Repeated surgical embryo recovery and embryo
production in rabbits
F. Forcada
∗, M. López
Departamento de Producción Animal y Ciencia de los Alimentos, Universidad de Zaragoza, Miguel Servet 177, 50013 Zaragoza, Spain
Received 7 April 2000; received in revised form 18 July 2000; accepted 2 August 2000
Abstract
The purpose of this study was to assess the embryo production after repeated surgical recovery of embryos in Gigante de España does. A total of 195 ovulatory treatments and embryo recoveries were performed from 1995 to 1999. Ovulation was induced by an intramuscular injection of 20g GnRH immediately after mating. Each doe was induced to ovulate up to four consecutive times at intervals of at least 50 days. Embryos were surgically collected from oviducts 68–69 h post-coitus. An average of 8.6 corpora lutea and 6.4 recovered embryos (90% of them classified as viable) were recorded from the 195 treatments. The process seemed to be less efficient in the fourth treatment, with a drop of more than two recovered and viable morulae with reference to the third (P <0.05 for both parameters) or the second recovery (P <0.1 andP <0.05, respectively). More than 20 recovered embryos and 18 viable embryos per donor doe were recorded considering the three first ovulatory treatments performed in 33% of the does (30/90). Results indicate that the methodology used in the present study could be an efficient way to maximize in vivo embryo production from rabbits. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Rabbit; Ovulation; Embryo production; GnRH
1. Introduction
Embryo transfer technology has enhanced the reproductive potential of several livestock species and has the potential for aiding in the assisted reproduction of wild animals. Although the embryo transfer was first accomplished in domestic rabbits (Heape, 1890), there has been no systematic collection of genetic resources in this species, as in most domestic farm animals. However, the rabbit was used as a model animal in a European Joint Action to elucidate the procedures for preservation of rabbit embryos (Joly et al., 1996).
∗Corresponding author. Tel.:+34-976-761600; fax:+34-976-761612. E-mail address: forcada@posta.unizar.es (F. Forcada).
The Gigante de España breed was first described in 1921. It is a local breed with good maternal and growth characteristics, being the base of many Spanish rabbit farms until 1960. The development of rabbit breeding and the appearance of competitive new breeds (New Zealand and Californian) and hybrids, caused the progressive disappearance and almost extinction of the Gigante de España breed. Since 1984, however, the Department of Animal Production of the Veterinary Faculty of Zaragoza (Spain) has been responsible for the recovery of the breed from a limited population size (Sierra and López, 1990).
Gigante de España does are characterized by a fertility around 80% and a litter size of 8.2 pups born (Sierra and López, 1990). After induction of ovulation with GnRH, 8.6 corpora lutea and 6.8 recovered embryos have been recorded (López et al., 1993). Breed preservation will allow useful genes to be conserved and support the diffusion of Gigante de España does in commercial farms. Artificial insemination, embryo production and cryopreservation are essential tools in the achievement of these objectives.
The major limitation to the development of embryo transfer technology in most mammals is the difficulty in traversing the cervix. Surgical intervention can solve this problem, al-though exteriorization of the reproductive tract often leads to the formation of post-operative adhesions inducing a reduction in embryo recovery after repeated surgery (Torres and Sevellec, 1987).
Rabbit embryos are usually collected post-mortem and, to our knowledge, no studies have been reported in the literature on successive surgical embryo collection by laparotomy. A method of a transcervical catheterization using fibre optic endoscopy in order to overcome the limitations of the surgical procedures has been recently reported (Kidder et al., 1999).
The objective of this study was to evaluate the possibility of using repeated surgical embryo recovery in rabbits. Gigante does underwent up to four surgical recovery following induction of ovulation with GnRH and natural mating. The study was performed over 5 consecutive years.
2. Materials and methods
The experiment was conducted at the experimental farm of the University of Zaragoza (Spain), which meets the requirements of the European Community Commission for Sci-entific Procedure Establishments (1986). Animals used in the present study were sexually mature Gigante de España does, aged 7–20 months and weighing 4.5–5.0 kg. They were individually housed in metal cages under artificial light conditions (16:8 h light:dark) and fed ad libitum.
Embryos were recovered by separate perfusion of the oviducts with pre-warmed (38◦C) phosphate-buffered saline (PBS; Sigma, St. Louis, MO, USA) supplemented with antibiotics (penicillin and streptomycin) and 0.3 mM sodium pyruvate. The oviducts were flushed from the utero-tubal junction to the fimbria. The number of corpora lutea and the number of embryos recovered were recorded. The embryos were washed (PBS), evaluated under a stereo microscope at a magnification of 20–40×and classified according to their stage of development, morphological appearance and quality. Only selected embryos without any imperfections and spherical in shape were deemed as viable in this experiment. Immediately after the surgical collection and before closure, the reproductive tract was washed with a 2.5% heparin solution in saline in order to minimize the post-operative development of abdominal adhesions. At the end of the laparotomy, the females were treated with an intramuscular injection of 100 mg oxytetracicline (Terramicina®, Pfizer Salud Animal, Madrid, Spain).
Most of the 195 embryo recoveries (134) were performed from autumn 1998 to summer of 1999. Although few data were obtained in 1995, 1996 and 1997, they were included in this study in order to test for a possible effect of year on the outcomes. Each doe was ovulated and subjected to surgical embryo recovery on one to four occasions at intervals of at least 50 days. Animals were only eliminated from the study if anatomical problems (adhesions, lesions, etc.) prevented embryo recovery, although data obtained for item previously were included in the analyses.
Ovulation rate and numbers of recovered and viable embryos were analysed by one-way analysis of variance (Tukey’s HSD test) to test the year effect on ovulation and embryo recov-ery. The significance of differences between years was analysed using t-tests. Percentages of recovered and viable embryos were analysed also by ANOVA after transformation of data by calculating the square root of each value. Data for all years were pooled to test the effect of the number of embryo recoveries within does using the same statistical procedures. Data were analysed using the GLM procedure of the Statistical Analysis System (SAS, 1987).
3. Results
The mean number of ovulations for Gigante de España does in the present study was 8.6±0.2 (mean±S.E.) corpora lutea. All treated donors ovulated from at least one of the two ovaries. Mean number of embryos recovered from the 195 does was 6.4±0.3 (74% recovery rate) and 7.0±0.23 from the 179 does from which at least one embryo was collected. Mean numbers of viable embryos were 5.8±0.2 and 6.3±0.2, respectively, representing the 90% of the embryos recovered. Embryos classified as non-viable were non-fertilized (28%), in an earlier stage of development than that of morula (23%), degenerate (23%) or with a non-spherical shape (26%).
Table 1
Effect of year on ovulatory response and embryo production in Gigante de España does (mean±S.E.)a
Year 1995 1996 1997 1998 1999 Total
Number of embryo recoveries 26 14 21 16 118 195
Number of corpora lutea 9.0±0.5 9.4±0.7 9.2±0.4 8.2±0.8 8.4±0.2 8.6±0.2 Number of embryos recovered 7.4±0.7 a 5.9±1.1 5.3±0.7 b 6.3±0.8 6.5±0.3 6.4±0.3 Percentage of ovulation rate 82.8 a 62.9 58.0 b 77.1 77.1 74.5 Number of viable embryos 6.7±0.6 a 5.6±1.1 4.4±0.7 b 6.3±0.8 5.8±0.3 5.8±0.2 Percentage of ovulation rate 74.7 a 59.8 47.7 b 77.0 68.6 66.9
aWithin a row, different letters indicate that the differences are significant (P <0.05).
Table 2
Effect of treatment number on ovulatory response and embryo recovery (mean±S.E.) in the 10 Gigante de España does used for four recoveriesa
Recovery number 1 2 3 4
Number of recoveries 90 65 30 10
Corpora lutea 8.7±0.2 8.7±0.2 8.5±0.3 7.4±0.6
Recovered embryos 6.0±0.4 6.9±0.4 7.2±0.5 a 4.8±1.0 b Percentage of ovulation rate 68.9 a 79.1 a,b 84.8 b 64.9 a Viable embryos 5.3±0.4 6.3±0.4 a 6.6±0.5 a 4.2±0.9 b Percentage of ovulation rate 61.0 a 72.1 b 77.0 b 56.8 a
aWithin a row, different letters indicate that the differences are significant (P <0.05).
the third (P < 0.05 for both parameters) or the second recovery (P < 0.1 andP < 0.05, respectively). This effect was confirmed when only the results obtained from the 10 rabbits used for four recoveries were analysed (Table 3), although no significant differences were found. In contrast, results from the first three recoveries showed that percentages of recovered and viable embryos were not affected by the treatment number.
4. Discussion
The percentage of does ovulating in the present study (100%) was similar to that reported in the literature using the same protocol, either in Gigante de España does (López et al.,
Table 3
Effect of treatment number on ovulatory response and embryo recovery (mean±S.E.) in the 10 Gigante de España does used for four recoveries
Recovery number 1 2 3 4
Number of recoveries 10 10 10 10
Corpora lutea 7.0±0.4 7.6±0.5 8.2±0.5 7.4±0.6
Recovered embryos 6.4±1.0 5.4±1.0 7.0±0.7 4.8±1.0
Percentage of ovulation rate 91.4 71.1 85.4 64.9
Viable embryos 5.9±1.2 5.0±1.0 6.6±0.7 4.2±0.9
1993) or in other strains (Théau-Clement et al., 1990; Viudes et al., 1995). The number of ovulations observed in the present study was similar to that of López et al. (1993) in Gigante de España does using the same experimental protocol (8.6 corpora lutea), but lower than that reported by Forcada and Abecia (1989) for the same breed and experimental protocol (10.4 corpora lutea). The embryo recovery rate (percentage of embryos recovered of corpora lutea) was similar to those reported by other authors (Carney and Foote, 1990; López et al., 1993; Viudes et al., 1995).
Superovulatory treatment with FSH increases by two- or three-fold the number of em-bryos recovered compared to the use of GnRH to induce ovulation (Carney and Foote, 1990; Kauffman et al., 1998), but several authors have reported the quality and viability of embryos from superovulated does to be low as a consequence of chromosomal (Fujimoto et al., 1974) or morphological (Carney and Foote, 1990) abnormalities. The major limitation of superovulatory treatment is the cost of labour, particularly for a 4-day treatment.
A new technique which allows repeated embryo recovery from rabbits is surgical catheter-ization of the oviducts. Results of the present study show that does can be induced to ovulate and embryo recovered up to four times without affecting ovulation rate. Moreover, num-ber of recovered and viable embryos did not decrease after three treatments. Viudes et al. (1995) reported both GnRH and hCG induce ovarian abnormalities (cystic and haemor-rhagic follicles) that do not modify ovulation rate but could impair the recovery rate of normal embryos (Garc´ıa-Ximénez and Vicente, 1992). This effect is observed at the first and subsequent induced ovulations, and probably is a consequence of the steroidogenic activity of the affected follicles (Eyestone and Ax, 1984). The incidence of ovarian abnor-malities was not recorded in the present study, but any possible negative effect on embryo recovery appears to be constant since the numbers of embryos recovered and viable did not differ between the first three recoveries. In ewes superovulated three times at 50-day intervals, Torres and Sevellec (1987) reported that post-operative adhesions had an effect on embryo recovery rate or sperm transport as indicated by the percentage of unfertilized ova. This contrasts with the observations of Forcada et al. (2000) using a similar protocol in ewes at the end of their reproductive life. In this latter study, and in the present study, females with adhesions that made flushing the oviducts impossible, were not re-used.
The results show that it was possible to induce ovulation and carry out embryo recovery on three occasions in 33% (30/90) of the does recovering a total of 20.1 embryos of which 18.2 were viable. Therefore, repeated use of does could be an efficient way to maximize in vivo embryo production. The non-surgical method of Kidder et al. (1999) allowed five flushes per doe to be performed, with a 71% recovery rate overall. The use of this technique is only limited by the high labour costs and its viability to allow the recovery of embryos from oviducts.
In conclusion, the results of the present study indicate that repeated embryo recovery by laparotomy under anaesthesia can be used to maximize in vivo embryo production.
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