, oviducts and ovaries, thus inducing a reduction in embryo recovery after repeated surgery (Torres and Sevellec, 1987). Although the use of flushed heparinized saline solutions can delay the development of such adhesions, recovery from genetically superior ewes often yields low numbers of embryos. However, the alternative, embryo production in vitro in sheep, has several limitations. The low efficiency of the technique, as measured by the proportion of blastocysts obtained using oocytes, especially from small and medium-sized antral follicles matured in vitro (Cognie et al., 1998), makes the selection of oocytes suitable for in vitro maturation, fertilization and subsequent development from the best females difficult (Cognie, 1999). A new and interesting possibility to obtain embryos of high genetic value involves identification of the best females for a given character, recorded throughout their productive life, and their use as embryo donors when they are candidates for culling for non-reproductive reasons (age, teeth, etc.). The present experiment was designed to evaluate the embryo yield from such selected ewes at the end of their productive life, undergoing up to three superovulatory hormonal treatments with oFSH during their final breeding season (autumn–winter). The study was performed over 3 consecutive years.
2. Materials and methods The experiment was conducted at the experimental farm of the University of Zaragoza, Spain (latitude 418409 N), which meets the requirements of the European Community Commission (1986). The Rasa Aragonesa breed is a local Spanish genotype that shows a short seasonal anoestrous period between May and July (Forcada et al., 1992) and a reduced prolificacy. Only mature Rasa Aragonesa ewes older than 8 years, with more than six lambings in their productive life and selected for prolificacy (mean litter size of at least 1.4 lambs per lambing), were used in the study. They were maintained on farms registered by ANGRA (Spanish Association of Rasa Aragonesa Breeders), where the mean litter size of these selected animals under an accelerated lambing management (three lambings in ´ and Ascaso, 1999), being 2 years) is 1.4 (Marıa brought to the University at the end of their productive life. Animals were housed in communal yards with an uncovered area and fed a concentrate ration, lucerne hay and barley straw at rates designed to provide 1.2 times their maintenance requirements. Fresh, clean water was available at all times. Oestrus was synchronized using intravaginal sponges containing 30 mg fluorogestone acetate (FGA) (Chrono-gest; Intervet, Salamanca, Spain) inserted for 14 days. Ewes were superovulated with a total dose of 176 NIH-FSH-S1 units of NIADDKoFSH-17 (Ovagen ICP-LTD Ltd., New Zealand) in eight doses administered i.m. at 12-h intervals starting 72 h before sponge removal; each animal received the total dose in 10 ml of solution subdivided into injections of 2, 2, 1, 1, 1, 1, 1 and 1 ml. In order to avoid any variability of fertilization rate due to the intrauterine insemination technique (operator effect, fresh or frozen-thawed semen, number of spermatozoa per dose, etc.), only natural mating was used. Rams of proven fertility were placed with the ewes at the time of sponge removal, and ewes were checked for oestrus every 8 h using different males. The embryos were collected via mid-ventral laparotomy 7 days after the onset of oestrus. The ewes were anesthetized by i.m. injection with 0.4 ml 2% xylazine and, 5 min later, 10 ml sodium thiopental (20 mg / ml) (Thiobarbital, Braun Medical, Jaen,
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269
Spain) were administered by i.v. injection. Both uterine horns were exposed and flushed with prewarmed phosphate buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) and antibiotics (penicillin and streptomycin). The reproductive tract was flushed with a 2.5% heparin solution in saline before closure in order to minimize the post-operative development of abdominal adhesions. The embryos were evaluated under a stereo microscope at a magnification of 20–403, and classified by their stage of development and morphological appearance according to Winterberger-Torres and Sevellec (1987). Given the excellent viability in vivo of vitrified ovine blastocysts (Szell and Windsor, 1994; Naitana et al., 1995, 1997), and even compacted ´ morulae (Martınez and Matkovic, 1998), only selected embryos in these two stages of development, without any imperfections and with spherical / symmetrical shape (Linder and Wright, 1983), were deemed freezable in this experiment. In order to optimize the costs of the superovulatory treatments, ewes underwent a pregnancy diagnosis by ultrasound procedures when they arrived at the experimental farm. A total of 211 superovulatory treatments were performed throughout the autumn and winter of 1996–1997 (n5109), 1997–1998 (n5 44) and 1998–1999 (n558). In each year the ewes were given the same superovulatory treatment up to three times at intervals of at least 50 days from September to February. Animals that did not show oestrus or presented regressing corpora lutea (with a pale and avascular morphology) after the first embryo recovery were eliminated from the study, although their previous performances in treatments 1 and / or 2 were included in subsequent analyses. Proportions of ewes showing oestrus, not ovulating or with pale corpora lutea were compared by chi-square analysis for the effect of the year in which the superovulatory treatments were performed. The incidences of ovarian response, and numbers and quality of embryos, were analysed by one-way analysis of variance. Because there was no effect of year on ovulation rate or embryo number, data for all years were pooled and the effect of the number of the superovulatory treatment in the same ewe on these variables was compared using the GLM pro-
265
cedure of the Statistical Analysis System (SAS Institute Inc., 1987).
3. Results Of the 211 ewes treated, only 141 (67%) ovulated with functional corpora lutea (Table 1) and were included in the analysis to test the effect of the number of the gonadotrophin treatment on embryo production variables. Despite exhaustive veterinary examination of the ewes on arrival to the experimental farm to establish that they were not pregnant, 20% of them did not show oestrus after the superovulatory protocol; moreover, the percentage of ewes in oestrus had a tendency to be influenced by year of the treatment (P 5 0.09). A high percentage of premature luteal regression (12.4%), which was not dependent on the year of breeding (Table 1), also was observed. An average of 4.0 freezable embryos (compacted morulae and blastocysts) were recovered from each of the 141 ovulating ewes (Table 1). As there were no differences among years in the response to the oFSH treatment protocol, data were pooled to test the effect of the superovulatory treatment number on ovarian response and embryo production (Table 2). Percentage of ewes in oestrus had a tendency to be influenced by the number of superovulatory treatment (P 5 0.08); however, there was no effect on percentage of ewes with regressed corpora lutea. The process seemed to become less efficient after the third treatment, especially due to the somewhat lower ovulation rate (8.5 corpora lutea vs. 11.4 and 11.6 after the first and second treatments; P , 0.10), which resulted in a reduced yield of embryos (2.4 freezable vs. 4.1 after the first treatment (P , 0.10) and 4.5 after the second treatment (P , 0.05)). However, neither the percentages of ova recovered (64.0, 69.0 and 63.5% for the first, second and third treatments, respectively) nor the percentages of embryos fertilized (79.4, 77.5 and 74.1%, respectively) nor the number of embryos considered as transferable (freezable plus non-compacted morulae and hatched blastocysts; 5.0, 5.4 and 3.4 embryos, respectively), were affected by the number of the superovulatory and embryo recovery procedures.
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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 No. of ewes in oestrus (%) No. of non-ovulating ewes (%) No. of ewes with regressed corpora lutea (%) No. of ovulating ewes with functional corpora lutea (%) No. of corpora lutea (mean6S.E.M.) No. of recovered ova (mean6S.E.M.) No. of fertilized embryos (mean6S.E.M.) No. of transferable embryos a (mean6S.E.M.) No. of freezable embryos b (mean6S.E.M.)
109 87 (79.8) 5 (5.7) 9 (10.3)
44 31 (70.4) 1 (3.2) 3 (9.7)
58 51 (87.9) 1 (2.0) 9 (17.6)
211 169 (80.1) 7 (4.1) 21 (12.4)
73 (83.9)
27 (87.1)
41 (80.4)
141 (83.4)
11.460.8
10.461.4
10.961.0
11.060.6
7.760.6
6.761.3
6.960.8
7.360.5
6.260.6
4.661.0
5.660.8
5.760.4
5.360.5
3.761.0
4.960.8
4.960.4
4.460.5
3.260.9
3.960.6
4.060.3
a
Early and compacted morulae, and early, expanded and hatched blastocysts. Compacted morulae and blastocysts (except hatched blastocysts). There were no significant differences between years in any of the parameters studied (P . 0.05). b
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 No. of ewes in oestrus (%) No. of non-ovulating ewes (%) No. of ewes with regressed corpora lutea (%) No. of ovulating ewes with functional corpora lutea (%) Ovulation rate (no. of corpora lutea) (mean6S.E.M.) No. of recovered ova (mean6S.E.M.) No. of fertilized embryos (mean6S.E.M.) No. of transferable embryos a (mean6S.E.M.) No. of freezable embryos b (mean6S.E.M.)
113 84 (74.3) 2 (2.4) 12 (14.3)
73 63 (86.3) 4 (6.3) 7 (11.1)
25 22 (88.0) 1 (4.5) 2 (9.1)
70 (83.3)
52 (82.5)
19 (86.4)
11.460.8 c
11.660.9 c
8.561.5 d
7.360.6 cd
8.060.7 c
5.461.2 d
5.860.6
6.260.7
4.061.1
5.060.6
5.460.7
3.560.8
4.160.5 c
4.560.6 C
2.460.9 Dd
a
Early and compacted morulae, and early, expanded and hatched blastocysts. Compacted morulae and blastocysts (except hatched blastocysts). Within row, different superscripts indicate differences of P , 0.10 (small letters) or P , 0.05 (capital letters). b
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269
4. Discussion Since the cost-effectiveness of superovulatory treatments is a critical factor for the MOET techniques in small ruminants, the genetic value of the produced embryos should more than cover the expense of the superovulation, recovery and cryopreservation procedures. The repeated use of selected ewes as donors at the end of their reproductive life, when they are candidates for culling, could be a useful tool to reduce the unit cost of high-quality embryos. The failure of 20% of the ewes receiving the gonadotrophin treatment to exhibit oestrus probably reflects the fact that they were at the end of their commercial life; a rate below 10% is normal in younger animals receiving pFSH (D’Alessandro et al., 1996). The high percentage of Rasa Aragonesa ewes showing regressed corpora lutea (12.4%) at laparotomy is broadly comparable with the incidence of up to 27% associated with superovulation in goats (Tervit et al., 1986). Prematurely regressed corpora lutea have been reported previously in small proportions of superovulated ewes (Trounson et al., 1976; Ryan et al., 1991; Schiewe et al., 1991). The cause of this phenomenon is uncertain but undernutrition or status of the follicular population at the start of the gonadotrophin treatment are not the only factors involved. The percentage of superovulated ewes with regressed corpora lutea seems to be greater in seasonal anoestrus than it is in the breeding season (Ryan et al., 1991), probably due to a reduced progesterone support before the superovulatory treatment in the anoestrous period (Pearce and Oldham, 1984) which is unable to prevent a premature release of PGF 2a from the uterus. The individual nature of the females used in the present study, associated with the stress induced either by the change of location or of breeding conditions, may have played a significant role. One common feature reported in the literature is the high variability in ovulation rate and the number of embryos recovered from small ruminants, especially when they are not subjected to a treatment with gonadotrophin-releasing hormone (Ishwar and Memon, 1996; Cognie, 1999) or are susceptible to social or nutritional influences on sexual seasonality and ovulation rate (Forcada et al., 1992; Lindsay,
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1996). However, the high ovulation rate obtained in our study (average of 11 corpora lutea from the 141 ovulating ewes) indicates a good response to the gonadotrophin treatment in the selected Rasa Aragonesa ewes when compared with others of the same breed or with other Mediterranean genotypes ´ ´ et al., 1993; (Folch et al., 1991; Lopez-Sebastian D’Alessandro et al., 1996). This was achieved in spite of their relatively high average age. The ovulatory response after repeated superovulation tended to decrease at the third oFSH treatment compared with the first or second treatments independently of the month in the breeding season in which the superovulatory protocol was performed. In a previous study, three successive treatments with pFSH at 50-day periods did not alter the number of recorded corpora lutea in sheep (Torres and Sevellec, 1987). In goats, repeated superovulation with porcine FSH seems to reduce the ovulation rate after the third treatment due to the effect of anti-FSH antibodies, but the superovulatory response was maintained in this species treated several times (up to five) with an ovine FSH preparation (Baril et al., 1993). Moreover, Torres and Sevellec (1987) reported that the formation of post-operative adhesions hardly impaired the percentage of embryo recovery or even sperm transport (increasing the percentage of unfertilized ova). As surgery procedures did not alter either variable after successive superovulation treatments in the present study, the low number of freezable embryos produced by the Rasa Aragonesa ewes after the third treatment seems to be mainly attributable to a reduced response to gonadotrophin. Thus, because an average of 11 freezable embryos can be obtained from ovulating ewes receiving the three successive gonadotrophin treatments, the technique seems to be economically sustainable, although this is true only for 27% (19 / 70) of the ewes. One major objective of the embryo production evaluation conducted in the present study was to successfully cryo-preserve (vitrify) selected embryos. Consequently, only compacted morulae, and early and expanded blastocysts, even with a cracked zona, were considered freezable at the time of recovery, and earlier stages, up to compacted morulae and hatched blastocysts, were rejected. On average, 7.4 and 11.0 functional corpora lutea per superovulatory treatment and ovulating ewe were recorded while the
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number of freezable embryos recovered was 2.7 and 4.0, respectively. Although the individual nature of the ewes used in this experiment would have impaired the real return on investment, the response obtained per ovulating ewe can be considered good. However, additional research on synchronization of the response induced by an oFSH treatment is needed in order to improve the percentages of recovered ova and fertilized embryos as well as the homogeneity of the embryo development stage obtained. In fact, 0.4 and 0.7 additional fully hatched blastocysts per superovulatory treatment and ovulating ewe were also recovered. Using excellent and good compacted morulae and blastocysts, we can expect an in vivo viability of 50% after transfer of vitrified-thawed sheep embryos to recipients (Szell and Windsor, 1994; Naitana et al., 1995, 1997; ´ Martınez and Matkovic, 1998). In conclusion, the results of the present study indicate that the use of prolific Rasa Aragonesa ewes at the end of their productive life as embryo donors seems to result in a lower percentage of ovulating ewes and fewer recovered ova and fertilized embryos than typically seen in younger animals. However, the good ovulatory response to the superovulatory treatment of the ovulating ewes in response to three successive gonadotrophin treatments, indicates that the methodology used in the present study is an efficient and inexpensive method for the collection of high-quality embryos from high-prolificacy ewes selected before culling.
Acknowledgements This study was supported by Grant 95-0032-OP from C.I.C.Y.T. (Spain). The authors thank ANGRA for providing the Rasa Aragonesa ewes used in the present study.
References Armstrong, D.T., Evans, G., 1983. Factors influencing success of embryo transfer in sheep and goats. Theriogenology 19, 31–43. ´ P., 1993. Training Manual for Baril, G., Brebion, P., Chesne, Embryo Transfer in Sheep and Goats. FAO, Rome, ISSN 1014-1019.
Cognie, Y., 1999. State of the art in sheep-goat embryo transfer. Theriogenology 51, 105–116. Cognie, Y., Benoit, F., Poulin, N., Khatir, H., Driancourt, M.A., 1998. Effect of follicle size and of the FecB Booroola gene on oocyte function in sheep. J. Reprod. Fertil. 112, 379–386. D’Alessandro, A., Martemucci, G., Toteda, F., Gambacorta, M., Manchisi, A., 1996. Superovulation and embryo production in ewes using a commercial p-FSH. Small Ruminant Res. 19, 255–261. Dattena, M., Vespignani, S., Branca, A., Gallus, M., Ledda, S., Naitana, S., Cappai, P., 1994. Superovulatory response and quality of embryos recovered from anestrous ewes after a single injection of porcine FSH dissolved in PVP. Theriogenology 42, 235–239. European Community Commission, 1986. Scientific Procedure and Breeding of Animals for use in Scientific Procedure Establishments, Directive 86 / 609 / EEC. ´ J.P., Fernandez-Arias, ´ Folch, J., Cocero, M.J., Ramon, A., Alabart, J.L., 1991. Embryo recovery in the oviduct improves the efficiency of superovulation in the ewe. In: 7th Scientific Meeting of the European Embryo Transfer Association, Cambridge, Vol. 1, p. 144, Abstract. Forcada, F., Abecia, J.A., Sierra, I., 1992. Seasonal changes in oestrous activity and ovulation rate in Rasa Aragonesa ewes maintained at two different body condition levels. Small Ruminant Res. 8, 313–324. Ishwar, A.K., Memon, M.A., 1996. Embryo transfer in sheep and goats: a review. Small Ruminant Res. 19, 35–43. Linder, G.M., Wright, W., 1983. Bovine embryo morphology and evaluation. Theriogenology 20, 407–416. Lindsay, D.R., 1996. Environment and reproductive behaviour. Anim. Reprod. Sci. 42, 1–12. ´ ´ A., Gomez-Brunet, ´ Lopez-Sebastian, A., Lishman, A.W., Johnson, S.K., Inskeep, E.K., 1993. Modification by propylene glycol of ovulation rate in ewes in response to a single injection of FSH. J. Reprod. Fertil. 99, 437–442. ´ G.A., Ascaso, M.S., 1999. Litter size, lambing interval and Marıa, lamb mortality of Salz, Rasa Aragonesa, Romanov and F 1 ewes on accelerated lambing management. Small Ruminant Res. 32, 167–172. ´ Martınez, A.G., Matkovic, M., 1998. Cryopreservation of ovine embryos: slow freezing and vitrification. Theriogenology 49, 1039–1049. Naitana, S., Dattena, M., Gallus, M., Loi, P., Branca, A., Ledda, S., Cappai, P., 1995. Recipient synchronization affects viability of vitrified ovine blastocysts. Theriogenology 43, 1371–1378. Naitana, S., Ledda, S., Loi, P., Leoni, G., Bogliolo, L., Dattena, M., Cappai, P., 1997. Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development. Anim. Reprod. Sci. 48, 247–256. Pearce, D.T., Oldham, C.M., 1984. The ram effect, its mechanism and application to the management of sheep. In: Lindsay, D.R., Pearce, D.T. (Eds.), Reproduction in Sheep. Australian Academy of Science, Canberra, pp. 26–34. Rexroad, C.E., Powell, A.M., 1991. FSH injections and intrauterine insemination in protocols for superovulation of ewes. J. Anim. Sci. 69, 246–251.
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269 Ryan, J.P., Hunton, J.R., Maxwell, M.C., 1991. Increased production of sheep embryos following superovulation of Merino ewes with a combination of pregnant mare serum gonadotrophin and follicle stimulating hormone. Reprod. Fertil. Dev. 3, 551–560. SAS Institute Inc, 1987. SAS / STATE. Guide for Personal Computers, Version 6 Edition. SAS Institute Inc, Cary, NC. Schiewe, M.C., Fitz, T.A., Brown, J.L., Stuart, L.D., Wildt, D.E., 1991. Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2a receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep. J. Reprod. Fertil. 93, 19–30. Szell, A.Z., Windsor, D.P., 1994. Survival of vitrified sheep embryos in vitro and in vivo. Theriogenology 42, 881–889.
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Tervit, H.R., Gold, P.G., McKenzie, R.D., 1986. Development of an effective goat embryo transfer regime. Proc. NZ Soc. Anim. Prod. 46, 233–236. Torres, S., Sevellec, C., 1987. Repeated superovulation and ´ surgical recovery of embryos in the ewe. Reprod. Nutr. Dev. 27, 859–863. Trounson, A.O., Willadsen, S.M., Moor, R.M., 1976. Effect of prostaglandin analogue cloprostenol on oestrus, ovulation and embryonic viability in sheep. J. Agric. Sci. (Camb.) 86, 609– 611. Winterberger-Torres, S., Sevellec, C., 1987. Atlas of the Early Development of the Sheep Embryo. INRA, Paris.
Repeated superovulation of high-prolificacy Rasa Aragonesa ewes before culling as an inexpensive way to obtain high-quality embryos ´˜ F. Forcada*, J.A. Abecia, J.M. Lozano, O. Zuniga ´ Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet, 177, 50013 Zaragoza, Departamento de Produccion 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 Limited prolificacy and a relatively long intergenerational interval are two of the most important *Corresponding author. Tel.: 134-976-761-600; fax: 134-976761-612. E-mail address: [email protected] (F. Forcada).
factors which limit genetic improvement in sheep. The techniques of multiple ovulation and embryo transfer (MOET) have been developed in this species to accelerate genetic improvement by increasing the number of offspring produced by each superior female. Several follicle stimulating hormone (FSH) preparations have been used in the last 15 years to induce
0301-6226 / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00168-8
264
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269
superovulation in sheep using protocols involving several injections during the follicular phase (Armstrong and Evans, 1983; Rexroad and Powell, 1991; D’Alessandro et al., 1996). The effects of single FSH ´ et injections also have been tested (Lopez-Sebastian al., 1993; Dattena et al., 1994). At present, only purified ovine FSH (oFSH) (OvagenE, ImmunoChemical Products Ltd., New Zealand) and porcine FSH (pFSH) (Folltropin -V, Vetrepharm, Canada; Pluset , Serono Veterinary, Rome) are commercially available. Although the ovulatory response of the oFSH can vary with batches, the commercial use of this pituitary preparation is recommended for induction of superovulation in donors and repeated treatments are not associated with a decreased ovarian response (Baril et al., 1993). Surgical embryo collection procedures have not changed in the last few decades. Exteriorization of the reproductive tract often leads to the formation of post-operative adhesions in the uterus, oviducts and ovaries, thus inducing a reduction in embryo recovery after repeated surgery (Torres and Sevellec, 1987). Although the use of flushed heparinized saline solutions can delay the development of such adhesions, recovery from genetically superior ewes often yields low numbers of embryos. However, the alternative, embryo production in vitro in sheep, has several limitations. The low efficiency of the technique, as measured by the proportion of blastocysts obtained using oocytes, especially from small and medium-sized antral follicles matured in vitro (Cognie et al., 1998), makes the selection of oocytes suitable for in vitro maturation, fertilization and subsequent development from the best females difficult (Cognie, 1999). A new and interesting possibility to obtain embryos of high genetic value involves identification of the best females for a given character, recorded throughout their productive life, and their use as embryo donors when they are candidates for culling for non-reproductive reasons (age, teeth, etc.). The present experiment was designed to evaluate the embryo yield from such selected ewes at the end of their productive life, undergoing up to three superovulatory hormonal treatments with oFSH during their final breeding season (autumn–winter). The study was performed over 3 consecutive years.
2. Materials and methods The experiment was conducted at the experimental farm of the University of Zaragoza, Spain (latitude 418409 N), which meets the requirements of the European Community Commission (1986). The Rasa Aragonesa breed is a local Spanish genotype that shows a short seasonal anoestrous period between May and July (Forcada et al., 1992) and a reduced prolificacy. Only mature Rasa Aragonesa ewes older than 8 years, with more than six lambings in their productive life and selected for prolificacy (mean litter size of at least 1.4 lambs per lambing), were used in the study. They were maintained on farms registered by ANGRA (Spanish Association of Rasa Aragonesa Breeders), where the mean litter size of these selected animals under an accelerated lambing management (three lambings in ´ and Ascaso, 1999), being 2 years) is 1.4 (Marıa brought to the University at the end of their productive life. Animals were housed in communal yards with an uncovered area and fed a concentrate ration, lucerne hay and barley straw at rates designed to provide 1.2 times their maintenance requirements. Fresh, clean water was available at all times. Oestrus was synchronized using intravaginal sponges containing 30 mg fluorogestone acetate (FGA) (Chrono-gest; Intervet, Salamanca, Spain) inserted for 14 days. Ewes were superovulated with a total dose of 176 NIH-FSH-S1 units of NIADDKoFSH-17 (Ovagen ICP-LTD Ltd., New Zealand) in eight doses administered i.m. at 12-h intervals starting 72 h before sponge removal; each animal received the total dose in 10 ml of solution subdivided into injections of 2, 2, 1, 1, 1, 1, 1 and 1 ml. In order to avoid any variability of fertilization rate due to the intrauterine insemination technique (operator effect, fresh or frozen-thawed semen, number of spermatozoa per dose, etc.), only natural mating was used. Rams of proven fertility were placed with the ewes at the time of sponge removal, and ewes were checked for oestrus every 8 h using different males. The embryos were collected via mid-ventral laparotomy 7 days after the onset of oestrus. The ewes were anesthetized by i.m. injection with 0.4 ml 2% xylazine and, 5 min later, 10 ml sodium thiopental (20 mg / ml) (Thiobarbital, Braun Medical, Jaen,
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269
Spain) were administered by i.v. injection. Both uterine horns were exposed and flushed with prewarmed phosphate buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA; Sigma, St. Louis, MO, USA) and antibiotics (penicillin and streptomycin). The reproductive tract was flushed with a 2.5% heparin solution in saline before closure in order to minimize the post-operative development of abdominal adhesions. The embryos were evaluated under a stereo microscope at a magnification of 20–403, and classified by their stage of development and morphological appearance according to Winterberger-Torres and Sevellec (1987). Given the excellent viability in vivo of vitrified ovine blastocysts (Szell and Windsor, 1994; Naitana et al., 1995, 1997), and even compacted ´ morulae (Martınez and Matkovic, 1998), only selected embryos in these two stages of development, without any imperfections and with spherical / symmetrical shape (Linder and Wright, 1983), were deemed freezable in this experiment. In order to optimize the costs of the superovulatory treatments, ewes underwent a pregnancy diagnosis by ultrasound procedures when they arrived at the experimental farm. A total of 211 superovulatory treatments were performed throughout the autumn and winter of 1996–1997 (n5109), 1997–1998 (n5 44) and 1998–1999 (n558). In each year the ewes were given the same superovulatory treatment up to three times at intervals of at least 50 days from September to February. Animals that did not show oestrus or presented regressing corpora lutea (with a pale and avascular morphology) after the first embryo recovery were eliminated from the study, although their previous performances in treatments 1 and / or 2 were included in subsequent analyses. Proportions of ewes showing oestrus, not ovulating or with pale corpora lutea were compared by chi-square analysis for the effect of the year in which the superovulatory treatments were performed. The incidences of ovarian response, and numbers and quality of embryos, were analysed by one-way analysis of variance. Because there was no effect of year on ovulation rate or embryo number, data for all years were pooled and the effect of the number of the superovulatory treatment in the same ewe on these variables was compared using the GLM pro-
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cedure of the Statistical Analysis System (SAS Institute Inc., 1987).
3. Results Of the 211 ewes treated, only 141 (67%) ovulated with functional corpora lutea (Table 1) and were included in the analysis to test the effect of the number of the gonadotrophin treatment on embryo production variables. Despite exhaustive veterinary examination of the ewes on arrival to the experimental farm to establish that they were not pregnant, 20% of them did not show oestrus after the superovulatory protocol; moreover, the percentage of ewes in oestrus had a tendency to be influenced by year of the treatment (P 5 0.09). A high percentage of premature luteal regression (12.4%), which was not dependent on the year of breeding (Table 1), also was observed. An average of 4.0 freezable embryos (compacted morulae and blastocysts) were recovered from each of the 141 ovulating ewes (Table 1). As there were no differences among years in the response to the oFSH treatment protocol, data were pooled to test the effect of the superovulatory treatment number on ovarian response and embryo production (Table 2). Percentage of ewes in oestrus had a tendency to be influenced by the number of superovulatory treatment (P 5 0.08); however, there was no effect on percentage of ewes with regressed corpora lutea. The process seemed to become less efficient after the third treatment, especially due to the somewhat lower ovulation rate (8.5 corpora lutea vs. 11.4 and 11.6 after the first and second treatments; P , 0.10), which resulted in a reduced yield of embryos (2.4 freezable vs. 4.1 after the first treatment (P , 0.10) and 4.5 after the second treatment (P , 0.05)). However, neither the percentages of ova recovered (64.0, 69.0 and 63.5% for the first, second and third treatments, respectively) nor the percentages of embryos fertilized (79.4, 77.5 and 74.1%, respectively) nor the number of embryos considered as transferable (freezable plus non-compacted morulae and hatched blastocysts; 5.0, 5.4 and 3.4 embryos, respectively), were affected by the number of the superovulatory and embryo recovery procedures.
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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 No. of ewes in oestrus (%) No. of non-ovulating ewes (%) No. of ewes with regressed corpora lutea (%) No. of ovulating ewes with functional corpora lutea (%) No. of corpora lutea (mean6S.E.M.) No. of recovered ova (mean6S.E.M.) No. of fertilized embryos (mean6S.E.M.) No. of transferable embryos a (mean6S.E.M.) No. of freezable embryos b (mean6S.E.M.)
109 87 (79.8) 5 (5.7) 9 (10.3)
44 31 (70.4) 1 (3.2) 3 (9.7)
58 51 (87.9) 1 (2.0) 9 (17.6)
211 169 (80.1) 7 (4.1) 21 (12.4)
73 (83.9)
27 (87.1)
41 (80.4)
141 (83.4)
11.460.8
10.461.4
10.961.0
11.060.6
7.760.6
6.761.3
6.960.8
7.360.5
6.260.6
4.661.0
5.660.8
5.760.4
5.360.5
3.761.0
4.960.8
4.960.4
4.460.5
3.260.9
3.960.6
4.060.3
a
Early and compacted morulae, and early, expanded and hatched blastocysts. Compacted morulae and blastocysts (except hatched blastocysts). There were no significant differences between years in any of the parameters studied (P . 0.05). b
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 No. of ewes in oestrus (%) No. of non-ovulating ewes (%) No. of ewes with regressed corpora lutea (%) No. of ovulating ewes with functional corpora lutea (%) Ovulation rate (no. of corpora lutea) (mean6S.E.M.) No. of recovered ova (mean6S.E.M.) No. of fertilized embryos (mean6S.E.M.) No. of transferable embryos a (mean6S.E.M.) No. of freezable embryos b (mean6S.E.M.)
113 84 (74.3) 2 (2.4) 12 (14.3)
73 63 (86.3) 4 (6.3) 7 (11.1)
25 22 (88.0) 1 (4.5) 2 (9.1)
70 (83.3)
52 (82.5)
19 (86.4)
11.460.8 c
11.660.9 c
8.561.5 d
7.360.6 cd
8.060.7 c
5.461.2 d
5.860.6
6.260.7
4.061.1
5.060.6
5.460.7
3.560.8
4.160.5 c
4.560.6 C
2.460.9 Dd
a
Early and compacted morulae, and early, expanded and hatched blastocysts. Compacted morulae and blastocysts (except hatched blastocysts). Within row, different superscripts indicate differences of P , 0.10 (small letters) or P , 0.05 (capital letters). b
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269
4. Discussion Since the cost-effectiveness of superovulatory treatments is a critical factor for the MOET techniques in small ruminants, the genetic value of the produced embryos should more than cover the expense of the superovulation, recovery and cryopreservation procedures. The repeated use of selected ewes as donors at the end of their reproductive life, when they are candidates for culling, could be a useful tool to reduce the unit cost of high-quality embryos. The failure of 20% of the ewes receiving the gonadotrophin treatment to exhibit oestrus probably reflects the fact that they were at the end of their commercial life; a rate below 10% is normal in younger animals receiving pFSH (D’Alessandro et al., 1996). The high percentage of Rasa Aragonesa ewes showing regressed corpora lutea (12.4%) at laparotomy is broadly comparable with the incidence of up to 27% associated with superovulation in goats (Tervit et al., 1986). Prematurely regressed corpora lutea have been reported previously in small proportions of superovulated ewes (Trounson et al., 1976; Ryan et al., 1991; Schiewe et al., 1991). The cause of this phenomenon is uncertain but undernutrition or status of the follicular population at the start of the gonadotrophin treatment are not the only factors involved. The percentage of superovulated ewes with regressed corpora lutea seems to be greater in seasonal anoestrus than it is in the breeding season (Ryan et al., 1991), probably due to a reduced progesterone support before the superovulatory treatment in the anoestrous period (Pearce and Oldham, 1984) which is unable to prevent a premature release of PGF 2a from the uterus. The individual nature of the females used in the present study, associated with the stress induced either by the change of location or of breeding conditions, may have played a significant role. One common feature reported in the literature is the high variability in ovulation rate and the number of embryos recovered from small ruminants, especially when they are not subjected to a treatment with gonadotrophin-releasing hormone (Ishwar and Memon, 1996; Cognie, 1999) or are susceptible to social or nutritional influences on sexual seasonality and ovulation rate (Forcada et al., 1992; Lindsay,
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1996). However, the high ovulation rate obtained in our study (average of 11 corpora lutea from the 141 ovulating ewes) indicates a good response to the gonadotrophin treatment in the selected Rasa Aragonesa ewes when compared with others of the same breed or with other Mediterranean genotypes ´ ´ et al., 1993; (Folch et al., 1991; Lopez-Sebastian D’Alessandro et al., 1996). This was achieved in spite of their relatively high average age. The ovulatory response after repeated superovulation tended to decrease at the third oFSH treatment compared with the first or second treatments independently of the month in the breeding season in which the superovulatory protocol was performed. In a previous study, three successive treatments with pFSH at 50-day periods did not alter the number of recorded corpora lutea in sheep (Torres and Sevellec, 1987). In goats, repeated superovulation with porcine FSH seems to reduce the ovulation rate after the third treatment due to the effect of anti-FSH antibodies, but the superovulatory response was maintained in this species treated several times (up to five) with an ovine FSH preparation (Baril et al., 1993). Moreover, Torres and Sevellec (1987) reported that the formation of post-operative adhesions hardly impaired the percentage of embryo recovery or even sperm transport (increasing the percentage of unfertilized ova). As surgery procedures did not alter either variable after successive superovulation treatments in the present study, the low number of freezable embryos produced by the Rasa Aragonesa ewes after the third treatment seems to be mainly attributable to a reduced response to gonadotrophin. Thus, because an average of 11 freezable embryos can be obtained from ovulating ewes receiving the three successive gonadotrophin treatments, the technique seems to be economically sustainable, although this is true only for 27% (19 / 70) of the ewes. One major objective of the embryo production evaluation conducted in the present study was to successfully cryo-preserve (vitrify) selected embryos. Consequently, only compacted morulae, and early and expanded blastocysts, even with a cracked zona, were considered freezable at the time of recovery, and earlier stages, up to compacted morulae and hatched blastocysts, were rejected. On average, 7.4 and 11.0 functional corpora lutea per superovulatory treatment and ovulating ewe were recorded while the
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number of freezable embryos recovered was 2.7 and 4.0, respectively. Although the individual nature of the ewes used in this experiment would have impaired the real return on investment, the response obtained per ovulating ewe can be considered good. However, additional research on synchronization of the response induced by an oFSH treatment is needed in order to improve the percentages of recovered ova and fertilized embryos as well as the homogeneity of the embryo development stage obtained. In fact, 0.4 and 0.7 additional fully hatched blastocysts per superovulatory treatment and ovulating ewe were also recovered. Using excellent and good compacted morulae and blastocysts, we can expect an in vivo viability of 50% after transfer of vitrified-thawed sheep embryos to recipients (Szell and Windsor, 1994; Naitana et al., 1995, 1997; ´ Martınez and Matkovic, 1998). In conclusion, the results of the present study indicate that the use of prolific Rasa Aragonesa ewes at the end of their productive life as embryo donors seems to result in a lower percentage of ovulating ewes and fewer recovered ova and fertilized embryos than typically seen in younger animals. However, the good ovulatory response to the superovulatory treatment of the ovulating ewes in response to three successive gonadotrophin treatments, indicates that the methodology used in the present study is an efficient and inexpensive method for the collection of high-quality embryos from high-prolificacy ewes selected before culling.
Acknowledgements This study was supported by Grant 95-0032-OP from C.I.C.Y.T. (Spain). The authors thank ANGRA for providing the Rasa Aragonesa ewes used in the present study.
References Armstrong, D.T., Evans, G., 1983. Factors influencing success of embryo transfer in sheep and goats. Theriogenology 19, 31–43. ´ P., 1993. Training Manual for Baril, G., Brebion, P., Chesne, Embryo Transfer in Sheep and Goats. FAO, Rome, ISSN 1014-1019.
Cognie, Y., 1999. State of the art in sheep-goat embryo transfer. Theriogenology 51, 105–116. Cognie, Y., Benoit, F., Poulin, N., Khatir, H., Driancourt, M.A., 1998. Effect of follicle size and of the FecB Booroola gene on oocyte function in sheep. J. Reprod. Fertil. 112, 379–386. D’Alessandro, A., Martemucci, G., Toteda, F., Gambacorta, M., Manchisi, A., 1996. Superovulation and embryo production in ewes using a commercial p-FSH. Small Ruminant Res. 19, 255–261. Dattena, M., Vespignani, S., Branca, A., Gallus, M., Ledda, S., Naitana, S., Cappai, P., 1994. Superovulatory response and quality of embryos recovered from anestrous ewes after a single injection of porcine FSH dissolved in PVP. Theriogenology 42, 235–239. European Community Commission, 1986. Scientific Procedure and Breeding of Animals for use in Scientific Procedure Establishments, Directive 86 / 609 / EEC. ´ J.P., Fernandez-Arias, ´ Folch, J., Cocero, M.J., Ramon, A., Alabart, J.L., 1991. Embryo recovery in the oviduct improves the efficiency of superovulation in the ewe. In: 7th Scientific Meeting of the European Embryo Transfer Association, Cambridge, Vol. 1, p. 144, Abstract. Forcada, F., Abecia, J.A., Sierra, I., 1992. Seasonal changes in oestrous activity and ovulation rate in Rasa Aragonesa ewes maintained at two different body condition levels. Small Ruminant Res. 8, 313–324. Ishwar, A.K., Memon, M.A., 1996. Embryo transfer in sheep and goats: a review. Small Ruminant Res. 19, 35–43. Linder, G.M., Wright, W., 1983. Bovine embryo morphology and evaluation. Theriogenology 20, 407–416. Lindsay, D.R., 1996. Environment and reproductive behaviour. Anim. Reprod. Sci. 42, 1–12. ´ ´ A., Gomez-Brunet, ´ Lopez-Sebastian, A., Lishman, A.W., Johnson, S.K., Inskeep, E.K., 1993. Modification by propylene glycol of ovulation rate in ewes in response to a single injection of FSH. J. Reprod. Fertil. 99, 437–442. ´ G.A., Ascaso, M.S., 1999. Litter size, lambing interval and Marıa, lamb mortality of Salz, Rasa Aragonesa, Romanov and F 1 ewes on accelerated lambing management. Small Ruminant Res. 32, 167–172. ´ Martınez, A.G., Matkovic, M., 1998. Cryopreservation of ovine embryos: slow freezing and vitrification. Theriogenology 49, 1039–1049. Naitana, S., Dattena, M., Gallus, M., Loi, P., Branca, A., Ledda, S., Cappai, P., 1995. Recipient synchronization affects viability of vitrified ovine blastocysts. Theriogenology 43, 1371–1378. Naitana, S., Ledda, S., Loi, P., Leoni, G., Bogliolo, L., Dattena, M., Cappai, P., 1997. Polyvinyl alcohol as a defined substitute for serum in vitrification and warming solutions to cryopreserve ovine embryos at different stages of development. Anim. Reprod. Sci. 48, 247–256. Pearce, D.T., Oldham, C.M., 1984. The ram effect, its mechanism and application to the management of sheep. In: Lindsay, D.R., Pearce, D.T. (Eds.), Reproduction in Sheep. Australian Academy of Science, Canberra, pp. 26–34. Rexroad, C.E., Powell, A.M., 1991. FSH injections and intrauterine insemination in protocols for superovulation of ewes. J. Anim. Sci. 69, 246–251.
F. Forcada et al. / Livestock Production Science 66 (2000) 263 – 269 Ryan, J.P., Hunton, J.R., Maxwell, M.C., 1991. Increased production of sheep embryos following superovulation of Merino ewes with a combination of pregnant mare serum gonadotrophin and follicle stimulating hormone. Reprod. Fertil. Dev. 3, 551–560. SAS Institute Inc, 1987. SAS / STATE. Guide for Personal Computers, Version 6 Edition. SAS Institute Inc, Cary, NC. Schiewe, M.C., Fitz, T.A., Brown, J.L., Stuart, L.D., Wildt, D.E., 1991. Relationship of oestrus synchronization method, circulating hormones, luteinizing hormone and prostaglandin F-2a receptors and luteal progesterone concentration to premature luteal regression in superovulated sheep. J. Reprod. Fertil. 93, 19–30. Szell, A.Z., Windsor, D.P., 1994. Survival of vitrified sheep embryos in vitro and in vivo. Theriogenology 42, 881–889.
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Tervit, H.R., Gold, P.G., McKenzie, R.D., 1986. Development of an effective goat embryo transfer regime. Proc. NZ Soc. Anim. Prod. 46, 233–236. Torres, S., Sevellec, C., 1987. Repeated superovulation and ´ surgical recovery of embryos in the ewe. Reprod. Nutr. Dev. 27, 859–863. Trounson, A.O., Willadsen, S.M., Moor, R.M., 1976. Effect of prostaglandin analogue cloprostenol on oestrus, ovulation and embryonic viability in sheep. J. Agric. Sci. (Camb.) 86, 609– 611. Winterberger-Torres, S., Sevellec, C., 1987. Atlas of the Early Development of the Sheep Embryo. INRA, Paris.