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ANIMAL GENETICS |
Departamento de Ciencia Animal, Universidad Politécnica de Valencia, PO Box 22012, 46071 Valencia, Spain
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: uterine capacity • selection • early embryo survival • embryo development • rabbit
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). These lines had similar ovulation (Mocé et al., 2004) and fertilization rate (Santacreu et al., 1996) but different numbers of implanted embryos (Santacreu et al., 2005) and numbers of embryos at 72 to 75 h of gestation (Mocé et al., 2004). The high line also had a more advanced embryonic stage of development. In mice, Ribeiro et al. (1996) observed greater embryo survival (ES) until the sixth day after mating, when the selection for uterine capacity was performed. In the same experiment, when selecting for uterine capacity, Al-Shorepy et al. (1992) observed increased embryo development and homogeneity before implantation.
In mice and rabbits, a large part of the difference in prenatal survival occurs in the early stages of gestation, when embryos are in the oviduct. The oviduct has been commonly considered as a passive tissue, but Killian (2004) and Buhi et al. (2000) found reasons to believe the oviduct is a biologically active tissue, providing an optimum environment for the developing embryo, as well as synthesizing and releasing proteins.
The aim of this work was to study the time at which differences in ES appear due to selection for uterine capacity, which is a characteristic of the female.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Animals belonged to a divergent selection experiment for uterine capacity. Selection was performed on the high and low lines through 10 generations (Argente et al., 1997). After 10 generations, selection was relaxed until the 18th generation. Uterine capacity was estimated as litter size in unilaterally ovariectomized does. Does were housed at the experimental farm of the Universidad Politécnica de Valencia in individual cages. Animals were fed with a commercial diet, and the photo-period used was 16-h light:8-h dark.
A total of 295 intact females (with both functional ovaries) belonging to high and low lines were slaughtered at several stages of gestation. All females came from the 18th generation. At 25 h of gestation, 69 high and 58 low females were slaughtered, whereas at 48 and 62 h of gestation, 50 and 43 females belonging to the high line and 39 and 36 females belonging to the low line, respectively, were slaughtered.
Embryo Recovery and Classification.
The ovulation rate (OR) was estimated as the number of corpora hemorrhagica in both ovaries. To recover embryos and oocytes, the oviduct and the first one-third of the uterine horn were flushed with 5 mL of Dulbecco’s phosphate buffered saline (DPBS, Sigma, Alcobendas, Madrid, Spain) supplemented with 0.132 g/L of calcium chloride, 0.2% BSA (Cod. A-3111, Sigma) and 0.2 mL of antibiotic (Penivet 1, Divasa Farmavic, Barcelona, Spain) at room temperature. At 25 h of gestation, 2 recovery techniques were used: tube or plate. All embryos and oocytes recovered were classified as no cleavage, 2-cell, or 4-cell stages. No cleavage was either an oocyte or a 1-cell embryo. To differentiate between the 2 cell types, they were dyed with Hoestch 33342 (B-2261, Sigma), 2 to 3% citrate sodium, and absolute ethanol (Cod. 131086, Panreac, Castellar del Valles, Barcelona, Spain) and evaluated by fluorescence microscopy. No cleavage embryos were classified as embryos of 1-cell stage when male and female pronuclei, 2 polar bodies, or spermatozoa in the periviteline space were observed. At 48 h of gestation, embryos were classified as abnormal embryos, 8- to 16-cell stages, or early morulae. At 62 h of gestation, embryos were classified morphologically as abnormal embryos, early morulae, or compacted morulae. Abnormal embryos were degenerated embryos or embryos with a stage of development not corresponding to that expected. No degenerated embryos or unexpected development embryos were found at 24 h of gestation, so this category was not included in the analysis for this gestation time. Embryo classification was always carried out by the same operator.
Traits
The following traits were recorded: OR estimated as number of corpora hemorrhagica, number of oocytes recovered (OOR), number of normal embryos recovered (ER), number of abnormal embryos recovered (AR), total recovered (TR = OOR + ER + AR), and BW at slaughter. The trait ES was analyzed as the number of normal embryos recovered fitting OR as a covariate. Traits calculated were: fertilization rate (FR) = [(ER + AR)/TR] x 100, percentage of 1-cell embryos [% 1-cell = (number of 1-cell embryos/ER) x 100], percentage of 2-cell embryos [% 2-cell = (number of 2-cell embryos/ER) x 100]; percentage of 4-cell embryos [% 4-cell = (number of 4-cell embryos/ER) x 100]; percentage of abnormal embryos {% AE = [AR/(AR+ER)] x 100}; percentage of normal embryos {% NE = [ER/(AR+ER)] x 100}; percentage of 8- to 16-cell embryos [% 8–16 cell = (number of 8–16 cell embryos/ER) x 100], percentage of early morulae [% EM = (number of early morulae/ER) x 100], and percentage of compacted morulae [% CM = (number of compacted morulae/ER) x 100].
Statistical Analyses
The analysis was based on Bayesian methods. Bounded uniform priors were used for all unknowns, and the data were assumed to be normally distributed. The following model was assumed for OR, FR, and embryonic stage of development:
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where Li is the effect of the line (high and low line) and LACTj is the effect of lactation status (lactating or nonlactating doe).
The following model was assumed for ER:
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where RECk is the effect of recovery method (tube or plate).
The model assumed for ES was the same as for ER but using OR as the covariate:
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At 25 h, all the effects were considered, but at 48 and 62 h, lactation and recovery effects were not considered because all females were nulliparous and the recoveries were in the plate. Marginal posterior distributions of all unknowns were estimated by Gibbs Sampling (Sorensen and Gianola, 2002). After some exploratory analyses, we used 1 chain of 100,000 samples, discarding the first 20,000 and saving every 10 thereafter. Convergence was tested using the Z criterion of Geweke. The Monte Carlo SE (MCse) was estimated as described by Sorensen and Gianola (2002).
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RESULTS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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. Fertilization rate was high (98%) in all gestation stages. The percentage of abnormal embryos was small in both lines at 48 and 62 h of gestation. The majority of embryos at 25 and 48 h were in the 2-cell stage (79%) and early morulae (75%) respectively. At 62 h of gestation, 46% of the embryos were compacted morulae. After the 2-cell stage, embryo development demonstrated high variability.
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. All MCse were very small, and lack of convergence was not detected by the Geweke test. Marginal posterior distributions were approximately normal; thus mode, mean, and median were similar, and only the posterior mean of the difference is given in the tables. An advantage of the Bayesian approach through MCMC procedures is the ease of computation of confidence intervals and probabilities (see review by Blasco, 2005). Thus, it is possible to calculate the shortest interval containing the true value with a probability of 95% (HPD95%). In our case, we are interested in estimating differences between lines, thus we can calculate not only the probability of this difference being greater than zero, P(D>0), but also the probability of this difference being economically or biologically relevant, P(D>b) when b is this relevant value.
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). Bayesian methods posterior probabilities, a measure of the uncertainty for the estimated parameter, and not a significance test or P-value. Ovulation rate was greater for lactating than for non-lactating females (D = 1.38 ova; Pr = 97%). No relevant differences were found between lactation status for FR, ER, and embryo developmental stages. Ovulation rate and BW at 25 h of gestation were greater than at 48 and 62 h of gestation. The difference in OR disappeared when using the variable BW as covariate.
In order to establish relevant differences for FR we considered that a difference of 1 embryo corresponds to 7% in FR. The high line had a greater FR than the low line, P(D > 0%) = 95%, but this difference between lines, although it exists, was not relevant (Pr = 0%, Table 2).
A difference of 0.5 embryos could be considered as a relevant difference for ER and ES. Although the high line had a greater number of embryos recovered than the low line (D = 0.57), when ES was analyzed this difference decreased to 0.27 embryos (Pr = 24%).
Differences between lines in percentage of 1-cell, 2-cell, and 4-cell stages were low and irrelevant (1.43, 1.51, and 2.94%, respectively). Using the same argument as was previously used for FR, the probability of a relevant difference in percentage of 1-cell, 2-cell, and 4-cell stages were 14, 16, and 9% respectively. Thus, both lines had a similar embryonic stage of development.
Features of the estimated marginal posterior distributions of the differences between high and low lines are presented in Tables 3 and 4 at 48 and 62 h of gestation, respectively. As in the previous analyses, all MCse were very small; lack of convergence was not detected by the Geweke test; and marginal posterior distributions were approximately normal. The assumed values for relevant differences were the same as in the analyses at 25 h of gestation. We did not find any relevant difference between high and low lines in OR. As in the previous analysis at 25 h, no relevant difference for FR was found between lines, either at 48 or at 62 h.
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At 62 h of gestation, the high line had a greater ER [P(D>0) is 77%] and a greater ES [P(D>0) is 99%] than the low line. After fitting by ovulation rate (ES), the high line had 0.57 embryos more, although the estimate had low precision; the HPD95% is large, from 0.15 to 1.12 embryos, and the probability of a relevant difference in favor of the high line was only 57%. Nevertheless, the interval [k, 
) of the marginal posterior distribution containing 80, 85, and 95% of the probability shows that the minimum values that the difference has with these probabilities are at least 0.40, 0.36, and 0.20 embryos in favor of the low line, respectively.
At 48 h of gestation, the high line had 9.94% (1.42 embryos) more early morulae than the low line. Although HPD95% included zero, the high line had a greater percentage of compacted morulae [P(D > 0) = 97%], which is the more advanced stage of development. The probability of a relevant difference was high [P(D > b) = 70%] and always in favor of the high line.
At 62 h of gestation, the high line had 16.45% (2.35 embryos) more compacted morulae than the low line. As at 48 h, the HPD95% included zero, but the probability of a relevant difference was high [P(D > b) is 85%] and always in favor of the high line. Moreover, the probability of a relevant difference in favor of the high line was greater than this probability at 48 h. Taken together, these results indicate that the difference between lines in ES appears between 48 and 62 h of gestation, whereas the difference in the embryonic stage of development appears between 25 and 48 h, and increased at 62 h of gestation.
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DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Nulliparous females (at 48 and 62 h) had a lower OR and BW than multiparous. Similar results were obtained by Hulot and Matheron (1980). Fertilization rate was high, near 100%, in agreement with results obtained by other authors in lines selected for reproductive traits in rabbits (Torres et al., 1987b; Garcia-Ximenez and Vicente, 1992; Bolet and Theau-Clement, 1994) and in pigs (Chang, 1969, Wrathall, 1977; Polge, 1978). At 48 and 62 h of gestation, both lines presented a high percentage of normal embryos (97 and 95%, respectively) when compared with other authors. For example, Viudes de Castro et al. (1995) obtained a lower percentage of normal embryos (85 to 90%) at 64 to 66 h of gestation in 3 lines selected by different criteria but more than 40% of their females had ovarian anomalies. Lavara et al. (2005) also found a similar percentage of normal embryos in these lines at 76 to 78 h. The percentage of each embryonic stage of development was similar to the results obtained by Ménézo and Renard (1991) and Garcia-Ximenez and Vicente (1992) in maternal rabbit lines: the majority of the embryos were 2-cell stages at 25 h and early morulae at 48 h of gestation.
When comparing the high and low line, we found that at 25 and 48 h of gestation both lines had similar ES, but the high line showed a more advanced embryonic stage of development at 48 h. Fourteen hours later, the difference in the percentage of compacted morulae was greater in the high line (16.45 vs. 9.94). At this moment of gestation, the difference of ES was relevant (0.57 embryos), and the probability of a relevant difference in favor of the high line was moderate-high (57%). Mocé et al. (2004) reported concordant results at 72 h of gestation; the high line had around 1 embryo more than the low line and a more advanced embryonic stage of development. All these results might indicate that the first step related to early survival is the embryonic stage of development. Thus, we observed that a greater embryonic stage of development took place at least at 48 h and later, with an increase in ES. Divergent selection for uterine capacity seems to have modified embryo development at least from 48 h of gestation and ES at least from 62 h.
There are few published reports about the effect of selection for uterine capacity, litter size, or its components on ES. In mice, Ribeiro et al. (1996) and Al-Shorepy et al. (1992) studied ES at 6 and 3.5 d of gestation, respectively, in 3 lines selected for 21 generations for different criteria. At 6 d, when embryos are implanted, a positive effect on ES was found in the line selected for litter size and in the line selected with an index on ovulation rate and ova success, with respect to the control line. In the line selected by uterine capacity, they did not find any relevant effect on ES. Contrary to our results, at 3.5 d of gestation, when embryos were in the uterus, Al-Shorepy et al. (1992) did not find differences in ES. However, the 3 lines selected showed a greater frequency of blastocyst vs. morulae, in agreement with our results. Durrant et al. (1980) also showed a greater percentage of embryos developed beyond the 8-cell stage at 2 d of gestation in a line selected for litter size. Likewise, Moler et al. (1981) studied the effect of selection on embryo development at 3.5 d of gestation in a line selected for high ES and another selected for small litter size. The number of embryos recovered was greater in the line selected for high ES. Moreover, this line had a more advanced embryonic stage of development, having a greater percentage of blastocyst (83 vs. 44%) and a lower percentage of morulae (10 vs. 36%) and degenerating embryos (10 vs. 20%). All these results demonstrate that it is possible to modify the embryonic stage of development and ES in the early gestation period by selection, although it is not clear which mechanisms are modified.
Previous studies with rabbits and pigs showed a relation between the stage of embryo development and ES. In rabbits, Torres et al. (1987b) found that the difference in prolificacy between 2 lines was due to embryonic loss before 96 h. Also, the line with lower prolificacy showed a greater percentage of unfertilized and delayed embryos. Torres et al. (1987a) found that larger blastocysts showed greater survival than small blastocysts at 27 d of gestation, after transferring embryos of different size in each horn of the female at 4 d of gestation.
Some authors proposed that differences in the embryonic stage of development could mainly be due to timing of ovulation and composition of oviductal and uterine fluid (Hunter et al., 2004). In our case, because the embryonic stage of development at 25 h of gestation are similar in both lines, the differences at 48 and 62 h could be due to changes in the composition of oviductal fluid. During fertilization and early cleavage-stage development of the embryos, the oviduct undergoes specific biochemical modifications, which contribute to an optimization of the microenvironment.
In conclusion, divergent selection for uterine capacity seems to modify embryo development and ES in the early stages of gestation. Wider knowledge of the pattern of oviductal fluids in these lines will provide an insight into the physiological events that regulate embryo development and ES.
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Footnotes |
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2 Corresponding author: ropeibar{at}dca.upv.es
Received for publication November 7, 2006. Accepted for publication February 27, 2007.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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