401. In vivo development of embryos derived from sex-sorted and non-sorted ram sperm

2008 ◽  
Vol 20 (9) ◽  
pp. 81
Author(s):  
K. H. Beilby ◽  
C. C. Grupen ◽  
W. M. C. Maxwell ◽  
G. Evans
Keyword(s):  
Y Chromosome ◽  
Embryo Development ◽  
X Chromosome ◽  
Developmental Stages ◽  
Domestic Animals ◽  
Embryo Production ◽  
Fertilisation Rate ◽  
Ram Sperm ◽  
In Pregnancy

The use of sex-sorted ram sperm results in pregnancy rates similar or superior to that of non-sorted sperm after laparoscopic insemination of synchronised ewes under defined conditions [1, 2]. To further assess the fertility of sex-sorted ram sperm, embryo production and development rates were examined after the insemination of superovulated ewes with either sex-sorted or non-sorted sperm. Merino ewes (n = 30) were synchronised in oestrus using progestagen sponges inserted for 14 days and hormonally stimulated with PMSG (600 IU) on sponge removal (SR), FSH (133 mg) at decreasing doses every 12 h for 4 days before insemination, and GnRH 24 h before insemination. Each ewe was inseminated in the uterus by laparoscopy 42–44 h after SR with 15 × 106 motile X- or Y-chromosome bearing or non-sorted spermatozoa. On day 6 after insemination, antegrade flushing of both uterine horns was performed. Overall, the fertilisation rate was higher using X-chromosome bearing sperm (70%) compared with both Y-chromosome enriched (59%) and non-sorted treatments (64%). Embryo development was more advanced after the insemination of non-sorted sperm with more hatching (hg) and expanded blastocysts (exb) recovered per animal (hg: 3.37 ± 1.19; exb: 5.00 ± 1.68) compared with sorted groups (X hg: 1.71 ± 0.92; exb: 3.28 ± 1.80; Y hg: 1.67 ± 1.67; exb: 1.50 ± 1.02). Moreover, embryos were recovered at earlier developmental stages after insemination with Y- compared with X-chromosome bearing sperm. In conclusion, the use of sex-sorted sperm did not affect the efficiency of embryo production. However, the development of embryos derived from sex-sorted sperm was delayed compared with those from non-sorted sperm. (1) de Graaf et al. 2007. Reproduction in Domestic Animals 42, 648–653 (2) Beilby et al. 2008. Theriogenology [IN PRESS]

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
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.

17 SPERM FERTILITY RATE ASSESSED BY EMBRYO PRODUCTION IN VIVO AND IN VITRO IN SOUTH AFRICAN BULLS

2017 ◽  
Vol 29 (1) ◽  
pp. 116
Author(s):  
M. H. Mapeka ◽  
F. V. Ramukhithi ◽  
C. M. Pilane ◽  
D. Norris ◽  
C. Banga ◽  
...  
Keyword(s):  
South African ◽  
Sperm Motility ◽  
Developmental Stages ◽  
Fertility Rate ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Recovery Methods

The aim of this study was to determine the sperm fertility rate by embryo production in vivo and in vitro in South African bulls and further compare the embryo quality developed from different oocyte recovery methods. A total of 15 frozen semen straws (5 Bonsmara; 5 Nguni; 5 Boran) were thawed and evaluated for sperm motility characteristics using sperm class analyzer. The fertilizing ability of frozen–thawed semen was assessed by performing AI and in vitro fertilization. For AI, 6 cows were superovulated and inseminated with frozen–thawed semen followed by flushing on Day 7 post-insemination and then evaluated for embryo developmental stages. For IVF, oocytes were retrieved using two recovery methods namely ovum pick-up (OPU) and ovary aspiration. A total of 383 (106, OPU; 277, ovary aspiration) oocytes were matured in M199 + 10% fetal bovine serum (FBS) maturation medium at 38.5°C for 24h. Oocytes were washed in Bracket and Oliphant’s fertilization medium, co-incubated with frozen–thawed (Boran) semen at 38.5°C for 6 h, and then cultured in SOF-BSA medium, incubated at 38.5°C, 5% CO2 for 7 days, and further evaluated for embryo development. Data were analysed by ANOVA. Total sperm motility was >70% in all breeds. Boran had a significantly (P < 0.05) higher total post-thaw sperm motility (93.2 ± 3.6) compared with Nguni (75.1 ± 4.2) and Bonsmara (80.7 ± 6.9). Furthermore, Boran had higher (P < 0.05) progressive motility (39.7 ± 3.4) and rapid motility (36.1 ± 5.9) compared with other breeds. Interestingly, Boran produced significantly (P < 0.05) higher blastocyst rate (56.34%) compared with Bonsmara (38.03%) Nguni (31.08%). Superovulation and OPU resulted in a significantly higher (P < 0.05) number of blastocysts (10.5 ± 3.3 and 10.5 ± 3.3) respectively, compared with aspiration (1.3 ± 3.3). Moreover, the OPU method yielded a significantly higher (P < 0.05) number of grade 2 blastocyst (3.0 ± 0.1) compared with aspiration (0.50 ± 0.1). However, there was no significant (P > 0.05) difference in the number of grade 1 and grade 3 blastocysts obtained when the 3 recovery methods were used. In conclusion, the Boran breed showed better a sperm fertility rate following in vivo and in vitro embryo production. The superovulation and OPU methods resulted in higher numbers and better quality blastocysts compared with aspiration.

scholarly journals In Vivo Analysis of Embryo Development and Behavioral Response of Medaka Fish under Static Magnetic Field Exposures

2019 ◽  
Vol 16 (5) ◽  
pp. 844 ◽  
Author(s):  
Weinong Sun ◽  
Yaqing He ◽  
Sai-Wing Leung ◽  
Yuen-Chong Kong
Keyword(s):  
Magnetic Field ◽  
Embryo Development ◽  
Static Magnetic Field ◽  
Behavioral Response ◽  
Prolonged Exposure ◽  
Developmental Stages ◽  
Control Group ◽  
Adult Fish ◽  
Medaka Fish

The static magnetic field (SMF) in human exposure has become a health risk concern, especially with respect to prolonged exposure. The International Commission on Non-Ionizing Radiation Protection (ICNIRP) has been considering cell or animal models to be adopted to estimate the possible human health impacts after such exposure. The medaka fish is a good animal model for human-related health assessment studies; this paper examines both the embryo development and behavioral responses in medaka fish in vivo to long-term SMF exposure at the mT level. SMF exposure was examined for the complete developmental period of embryos until hatched; the embryos were monitored and recorded every 24 h for different morphological abnormalities in their developmental stages. The behavioral response of adult fish was also examined by analyzing their swimming velocities and positioning as compared with that of the control group. It was observed that there were no impacts on embryo development under prolonged exposure up to about 100 mT while the swimming behavior of the adult fish under exposure was different to the control group—the swimming movement of the treated group was more static, with an average velocity of 24.6% less as observed over a 24-h duration.

204 RELATIVE MESSENGER RNA ABUNDANCE OF HYALURONAN RECEPTORS AND SYNTHASES ON IN VITRO- AND IN VIVO-DERIVED DAY 7 AND DAY 13 BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
M. Clemente ◽  
A. T. Palasz ◽  
J. de La Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Messenger Rna ◽  
Developmental Stages ◽  
Transcript Abundance ◽  
Early Embryo ◽  
Bovine Embryos ◽  
Cd44 Receptor ◽  
Mrna Extraction

Hyaluronan (HA), which progressively increases during embryogenesis, is a glycosaminoglycan that plays a major role in oocyte/embryo development (Fenderson et al. 1993 Differentiation 54, 85–95). One of the main functions of HA is to participate in the cell proliferation and migration that are controlled by HA receptors, RHAMM and C44, and by the presence of different HA synthases, Has1, Has2, and Has3. All have very distinctive features and functions at different stages of embryo development. The objective of this study was to determine the relative mRNA abundance of HA receptors and synthases in Day 7 and 13 bovine embryos derived in vitro or in vivo. In vitro embryos were produced by standard oocyte maturation and fertilization procedures. Presumptive zygotes were cultured in groups of 25 in 25-μL droplets of synthetic oviduct fluid supplemented with 5% FCS at 39°C, 5% CO2, and 5%O2 with maximum humidity. In vivo blastocysts were collected from superovulated heifers on Day 7 (estrus = Day 0) by uterine flushing and on Day 13 immediately after slaughter by flushing the dissected reproductive tracts. All embryos were frozen in LN2 and stored at –80°C for mRNA extraction. Quantification of transcripts for RHAMM and CD44 receptors and Has2 and Has3 synthases was performed on groups of ten Day 7 blastocysts (3 groups for in vivo or in vitro) and individual Day 13 embryos (7 embryos in vivo or in vitro) by real-time quantitative RT-PCR. Data on differences in transcript abundance were analyzed by ANOVA. The relative abundance of the Has2 and Has3 synthases was similar between in vivo and in vitro embryos, irrespective of their developmental stage. The quantity of CD44 was significantly higher in in vitro compared with in vivo embryos only on Day 7. However, the quantity of RHAMM receptor was higher on Day 13 in in vitro compared with in vivo embryos. When the comparison was done between developmental stages (Day 7 v. Day 13) for in vivo and in vitro embryos, we found that in vivo-produced Day 7 blastocysts expressed significantly more RHAMM receptor than embryos on Day 13. The reverse pattern of expression was shown for CD44 receptor. For in vitro embryos, the only difference observed was for Has3, which was up-regulated on Day 13 compared with Day 7 embryos. In conclusion, the present study demonstrates, for the first time, developmental changes in the abundance of RHAMM and CD44 receptor mRNA and Has2 and Has3 synthase mRNA in in vivo and in vitro bovine-derived embryos on Day 7 and 13. We believe that our results will provide new insight into the potential role of this intriguing multifunctional molecule in bovine early embryo development.

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

scholarly journals Dynamics of the transcriptome during chicken embryo development based on primordial germ cells

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Aleksandra Dunislawska ◽  
Agata Szczerba ◽  
Maria Siwek ◽  
Marek Bednarczyk
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Germ Cells ◽  
Developmental Stages ◽  
Single Cells ◽  
Primordial Germ Cells ◽  
Regulation Of Gene Expression ◽  
Cell Number ◽  
Early Developmental

Abstract Objective Regulation of gene expression during embryo development on the basis of migration of primordial germ cells (PGCs) in vivo has been rarely studied due to limited cell number and the necessity to isolate PGCs from a large number of embryos. Moreover, little is known about the comprehensive dynamics of the transcriptome in chicken PGCs during early developmental stages. The current study investigated transcriptome dynamics of chicken PGCs at key developmental stages: 4.5, 8 and 12 days of embryo incubation. PGCs were collected, and RNA was isolated using a commercial kit for single cells. The isolated RNA was subjected to microarray analysis (Agilent Technologies). Results Between 8 and 12 days of incubation, the highest number of genes was regulated. These data indicate that the most intense biological activity occurs between 8 and 12 days of embryo development. Heat map showed a significant decrease in gene expression on day 8, while it increased on day 12. The development of a precise method to isolate bird PGCs as well as the method to isolate RNA from single cells isolated from one embryo allows for early molecular analysis and detection of transcriptome changes during embryonic development.

Piezo-actuated zona-drilling improves the fertilisation of ops vitrified mouse oocytes

2007 ◽  
Vol 55 (3) ◽  
pp. 369-378 ◽  
Author(s):  
Qinggang Meng ◽  
Xiuwei Li ◽  
Tongyi Wu ◽  
A. Dinnyés ◽  
Shien Zhu
Keyword(s):  
Cell Number ◽  
Blastocyst Formation ◽  
Rate Of Development ◽  
Mouse Oocytes ◽  
Fertilisation Rate ◽  
Developmental Capacity ◽  
Zona Drilling ◽  
In Pregnancy

The present study was designed to investigate fertilisation of open pulled straw (OPS) vitrified mouse oocytes drilled with piezo-micromanipulation method and their subsequent in vitro and in vivo developmental capacity. Ovulated mouse oocytes were vitrified using the OPS method. After warming, the zona pellucida of a group of vitrified-warmed oocytes was drilled by piezo-micro-manipulation. Groups of (a) vitrified, (b) vitrified/drilled and (c) fresh control oocytes were fertilised in vitro . The fertilisation rate of vitrified-warmed oocytes was significantly lower than that of fresh oocytes (45.0 ± 12.6% vs. 85.2 ± 6.8%, P < 0.05), and was significantly improved by zona-drilling (85.4 ± 7.3%). However, blastocyst formation rates of the vitrified and vitrified/drilled groups were significantly lower than those of the fresh controls (65.7 ± 7.0% and 66.4 ± 2.5% vs. 86.6 ± 4.3%, respectively, P < 0.05). The cell number of blastocysts from the vitrified/drilled or the vitrified group was not different from that of the controls. Embryo transfer resulted in pregnancy in all three groups, but the rate of development to term was lower in the vitrified/drilled or vitrified groups than in the controls (16.6 ± 0.7% or 36.0 ± 2.4% vs. 51.3 ± 2.9%, respectively). Our results demonstrated that zona-drilling with piezo-micromanipulation could improve fertilisation in OPS vitrified mouse oocytes but did not increase the overall number of vitrified oocytes developing to term.

Towards the use of microfluidics for individual embryo culture

2010 ◽  
Vol 22 (1) ◽  
pp. 32 ◽  
Author(s):  
R. L. Krisher ◽  
M. B. Wheeler
Keyword(s):  
Embryo Development ◽  
Embryo Culture ◽  
Physical Culture ◽  
Embryo Production ◽  
Mammalian Embryo ◽  
Future Directions ◽  
Initial Work ◽  
In Vitro Embryo Production

Mammalian embryo development is still relatively inefficient in vitro. Much research has been conducted on the chemical environment, or culture medium, surrounding the embryo, but little attention has been given to the actual physical culture environment, which has changed very little over the years. The application of microfluidics to embryo production in vitro is a tantalising approach that may alleviate some of the limits that traditional microdrop culture places on embryo development and research into gamete and embryo physiology. These devices may lead to enhanced in vitro embryo development and quality by more closely mimicking the in vivo environment. Initial work in this area is promising and gives us proof-of-principle that these unique microfluidic systems may indeed be applicable to in vitro culture of gametes and embryos. The present paper reviews the advantages of microfluidics for in vitro embryo production: how the platforms are manufactured, the current uses of microfluidics in assisted reproduction, static v. dynamic culture environments, individual gamete and embryo culture and the future directions of microfluidic application to in vitro embryo production and manipulation. Finally, preliminary data from our laboratory using a new microfluidic well insert for porcine, bovine and murine embryo culture is discussed.

Evolution of a Y Chromosome from an X Chromosome

2019 ◽  
Author(s):  
Roberta Bergero ◽  
Jim Gardner ◽  
Deborah Charlesworth
Keyword(s):  
Y Chromosome ◽  
X Chromosome

scholarly journals Genome-Wide Transcriptomic Identification and Functional Insight of Lily WRKY Genes Responding to Botrytis Fungal Disease

Plants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 776
Author(s):  
Shipra Kumari ◽  
Bashistha Kumar Kanth ◽  
Ju young Ahn ◽  
Jong Hwa Kim ◽  
Geung-Joo Lee
Keyword(s):  
Abiotic Stress ◽  
Biotic Stress ◽  
Developmental Stages ◽  
Expression Patterns ◽  
Lilium Longiflorum ◽  
Biotic And Abiotic Stress ◽  
Biotic Stresses ◽  
Genome Wide

Genome-wide transcriptome analysis using RNA-Seq of Lilium longiflorum revealed valuable genes responding to biotic stresses. WRKY transcription factors are regulatory proteins playing essential roles in defense processes under environmental stresses, causing considerable losses in flower quality and production. Thirty-eight WRKY genes were identified from the transcriptomic profile from lily genotypes, exhibiting leaf blight caused by Botrytis elliptica. Lily WRKYs have a highly conserved motif, WRKYGQK, with a common variant, WRKYGKK. Phylogeny of LlWRKYs with homologous genes from other representative plant species classified them into three groups- I, II, and III consisting of seven, 22, and nine genes, respectively. Base on functional annotation, 22 LlWRKY genes were associated with biotic stress, nine with abiotic stress, and seven with others. Sixteen unique LlWRKY were studied to investigate responses to stress conditions using gene expression under biotic and abiotic stress treatments. Five genes—LlWRKY3, LlWRKY4, LlWRKY5, LlWRKY10, and LlWRKY12—were substantially upregulated, proving to be biotic stress-responsive genes in vivo and in vitro conditions. Moreover, the expression patterns of LlWRKY genes varied in response to drought, heat, cold, and different developmental stages or tissues. Overall, our study provides structural and molecular insights into LlWRKY genes for use in the genetic engineering in Lilium against Botrytis disease.

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