scholarly journals Effect of Ram Breed on the Efficiency of in vitroDevelopment of Sheep Embryos

2017 ◽  
Vol 14 (4) ◽  
pp. 1309-1313
Author(s):  
Y. Al-Anazi ◽  
M. G. Al-Mutary ◽  
M. M. Alfuraiji ◽  
M. Al-Ghadi ◽  
A. R. Al-himaidi ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Cell Stage ◽  
In Vitro Development ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Stage 4 ◽  
Before And After ◽  
Vitro Development ◽  
Fresh Semen

The aim of this work was to investigate the impacts of ram breed on in vitro embryo development from fresh or frozen semen. Semen was collected from Najdi and Naimi rams and frozen; the mass and progressive motility of the spermwere assessed in each trial before and after freezing. Then, 970 oocytes in six replicates were fertilized with fresh and frozen semen in vitro. Different stages of sheep embryos were recorded. There were no significant differences in mass and progressive sperm motility of fresh or frozen ram semen between Najdi and Naimi,but there were significant differences between frozen and fresh semen within each breed. Our results showed significant (P<0.05) differences in 2-cell stage, 4-cell stage, 8-cell stage, morula, fragmented embryos, cleavage and blastocyst rates in the frozen semen group compared to fresh semen group in both breeds. In addition, significant (P<0.05) differencesbetween the two breeds were shown in 8-cell and16-cell embryonic stages.In conclusion, there were slight breed effects on the efficiency of in vitro development of sheep embryos.

198 THE EFFECT OF SOURCE AND IN VITRO MATURATION ON THE ABUNDANCE OF MATERNAL mRNA OF SELECTED GENES IN FOLLICULAR BOVINE OOCYTES AND THEIR INFLUENCE ON IN VITRO DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 199
Author(s):  
T. Somfai ◽  
K. Imai ◽  
M. Kaneda ◽  
S. Akagi ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Before And After ◽  
Vitro Development ◽  
Bovine Oocytes

The aim of the present study was to investigate the effect of oocyte source and in vitro maturation (IVM) on the expression of selected genes in bovine oocytes and their contribution to in vitro embryo development. Follicular oocytes were collected either by ovum pick-up from live cows or by the aspiration of ovaries of slaughtered cows following storage in Dulbecco’s PBS at 15°C for overnight. In vitro maturation was performed according to the method of (Imai et al. 2006 J. Reprod. Dev. 52, 19–29 suppl.). Gene expression was assessed before and after IVM by real-time PCR. The following genes were investigated: GAPDH, G6PDH, ACTB, H2A, CCNB1, MnSOD, OCT4, SOX2, CX43, HSP70, GLUT8, PAP, GDF9, COX1, ATP1A1, CDH1, CTNNB1, AQP3, DYNLL1, DYNC 1/1, and PMSB1. In brief, mRNA was extracted from 20 oocytes per sample using a Qiagen RNeasy Micro Kit (Qiagen, Valencia, CA). Gene expression was analysed by a Roche Light Cycler 480 device and software (Roche, Indianapolis, IN). Relative expression of each gene was normalized to CCNB1, which in preliminary experiments appeared the most stably expressed irrespective of oocyte source and meiotic stage. Three replications were performed. Data were analysed by paired t-test. In immature ovum pick-up oocytes, genes related to metabolism (GAPDH, G6PDH, GLUT8) and stress (MnSOD, HSP70), and also OCT4, ATP1A1, and DYNC1/1 showed significantly (P < 0.05) higher expression compared with immature oocytes collected from slaughtered-stored ovaries. The expression of GDF9, GLUT8, CTNNB1, and PMSB1 was significantly (P < 0.05) reduced during IVM irrespective of the oocyte source. In a second experiment, IVF IVM oocytes showing an early (at 22 to 25 h after IVF) or late (at 27 to 30 h after IVF) first cleavage were either cultured in vitro or analysed for gene expression at the 2-cell stage. A higher (P < 0.05) rate of early-cleaving oocytes developed to the blastocyst stage compared with the rate of late-cleaving ones (46.2% v. 15.6%, respectively). Nevertheless, only ATP1A1 showed significantly reduced (P < 0.05) expression in late-cleaving embryos compared with early-cleaving ones. Our results suggest that although removal and storage of ovaries and IVM caused a reduction in the relative abundance of several genes in oocytes, in most cases, this did not affect embryo development. Among the genes studied, only ATP1A1 was correlated with in vitro development.

297 PRODUCTION OF CHIMERIC PORCINE FETUSES BY AGGREGATION METHOD USING PARTHENOGENETIC EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 296
Author(s):  
K. Nakano ◽  
M. Watanabe ◽  
H. Matsunari ◽  
T. Matsuda ◽  
K. Honda ◽  
...  
Keyword(s):  
Cell Mass ◽  
Ips Cells ◽  
Large Animal ◽  
Aggregation Method ◽  
Cell Stage ◽  
In Vitro Development ◽  
Donor Cells ◽  
Vitro Development ◽  
Parthenogenetic Embryos

Porcine induced pluripotent stem (iPS) cells are considered to be an invaluable research tool in translational research with pigs as a large animal model. Pluripotency of the iPS cells needs to be verified by their competence to contribute to chimera formation. The aim of the present study is to establish feasible system to create chimeric pig fetuses using parthenogenetic embryos. In Experiment 1, inner cell mass (ICM) was isolated by immunosurgery from Day 6 blastocysts obtained by parthenogenetic activation of in vitro matured (IVM) oocytes. Isolated ICM were used as the donor cells after staining with fluorescent carbocyanine dye (DiI). Using parthenogenetic morulae or 4- to 8-cell embryos as the host embryos, chimeric embryos were prepared by injection or aggregation method. Injection of ICM was performed by micromanipulation: a single ICM was directly injected into the centre portion of the host morulae. In the aggregation method, a single ICM was aggregated with blastomeres isolated from 2 host embryos at the morula or 4- to 8-cell stage in a micro-well (400 µm diameter, 300 µm deep). The chimeric embryos were cultured in PZM-5 (Yoshioka et al. 2008) for 2 to 3 days to examine development to blastocysts and incorporation of donor ICM cells into the resultant blastocysts ICM (ICM chimerism). In Experiment 2, donor blastomeres isolated from a parthenogenetic morula or 4- to 8-cell embryo were stained by DiI and aggregated with a parthenogenetic host embryo at the morula or 4- to 8-cell stage, and the in vitro development to the blastocyst stage and the ICM chimerism were examined. In Experiment 3, ICM isolated from IVF blastocysts harboring humanized Kusabira-Orange (huKO) gene were used as donor cells. Donor ICM were aggregated with the host embryos at the morula or 4- to 8-cell stage, and the resultant blastocysts were transferred to 4 recipient gilts to collect fetuses on Day 18. Results of Experiments 1 and 2 are summarised in Table 1. Combination of the donor ICM and host morulae yielded high rates of blastocyst formation (~95%) and ICM chimerism (~85%), regardless of the method used (injection or aggregation). Transfer of 73 blastocysts developed from host morulae to 2 recipients (Experiment 3) gave rise to 25 (34.2%) fetuses, of which 6 (24.0%) were confirmed to be chimeric by their clear orange fluorescence and immunostaining by anti-huKO antibody. Of 22 (40.7%) fetuses obtained after transfer of 54 blastocysts derived from 4- to 8-cell host embryos to 2 recipients, 3 (13.6%) were chimeric. Contribution of the donor cells in the tissues of the chimeric fetuses measured by image analysis software (ImageJ, NIH, Bethesda, MD, USA) ranged between 16.1 and 65.2%. These results demonstrate that the aggregation method using parthenogenetic host embryos is an efficient means to produce chimeric pig fetuses, and thereby feasible for verification of pluripotent cells such as iPS cells. Table 1.In vitro development of injected or aggregated porcine embryos

scholarly journals 25 IN VITRO DEVELOPMENT OF AGGREGATED NUCLEAR TRANSFERRED EMBRYOS DERIVED FROM BOVINE CUMULUS CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 162
Author(s):  
S. Akagi ◽  
B. Tsuneishi ◽  
S. Watanabe ◽  
S. Takahashi
Keyword(s):  
Zona Pellucida ◽  
Cumulus Cells ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Cell Stage ◽  
In Vitro Development ◽  
Functional Peptide ◽  
Vitro Development

It has been reported that aggregation of two nuclear transfer (NT) mouse embryos shows an improvement in full-term development (Boiani M et al. 2003 EMBO J. 22, 5304–5312). In this study, we examined the effect of aggregation on in vitro development of bovine NT embryos. As donor cells for NT, cumulus cells of passage 3–5 were used following culture in serum-starved medium for 5–7 days. NT was performed as previously described (Akagi S et al. 2003 Mol. Reprod. Dev. 66, 264–272). NT embryos were cultured in a serum-free medium (IVD-101, Research Institute of Functional Peptide Co., Ltd., Shimojo, Yamagat, Japan). Eight-cell-stage embryos on Day 2 or 16- to 32-cell-stage embryos on day 4 were used for embryo aggregation after removal of the zona pellucida. A small depression was made in a 25-μL drop of TCM-199 with 50 μg/mL phytohemagglutinin (TCM199/PHA) or IVD-101 using a darning needle. Two or three NT embryos were placed into the depression in the drop of TCM199/PHA for 20 min. NT aggregates were then moved into the depression in the drop of IVD-101 and cultured until Day 7. In vitro development of NT aggregates was summarized in Table 1. There were no differences in the cell number and ICM ratio of blastocysts between non-aggregated zona-intact and zona-free embryos. All aggregates of three NT embryos developed to the blastocyst stage and the cell number of these blastocysts was significantly higher than that of non-aggregated NT blastocysts. These results indicate that removal of the zona pellucida does not affect the cell number and ICM ratio of blastocysts and that aggregates of three NT embryos can develop to blastocysts with high cell numbers which are equivalent to in vivo-derived embryos (166 ± 11, Knijn HM et al. 2003 Biol. Reprod. 69, 1371–1378). Table 1. Development, cell number, and ICM ratio of NT aggregates

scholarly journals 331INSEMINATION OF OOCYTES WITH AGED SEMEN ALTERS SEX RATIO OF BOVINE EMBRYOS PRODUCED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 285
Author(s):  
J.L. Edwards ◽  
P. Bredbacka ◽  
A.M. Saxton ◽  
F.N. Schrick
Keyword(s):  
Sex Ratio ◽  
Sperm Motility ◽  
Block Design ◽  
Water Bath ◽  
Bovine Embryos ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Frozen Semen ◽  
Effects Of Aging

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14h post-thaw in a 34.4°C water bath, or after aging for 23h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14h, respectively (SEM=10). Cleavage of inseminated oocytes (67hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14h post-thaw, respectively). Insemination of oocytes with semen aged for 14h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14h v. 8h or control, respectively;; P=0.08; SEM=9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P&lt;0.05) by Fisher’s Exact test. Bull or replicate variation was noted but the direction of aging for increasing proportion of females was consistent. Preliminary observations suggest that biological differences between X- and Y-bearing sperm may exist such that alternative strategies for altering sex ratio in livestock species may be possible.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

Gene expression profiles and in vitro development following vitrification of pronuclear and 8-cell stage mouse embryos

2006 ◽  
Vol 73 (6) ◽  
pp. 700-708 ◽  
Author(s):  
Duangjai Boonkusol ◽  
Arpad Baji Gal ◽  
Szilard Bodo ◽  
Botond Gorhony ◽  
Yindee Kitiyanant ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Mouse Embryos ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

scholarly journals Human Sperm Motility, Viability, and Morphology Decreased after Cryopreservation

2019 ◽  
Vol 55 (3) ◽  
pp. 198
Author(s):  
Ninik Darsini ◽  
Berliana Hamidah ◽  
Seso Sulijaya Suyono ◽  
Faisal Yusuf Ashari ◽  
R Haryanto Aswin ◽  
...  
Keyword(s):  
Experimental Study ◽  
Informed Consent ◽  
Sperm Motility ◽  
Human Sperm ◽  
Sperm Viability ◽  
Group Design ◽  
Progressive Motility ◽  
Before And After ◽  
Immotile Spermatozoa ◽  
Fresh Semen

The aim of this study was to analyze human sperm motility, viability, and morphology before and after cryopreservation. This true laboratory experimental study had pre and post randomized one group design. The study was conducted at the Embryology, Andrology, and Genetics Laboratory, Department of Medical Biology, Faculty of Medicine, Universitas Airlangga from August to November 2017. The eighteen samples of fresh semen were collected from male volunteers who agreed and signed the informed consent of the study. Samples were analyzed their motility, viability, and morphology before and after cryopreservation. Results of this study indicated differentiation between motility before and after cryopreservation. Cryopreservation process decreased progressive motility (42.22 + 9.46%; 17.83 + 6.24%; p< 0.0001) and increased the number of immotile spermatozoa (35.44 + 10.15%; 60.11 + 12.53%; p< 0.0001). Cryopreservation also decreased human sperm viability (73.78 + 8.91%; 40.83 + 12.89%; p< 0.0001) and morphology (10.94 + 4.96%; 7.39 + 3.90%; p< 0.0001). Cryopreservation of human spermatozoa caused the decreased of motility, viability, and morphology.

scholarly journals 142 IN VITRO DEVELOPMENT OF BOVINE EMBRYOS CULTURED IN KSOM, CR1AA, OR KSOM/CR1aa

2005 ◽  
Vol 17 (2) ◽  
pp. 221 ◽  
Author(s):  
M.R.B. Mello ◽  
C.E. Ferguson ◽  
A.S. Lima ◽  
M.B. Wheeler
Keyword(s):  
Embryo Culture ◽  
Cumulus Cells ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Cell Stage ◽  
In Vitro Development ◽  
Significant Difference ◽  
Developmental Rates ◽  
Vitro Development

In vitro embryo culture is an important step of in vitro production of bovine embryos. It has been shown that IVF-derived bovine embryos cultured in KSOM or CR1aa have high development rates. In our laboratory, we have observed that 8-cell embryos are morphologically superior when embryos are cultured in KSOM whereas blastocysts are morphologically superior when embryos are cultured in CR1aa. Based on these observations, we hypothesized that development of IVF-derived bovine embryos can be improved by sequential use of these media (KSOM and CR1aa). The aim of this experiment was to compare the in vitro development of bovine embryos cultured in KSOM, CR1aa or KSOM/CR1aa supplemented with BSA at Day 0 and BSA and FBS at Day 3. In order to accomplish the sequential culture, fertilized oocytes where cultured in KSOM to the 8-cell stage and then transferred to CR1aa for further development. Oocytes were purchased from Bomed (Madison, WI, USA), and after 22 hours of maturation were fertilized with frozen-thawed semen for 5 hours at 39°C in 5% CO2. After fertilization, the presumptive zygotes were denuded from cumulus cells by votexing and were randomly allotted to one of 3 treatments: (1) cultured only in KSOM (n = 110), (2) cultured only in CR1aa (n = 102), and (3) cultured in KSOM in the first 3 days and then in CR1aa from Day 3 to Day 9 (n = 110). The embryo culture was carried out in 50-μL droplets of medium that were placed in an airtight modular incubator filled with 5% CO2, 5% O2 and 90% N2. The embryos were evaluated on Days 6 to 9 post insemination. All embryo developmental rates were calculated from presumptive zygotes. The Day 6 morula rates were 52%, 40%, and 47% for KSOM, CR1aa, and KSOM/CR1aa, respectively. The Day 7 blastocyst rates for KSOM (40%), CR1aa (25%), and KSOM/CR1aa (30%) were not significantly different; however, Day 9 hatched blastocyst rates were significantly higher (P < 0.05) for KSOM (22%) compared to CR1aa (9%) but not different from KSOM/CR1aa (14%). Regarding embryo quality, Day 7 transferable embryos rates (Grade 1 and Grade 2) were 35%, 25%, and 30%, respectively for KSOM, CR1aa, and KSOM/CR1aa; however, no significant difference was observed. These results indicate that IVF-derived bovine embryos can develop in KSOM, CR1aa, or KSOM/CR1aa with no significant difference among morula, blastocyst and hatched blastocyst rates. However, the combination of KSOM and CR1aa during in vitro culture did not decrease the morula and blastocyst rates.

Gene expression profile differences in embryos derived from prepubertal and adult Japanese Black cattle during in vitro development

2012 ◽  
Vol 24 (2) ◽  
pp. 370 ◽  
Author(s):  
Dorji ◽  
Yukihiro Ohkubo ◽  
Kazuchika Miyoshi ◽  
Mitsutoshi Yoshida
Keyword(s):  
Gene Expression ◽  
Developmental Stages ◽  
Expression Profiles ◽  
Bovine Genome ◽  
Gene Expression Profiles ◽  
Japanese Black Cattle ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The present study was carried out to compare the gene expression profiles of in vitro-generated embryos derived from adult and prepubertal Japanese Black cattle oocytes using GeneChip Bovine Genome Array (containing 24 072 probe sets representing over 23 000 transcripts). Microarray experiments were performed on populations of 8- to 16-cell stage embryos and blastocysts derived from adult (24–35 months old) versus prepubertal (9–10 months old) Japanese Black cattle oocytes matured and fertilised in vitro. In total, 591 (2.4%) and 490 (2.0%) genes were differentially expressed in prepubertal and adult bovine in 8- to 16-cell and blastocyst stage embryos, respectively. Out of these, 218 and 248 genes were upregulated, while 373 and 242 were downregulated in prepubertal and adult 8- to 16-cell and blastocysts stage embryos, respectively. Gene ontology classification regarding biological process, molecular functions and cellular component revealed diversity in transcript abundances between prepubertal and adult groups in both the distinct developmental stages. Quantitative reverse transcription–PCR validated the expression differences of some selected transcripts as identified by microarray analysis. To our knowledge, this is the first report indicating the significant number of genes differentially expression (>2-fold, P < 0.01) in preimplantition embryos between adult and prepubertal Japanese Black cattle during in vitro development.

213 ASSESSMENT OF THE SUSCEPTIBILITY OF PORCINE PRE-IMPLANTATION EMBRYOS TO PSEUDORABIES VIRUS (PRV) AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV) USING SUBZONAL MICROINJECTION

2006 ◽  
Vol 18 (2) ◽  
pp. 214 ◽  
Author(s):  
B. Mateusen ◽  
A. Van Soom ◽  
D. Maes ◽  
H. Nauwynck
Keyword(s):  
Embryo Development ◽  
Pseudorabies Virus ◽  
Respiratory Syndrome Virus ◽  
Direct Immunofluorescence ◽  
Embryonic Cells ◽  
Lelystad Virus ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

It is known that porcine pre-implantation embryos before the morula stage are refractory to infection with pseudorabies virus (PRV) and porcine reproductive and respiratory syndrome virus (PRRSV) (Bolin et al. 1981 Am. J. Vet. Res. 42, 1711-1712; Prieto et al. 1996 Theriogenology 46, 687-693, respectively). The effects of PRV and PRRSV on embryonic cells of morulae and blastocysts are unknown. Therefore, the objectives of the present study were to (1) assess the effects of PRV and PRRSV exposure on further embryo development, and (2) determine whether PRV and PRRSV are able to replicate in embryonic cells. Zona pellucida (ZP)-intact morulae (6 days post-insemination, 6 dpi) and early blastocysts (7 dpi) were microinjected subzonally with approximately 3 nL of 109 TCID50/mL PRV (strain 89v87, second passage in swine testicle cells) or 108.6 TCID50/mL PRRSV (Lelystad virus strain, 13th passage in swine alveolar macrophages). Control embryos were microinjected under the same circumstances with phosphate-buffered saline (PBS). Hatched blastocysts (8 dpi) were exposed for 1 h at 39�C to 105 TCID50/mL of PRV or PRRSV of the same strains used for injecting earlier embryonic stages. Control hatched blastocysts were incubated with PBS. Each group of morulae and blastocysts consisted of approximately 20 embryos. Embryonic development was assessed every 12 h. At 48 h post injection, the percentage of infected embryos and the percentage of viral antigen positive cells per embryo were determined by immunofluorescence. Subzonal microinjection of ZP-intact morulae and blastocysts with PRV inhibited in vitro development in comparison to the controls. Moreover, under direct immunofluorescence, PRV antigen-positive cells were detected in association with the embryos. Exposure of hatched blastocysts to PRV inhibited further embryo development; the majority (16/20) of the embryos degenerated 24 h after incubation. Perivitelline microinjection of ZP-intact morulae and blastocysts with PRRSV and incubation of hatched blastocysts with PRRSV did not inhibit in vitro development in comparison to the controls. No PRRSV antigen positive cells were detected in association with the embryos. Based on these results, it can be deduced that embryonic cells of morulae and blastocysts are susceptible to PRV infection but refractory to PRRSV infection. Another argument substantiating insusceptibility of embryos to certain viral pathogens is the demonstration of the lack of virus receptors at a given embryonic cell stage. Therefore, the expression of sialoadhesin, the receptor that mediates the internalization of PRRSV in cells, was investigated in hatched blastocysts (n = 10). By indirect immunofluorescence using monoclonal antibody 41D3 directed against porcine sialoadhesin, no positive signals were detected. The result of this experiment strengthens the statement that embryonic stages up to the hatched blastocyst stage are refractory to PRRSV infection.

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