The hormone progesterone is essential for proper embryonic development. The objective of this study was to examine the relationship between recipient serum concentrations of progesterone, at the time ...of embryo transfer and at conceptus recovery, on conceptus development from in vivo- or in vitro-produced embryos. Embryos were produced in vivo by superovulation of Holstein cows (IVO; n = 17) or in vitro with either serum-containing (IVPS; n = 27) or serum-restricted medium (IVPSR; n = 34). Single grade I blastocysts from each embryo production system were transferred into heifers on day 7 of development. Conceptuses were recovered on day 17 of gestation and classified as complete, degenerated, or no conceptus. Compared with the IVO group, in vitro-produced embryos had more (P = 0.055) degenerated conceptuses (IVO, 0%; IVPS, 18.5%; and IVPSR, 20.6%). There were no differences in progesterone concentrations at the time of transfer when recipients received either male or female embryos (P > 0.05). Progesterone concentrations in recipients receiving in vivo-produced embryos were higher (P < 0.05; 3.74 ± 0.4 ng/mL; least-squares mean ± standard error of the mean) on day 7 compared with those receiving in vitro-produced embryos (IVPS, 2.4 ± 0.2; IVPSR, 2.58 ± 0.3 ng/mL). However, there was no difference in progesterone concentration on day 7 between treatment groups for heifers from which short conceptuses (≤194 mm) were recovered on day 17. In contrast, when longer (>194 mm) conceptuses were recovered on day 17, heifers receiving in vitro-produced embryos had lower (P = 0.05) serum concentrations of progesterone on day 7 compared with those receiving in vivo-produced embryos (IVPS, 2.2 ± 0.5; IVPSR, 2.3 ± 0.5; IVO, 3.9 ± 0.5 ng/mL). In conclusion, differences in autonomy may exist between in vitro- and in vivo-produced embryos during the period of conceptus elongation with in vitro-produced embryos relying more on intrinsic factors to influence elongation.
Development of the post-hatching conceptus in ruminants involves a period of morphological expansion that is driven by complex interactions between the conceptus and its intrauterine environment. As ...a result of these interactions, endometrial physiology is altered, leading to establishment of the pregnancy and continued development of the placenta. Disruption of normal fetal and placental development can occur when embryos are exposed to manipulations in vitro or when inappropriate endocrine sequencing occurs in vivo during the pre- and peri-implantation periods. The present review addresses the development of the post-hatching bovine conceptus, its interactions with the maternal system and changes in development that can occur as a result of in vivo and in vitro manipulations of the bovine embryo.
In vitro systems are commonly used for the production of bovine embryos. Comparisons between in vivo and in vitro produced embryos illustrate that the morphology of preimplantation-stage embryos ...differ significantly, the survival of embryos and fetuses is decreased, the size distributions of the populations of conceptuses and fetuses are altered throughout gestation, and placental development is significantly changed. Taken together these findings indicate that exposure to some in vitro environments during the first 7 days of life can profoundly influence fetal and placental development in cattle. An understanding of how in vitro oocyte maturation, in vitro fertilization, and embryo culture systems influence both fetal and placental development should result in systems that consistently produce normal embryos, fetuses, and calves.
The objectives of the present experiment were to compare survival after transfer of bovine embryos produced in vivo with those produced in vitro and to examine the physical characteristics of fetuses ...produced from these transfers. Embryos produced in vivo (Holstein X Angus) were recovered from uterine flushings of superovulated heifers 7 days after first artificial insemination, and embryos produced in vitro (Holstein X beef breeds) were collected 7 days after insemination. Embryos were paired by source (in vivo, in vitro), stage (compact morula, blastocyst), and quality grade (excellent = 1, good = 2), and transferred nonsurgically to recipient heifers on Day 7 (+/- 1 day) of the estrous cycle. Pregnancy status was monitored by determination of serum progesterone concentrations, ultrasonography, and palpation through 7 mo of gestation, at which time fetuses were recovered. In comparison with grade 1 embryos produced in vivo, the risk of embryonic death after transfer was similar for grade 2 embryos produced in vivo (p = 0.56) and for grade 1 embryos produced in vitro (p = 0.88). By contrast, grade 2 embryos produced in vitro were at greater (p = 0.04) risk of embryonic death. Embryo loss was associated (p = 0.01) with increased serum concentrations of progesterone in recipients at the time of transfer. At 7 mo of gestation, fetuses from embryos produced in vitro were heavier (p = 0.02) than fetuses from embryos produced in vivo and had skeletal measurements that were disproportionate (p less than or equal to 0.04) to body weight
The insulin-like growth factor type 2 receptor (IGF2R) regulates fetal growth by removing IGF2 from circulation. In mice, expression of the Igf2r gene is only imprinted after implantation and is ...associated with expression of the antisense non-coding (nc)RNA, Airn. The objectives of this study were, first, to determine if bovine AIRN was expressed during developmentally important stages of gestation, and second, to determine if expression of bAIRN was affected by method of embryo production. Control reactions confirmed that sequence verified bAIRN PCR amplicons resulted from RNA within the sample and not from genomic DNA contamination. IGF2R mRNA was expressed in all fetal liver samples at Days 35–55 and 70 of gestation as well as in 8 of 9 Day 15 conceptuses, 10 of 10 Day 18 conceptuses, and in all day 7 blastocyst pools. bAIRN was expressed in all samples of fetal liver at Days 35–55 and 70 of gestation. The proportion of conceptuses that expressed bAIRN increased from 1 of 9 at Day 15 of gestation to 8 of 10 at Day 18 of gestation. No bAIRN was expressed in any blastocyst pools. The relative level of bAIRN was greater (P<0.05) in fetal liver from embryos produced in vivo compared to that from embryos produced in vitro. In summary bAIRN was not expressed in blastocyst-stage embryos, was expressed in an increasing proportion of embryos around the time of maternal recognition of pregnancy and was expressed following implantation. Furthermore, relative levels of bAIRN in bovine fetal liver can be altered by method of embryo production.
The objective of this study was to compare the ultrastructure of bovine blastocysts produced in vivo or in vitro by using
morphometric analysis. Blastocysts produced in vivo (multiple ovulations, MO) ...were obtained from superovulated Holstein cows.
For blastocysts produced in vitro, cumulus-oocyte complexes aspirated from ovaries of Holstein cows were matured and fertilized
in vitro. At 20 h postinsemination (hpi), zygotes were distributed into one of three culture media: 1) IVPS (in vitro produced
with serum): TCM-199 + 10% estrous cow serum (ECS); 2) IVPSR (in vitro produced with serum restriction): TCM-199 + 1% BSA
until 72 hpi, followed by TCM-199 + 10% ECS from 72 to 168 hpi; and 3) mSOF (modified synthetic oviductal fluid): mSOF + 0.6%
BSA. At 168 hpi, six or seven grade 1 blastocysts from each of the four treatments (MO, IVPS, IVPSR, and mSOF) were fixed
and prepared for transmission electron microscopy. Random micrographs of each blastocyst were used to determine the volume
density of cellular components. Overall, as blastocysts progressed in development, the volume densities of cytoplasm and intercellular
space decreased ( P < 0.05) and the volume densities of mature mitochondria, nuclei, blastocoele, and apoptotic bodies increased ( P < 0.05). Across treatments, the proportional volumes of nuclei and inclusion bodies were increased in inner cell mass cells
compared with trophectoderm cells for mid- and expanded blastocysts. For blastocysts produced in vitro, the volume density
of mitochondria was decreased ( P < 0.05) as compared with that of blastocycts produced in vivo. The proportional volume of vacuoles was increased ( P < 0.05) in blastocysts from the mSOF treatment as compared with blastocysts produced in vivo. For mid- and expanded blastocysts
from all three in vitro treatments, the volume density of lipid increased ( P < 0.05) and the volume density of nuclei decreased ( P < 0.05) compared with those of blastocysts produced in vivo. In conclusion, blastocysts produced in vitro possessed deviations
in volume densities of organelles associated with cellular metabolism as well as deviations associated with altered embryonic
differentiation. However, the specific nature of these deviations varied with the type of culture conditions used for in vitro
embryo production.
The main objective was to determine the efficacy of using X-sorted sperm to produce embryos in vitro for transfer into lactating dairy cows. Cows were bred by timed artificial insemination (TAI) ...using nonsorted semen or X-sorted sperm, or they received a fresh embryo produced in vitro by fertilization with X-sorted or nonsorted sperm using timed embryo transfer (TET). Pregnancy rates at approximately Day 32 averaged over all dairies were 39.3 ± 3.2% (least-squares mean ± SEM) for TAI nonsorted, 27.3 ± 3.4% for TET nonsorted fresh embryos, and 30.2 ± 3.3% for TET X-sorted fresh embryos (TAI vs. both TET groups, P < 0.05; 206 to 233 cows per group). Pregnancy losses between approximately Day 32 and term ranged from 16% to 37%, the latter from TET with X-sorted sperm. Pregnancy losses to term were higher for cows receiving embryos produced in vitro than for cows bred by TAI. Calves produced via TET were not substantively different from AI controls in physical measurements or standard blood chemistry profiles.
The establishment of in vitro fertilization and culture systems for mammalian embryos has facilitated the application of embryo technologies in research, industry, and clinical settings. Furthermore, ...the advent of cloning by nuclear transfer has significantly enhanced the potential for genetic modification of livestock. Based on studies in cattle, sheep, and mice, it has become apparent that embryos produced using these systems can differ in morphology and developmental potential compared with embryos produced in vivo. Referred to as "large offspring syndrome," these abnormalities in the development of fetuses, placentas, and offspring are particularly evident following transfer of cloned embryos, but they also occur in pregnancies from embryos produced using in vitro culture alone. The objective of this review is to examine the effects of in vitro production and cloning on bovine embryo and fetal development. Literature pertaining to preimplantation embryo, conceptus, and fetal development, as well as gene expression occurring at each of these three stages, is reviewed. Physiologic and genetic mechanisms that contribute to large offspring syndrome also are discussed.
Contents
Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of ...today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll‐like receptors (TLR) mediate innate immune mechanisms via the production of pro‐inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30–50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll‐like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN‐G), and interleukin (IL)‐12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL‐6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro‐inflammatory cytokine mRNA expression.
The objective of this study was to determine the effects of
in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, ...IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation.
In vivo embryos were recovered from superovulated Holstein cows. For production of embryos
in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (
in vivo, n = 14;
in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos
in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the
in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the
in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced
in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced
in vivo. In conclusion,
in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.
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