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ANIMAL GROWTH, PHYSIOLOGY, AND REPRODUCTION |
Departamento de Ciencia Animal, Universidad Politécnica de Valencia, 46071 Valencia, Spain
Abstract
The aim of this work was to study the effects of the genotype of the dam, the embryo, or their interactions on prenatal growth by performing double-reciprocal embryo transfers between two lines of rabbits divergently selected for uterine capacity. Females from high (n = 53) and low (n = 48) lines were slaughtered at 72 h of gestation, and recovered embryos were transferred to the oviducts of recipient does from the high (n = 23) and low (n = 19) lines. Each recipient doe received eight embryos from the high line into one oviduct and eight embryos from the low line into the other. Recipient does were slaughtered on d 28 of gestation. The percentages of live fetuses at 28 d of gestation were 89.2 and 74% for high and low recipient lines, respectively. Length and weight of the empty uterine horn and weight of the full uterine horn were not affected by either the recipient or by donor line. Fetal weight was affected by the recipient line but not by the donor line. Fetuses gestated in high recipient does were 7% heavier (P < 0.10) than those in the low recipient does. There was a donor and a donor x recipient interaction effect on fetal placental weight. Fetal placental weight was heavier (7%, P < 0.01) for embryos from the low line. Embryos from the high line gestated in low-line uteri showed a lower fetal placenta weight than did low-line embryos gestated in high-line uteri and low-line uteri (P < 0.05). Linear regression coefficients of fetal weight at term on fetal placental weights differed (P < 0.05) for the high and low donors (4.33 ± 0.28 and 3.41 ± 0.29 respectively). A significant effect of the donor genotype on individual placental length was observed (P < 0.05), which might have resulted from a smaller individual placental length of low-line embryos gestated high-line uteri (P < 0.10). Neither donor nor recipient lines affected maternal placental weight or available space for fetuses. Fetuses and their fetal placentae were heavier when receiving more than four blood vessels than when receiving less than three blood vessels (13 and 17% respectively, P < 0.05). Neither recipient nor donor genotype affected the number of blood vessels arriving at each live fetus. Thus, fetal weight depends on the maternal genotype, whereas fetal placental weight depends on the embryo genotype in these two lines of rabbits divergently selected for uterine capacity.
Key Words: Embryonic Effects • Fetal Development • Maternal Effects • Placenta • Rabbit • Uterine Capacity
Introduction
Embryos produced by reproductive techniques such as pronuclear injection or nuclear transfer often show unusual fetal and placental development, which compromise their survival and therefore the success of these new reproductive techniques (Barnes, 2000
; Young and Fairburn, 2000; Renard et al., 2002). Moreover, fetal mortality limits litter size in livestock species (Dziuk, 1992; Blasco et al., 1993; Vallet et al., 2002). Knowledge of the effects of the embryo and the uterine genotype on fetal and placental development will be helpful in better controlling those factors that are limiting both litter size and success of new reproductive technologies.
Several reciprocal embryo transfer experiments have been carried out to study prenatal growth in mice (Al-Murrani and Roberts, 1978; Ernst et al., 2000) and in pigs (Asworth et al., 1990; Biensen et al., 1998). Reciprocal embryo transfers between lines enable the differentiation of maternal and fetal genetic factors, but results depend on genetic origin and day of gestation. Rabbit does are particularly appropriate for studying genetic control of prenatal growth because ovulation is induced by coitus and it is easier to synchronize recipient and donor does. Furthermore, because there is no embryonic migration between uterine horns in rabbits, it is possible to have two different embryo genotypes within the same uterine genotype.
The aim of this work was to study the effects of the genotype of the dam (recipient), the embryo (donor), and their interaction on prenatal growth by performing double-reciprocal embryo transfers between two lines of rabbits divergently selected for uterine capacity. This report follows that of Mocé et al. (2004), in which we studied the effect of the genotype of the dam and the embryo on prenatal survival.
Materials and Methods
Animals
Animals used as donors and recipients came from an experiment on divergent selection for uterine capacity. In rabbits, Blasco et al. (1994) proposed that litter size in unilaterally ovariectomized females could be an estimator of uterine capacity. As transuterine migration is not found in rabbits, unilateral ovariectomy and consequent ovarian hypertrophy results in overcrowding of the uterine horn corresponding to the functional ovary. The two lines were generated from a synthetic population bred at the experimental farm of the Universidad Politécnica de Valencia. Both were divergently selected for 10 generations, and selection was relaxed from the 11th to the 15th generation. A detailed description of the selection procedure was reported by Argente et al. (1997). Animals were housed at the experimental farm of the Universidad Politécnica de Valencia in individual metal cages. Animals were kept under controlled (16 h light:8 h dark) photoperiod and fed a pelleted commercial diet (Cunilactal, NANTA, S. A., Valencia, Spain). Differences between lines for uterine capacity and litter size were published by Blasco et al. (2001) and by Santacreu et al. (2000), respectively.
Embryo Recovery.
Does came from the 14th and the 15th generations: 53 does from the line selected for high uterine capacity and 48 does from the line selected for low uterine capacity. Natural matings were carried out with males from the same line as the donor females. Does were slaughtered by stunning and exsanguination at 72 to 75 h postcoitum. The entire reproductive tract was removed after slaughter. The oviducts and the first one third of the uterine horns were excised and flushed once with 5 mL of Dulbecco’s PBS (Sigma, Alcobendas, Madrid, Spain) supplemented with Cl2Ca (0.132 g/L), 0.2% BSA (Sigma, Alcobendas, Madrid, Spain), and antibiotics (300,000 IU of penicillin G sodium, 700,000 IU of penicillin G procaine, and 1,250 mg of dihydrostreptomycin sulfate; Penivet 1, Divasa Farmavic, Barcelona, Spain) at room temperature. Embryos were counted and classified as normal or abnormal according to morphological criteria; early morulae, compact morulae, and blastocysts were classified as normal when they presented homogenous cellular masses and intact zona pellucidae (Hafez, 1993). Embryo classification was always carried out by the same operator.
Embryo Transfers.
Nulliparous females from the 15th generation, 19 to 20 wk of age, were used as recipients: 23 does from the high line and 19 does from the low line. Twenty-one days before the transfer, recipients were synchronized by i.m. administration of 1 µg of busereline acetate (Hoechst, Marion Roussel, Madrid, Spain). In rabbits, vulva color allows determination of whether a female is receptive; hence, females that presented a vulva color associated with receptive status were induced to ovulate with a second injection of busereline acetate 72 h before transfer. To perform the transfers, rabbits were anaesthetized with an i.m. injection of xylazine (Rompun 2%, Bayer AG, Leverkusen, Germany) at a rate of 0.2 mL/kg body weight; 5 min later, this injection was followed by an i.v. dose of 2 to 3 mL of Ketamine HCL and clorbutol (Imalgéne 500, Merial, Lyon, France) in the marginal ear vein. Embryo transfers were performed using the laparoscopic technique described by Besenfelder and Brem (1993). This technique allows for transfer of embryos with minor abdominal surgery. Only embryos that were classified as normal were transferred. The number of embryos transferred per Fallopian tube was standardized to eight, such that each recipient received 16 embryos (eight embryos from the high line into one oviduct and eight embryos from the low line into the other one). Transfers to right or left uterine horns were randomized. Embryo transfers were performed with an average of 2.9 blastocysts, 4.5 compact morulae, and 0.6 early morulae per oviduct.
Measurement of Uterine and Fetal Variables
Laparoscopy was performed on the recipient females at 13.78 ± 0.24 d of gestation and number of live fetuses for each uterine horn was recorded. The number of implanted embryos was estimated as the total number of embryos in the uterus (live fetuses + dead fetuses). Recipient females were slaughtered by stunning and exsanguination at d 28 of gestation (birth takes place at 30 d of gestation). Each uterine horn with its fetuses was weighed (weight of the full uterine horn, WFU) to the nearest centigram. For each uterine horn, fetuses were classified according to their status as live fetuses, dead fetuses, atrophic fetal and maternal placentae, atrophic maternal placentae, and decidual reactions. Live fetuses at d 28 of gestation were weighed to the nearest centigram (IWF) after placental membranes and fluids were removed. Each fetal placenta and adjacent maternal placenta was dissected separately and weighed to the nearest centigram (IWFP and IWMP, respectively). Placental length was measured for each implantation site (IPL) (Figure 1). Uterine space available for each fetus (ISF) was estimated as the length of its maternal placenta plus half of the distance from its right and left boundaries to the nearest placenta. For embryos located at the beginning of the cervix, distance was calculated as the length of its maternal placenta plus the distance from the boundary of the placenta to the beginning of the cervix, plus one-half of the distance from the other boundary to the nearest placenta. The same procedure was used for embryos located at the beginning of the oviduct. The number of blood vessels arriving at each implantation site was used to estimate the vascular supply to each fetus, as in Argente et al. (2003). Pictures from each reproductive tract were taken and the number of blood vessels arriving at each live fetus was counted on the picture. Live fetuses were classified as receiving less than three blood vessels, three blood vessels, four blood vessels and more than four blood vessels. Empty uterine horns were weighed to the nearest centigram (WEU) and measured to the nearest millimeter (LEU; Figure 1).
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2 test was performed to determine the effect of the recipient line (HR = high recipient, LR = low recipient) and the donor line (HD = high donor, LD = low donor) on the distribution of fetal status. The effect of the recipient line, effect of the donor line, and the donor x recipient interaction on the number of blood vessels arriving at each live fetus was analyzed using a 2 test. The 2 tests were performed using STATGRAPHICS (Manugistic, Inc., Rockville, MD). Only data from live fetuses were used. The variables IWF, IWFP, IWMP and IPL were analyzed fitting the model:
 | [1] |
where µ was the general mean, RDi had four levels (high recipient with high donor, high recipient with low donor, low recipient with high donor, low recipient with low donor), mij was the random effect of dam of fetuses and eijk was the random residual term. The variables IWF, IWFP, and IWMP were analyzed with the same model, including the number of live fetuses in the corresponding uterine horn (LF28) as a covariate, the number of live fetuses in both uterine horns (LS) as a covariate, and both LF28 and LS as covariates. Because results were similar for the three models proposed, we only reported those obtained when fitting LS as a covariate. The variable IPL was also analyzed with the same model, including number of implanted embryos as a covariate. For LEU, WFU, and WEU, the number of live fetuses in the corresponding uterine horn was added to Model [1] as covariate. Data from uteri with only one fetus were removed to study ISF, and Model [1] included the number of implanted embryos as a covariate. The MIXED procedure of SAS (SAS Inst., Inc., Cary, NC) was used for these analyses.
To assess the relationships between IWF and IWFP, the following models were fitted:
 | [2] |
 | [3] |
where Ri was the recipient effect (HR and LR), Dj the donor effect (HD and LD), Ri x Dj was the interaction of the recipient line x donor line, mijk was the random effect of dam of fetuses, b was the regression coefficient of IWFP on IWF, (b x R)i was the interaction between the regression coefficient and the effect of recipient line, (b x D)j was the interaction between the regression coefficient and the effect of donor line, and eijkl was the random residual term. The MIXED procedure of SAS was used for these analyses.
Results and Discussion
Recipient genotype affected the distribution of the fetuses according to their fetal status at 28 d of gestation, but donor genotype did not (Table 1). High-recipient does had a higher percentage of live fetuses at 28 d of gestation and a lower percentage of fetuses for any of the other statuses (Table 1). The difference between recipient lines seemed to be greater for decidual reactions and atrophic maternal placentas than for atrophic fetal and maternal placentas or dead fetuses; thus, it seems that most of the difference for fetal mortality between both recipient lines appears soon after implantation. Additional results of the experiment regarding donor and recipient effects on prenatal survival were reported by Mocé et al. (2004). They observed that fetal survival was mainly affected by the recipient genotype, whereas the donor genotype only affected fetal survival when embryo transfers were performed to HR does.
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did not find differences between the high and the low line for WFU and WEU at 25 d of gestation using unilaterally ovariectomized does from the second and the third generation of this selection experiment. Nevertheless, Argente et al. (2003) observed that females from the high line showed a larger LEU than did females from the low line, probably due to a larger litter size in previous parities since they used fifth gestation does instead of nulliparous does.
Fetal weight at 28 d of gestation was not affected by either the donor or by the recipient; however, recipient line affected fetal weight at 28 d of gestation after adjusting for the number of live fetuses in both uterine horns (Table 2). This slightly higher fetal weight (7%) may be related to a more advanced development of embryos gestated in the HR does. Preliminary studies showed that embryos from the high line showed a more advanced embryonic stage of development at 72 h of gestation, having a higher percentage of blastocysts and a lower percentage of compact morulae (Mocé et al., 2004). In pigs, the Meishan breed exhibits reduced growth rate throughout gestation compared with U.S. or European breeds (Ashworth et al., 1990; Youngs et al., 1994; Biensen et al., 1998). Furthermore, Lewis et al. (1992) reported that IGF-I could increase early development in the pig, and concentrations of IGF-I in uterine flushings were reported to be lower in Meishan gilts on d 11 to 12 of gestation (Wilson and Ford, 1997). Thus, HR does may secrete some growth factor that leads to differences in both embryonic and fetal development. This higher fetal weight also might be due to maternal or fetal effects indirectly related to the rate of development, such as a greater uterine blood flow in the HR dams. In rabbits, Bruce and Abdul-Karim (1973) reported that fetal weight and placental blood flow were positively related at d 28 of gestation. Neither donor line nor donor x recipient interaction affected fetal weight at 28 d. Several authors have studied the role of maternal and embryonic genotype on fetal growth by performing embryo transfers among inbred mouse lines (Pomp et al., 1989), genetically selected mouse lines (Al-Murrani and Roberts, 1978; Rhees et al., 1999; Ernst et al., 2000), and different pig breeds (Asworth et al., 1990; Wilson et al., 1998; Biensen et al., 1998). Results obtained did not show a clear pattern because they depend on the genotypes used to perform the studies. For example, Pomp et al. (1989), using two inbred lines differing in adult body size, reported that prenatal growth was mainly determined by the genotype of the recipient female, but was not affected by the genotype of the donor line or the donor x recipient interaction. Conversely, Al-Murrani and Roberts (1978), working with two lines of mice selected for high and low 6-wk weight, reported that the donor effect was more important than the maternal effect. In pigs, Biensen et al. (1998) observed that fetal weight throughout late gestation was determined by both the recipient and the donor. Fetuses gestated in Yorkshire uteri were heavier than fetuses gestated in Meishan uteri, and moreover, Yorkshire fetuses, regardless of the uterine environment in which they were gestated, tended to weigh more. Similar results have been obtained in earlier stages of gestation (Ashworth et al., 1990).
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). It could be that fetuses from the low line may need a heavier fetal placenta to meet nutritional demands since both donor genotypes showed similar fetal weight but different placental fetal weight. Fetuses from the high line gestated in low-line uteri showed a lower fetal placental weight than did fetuses from the low line gestated in both high- and low-line uteri (Table 3). In mice, the effect of antigenic differences between the dam and fetus on placental development has been studied by performing crosses between different genotypes, but results obtained are controversial. For example, Billington (1964) and Billington et al. (1968) reported that genetic dissimilarities between dams and fetuses led to heavier placentas, whereas Hetherington (1971) observed that results depended on the genetic cross used to study it. Furthermore, Hetherington (1971) suggested that when dam and conceptus differed antigenically, an immunological interaction took place that brought about a reduction of the decidual weight at d 7 of gestation. In pigs, Biensen et al. (1998) and Wilson et al. (1998), using Meishan and Yorkshire gilts, observed that placental weight throughout late gestation was determined by both the donor and the recipient genotype. Meishan fetuses developed lighter placetas, and fetuses recovered from Meishan uteri developed lighter placentas.
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also observed differences in the linear regression coefficients of the high and low lines (2.56 ± 0.47 vs. 1.27 ± 0.57, respectively).
A significant effect of the donor genotype on individual placental length was observed after adjusting for the number of implanted embryos in the corresponding uterine horn (Table 2). This could be due to the smaller individual placental length of low-line embryos gestated in HR uteri (Table 3). Placentation is a complex phenomenon that involves a large number of factors and interactions, thus it is a difficult issue to determine factors that explain the differences observed and it requires further study.
It was observed that neither the recipient line nor the donor line affected maternal placental weight (Table 2). The recipient x donor interaction did not affect maternal placental weight (Table 3). The available space for fetuses was not affected by donor, recipient, or the donor x recipient interaction after removing data of uteri with only one fetus (Tables 2 and 3).
Number of blood vessels arriving at the implantation site affected individual fetal weight and individual fetal placental weight but did not affect maternal placental weight (Table 4). Fetuses and their placentas were heavier (13 and 17%, respectively) when receiving more than four blood vessels than when receiving less than three blood vessels. Similar results were obtained by Argente et al. (2003), but they observed a slight effect of the blood supply on maternal placental weight at 25 d of gestation. We did not obtain significant differences for maternal placental weight between fetuses receiving more than four blood vessels and fetuses receiving less than three blood vessels, although the difference (6%) was similar to the difference reported as significant by Argente et al. (2003). Recipient and donor genotype (Table 5) or their interaction (2 = 10.82, P = 0.29) had no affect on the number of blood vessels arriving at each live fetus. However, blood supply is determined by a large number of factors (Ford, 1995; Reynolds and Redmer, 2001); thus, new studies are needed to assess the importance of the genotype on blood supply.
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Divergent selection for uterine capacity in rabbits modifies fetal weight at term and fetal placental weight. Differences in fetal weight depend on recipient genotype, whereas differences on fetal placental weight depend on donor genotype. Knowing the uterine and fetal factors determining differences between both lines will allow for an understanding of physiological events regulating fetal survival, and it will therefore be easier to control detrimental factors affecting fetal survival.
Footnotes
1 This study was supported by the Comisión Interministerial de Ciencia y Tecnología (CICYT-AGF98-0382-C02-01). The authors thank the European Cooperation in the Field of Scientific and Technical Research Action 848 for providing the financial support to learn about laparoscopic embryo transfers. We would also thank U. Besenfelder, J. S. Vicente, E. Mocé, and R. Lavara for their technical support. 
2 Correspondence and present address: Departamento de Producción Animal y Ciencia y Tecnología de los Alimentos, Universidad Cardenal Herrera-CEU, Edificio Seminario, 46113 Moncada, Valencia, Spain (phone: +34961369000; fax: +34961395272; e-mail: mmoce{at}uch.ceu.es).
Received for publication September 8, 2003. Accepted for publication December 16, 2003.
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