@article{barnwell_farin_whisnant_alexander_farin_2015, title={Maternal serum progesterone concentration and early conceptus development of bovine embryos produced in vivo or in vitro}, volume={52}, ISSN={["1879-0054"]}, DOI={10.1016/j.domaniend.2015.03.004}, abstractNote={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.}, journal={DOMESTIC ANIMAL ENDOCRINOLOGY}, author={Barnwell, C. V. and Farin, P. W. and Whisnant, C. S. and Alexander, J. E. and Farin, C. E.}, year={2015}, month={Jul}, pages={75–81} }
 @article{farin_alexander_farin_2010, title={Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro}, volume={74}, ISSN={["0093-691X"]}, DOI={10.1016/j.theriogenology.2010.05.035}, abstractNote={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.}, number={7}, journal={THERIOGENOLOGY}, author={Farin, C. E. and Alexander, J. E. and Farin, P. W.}, year={2010}, month={Oct}, pages={1288–1295} }
 @article{farin_rodriguez_alexander_hockney_herrick_kennedy-stoskopf_2007, title={The role of transcription in EGF- and FSH-mediated oocyte maturation in vitro}, volume={98}, ISSN={["1873-2232"]}, DOI={10.1016/j.anireprosci.2006.10.007}, abstractNote={Understanding mechanisms responsible for meiotic resumption in mammalian oocytes is critical for the identification of strategies to enhance developmental competence of in vitro-matured oocytes. Improvement of in vitro oocyte maturation systems is dependent on a better understanding of mechanisms that regulate oocyte maturation both in vivo and in vitro as well as on the identification of methods to manipulate the meiotic progression of oocytes matured in vitro in a physiological manner. The purpose of this review is two-fold: first, to examine the mechanisms that underlie the acquisition of oocyte developmental competence and regulation of oocyte maturation in vivo and in vitro; second, to present data examining the role of transcription in mediating the ability of EGF and FSH to induce oocyte maturation in vitro. Results presented support the conclusions that (1) EGF-induced oocyte maturation does not require nascent gene transcription in both mice and domestic cats; (2) FSH requires gene transcription to induce oocyte maturation in both species; (3) EGF must be present in the maturation medium to optimize the effectiveness of FSH to promote oocyte maturation; (4) the mechanism used by FSH to induce oocyte maturation in vitro appears to predominate over that used by EGF when both EGF and FSH are present in maturation medium used for either murine or feline cumulus oocyte complexes.}, number={1-2}, journal={ANIMAL REPRODUCTION SCIENCE}, author={Farin, C. E. and Rodriguez, K. F. and Alexander, J. E. and Hockney, J. E. and Herrick, J. R. and Kennedy-Stoskopf, S.}, year={2007}, month={Mar}, pages={97–112} }
 @article{rodriguez_blomberg_zuelke_miles_alexander_farin_2006, title={Identification of candidate mRNAs associated with gonadotropin-induced maturation of murine cumulus oocyte complexes using serial analysis of gene expression}, volume={27}, ISSN={["1094-8341"]}, DOI={10.1152/physiolgenomics.00309.2005}, abstractNote={In cultured cumulus oocyte complexes (COC), FSH induces gene transcription required for germinal vesicle breakdown (GVBD). Experiments were performed to determine the critical period when gene transcription is required for GVBD and to identify candidate mRNAs involved. Experiment I: murine COC were cultured 4 h in the presence of FSH with 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) added at different intervals after the start of culture. COC cultured with FSH underwent GVBD (82 ± 7%). When DRB was added at 0, 5, or 10 min after culture initiation, oocyte maturation was blocked (17 ± 7, 14 ± 6, and 21 ± 6% GVBD, respectively). When DRB was added after 15, 20, or 30 min, progressively more COC underwent GVBD (37 ± 6, 39 ± 6, and 66 ± 6%, respectively). The critical period of transcription required for GVBD occurred between 15 and 30 min after culture initiation. Experiment II: COC were cultured for 25 min in the presence (plusDRB) or absence (minusDRB) of DRB. SAGE libraries were generated from COC RNA of each treatment group. A total of 48,431 and 45,367 tags were sequenced for the plusDRB and minusDRB libraries, respectively. Criteria used to identify transcripts of interest included a total tag count of at least 10 across both libraries and a threefold or greater difference in expression between libraries. Using these criteria, 39 and 27 transcripts were identified as differentially expressed at the P ≤ 0.01 and P ≤ 0.001 levels, respectively. Differentially expressed transcripts were classed into major categories that included cell growth, development, and regulation of gene expression. Differentially expressed transcripts represent candidates potentially involved in regulating maturation of murine COC.}, number={3}, journal={PHYSIOLOGICAL GENOMICS}, author={Rodriguez, K. F. and Blomberg, L. A. and Zuelke, K. A. and Miles, J. R. and Alexander, J. E. and Farin, C. E.}, year={2006}, month={Nov}, pages={318–327} }
 @article{miles_farin_rodriguez_alexander_farin_2005, title={Effects of embryo culture on angiogenesis and morphometry of bovine placentas during early gestation}, volume={73}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod.105.040808}, abstractNote={Abstract The objective of this study was to determine the effects of undefined and semidefined culture systems for in vitro embryo production on angiogenesis and morphometry of bovine placentas during early gestation. Blastocysts produced in vivo were recovered from superovulated Holstein cows and served as controls. Blastocysts produced in vitro were exposed to either serum-supplemented medium with cumulus cell coculture (in vitro-produced with serum; IVPS) or modified synthetic oviductal fluid medium without serum or coculture (mSOF). Single blastocysts from each production system were transferred into heifers. Fetuses and placentas were recovered on Day 70 of gestation. Cotyledonary tissues were obtained for quantification of vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor-gamma (PPARG) mRNA and protein. Samples of placentomes were prepared for immunocytochemistry and histological analysis. Placentas from the mSOF group were heavier and had the fewest placentomes, least placental fluid, and lowest placental efficiency (fetal weight/placental weight) compared with the in vivo and IVPS groups. There was no effect of embryo culture system on volume densities of fetal villi or maternal endometrium within placentomes. The volume density of fetal pyknotic cells was increased in placentomes in the mSOF group compared with the in vivo and IVPS groups. Placentomes in the mSOF group had decreased densities of blood vessels and decreased levels of VEGF mRNA in cotyledonary tissue. In conclusion, compared with placentas from embryos produced in vivo or in vitro using an undefined culture system, placentas from embryos produced in vitro using a semidefined culture system exhibited a greater degree of aberrant development of the placenta during early gestation.}, number={4}, journal={BIOLOGY OF REPRODUCTION}, author={Miles, JR and Farin, CE and Rodriguez, KF and Alexander, JE and Farin, PW}, year={2005}, month={Oct}, pages={663–671} }
 @article{miles_farin_rodriguez_alexander_farin_2004, title={Angiogenesis and morphometry of bovine placentas in late gestation from embryos produced in vivo or in vitro}, volume={71}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod.104.031427}, abstractNote={Abstract The objective of this study was to determine the effects of in vitro embryo production on angiogenesis and morphometry of the bovine placenta during late gestation. Blastocysts produced in vivo were recovered from superovulated Holstein cows. Blastocysts produced in vitro were obtained after culture of in vitro-matured and -fertilized Holstein oocytes. Single blastocysts from each production system were transferred into heifers. Fetuses and placentas were recovered on Day 222 of gestation (in vivo, n = 12; in vitro, n = 12). Cotyledonary and caruncular tissues were obtained for quantification of vascular endothelial growth factor (VEGF) and peroxisome proliferator-activated receptor-gamma (PPARγ) mRNA and protein. Tissue sections of placentomes were prepared for morphometric analysis. Fetuses and placentas were heavier from embryos produced in vitro than from embryos produced in vivo. More placentas from embryos produced in vitro had an excessive volume of placental fluid. There was no effect of treatment on the expression of mRNA for VEGF and PPARγ in either cotyledonary or caruncular tissues. The expression of VEGF protein in cotyledons and caruncles as well as the expression of PPARγ protein in cotyledons were not different between the in vitro and in vivo groups. However, caruncles from the in vitro group had increased expression of PPARγ protein. The total surface area of endometrium was greater for the in vitro group compared with controls. In contrast, the percentage placentome surface area was decreased in the in vitro group. Fetal villi and binucleate cell volume densities were decreased in placentomes from embryos produced in vitro. The proportional tissue volume of blood vessels in the maternal caruncles was increased in the in vitro group. Furthermore, the ratios of blood vessel volume density-to-placentome surface area were increased in the in vitro group. In conclusion, these findings are consistent with the concept that compensatory mechanisms exist in the vascular beds of placentas from bovine embryos produced in vitro.}, number={6}, journal={BIOLOGY OF REPRODUCTION}, author={Miles, JR and Farin, CE and Rodriguez, KF and Alexander, JE and Farin, PW}, year={2004}, month={Dec}, pages={1919–1926} }
 @article{crosier_farin_rodriguez_blondin_alexander_farin_2002, title={Development of skeletal muscle and expression of candidate genes in bovine fetuses from embryos produced in vivo or in vitro}, volume={67}, ISSN={["0006-3363"]}, DOI={10.1095/biolreprod67.2.401}, abstractNote={Abstract The objectives of this study were to determine the effects of in vitro embryo production on histological development and gene expression in the skeletal muscle of bovine fetuses during late gestation. Blastocysts produced in vivo were obtained from superovulated Holstein cows. Blastocysts produced in vitro were obtained from oocytes of Holstein cows that were matured and fertilized in vitro. Single blastocysts were transferred into heifers at a synchronized estrous and fetuses were recovered at Day 222 of gestation (n = 12 each for in vivo and in vitro). Samples of semitendinosus muscle were obtained for histological analysis and assessment of gene expression. Individual muscle sections were stained for the assessment of primary muscle fibers, secondary muscle fibers, or total muscle fibers. Semiquantitative reverse transcription-polymerase chain reaction assays were performed for 5 different candidate genes. The ratio of secondary-to-primary fiber number was greater in fetuses from embryos produced in vitro compared with fetuses from embryos produced in vivo. Similarly, the ratio of secondary-to-primary fiber volume density tended to be greater in fetuses from embryos produced in vitro. The proportional volume of tissue present between myofibrils was greater in fetuses from embryos produced in vitro. The expression of mRNA for myostatin was decreased in skeletal muscle of fetuses in the in vitro group compared with controls. The expression of mRNA for glyceraldehyde-3-phosphate dehydrogenase tended to be increased in skeletal muscle of fetuses in the in vitro treatment group. There was no effect of treatment on the expression of mRNAs for myf-5, myoD, or myogenin. In conclusion, in vitro production of embryos resulted in fetuses with altered development of skeletal muscle fibers. Myostatin was identified as the candidate gene whose expression may contribute to the observed changes in muscle development of these fetuses.}, number={2}, journal={BIOLOGY OF REPRODUCTION}, author={Crosier, AE and Farin, CE and Rodriguez, KF and Blondin, P and Alexander, JE and Farin, PW}, year={2002}, month={Aug}, pages={401–408} }
 @article{crosier_farin_dykstra_alexander_farin_2001, title={Ultrastructural morphometry of bovine blastocysts produced in vivo or in vitro}, volume={64}, ISSN={["0006-3363"]}, DOI={10.1095/biolreprod64.5.1375}, abstractNote={Abstract 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.}, number={5}, journal={BIOLOGY OF REPRODUCTION}, author={Crosier, AE and Farin, PW and Dykstra, MJ and Alexander, JE and Farin, CE}, year={2001}, month={May}, pages={1375–1385} }
 @article{blondin_farin_crosier_alexander_farin_2000, title={In vitro production of embryos alters levels of insulin-like growth factor-II messenger ribonucleic acid in bovine fetuses 63 days after transfer}, volume={62}, ISSN={["0006-3363"]}, DOI={10.1095/biolreprod62.2.384}, abstractNote={Abstract The objective of this study was to determine the effect of embryo production systems on the expression of insulin-like growth factor (IGF)-II mRNA in fetal bovine tissues at Day 70 of gestation (63 days after transfer). Oocytes aspirated from ovaries of Holstein cows were matured and fertilized in vitro. Zygotes were cultured in either tissue culture medium (TCM)-199 + 10% estrous cow serum (ECS; in vitro-produced with serum [IVPS]) or TCM-199 + 1% BSA (in vitro-produced with serum restriction [IVPSR]). At 72 h postinsemination, IVPSR embryos were transferred into fresh TCM-199 + 10% ECS whereas IVPS embryos had fresh medium replaced. All embryos were cultured for an additional 96 h. In vivo-produced embryos were harvested from superovulated Holstein cows (multiple ovulations [MO]). Grade 1 blastocysts from all groups were transferred singly into Angus heifers. At Day 70 of gestation, fetuses (n = 14, 13, and 11 for MO, IVPS, and IVPSR, respectively) were collected; liver and skeletal muscle samples were snap frozen, and whole-cell RNA (wcRNA) was extracted. Levels of IGF-II mRNA were determined by RNase protection assay and quantified relative to 18S rRNA (mean arbitrary units ± SEM). WcRNA from adult and Day 90 fetal bovine liver were used as controls. Adult liver contained 9-fold less IGF-II mRNA than liver from Day 90 fetuses (P < 0.05). Fetal livers of males originating from IVPS and IVPSR groups possessed approximately 2-fold greater levels of mRNA for IGF-II than those from MO males (0.25 ± 0.07, 0.33 ± 0.04, and 0.14 ± 0.03, respectively; P < 0.05). Levels of mRNA for IGF-II tended to be lower (P = 0.07) in skeletal muscle of fetuses originating from the IVPSR group (0.043 ± 0.005) compared to MO controls (0.070 ± 0.008). In conclusion, at Day 70 of gestation, fetuses originating from in vitro production systems possessed altered levels of IGF-II mRNA in both liver and skeletal muscle.}, number={2}, journal={BIOLOGY OF REPRODUCTION}, author={Blondin, P and Farin, PW and Crosier, AE and Alexander, JE and Farin, CE}, year={2000}, month={Feb}, pages={384–389} }
 @article{crosier_farin_dykstra_alexander_farin_2000, title={Ultrastructural morphometry of bovine compact morulae produced in vivo or in vitro}, volume={62}, ISSN={["1529-7268"]}, DOI={10.1095/biolreprod62.5.1459}, abstractNote={Abstract The objective of this study was to compare the ultrastructure of bovine compact morulae produced in vivo or in vitro using morphometric analysis. Compact morulae produced in vivo were obtained from superovulated Holstein cows. Compact morulae produced in vitro were obtained from cumulus-oocyte complexes aspirated from ovaries of Holstein cows. The complexes were matured and fertilized in vitro. At 20 h postinsemination (hpi), zygotes were distributed into 1 of 3 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 144 hpi; 3) mSOF (modified synthetic oviductal fluid): SOF + 0.6% BSA. At 144 hpi, five grade 1 compact morulae from each of the four treatments were prepared for transmission electron microscopy. The volume density occupied by cellular components was determined by the point-count method using a sampling of seven to nine random micrographs from each compact morula. The volume density of lipid was greater (P < 0.05) in compact morulae from IVPS, IVPSR, and mSOF treatments compared with those produced in vivo. There was a reduced proportional volume of total mitochondria in compact morulae from the IVPS treatment compared with those produced in vivo (P < 0.05). For compact morulae from the IVPS culture treatment, the volume density of vacuoles was greater than that for compact morulae produced in vivo (P < 0.05). The cytoplasmic-to-nuclear ratio for compact morulae from the IVPS treatment was increased (P < 0.05) compared with the ratio for those produced in vivo. In conclusion, compact morulae produced in vitro differed ultrastructurally from those produced in vivo. Compact morulae produced in IVPS culture medium possessed the greatest deviations in cellular ultrastructure.}, number={5}, journal={BIOLOGY OF REPRODUCTION}, author={Crosier, AE and Farin, PW and Dykstra, MJ and Alexander, JE and Farin, CE}, year={2000}, month={May}, pages={1459–1465} }
 @article{blondin_farin_crosier_alexander_farin_1999, title={Does in vitro culture affect the expression of insulin-like growth factor-II (IGF-II) messenger RNA in fetal bovine liver?}, volume={60}, number={1999}, journal={Biology of Reproduction}, author={Blondin, P. and Farin, P. W. and Crosier, A. E. and Alexander, J. E. and Farin, C. E.}, year={1999}, pages={248–249} }