scholarly journals Impairment on nuclear maturation rate in oocytes from cows naturally infected by bovine herpesvirus 1 (BoHV-1)

2018 ◽  
Vol 38 (12) ◽  
pp. 2207-2212
Author(s):  
Vívian R.A. Mendes ◽  
Eduardo P. Costa ◽  
Vanessa L.D. Queiroz ◽  
Abelardo Silva Júnior ◽  
Saullo V.P. Alves ◽  
...  
Keyword(s):  
Bovine Herpesvirus ◽  
Cumulus Cells ◽  
Control Group ◽  
Viral Dna ◽  
Bovine Herpesvirus 1 ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Developmental Capacity ◽  
Herpesvirus 1

ABSTRACT Bovine herpesvirus 1 (BoHV-1) is an important bovine pathogen that is responsible for causing respiratory diseases and reproductive failures. The presence of BoHV-1 in an in vitro embryo production system affects fertilization, maturation, and embryonic development. The objective of this study was to evaluate the developmental capacity of oocytes from naturally infected cows with no reproductive history. Moreover, this study investigated the presence of viral DNA in cumulus oophorus complexes (COCs). Experimental groups were differentiated by titrating the antibodies detected through seroneutralization assays, establishing three groups: seronegative animals (titer lower than 2), low titer (2 to 8), and animals with a titer above or equal to 16. COCs were obtained from 15 donors during 22 sessions of ultrasound-guided follicular aspiration. DNA was extracted from a pool of COCs obtained from all aspirations from the same donor as well as from whole blood and nested PCR reactions were performed. Only COCs with a compact layer of cumulus cells, an intact zona pellucida, and homogeneous cytoplasm were selected for in vitro culture and evaluation of nuclear maturation rate. After culturing for 24 hours, the oocytes were fixed and stained to evaluate the meiotic cell cycle stage. Oocytes that showed a chromosomal configuration in metaphase II were considered to have reached nuclear maturation. Compared with the other groups, the oocyte nuclear maturation rate in animals with a titer greater than or equal to 16 (50%) was compromised (P< 0.05). However, the viral titer did not influence the maturation rate of bovine oocytes in animals exhibiting low titration (62.2%) when compared with the control group (76.7%). Viral DNA was not observed in the blood samples but was detected in the COC pool from three seropositive donors. In view of the results obtained, we conclude that natural infections by the BoHV-1 virus can compromise the nuclear maturation rate in cows, depending on the titration levels of antibodies against the virus. Moreover, viral DNA could be present in COCs, contradicting the hypothesis that seropositive animals with no history of clinical symptomatology pose a negligible risk of transmitting BoHV-1 by COCs.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

scholarly journals Prediction of maturational competence of feline oocytes using supravital staining of cumulus cells by propidium iodide

Zygote ◽  
2011 ◽  
Vol 20 (4) ◽  
pp. 333-337 ◽  
Author(s):  
Kenzo Uchikura ◽  
Masashi Nagano ◽  
Mitsugu Hishinuma
Keyword(s):  
Propidium Iodide ◽  
Cumulus Cell ◽  
Membrane Integrity ◽  
Cumulus Cells ◽  
Nuclear Maturation ◽  
Non Invasive ◽  
Maturation Rate ◽  
Cell Layers ◽  
The Relationship

SummaryWe examined the relationship between integrity of cumulus cells and nuclear maturation rate after in vitro culture to determine a non-invasive prediction of the maturational competence of feline oocytes. Feline cumulus–oocyte complexes (COCs) were collected from either small (400–800 μm) or large (≥800 μm) follicles. Immediately after collection, cumulus cells were evaluated morphologically (thickness of cumulus cell layers) and stained with propidium iodide (PI), which penetrates only non-viable cells. Cumulus cells without PI staining were judged as having good membrane integrity. After evaluation, COCs were cultured for 30 h and their nuclear maturation rate was determined. The nuclear maturation rate of oocytes derived from large follicles (89.8%) was higher (p < 0.05) than that from small follicles (60.8%). There was no difference in the maturation rate of oocytes from follicles with the same size regardless of cumulus morphology. In contrast, oocytes that had cumulus cells with good membrane integrity showed a higher maturation rate (93.8%) than oocytes with poor cumulus integrity (76.9%) in large follicles (p < 0.05). We conclude that evaluation of membrane integrity of cumulus cells by propidium iodide staining can be used to predict the maturational competence of oocytes.

211 LACTOFERRIN INHIBITS BOVINE HERPESVIRUS-1 IN CELL CULTURE AND ALLOWS NORMAL DEVELOPMENT OF IN VITRO-PRODUCED EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 213 ◽  
Author(s):  
M. Givens ◽  
M. Marley ◽  
P. Galik ◽  
K. Riddell ◽  
D. Stringfellow
Keyword(s):  
Cell Culture ◽  
Cell Count ◽  
Bovine Milk ◽  
Control Sample ◽  
Bovine Herpesvirus ◽  
Blastocyst Development ◽  
Bovine Herpesvirus 1 ◽  
Herpesvirus 1 ◽  
In Vitro Embryo Production

Lactoferrin is an iron-binding glycoprotein found in milk, saliva, tears, and other exocrine secretions. It is known to have in vitro antiviral effects against human, feline, and canine herpesviruses. In addition, lactoferrin is known to be safe in cell culture. Bovine herpesvirus-1 (BHV-1) is a likely contaminant of in vitro embryo production. Further, trypsin treatment is not completely effective in removing the virus from these embryos. We hypothesized that a nontoxic concentration of lactoferrin might prevent replication of BHV-1 within in vitro embryo production systems. Thus, the specific objectives of this research were to determine if lactoferrin from bovine milk would inhibit BHV-1 in cell culture and to determine if in vitro-produced embryos could develop normally when cultured in lactoferrin. Two-fold dilutions of lactoferrin (from 10 to 0.625 mg/mL) were added to Madin Darby bovine kidney cells, followed in 15 min by the addition 104 PFU/mL of BHV-1 (Colorado strain). Samples of cell lysate were taken at Day 2 and virus was quantified by plaque assay. The percent of virus inhibited by the antiviral agent at each concentration was determined by comparison to equivalent samples from temporal control cultures in which no compound was added before or after inoculation (Percentage of virus inhibited = [Quantity of virus in the control sample - Quantity of virus in the compound sample]/Quantity of virus in the control sample � 100). Next, the effect of lactoferrin was determined on in vitro-produced embryos. Cumulus oocyte complexes were received from an abattoir, matured in transit, placed in fertilization drops for 6 h, and then placed in culture drops containing lactoferrin (10, 5, and 2.5 mg/mL). At Day 3.5, embryos > 4 cell stage were placed into fresh culture drops containing lactoferrin. On Day 7.5, blastocyst development was noted and the developed embryos were stained to count viable cells. Blastocyst development rate and nucleated cell count of the treated embryos were compared to those of the controls using Chi square test, and ANOVA and Tukey-Kramer HSD, respectively. Lactoferrin (10 mg/mL) inhibited 2 to 5 logs of virus. At concentrations of 5 and 2.5 mg/mL, 1 to 3 logs of virus were inhibited, and concentrations of 1.25 and 0.625 mg/mL inhibited 0 to 2 logs of virus. Lactoferrin did not affect the nucleated cell count of the treated embryos. In addition, unlike 10 and 5 mg/mL, 2.5 mg/mL of lactoferrin did not affect blastocyst development. These preliminary results indicate that lactoferrin from bovine milk can significantly inhibit BHV-1 in cell culture. Furthermore, supplementation of in vitro culture with 2.5 mg/mL of lactoferrin does not affect blastocyst development or cell count of in vitro-produced embryos.

148 RISK OF TRANSMISSION OF BOVINE HERPESVIRUS (BHV-1) BY INFECTED SEMEN TO RECIPIENTS AND EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 173 ◽  
Author(s):  
A. Bielanski ◽  
A. Lalonde ◽  
J. Algire
Keyword(s):  
Reproductive Tract ◽  
Bovine Herpesvirus ◽  
Corpora Lutea ◽  
Bovine Herpesvirus 1 ◽  
Bull Semen ◽  
Stage Embryo ◽  
Morula Stage ◽  
Herpesvirus 1 ◽  
Isolation Test

Bovine herpesvirus-1 (BHV-1) causes a variety of economically important respiratory and reproductive problems, the latter including vulvovaginitis, endometritis and infertility. For that reason, several countries have eradicated the disease and others have schemes in progress to achieve freedom. Although there is a considerable amount of information about the risk of BHV-1 transmission through contaminated semen used for artificial insemination, there is no available evidence to indicate whether the resulting embryos, when used for embryo transter (ET), can lead to the transmission of BHV-1 to recipients and offspring. For this experiment, bull semen contaminated in vitro with BHV-1 at 102 TCID50 mL–1 (Colorado strain) and then cryopreserved was used for insemination (2 times at estrus) of BHV-1 seronegative, superovulated heifers (N = 18). Embryos were collected postmortem 7 days post-insemination and were washed according to the IETS recommendations (however without trypsin treatment) or left unwashed. On 4 occasions, washed embryos were transferred to BHV-1 seronegative recipients. The remaining embryos and other samples collected from the reproductive tract were tested for BHV-1 presence using the standard virus isolation test. In total, out of 144 unfertilized oocytes and embryos collected, 9 were ET quality. Most of the embryos were degenerated (N = 79) or unfertilized (N = 56). The 4 heifers, which each received a single morula-stage embryo, maintained seronegative status, but did not become pregnant. BHV-1 was detected in 43% (23/53) unwashed and 0% (0/57) of washed embryos, 78% (14/18) of follicular fluid samples, 89% (16/18) of oviductal epithelial cells, 78% (14/18) of endometrium, and 89% (16/18) of corpora lutea tissues. Results herein suggest that BHV-1 can be transmitted by infected semen to embryo donors. The resulting unwashed embryos may remain infectious. However, whether BHV-1 uninfected offspring can be produced by ET of BHV-1 contaminated embryos that are washed according to the IETS guidelines, remains to be determined.

174 Effects of Melatonin on In Vitro Maturation of Bovine Oocytes and Gene Expression in Cumulus Cells: Preliminary Results

2018 ◽  
Vol 30 (1) ◽  
pp. 226
Author(s):  
F. C. Castro ◽  
L. Schefer ◽  
K. L. Schwarz ◽  
H. Fernandes ◽  
R. C. Botigelli ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Culture ◽  
Reverse Transcription ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Nuclear Maturation ◽  
Maturation Rate ◽  
Bovine Oocytes

Melatonin mediates several processes in animal reproduction and has drawn attention for its potent antioxidant, anti-apoptotic, anti-inflammatory action and, more recently, for its benefits on oocyte maturation and embryo development in vitro. The aim of this study was to assess the effect of melatonin during the in vitro maturation (IVM) on nuclear maturation of bovine oocytes and gene expression in their corresponding cumulus cells (CC). Bovine cumulus–oocyte complexes (COC) were obtained by aspiration of follicles (2-6 mm) from slaughterhouse ovaries, selected (grades I and II) and transferred to 4 well plates (25-30 COC/well) containing IVM medium [TCM-199 supplemented with sodium bicarbonate (26 mM), sodium pyruvate (0.25 mM), FSH (0.5 µg mL−1), LH (5.0 µg mL−1), 0.3% BSA, and gentamicin (50 µg mL−1)] with 0, 10−5, 10−7, 10−9 or 10−11 M melatonin and cultured for 24 h at 38.5°C and 5% CO2. At the end of IVM, oocytes were stained with Hoechst 33342 (10 μg mL−1) and evaluated for nuclear maturation rate. The CC were evaluated for the expression of antioxidant (SOD1, SOD2, GPX4), pro-apoptotic (P53, BAX) and expansion-related genes (PTX3, HAS1, HAS2). For transcript detection in CC, RNA isolation was performed with TRIzol®Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcription with High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). Relative quantification of transcripts was performed by RT-qPCR using 3 endogenous controls (β-actin, GAPDH, PPIA). Nuclear maturation rate and gene expression were tested by ANOVA and means were compared by Tukey’s test (6 replicates). In CC, the different concentrations of melatonin did not significantly alter expression of the investigated genes (P > 0.05), although all concentrations provided a numerical increase in the expression of the antioxidant SOD1 and of the expansion-related genes PTX3 and HAS2. Regarding the pro-apoptotic genes, concentrations of 10−11 and 10−9 M were able to reduce only numerically the expression of BAX and P53, respectively. In oocytes, the rate of nuclear maturation was not different among the tested treatments (P > 0.05), but it was numerically higher in the 10−7 M melatonin treated group compared with the control (69.71 ± 13.76% v. 88.1 ± 12.54%). In conclusion, under the studied conditions, melatonin was unable to improve maturation rate or to affect the expression of antioxidant, pro-apoptotic, and expansion-related genes in CC. Melatonin during IVM has shown variable results in different studies and appears to show different effects depending on culture conditions and parameters studied. In order to take advantage of the possible positive antioxidant effects of melatonin, other culture conditions and parameters should be investigated. In a next step, melatonin will be included during in vitro culture of embryos to evaluate its possible cytoprotective role, because such embryos are more exposed to oxidative stress during in vitro culture, and to investigate its benefits on developmental competence in vitro. This reaesrch was funded by FAPESP (2015/20379-0; 2014/17181-0).

scholarly journals Bovine Herpesvirus 1 UL3.5 Interacts with Bovine Herpesvirus 1 α-Transinducing Factor

2000 ◽  
Vol 74 (6) ◽  
pp. 2876-2884 ◽  
Author(s):  
Ngan Lam ◽  
Geoffrey J. Letchworth
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bovine Herpesvirus ◽  
Mass Spectrometry Analysis ◽  
Bovine Herpesvirus 1 ◽  
Subcellular Compartment ◽  
Transfected Cells ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACT The bovine herpesvirus 1 (BHV-1) UL3.5 gene encodes a 126-amino-acid tegument protein. Homologs of UL3.5 are present in some alphaherpesviruses and have 20 to 30% overall amino acid homology that is concentrated in the N-terminal 50 amino acids. Mutant pseudorabies virus lacking UL3.5 is deficient in viral egress but can be complemented by BHV-1 UL3.5 (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886–8892, 1997). The function of BHV-1 UL3.5 in BHV-1 replication is not known. To get a better understanding of its function, we sought to identify the proteins that interact with the BHV-1 UL3.5 protein. By using an in vitro pull-down assay and matrix-assisted laser desorption ionization mass spectrometry analysis, we identified BHV-1 α-transinducing factor (αBTIF) as a BHV-1 UL3.5-interacting protein. The interaction was verified by coimmunoprecipitation from virus-infected cells using an antibody to either protein, by indirect immunofluorescence colocalization in both virus-infected and transfected cells, and by the binding of in vitro-translated proteins. In virus-infected cells, UL3.5 and αBTIF colocalized in a Golgi-like subcellular compartment late in infection. In transfected cells, they colocalized in the nucleus. Deletion of 20 amino acids from the N terminus of UL3.5, but not 40 amino acids from the C terminus, abolished the UL3.5-αBTIF interaction both in vitro and in vivo. The interaction between UL3.5 and αBTIF may be important for BHV-1 maturation and regulation of αBTIF transactivation activity.

scholarly journals Persistence and Reactivation of Bovine Herpesvirus 1 in the Tonsils of Latently Infected Calves

2000 ◽  
Vol 74 (11) ◽  
pp. 5337-5346 ◽  
Author(s):  
M. T. C. Winkler ◽  
A. Doster ◽  
C. Jones
Keyword(s):  
Ribonucleotide Reductase ◽  
Virus Transmission ◽  
Bovine Herpesvirus ◽  
Rt Pcr ◽  
Dexamethasone Treatment ◽  
Viral Dna ◽  
Bovine Herpesvirus 1 ◽  
Lymphoid Follicles ◽  
Latently Infected ◽  
Herpesvirus 1

ABSTRACT Bovine herpesvirus 1 (BHV-1), like other members of theAlphaherpesvirinae subfamily, establishes latent infection in sensory neurons. Reactivation from latency can occur after natural or corticosteroid-induced stress culminating in recurrent disease and/or virus transmission to uninfected animals. Our previous results concluded that CD4+ T cells in the tonsil and other adjacent lymph nodes are infected and undergo apoptosis during acute infection (M. T. Winkler, A. Doster, and C. Jones, J. Virol. 73:8657–8668, 1999). To test whether BHV-1 persisted in lymphoreticular tissue, we analyzed tonsils of latently infected calves for the presence of viral DNA and gene expression. BHV-1 DNA was consistently detected in the tonsils of latently infected calves. Detection of the latency-related transcript (LRT) in tonsils of latently infected calves required nested reverse transcription-PCR (RT-PCR) suggesting that only a few cells contained viral DNA or that LRT is not an abundant transcript. bICP0 (immediate-early and early transcripts), ribonucleotide reductase (early transcript), and glycoprotein C (late transcript) were not detected by RT-PCR in latently infected calves. When reactivation was initiated by dexamethasone, bICP0 and ribonucleotide reductase transcripts were detected. Following dexamethasone treatment, viral nucleic acid was detected simultaneously in trigeminal ganglionic neurons and lymphoid follicles of tonsil. LRT was detected at 6 and 24 h after dexamethasone treatment but not at 48 h. Dexamethasone-induced reactivation led to apoptosis that was localized to tonsillar lymphoid follicles. Taken together, these findings suggest that the tonsil is a site for persistence or latency from which virus can be reactivated by dexamethasone. We further hypothesize that the shedding of virus from the tonsil during reactivation plays a role in virus transmission.

scholarly journals Intraspecific bovine herpesvirus 1 recombinants carrying glycoprotein E deletion as a vaccine marker are virulent in cattle

2006 ◽  
Vol 87 (8) ◽  
pp. 2149-2154 ◽  
Author(s):  
Benoît Muylkens ◽  
François Meurens ◽  
Frédéric Schynts ◽  
Frédéric Farnir ◽  
Aldo Pourchet ◽  
...  
Keyword(s):  
High Frequency ◽  
Bovine Herpesvirus ◽  
Clinical Score ◽  
Natural Host ◽  
Field Strain ◽  
Bovine Herpesvirus 1 ◽  
Glycoprotein E ◽  
Control Programmes ◽  
Herpesvirus 1

Vaccines used in control programmes of Bovine herpesvirus 1 (BoHV-1) utilize highly attenuated BoHV-1 strains marked by a deletion of the glycoprotein E (gE) gene. Since BoHV-1 recombinants are obtained at high frequency in experimentally coinfected cattle, the consequences of recombination on the virulence of gE-negative BoHV-1 were investigated. Thus, gE-negative BoHV-1 recombinants were generated in vitro from several virulent BoHV-1 and one mutant BoHV-1 deleted in the gC and gE genes. Four gE-negative recombinants were tested in the natural host. All the recombinants were more virulent than the gE-negative BoHV-1 vaccine and the gC- and gE-negative parental BoHV-1. The gE-negative recombinant isolated from a BoHV-1 field strain induced the highest severe clinical score. Latency and reactivation studies showed that three of the recombinants were reexcreted. Recombination can therefore restore virulence of gE-negative BoHV-1 by introducing the gE deletion into a different virulence background.

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