Exposure to high ambient temperatures alters embryology in rabbits.

High ambient temperatures are a determining factor in the deterioration of embryo quality and survival in mammals. The aim of this study was to evaluate the effect of heat stress on embryo development, embryonic size and size of the embryonic coats in rabbits. A total of 310 embryos from 33 females in thermal comfort zone and 264 embryos of 28 females in heat stress conditions were used in the experiment. The traits studied were ovulation rate, percentage of total embryos, percentage of normal embryos, embryo area, zona pellucida thickness and mucin coat thickness. Traits were measured at 24 and 48 h post-coitum (hpc); mucin coat thickness was only measured at 48 hpc. The embryos were classified as zygotes or two-cell embryos at 24 hpc, and 16-cells or early morulae at 48 hpc. The ovulation rate was one oocyte lower in heat stress conditions than in thermal comfort. Percentage of normal embryos was lower in heat stress conditions at 24 hpc (17.2%) and 48 hpc (13.2%). No differences in percentage of zygotes or two-cell embryos were found at 24 hpc. The embryo development and area was affected by heat stress at 48 hpc (10% higher percentage of 16-cells and 883 µm2 smaller, respectively). Zona pellucida was thicker under thermal stress at 24 hpc (1.2 µm) and 48 hpc (1.5 µm). No differences in mucin coat thickness were found. In conclusion, heat stress appears to alter embryology in rabbits.

Correlated response in litter size components in rabbits selected for litter size variability.

A divergent selection experiment for the environmental variability of litter size (Ve) over seven generations was carried out in rabbits at the University Miguel Hernández of Elche. The Ve was estimated as the phenotypic variance within the female, after correcting for year-season and parity-lactation status. The aim of this study was to analyse the correlated responses to selection in litter size components. The ovulation rate (OR) and number of implanted embryos (IE) in females were measured by laparoscopy at 12 day of the second gestation. At the end of the second gestation, the total number of kits born was measured (TB). Embryonic (ES), foetal (FS) and prenatal (PS) survival were computed as IE/OR, TB/IE and TB/OR, respectively. A total of 405 laparoscopies were performed. Data were analysed using Bayesian methodology. The correlated response to selection for litter size environmental variability in terms of the litter size components was estimated as either genetic trends, estimated by computing the average estimated breeding values for each generation and each line, or the phenotypic differences between lines. The OR was similar in both lines. However, after seven generations of selection, the homogenous line showed more IE (1.09 embryos for genetic means and 1.23 embryos for phenotypic means) and higher ES than the heterogeneous one (0.07 for genetic means and 0.08 for phenotypic means). The probability of the phenotypic differences between lines being higher than zero (p) was 1.00 and .99, respectively. A higher uterine overcrowding of embryos in the homogeneous line did not penalize FS; as a result, this line continued to show a greater TB (1.01 kits for genetic means and 1.30 kits for phenotypic means, p = .99, in the seventh generation). In conclusion, a decrease in litter size variability showed a favourable effect on ES and led to a higher litter size at birth.

Identification of differentially expressed genes in the oviduct of two rabbit lines divergently selected for uterine capacity using suppression subtractive hybridization.

Suppressive subtractive hybridization libraries from oviduct at 62 h post-mating of two lines of rabbits divergently selected for uterine capacity were generated to identify differentially expressed genes. A total of 438 singletons and 126 contigs were obtained by cluster assembly and sequence alignment of 704 expressed sequence tags (ESTs), of which 54% showed homology to known proteins of the non-redundant NCBI databases. Differential screening by dot blot validated 71 ESTs, of which 47 showed similarity to known genes. Transcripts of genes were functionally annotated in the molecular function and the biological process gene ontology categories using the BLAST2GO software and were assigned to reproductive developmental process, immune response, amino acid metabolism and degradation, response to stress and apoptosis terms. Finally, three interesting genes, PGR, HSD17B4 and ERO1L, were identified as overexpressed in the low line using RT-qPCR. Our study provides a list of candidate genes that can be useful to understanding the molecular mechanisms underlying the phenotypic differences observed in early embryo survival and development traits.

Identification of single-nucleotide polymorphism in the progesterone receptor gene and its association with reproductive traits in rabbits.

A total of 598 F2 does from a cross between the high and low lines selected divergently for uterine capacity during 10 generations were used in a candidate gene analysis. The presence of major genes affecting the number of implanted embryos and uterine capacity has been suggested in lines divergently selected for uterine capacity. Uterine capacity is a main component of litter size. The progesterone receptor gene was tested as a candidate gene to determine whether polymorphisms explain differences in litter size and its components. Fragments of the promoter region and exons 1-8 were amplified and sequenced. One SNP was found in the promoter region, 2464G>A, three SNPs in the 5'-UTR exon 1, and a silence SNP in exon 7. The first four SNPs were segregated in two haplotypes. The allele G found in the promoter region was found in 75% of the high-line parental animals and in 29% of the low-line parental animals. The GG genotype had 0.5 kits and 0.5 implanted embryos more than the AA genotype. At 48 hr of gestation, the difference in early embryo survival and embryonic stage of development was small. However, at 72 hr of gestation, the GG genotype had 0.36 embryos more than the AA genotype and also had a more advanced embryonic stage of development, showing a lower percentage of compacted morulae and a higher percentage of blastocysts. The difference in litter size between the GG and GA genotypes was similar to the difference found between homozygote genotypes; however, differences in implanted embryos, early embryo survival, and embryo development were not detected between the GG and GA genotypes.

Analysis of the oviductal glycoprotein 1 polymorphisms and their effects on components of litter size in rabbits.

The objective of this work was to study the effect of the oviductal glycoprotein 1 (OVGP1) genotype and mRNA expression on litter size and other fertility measures, as OVGP1 has positive effects on fertilization and early embryo development. We have analysed an F(2) cross of two lines of rabbits divergently selected for uterine capacity. The OVGP1 mRNA expression was analysed in both lines, but no differences were observed between them. The promoter region and mRNA were sequenced in the F(0) generation, and 17 polymorphic sites were found to co-segregate in three haplotypes (A, B and C). An association study was performed between several reproductive traits and a triallelic microsatellite identified in the promoter region as well as a non-synonymous SNP located in exon 11 [g.12944C>G (p.Arg468Gly)]. The alleles g.12944G and g.325(GT)(14)T(G)(5) of the B haplotype have a positive effect on the total number of kits born, number born alive, number of implanted embryos and foetal and prenatal embryo survival.

Correlated response in body condition and energy mobilisation in rabbits selected for litter size variability.

A divergent selection experiment on litter size variability (high and low lines) was performed in rabbits over seven generations. The aim of this study was to evaluate the correlated responses to selection in body condition and fat reserves mobilisation. Litter size variability was estimated as phenotypic variance of litter size within female after correcting for the year-season and the parity-lactation status effects. A total of 226 females were used in this study, of which 158 females were used to measure body condition and energy mobilisation. Body condition was measured as BW and perirenal fat thickness. Females were stimulated with the adrenergic isoproterenol. Mobilisation capacity of fat reserves was measured by the lipolytic potential, defined as the increment in non-esterified fatty acids (NEFA) levels from basal concentration until adrenergic stimulation at mating, delivery and 10 days after delivery of the second reproductive cycle. Females were classified as survivor or non-survivor when they were culled for sanitary reasons or died before the third kindling. Data were analysed using Bayesian methodology. Survivor females presented higher BW than the non-survivor females at delivery (238 g, P=1.00) and 10 days after delivery (276 g, P=1.00). They also showed higher perirenal fat thickness at 10 days after delivery (0.62 mm, P=1.00). At delivery, basal NEFA levels was lower in survivor than non-survivor females (-0.18 mmol/l, P=1.00), but their lipolytic potential (∆NEFA) was higher (0.08 mmol/l, P=0.94). Body weight was similar between lines in survivor females. Perirenal fat thickness was lower in the high line than in the low line at delivery (-0.23 mm, P=0.90) and 10 days after delivery (-0.28 mm, P=0.92). The high line exhibited higher NEFA (0.10 mmol/l, P=0.93) and lower ∆NEFA (-0.08 mmol/l, P=0.92) than the low line at delivery. The low line showed a favourable correlated response to selection on body condition and fat reserves mobilisation. In conclusion, the low line selected for litter size variability seems to adapt better to adverse conditions, as it has a greater capacity to mobilise energy reserves at delivery than the high line. Females that adequately manage their body reserves and perform energy mobilisation correctly have a lower risk of dying or being culled.

Correlated response to selection for litter size environmental variability in rabbits' resilience.

Resilience is the ability of an animal to return soon to its initial productivity after facing diverse environmental challenges. This trait is directly related to animal welfare and it plays a key role in fluctuations of livestock productivity. A divergent selection experiment for environmental variance of litter size has been performed successfully in rabbits over ten generations. The objective of this study was to analyse resilience indicators of stress and disease in the divergent lines of this experiment. The high line showed a lower survival rate at birth than the low line (-4.1%). After correcting by litter size, the difference was -3.2%. Involuntary culling rate was higher in the high than in the low line (+12.4%). Before vaccination against viral haemorrhagic disease or myxomatosis, concentration of lymphocytes, C-reactive protein (CRP), complement C3, serum bilirubin, triglycerides and cholesterol were higher in the high line than in the low line (difference between lines +4.5%, +5.6 µg/ml, +4.6 mg/ml, +7.9 mmol/l, +0.3 mmol/l and +0.4 mmol/l). Immunological and biochemical responses to the two vaccines were similar. After vaccination, the percentage of lymphocytes and CRP concentration were higher in the low line than in the high one (difference between lines +4.0% and +13.1 µg/ml). The low line also showed a higher increment in bilirubin and triglycerides than the high line (+14.2 v. +8.7 mmol/l for bilirubin and +0.11 v. +0.01 mmol/l for triglycerides); these results would agree with the protective role of bilirubin and triglycerides against the larger inflammatory response found in this line. In relation to stress, the high line had higher basal concentration of cortisol than the low line (+0.2ng/ml); the difference between lines increased more than threefold after the injection of ACTH 1 to 24, the increase being greater in the high line (+0.9 ng/ml) than in the low line (+0.4 ng/ml). Selection for divergent environmental variability of litter size leads to dams with different culling rate for reproductive causes and different kits' neonatal survival. These associations suggest that the observed fitness differences are related to differences in the inflammatory response and the corticotrope response to stress, which are two important components of physiological adaptation to environmental aggressions.

Embryologic changes in rabbit lines selected for litter size variability.

A divergent selection experiment on litter size variability was carried out. Correlated response in early embryo survival, embryonic development, size of embryos, and size of embryonic coats after four generations of selection was estimated. A total of 429 embryos from 51 high-line females and 648 embryos from 80 low-line females were used in the experiment. The traits studied were percentage of normal embryos, embryo diameter, zona pellucida thickness, and mucin coat thickness. Traits were measured at 24, 48, and 72 hours postcoitum (hpc); mucin coat thickness was only measured at 48 and 72 hpc. The embryos were classified as zygotes or two-cell embryos at 24 hpc; 16-cell embryos or early morulae at 48 hpc; and early morulae, compacted morulae, or blastocyst at 72 hpc. At 24 hpc, the percentage of normal embryos in the high line was lower than in the low line (-2.5%), and embryos in the high line showed 10% higher zona pellucida thickness than those of the low line. No differences in percentage of zygotes or two-cell embryos were found. At 48 hpc, the high-line embryos were less developed, with a higher percentage of 16-cell embryos (23.4%) and a lower percentage of early morulae (-23.4%). At 72 hpc, high-line embryos continued to be less developed, showing higher percentages of early morulae and compact morulae and lower percentages of blastocyst (-1.8%). No differences in embryo diameter or mucin coat thickness were found at any time. In conclusion, selection for litter size variability has consequences on early embryonic survival and development, with embryos presenting a lower state of development and a lower percentage of normal embryos in the line selected for higher variability.

Analyses for the presence of a major gene affecting uterine capacity in unilaterally ovariectomized rabbits.

The presence of a major gene for uterine capacity (UC), ovulation rate (OR), number of implanted embryos (IE), embryo survival (ES), fetal survival (FS), and prenatal survival (PS) was investigated in a population of rabbits divergently selected for UC for 10 generations. Selection was performed on estimated breeding values for UC up to four parities. UC was estimated as litter size in the remaining overcrowded horn of unilaterally ovariectomized does. OR and IE were counted by means of laparoscopy. Bartlett's test, Fain's test, and a complex segregation analysis using Bayesian methods were used to test for the presence of a major gene. All three tests showed that the data appeared consistent with the presence of a major gene affecting UC and IE. The results of the complex segregation analysis suggested the presence of a major gene with large effect on IE and ES (a > 1sigma(p)), at high frequency (p = 0.70 and 0.68, respectively), and with a large contribution to the total variance (R(g) = 0.39 and 0.47, respectively); and the presence of a major gene with moderate effect on each of OR, FS, PS, and UC. The results suggest that the studied reproductive traits are determined genetically by at least one gene of large effect.

Relationships between components of litter size in unilaterally ovariectomized and intact rabbit does.

A study was performed to evaluate the use of unilateral ovariectomy for the measurement of uterine capacity in rabbits through a comparison of the relationships between ovulation rate, number of implanted embryos, and litter size in unilaterally ovariectomized (ULO) and intact does. Data from 211 ULO and 323 intact does were analyzed. The animals were derived from a synthetic line previously selected on litter size. Laparoscopy was performed on all does during their second gestation 12 d after mating and the number of corpora lutea and implantation sites were recorded. Intact and ULO does had the same ovulation rate, confirming the presence of compensatory ovarian hypertrophy in the remaining ovary of the ULO does. The number of implantation sites in the ULO group (11.3) approached the number found in the control group (12.6). Embryonic survival (until implantation) was lower (P < .01) in ULO does (.77) than in intact does (.88), but fetal survival (after implantation) was the same in both groups. The ULO females produced litters 77% of the size of those of the normal control females. Pre- and postimplantation survivals were not related in intact does but seemed to be related in ULO does through an effect on the number of implantation sites. The coefficient of the regression of number of implantation sites on ovulation rate was positive in control does (.62 +/- .06) and was also positive in ULO does (.31 +/- .07), showing that a higher ovulation rate would have resulted in a higher number of embryos being implanted in both groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationships between uterine and fetal traits in rabbits selected on uterine capacity.

The aim of this study was to investigate whether uterine capacity (UC) in rabbits was related to uterine horn length and weight and whether these uterine traits and vascular supply were related to fetal development and survival. Data from 48 unilaterally ovariectomized (ULO) does of the High and 52 ULO does of the Low UC lines of a divergent selection experiment on UC were used. Does were slaughtered on d 25 of fifth gestation. The High line showed higher ovarian weight (0.08 g, P < 0.05) linked to a higher ovulation rate (1 ovum, P < 0.05) and greater length of the empty uterine horn. There were no differences between lines in the remaining doe traits. The number of implanted embryos and live fetuses, fetal survival, and uterine weight and length were positively associated and explained most of the observed variation. Average weights of the live fetuses and their fetal and maternal placentae were not related to uterine weight and length. The linear regression coefficient of full uterine horn length on the number of live fetuses was 2.43 +/- 0.21. The weight of the full uterine horn showed a small quadratic relationship (P < 0.05) with the number of live fetuses. Full uterine horn length, after adjusting for the number of embryos, was negatively associated (P < 0.001) with the number of dead fetuses. The linear regression coefficient of average fetal placental weight of the live fetuses on number of implanted embryos was higher (P < 0.10) in the Low line (-0.23 +/- 0.04 vs. -0.12 +/- 0.04). The linear regression coefficient of average weight of the live fetuses on the average weight of their fetal placentae was higher (P < 0.10) in the High line (2.56 +/- 0.47 vs. 1.27 +/- 0.57). The High line was more efficient, most likely because an increase in intrauterine crowding has a lesser effect on the development of fetal placentae and fetuses. The fetal position within the uterus did not affect the proportion of dead embryos. Fetuses with placentae receiving a single blood vessel had a higher probability of death (P < 0.001) and the lowest weight. There was no difference between lines for individual weight of the live fetuses, but the High line showed higher individual weights of fetal (P < 0.01) and maternal placentae (P < 0.10). Live fetuses in the midportion of the uterus were lighter in weight (P < 0.05) than in the oviductal and cervical regions (20.3 vs. 21.6 and 21.7g). Increasing uterine capacity increases uterine length and decreases weights of fetus and fetal placenta in rabbits.

Divergent selection for uterine capacity in rabbits.

To examine the genetic and phenotypic parameters of uterine capacity and its components in rabbits, we performed a divergent selection experiment for uterine capacity. Rabbit does were unilaterally ovariectomized, and a laparoscopy was performed at midgestation to count the number of corpora lutea and implanted embryos. Selection was performed on litter size in all parities. Genetic parameters were estimated by residual maximum likelihood. For most traits, phenotypic and genetic correlations were in the same range. In unilaterally ovariectomized rabbit does, litter size had a low genetic (.34) and phenotypic (-.08) correlation with ovulation rate, a high genetic (.71) and phenotypic (.60) correlation with the number of implanted embryos, and a high genetic correlation (.89) and moderate phenotypic correlation (.51) with fetal survival. Embryonal survival had a low phenotypic (-.26) or genetic (.12) correlation with fetal survival. A component of uterine capacity, fetal survival in one overcrowded uterine horn, had a low heritability (.05), and consequently limited possibilities for improvement by selection. Uterine capacity, estimated as litter size in one overcrowded uterine horn, had a moderately low heritability (.16). Divergent selection on uterine capacity was effective and a correlated response was found in implanted embryos. No correlated response was found in fetal survival and number of dead fetuses between implantation and birth.

Expression of progesterone receptor related to the polymorphism in the PGR gene in the rabbit reproductive tract.

The association of the 2464G > A SNP found in the promoter region of the rabbit progesterone receptor gene with progesterone receptor (PR) expression was evaluated by Western blot analysis. This SNP was associated with 2 lines divergently selected for uterine capacity, the high line selected to increase uterine capacity and the low line selected to decrease uterine capacity. Two progesterone isoforms were obtained using a commercial monoclonal antibody: the PR-B isoform described previously in rabbits, and the PR-A isoform, not described previously in rabbits. The GG genotype, the genotype more frequent in the high line, showed less PR-B and PR-A expression than the AA genotype in the oviduct (GG/AA(PR-B) = 0.81 and GG/AA(PR-A) = 0.73) and uterus (around 0.70 in both isoforms). The GA genotype showed similar PR-A expression in both tissues and also similar PR-B expression in the oviduct to the GG genotype. Conversely, the GG genotype showed less PR-B expression than the GA genotype in the uterus (GG/GA(PR-B) = 0.79). Similar expression of both PR isoforms was found in the uterus at d 2 and 3 of gestation; meanwhile, an increase of both isoforms was observed in the oviduct. Similar PR-A expression was observed in the ampulla and isthmus; meanwhile, the PR-B expression in the isthmus was double that in the ampulla.

Candidate gene analysis for reproductive traits in two lines of rabbits divergently selected for uterine capacity.

The objective of this work was to analyze 3 functional candidate genes for reproduction in 2 lines of rabbits divergently selected by uterine capacity. Both lines were selected for 10 generations. The selection was then relaxed until the 17th generation, when it was compounded by 61 and 63 does of the High and Low lines, respectively. We sequenced the SCGB1A1 gene, which encodes the main protein secreted by the rabbit in the uterus and seems to play an important role in implantation. We found 6 SNP in the promoter region cosegregating in 2 haplotypes in both lines with similar frequency. We also analyzed IGF1 mRNA because of its effects on embryo development, but we did not find any polymorphism between individuals of the 2 lines. The third gene analyzed was the TIMP1, which encodes a protein involved in many biological processes related to reproduction. We determined the sequence of its promoter region and found 1 SNP (g.1423A>G) segregating with different frequencies in both lines (0.60 for allele A in the High line and 0.82 for allele G in the Low line). The association study performed in an F(2) population (n = 598) generated by the cross of the 2 lines of rabbits revealed that the AA genotype had 0.88 embryos more than the GG genotype at 72 h of gestation. The difference increased to 2.23 embryos at implantation, but no difference was found between genotypes at birth. These results suggest that TIMP1 could be a candidate gene for embryo implantation and embryo survival.

Investigation of the oviductal glycoprotein 1 (OVGP1) gene associated with embryo survival and development in the rabbit.

An association study was performed in rabbits between early embryo survival and development, and the nonconservative SNP 12944C>G located in exon 11 and the triallellic microsatellite [(GT)(15)T(G)(5), (GT)(14)T(G)(5), and (GT)(11)T(G)(7))] located in the promoter region of the oviductal glycoprotein 1 (OVGP1) gene. We analyzed an F(2) cross of 2 lines of rabbits divergently selected for uterine capacity. A total of 172 and 159 females were slaughtered at 48 and 72 h of gestation, respectively, to determine whether OVGP1 influences ovulation rate, fertilization rate, early embryo survival, and embryonic stage of development. The results of the SNP indicated that all genotypes showed similar early embryo survival and a similar embryonic stage of development at 48 h of gestation. However, at 72 h of gestation, the GG genotype showed greater early embryo survival than the CC genotype (0.56 embryos) and their embryos presented less embryonic development. Analysis of the microsatellite was performed to ascertain the presence or absence of the allele (GT)(14)T(G)(5). At both stages of gestation, the (GT)(14)T(G)(5)/(GT)(14)T(G)(5) genotype showed greater early embryo survival (0.94 and 1.54 embryos at 48 and 72 h of gestation, respectively) and less embryonic development than the homozygous genotypes without the allele (GT)(14)T(G)(5).