168 Improvement of in vivo-produced bovine embryo competence by using a bovine recombinant FSH in a superovulation protocol

Genomic tools are now available for most livestock species and used routinely for marker-assisted selection (MAS) in cattle. The detection of a large number of markers that are widespread over the genome is generally limited by the amount of genomic DNA available in an embryo biopsy of a small size not to be detrimental to embryonic survival. Amplification of DNA from such a biopsy is then necessary. In this study, the efficiency of embryo genotyping for 45 microsatellites (MS) following whole-genome amplification (WGA) was evaluated from samples of a variable number of cells isolated from cattle embryos. In a second part, this work aims to test the reliability of the MAS method for 45 MS and 13 single nucleotide polymorphisms (SNP) from bovine embryo biopsies under field conditions. In experiment 1, in vitro bovine morulae (n = 10) were produced, and 1, 5, and 10 embryonic cells were removed from each morula. Cells were dry frozen in tubes before further processing. Whole-genome amplification was performed using the commercial Qiagen REPLI-g® Mini Kit according to the manufacturer instructions (Qiagen, Valencia, CA, USA). WGA solution was then diluted, processed by PCR with 45 markers, and the resulting data were genotyped with GeneMapper software® (Applied Biosystems Europe). Accuracy and reliability of genotyping were assessed using different samples of cells from the same embryo. In experiment 2, after superovulation (10 cows), bovine embryos were in vivo-produced and collected at day 6 or day 7 of pregnancy. Only grade 1 embryos were washed and biopsied using a microblade. Biopsied embryos were either frozen or transferred back to synchronized recipients. Individual biopsies were transferred as dry samples to the laboratory. Genomic DNA was amplified using WGA, and embryos were genotyped. The results of experiment 1 clearly indicate that a conventional biopsy of 5 to 10 cells was sufficient for multi-markers detection after whole-genome amplification as 98% of the 45 markers were detected compared to 45% of marker detection using 1 cell (P < 0.01). In experiment 2, from 123 collected embryos, 79 were classified as grade I or II transferable embryos (64.2%) and 57 were biopsied (34 were classified as stage 4–5 and 23 as stage 5–6, according to the IETS criteria). Using the stereomicroscopic analysis, 44 biopsies had a number of cells ranging from 4 to 7 (5.6 ± 1.4) and 13 biopsies from 8 to 10 (8.4 ± 1.6). Overall, at least 95% of markers (MS + SNP) were detected in 49.1% of biopsies (28/57). The total detection rate for SNP was significantly higher than for MS; 70.2% (40/57) v. 31.6% (18/57), respectively, (chi-square, P < 0.01). The detection rate of the markers was not significantly affected by the embryo stage or the biopsy size. Our results confirm that genotyping a large number of markers from biopsy samples after whole-genome amplification is possible under field conditions. A larger number of biopsies is required to assess the reliability of this method that may allow the development of MAS from early embryo. This work has been performed through the programme TYPAGENAE (GENANIMAL 4-03) with the financial support of FRT/ANR and Apis-Genes.

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293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab293 ◽

2007 ◽

Vol 19 (1) ◽

pp. 262 ◽

Cited By ~ 1

Author(s):

I. Dimitriadis ◽

E. A. Rekka ◽

E. Vainas ◽

G. S. Amiridis ◽

C. A. Rekkas

Keyword(s):

Lipid Peroxidation ◽

Embryo Development ◽

Antioxidant Properties ◽

Thiobarbituric Acid ◽

Early Embryo ◽

Bovine Embryo ◽

Embryo Production ◽

Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples' ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P < 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

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7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab7 ◽

2009 ◽

Vol 21 (1) ◽

pp. 104

Author(s):

J. T. Aaltonen ◽

K. J. Mattson ◽

N. M. Loskutoff

Keyword(s):

Embryo Development ◽

Disease Transmission ◽

Bos Taurus ◽

Gradient Centrifugation ◽

Control Group ◽

Bovine Embryo ◽

Human Sequence ◽

Bovine Sperm

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

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130 A CATALOG OF REFERENCE GENES WITH HIGH, MEDIUM, AND LOW LEVELS OF EXPRESSION DURING BOVINE IN VIVO PRE-IMPLANTATION DEVELOPMENT

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab130 ◽

2017 ◽

Vol 29 (1) ◽

pp. 173

Author(s):

Z. Jiang ◽

J. Sun ◽

S. Marjani ◽

H. Dong ◽

X. Zheng ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Target Genes ◽

Stable Expression ◽

Bovine Embryo ◽

Rna Seq ◽

Rt Pcr ◽

Data Set ◽

Low Expression

Appropriate reference genes for accurate normalization in RT-PCR are essential for the study of gene expression. Ideal reference genes should not only have stable expression across stages of embryo development, but also be expressed at comparable levels to the target genes. Using RNA-seq data from in vivo-produced bovine oocytes and embryos from the 2-cell to blastocyst stage (Jiang et al., 2014 BMC Genomics 15, 756), we tried to establish a catalogue of all reference genes for RT-PCR analysis. One-way ANOVA generated 4055 genes that did not differ across stages. To reduce this list, we used the entire RNA-seq data set and first removed genes with a FPKM (fragments per kilobase of transcript per million mapped reads) of <1, and then rescaled each gene’s expression values within a range of 0 to 1. We subsequently calculated the expression variance for each gene across all stages. By assuming that the calculated variances follow a Gaussian distribution and that the majority of the genes do not have a stable expression level, a gene was classified as a reference if its variance significantly deviated (P < 0.05) from these assumptions. We identified 346 potential reference genes, all of which were among the candidates from the ANOVA analysis. We arbitrarily assigned genes in this list to high (FPKM ≥ 100), medium (10 < FPKM < 100), and low expression levels (FPKM ≤ 10), and 37, 154, and 155 genes, respectively, fell into these groups. Surprisingly, none of the commonly used reference genes, such as GAPDH, PPIA, ACTB, PRL15, GUSB, and H3F2A, were identified as being stably expressed across in vivo development. This is consistent with findings of prior RT-PCR studies (Robert et al. 2002 Biol. Reprod. 67, 1465–1472; Ross et al. 2010 Cell Reprogram. 12, 709–717). The following gene ontology terms were significantly enriched for the 346 genes: cell cycle, translation, transport, chromatin, cell division, and metabolic process, indicating that the early embryos maintained constant levels of genes involved in fundamental biological functions. Finally, we performed RT-PCR to validate the RNA-seq results using different bovine in vivo-derived oocytes and embryos (n = 3/stage). We successfully validated 10 selected genes, including those in the high (CS, PGD, and ACTR3), medium (CCT5, MRPL47, COG2, CRT9, and HELLS), and low expression groups (CDC23 and TTF1). In conclusion, we recommend the use of reference genes that are expressed at comparable levels to target genes. This study offers a useful resource to aid in the appropriate selection of reference genes, which will improve the accuracy of quantitative gene expression analyses across bovine embryo pre-implantation development.

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Apoptosis in the early bovine embryo

Zygote ◽

10.1017/s0967199400000836 ◽

2000 ◽

Vol 8 (1) ◽

pp. 57-68 ◽

Cited By ~ 114

Author(s):

Christie Matwee ◽

Dean H. Betts ◽

W. Allan King

Keyword(s):

Dna Fragmentation ◽

Early Development ◽

P53 Protein ◽

Chromatin Condensation ◽

Terminal Deoxynucleotidyl Transferase ◽

Bovine Embryo ◽

Neighbouring Cell ◽

Tunel Labelling

Cell death occurs during early development in vivo and in vitro, although little is known about the mechanism of blastomere death and the relation to embryonic loss. Apoptosis, characterised by chromatin condensation, DNA fragmentation and membrane blebbing, occurs without damage to surrounding cells in contrast to necrosis. Bovine oocytes and in vitro fertilised embryos (total n = 449) were analysed for (1) DNA fragmentation using terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and (2) morphological features of apoptosis. TUNEL labelling was detected in immature and mature oocytes (7%, n = 57 and 23%, n = 60, respectively), and at least one cell of 8- to 16-cell embryos (5%, n = 57), morulae/early blastocysts (79%, n = 39) and expanded/hatched blastocysts (100%, n = 48). In contrast, TUNEL labelling was not detected in zygotes (n = 61), 2-cell embryos (n = 46) or 3- to 7-cell embryos (n = 81). Chromatin condensation, nuclear fragmentation, absence of neighbouring cell destruction and extrusion of cells was frequent among advanced stage embryos. Although not detected during early cleavage under standard conditions, TUNEL labelling indicative of apoptosis was induced by treatment with 10 μM staurosporine for 30 h in 95% of cleavage stage embryos (n = 59). Determination of the expression and localisation of the p53 tumour suppressor gene using reverse transcription polymerase chain reaction and whole-mount immunofluorescence revealed that although p53 transcripts were present throughout early development, nuclear localisation of p53 protein could not be detected in any blastocyst suggesting p53-independent apoptosis. This study has shown that apoptosis is dependent on embryonic developmental stage after standard culture. This suggests that bovine embryos become more capable of accommodating damaged or abnormal cells as development proceeds.

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Gonadotropins Regulate Rat Testicular Tight Junctions in Vivo

Endocrinology ◽

10.1210/en.2009-1278 ◽

2010 ◽

Vol 151 (6) ◽

pp. 2911-2922 ◽

Cited By ~ 32

Author(s):

Mark J. McCabe ◽

Gerard A. Tarulli ◽

Sarah J. Meachem ◽

David M. Robertson ◽

Peter M. Smooker ◽

...

Keyword(s):

Tight Junctions ◽

Sertoli Cell ◽

Gnrh Antagonist ◽

Hormone Replacement ◽

Recombinant Fsh ◽

Short Term ◽

Adult Rat ◽

Serum Androgens

Sertoli cell tight junctions (TJs) are an essential component of the blood-testis barrier required for spermatogenesis; however, the role of gonadotropins in their maintenance is unknown. This study aimed to investigate the effect of gonadotropin suppression and short-term replacement on TJ function and TJ protein (occludin and claudin-11) expression and localization, in an adult rat model in vivo. Rats (n = 10/group) received the GnRH antagonist, acyline, for 7 wk to suppress gonadotropins. Three groups then received for 7 d: 1) human recombinant FSH, 2) human chorionic gonadotropin (hCG) and rat FSH antibody (to study testicular androgen stimulation alone), and 3) hCG alone (to study testicular androgen and pituitary FSH production). TJ proteins were assessed by real-time PCR, Western blot analysis, and immunohistochemistry, whereas TJ function was assessed with a biotin permeation tracer. Acyline treatment significantly reduced testis weights, serum androgens, LH and FSH, and adluminal germ cells (pachytene spermatocyte, round and elongating spermatids). In contrast to controls, acyline induced seminiferous tubule permeability to biotin, loss of tubule lumens, and loss of occludin, but redistribution of claudin-11, immunostaining. Short-term hormone replacement stimulated significant recoveries in adluminal germ cell numbers. In hCG ± FSH antibody-treated rats, occludin and claudin-11 protein relocalized at the TJ, but such relocalization was minimal with FSH alone. Tubule lumens also reappeared, but most tubules remained permeable to biotin tracer, despite the presence of occludin. It is concluded that gonadotropins maintain Sertoli cell TJs in the adult rat via a mechanism that includes the localization of occludin and claudin-11 at functional TJs.

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Pregnancy recognition and abnormal offspring syndrome in cattle

Reproduction Fertility and Development ◽

10.1071/rd09217 ◽

2010 ◽

Vol 22 (1) ◽

pp. 75 ◽

Cited By ~ 34

Author(s):

C. E. Farin ◽

W. T. Farmer ◽

P. W. Farin

Keyword(s):

Present Review ◽

Bovine Embryo ◽

Placental Development ◽

Intrauterine Environment ◽

Complex Interactions ◽

Pregnancy Recognition

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.

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Revealing the bovine embryo transcript profiles during early in vivo embryonic development

Reproduction ◽

10.1530/rep-08-0533 ◽

2009 ◽

Vol 138 (1) ◽

pp. 95-105 ◽

Cited By ~ 21

Author(s):

Maud Vallée ◽

Isabelle Dufort ◽

Stéphanie Desrosiers ◽

Aurélie Labbe ◽

Catherine Gravel ◽

...

Keyword(s):

Gene Expression ◽

Embryonic Development ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Current Knowledge ◽

Mrna Level ◽

Early Embryonic Development ◽

Bovine Embryo ◽

Rna Content

Gene expression profiling is proving to be a powerful approach for the identification of molecular mechanisms underlying complex cellular functions such as the dynamic early embryonic development. The objective of this study was to perform a transcript abundance profiling analysis of bovine early embryonic development in vivo using a bovine developmental array. The molecular description of the first week of life at the mRNA level is particularly challenging when considering the important fluctuations in RNA content that occur between developmental stages. Accounting for the different intrinsic RNA content between developmental stages was achieved by restricting the reaction time during the global amplification steps and by using spiked controls and reference samples. Analysis based on intensity values revealed that most of the transcripts on the array were present at some point during in vivo bovine early embryonic development, while the varying number of genes detected in each developmental stage confirmed the dynamic profile of gene expression occurring during embryonic development. Pair-wise comparison of gene expression showed a marked difference between oocytes and blastocysts profiles, and principal component analysis revealed that the majority of the transcripts could be regrouped into three main clusters representing distinct RNA abundance profiles. Overall, these data provide a detailed temporal profile of the abundance of mRNAs revealing the richness of signaling processes in early mammalian development. Results presented here provide better knowledge of bovine in vivo embryonic development and contribute to the progression of our current knowledge regarding the first week of life in mammals.

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Genetic influence on the reduction in bovine embryo lipid content by L-carnitine

Reproduction Fertility and Development ◽

10.1071/rd14215 ◽

2016 ◽

Vol 28 (8) ◽

pp. 1172 ◽

Cited By ~ 5

Author(s):

Luis Baldoceda ◽

Dominic Gagné ◽

Christina Ramires Ferreira ◽

Claude Robert

Keyword(s):

Lipid Content ◽

Culture Medium ◽

Cell Damage ◽

Mitochondrial Activity ◽

Bovine Embryo ◽

Bovine Embryos ◽

Intracellular Lipid ◽

Embryo Culture Medium

The decreased rate of pregnancy obtained in cattle using frozen in vitro embryos compared with in vivo embryos has been associated with over-accumulation of intracellular lipid, which causes cell damage during cryopreservation. It is believed that the higher lipid content of blastomeres of bovine embryos produced in vitro results in darker-coloured cytoplasm, which could be a consequence of impaired mitochondrial function. In this study, l-carnitine was used as a treatment to reduce embryonic lipid content by increasing metabolism in cultured bovine embryos. We have observed previously that in vivo embryos of different dairy breeds collected from cows housed and fed under the same conditions differed in lipid content and metabolism. As such, breed effects between Holstein and Jersey were also examined in terms of general appearance, lipid composition, mitochondrial activity and gene expression. Adding l-carnitine to the embryo culture medium reduced the lipid content in both breeds due to increased mitochondrial activity. The response to l-carnitine was weaker in Jersey than in Holstein embryos. Our results thus show that genetics influence the response of bovine embryos to stimulation of mitochondrial metabolism.

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Gene expression and metabolic response of bovine oviduct epithelial cells to the early embryo

Reproduction ◽

10.1530/rep-18-0561 ◽

2019 ◽

Vol 158 (1) ◽

pp. 85-94 ◽

Cited By ~ 7

Author(s):

Meriem Hamdi ◽

María J Sánchez-Calabuig ◽

Beatriz Rodríguez-Alonso ◽

Sandra Bagés Arnal ◽

Kalliopi Roussi ◽

...

Keyword(s):

Epithelial Cells ◽

Metabolic Response ◽

Early Embryo ◽

Bovine Embryo ◽

Cell Stage ◽

Transcriptomic Response ◽

Early Embryos ◽

Oviduct Epithelial Cells

During its journey through the oviduct, the bovine embryo may induce transcriptomic and metabolic responses, via direct or indirect contact, from bovine oviduct epithelial cells (BOECs). An in vitro model using polyester mesh was established, allowing the study of the local contact during 48 h between a BOEC monolayer and early embryos (2- or 8-cell stage) or their respective conditioned media (CM). The transcriptomic response of BOEC to early embryos was assessed by analyzing the transcript abundance of SMAD6, TDGF1, ROCK1, ROCK2, SOCS3, PRELP and AGR3 selected from previous in vivo studies and GPX4, NFE2L2, SCN9A, EPSTI1 and IGFBP3 selected from in vitro studies. Moreover, metabolic analyses were performed on the media obtained from the co-culture. Results revealed that presence of early embryos or their CM altered the BOEC expression of NFE2L2, GPX4, SMAD6, IGFBP3, ROCK2 and SCN9A. However, the response of BOEC to two-cell embryos or their CM was different from that observed to eight-cell embryos or their CM. Analysis of energy substrates and amino acids revealed that BOEC metabolism was not affected by the presence of early embryos or by their CM. Interestingly, embryo metabolism before embryo genome activation (EGA) seems to be independent of exogenous sources of energy. In conclusion, this study confirms that early embryos affect BOEC transcriptome and BOEC response was embryo stage specific. Moreover, embryo affects BOEC via a direct contact or via its secretions. However transcriptomic response of BOEC to the embryo did not manifest as an observable metabolic response.

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