Adherence of bovine viral diarrhea virus to bovine oocytes and embryos with a hardened zona pellucida cultured in vitro

The purpose of this study was to investigate the adherence of bovine viral diarrhea virus (BVDV) to bovine mature, or immature, cumulus-free oocytes and to in vitro fertilized embryos, maintained in vitro in a ligated bovine oviduct to allow for the hardening of the zona pellucida. Incubation of the oocytes and embryos in the oviduct for 5 h caused hardening of the zona pellucida as measured by resistance to pronase digestion (which increased from approximately 3 min to 7 h; P >0.001). However, there was no difference between the number of infected oocytes and embryos (n = 965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P > 0.05). It was concluded that the modification of the proteolytic resistance properties of the zona pellucida during in vitro oviductal incubation did not influence the adherence of BVDV to zona pellucida of oocytes or in vitro fertilized embryos.     

Effect in vitro of bovine viral diarrhea virus on bovine embryos with the zona pellucida intact,damaged and removed

The in vitro effect of bovine viral diarrhea virus (BVDV) on the survival of day 7 to day 7.5 bovine embryos collected from superovulated donors was studied. Fifty-four experimental embryos with the zona pellucida (ZP) intact, damaged or removed were exposed to 1×10⁴ TCD₅₀/ml of the NADL cytopathic strain of BVDC at 37°C for 24 hrs and compared to 36 control embryos that were cultured for 24 hr. Seven embryos with the ZP-removed were similarly exposed for 48 hrs and compared to five control embryos. The overall survival rate was 68% for embryos exposed to BVDV for 24 hrs and 77% for embryos not exposed (P>0.05). Extended exposure of the embryos with the ZP removed to virus for 48 hrs did not affect their survival rate compared to controls. Damage to the ZP by cracking or total removal of the ZP by micromanipulation or acidic Tyrode's solution had no effect on subsequent embryonic survival in the presence of BVDV. It was concluded that exposure to BVDV in vitro is not cytopathic for morula and blastocyst stage bovine embryos over a 48 hr period, even when they are not protected by the ZP.     

Prevalence of bovine immunodeficiency-like virus in bulls as determined by serology and proviral detection.

We found the rate of bovine immunodeficiency-like virus (BIV) infection among bulls to be 9.6% using serology and 12.6% when tested by the polymerase chain reaction (PCR) for the presence of BIV provirus in peripheral blood leukocytes. Previously, we determined the frequency of BIV infection among the general dairy cow population in Ontario to be 5.5% based on serological analysis. Apparently, serological testing detects only 77% of BIV-infected bulls. Since almost a quarter of BIV-infected bulls may be seronegative, it is recommended that the PCR test be used to identify BIV-infected individuals. It is clear from the data presented here and supported by experimental studies that the latent carrier state is a relatively common outcome in ruminants infected with BIV.     

On the Species Specificity of Sperm Binding and Sperm Penetration of the Zona Pellucida

Sperm binding and sperm penetration of the zona pellucida (zp) are regarded as species‐specific. In this investigation, the interactions between bovine oocytes and porcine, respectively, equine spermatozoa have been studied under in vitro conditions and compared with the normal in vitro fertilization of bovine oocytes by bovine sperm. Surprisingly, many of the heterologous spermatozoa adhered firmly to the bovine oocytes and could not be removed by intense washing. On average, more than 100 boar or equine spermatozoa were bound to the zp of bovine oocytes. Electron microscopic studies clearly demonstrated that porcine sperm attached to the zona and underwent the acrosome reaction. Equine spermatozoa displayed a similar binding affinity, but unlike the porcine spermatozoa even penetrated the zp and were taken up into the oocyte after a longer period of co‐incubation. Considering these new results the dogma of a strict species specificity of sperm zona interactions under in vitro conditions has to be reconsidered.     

Serological evidence of bovine immunodeficiency-like virus infection in a sheep.

A six month-old sheep was entered into a control group in an experiment designed to study the effects of exposure to the bovine immunodeficiency-like virus (BIV). Anti-BIV antibodies were detected in the serum of this sheep prior to the start of the study; these antibodies persisted for 12 months at which time the animal was destroyed. The sheep was normal clinically and was grossly normal at postmortem examination. Blood from this sheep was inoculated into a recipient sheep which subsequently showed a transient anti-BIV antibody response beginning two months postinoculation. Sheep have been previously shown to produce anti-BIV antibodies after experimental inoculation with infected cell culture material or infected bovine blood and BIV infection was found in a sheep pastured with BIV-infected cattle. In the present case there was no contact with cattle; the source of the infection was not identified.     

Calves Born after Direct Transfer of Vitrified Bovine In Vitro-produced Blastocysts Derived from Vitrified Immature Oocytes

Vitrification has been the method of choice for the cryopreservation of bovine oocytes, as rapid cooling decreases chilling sensitivity. The aim of this study was to determine the in vitro and in vivo survival and the viability of immature oocytes vitrified using super‐cooled liquid nitrogen. Immature oocytes were randomly allocated to three groups: (i) non‐vitrified control group, (ii) vitrified in normal (?196°C) liquid nitrogen (LN₂) and (iii) vitrified in super‐cooled LN₂ (≤?200°C). Open‐pulled glass micropipettes were used as vitrification containers. Immature oocytes were in vitro‐matured, fertilized and cultured to the blastocyst stage. In vitro viability was assessed by cleavage and blastocyst rates on days 2 and 7 of culture respectively. Vitrified blastocysts derived from the immature vitrified oocytes were directly transferred to synchronous recipients. The in vitro embryo development of vitrified immature oocytes was not influenced by the LN₂ state. After direct transfer (one embryo per recipient) of 16 embryos obtained from immature vitrified oocytes (eight from each vitrified group), two healthy calves were born in each group. These results indicated that vitrification of immature bovine oocytes using glass micropipettes under normal or super‐cooled LN₂, resulted in viable blastocysts and live calves following in vitro embryo production.     

Activation of Zona‐Free Buffalo (Bubalus bubalis) Oocytes by Chemical or Electrical stimulation,and Subsequent Parthenogenetic Embryo Development

Parthenogenetic activation using zona‐free oocytes offers an alternative model that could be applied to develop protocols for the activation of reconstructed embryos for cloning. The aim of this study was to compare the efficacy of different methods for the activation of zona‐free buffalo oocytes in terms of their effects on the developmental competence of parthenogenetic embryos. The effects of zona removal on parthenogenetic activation and in vitro developmental competence of metaphase II oocytes were also examined. All activation methods were followed by incubation of 2 mm 6‐dimethylaminopurine (6‐DMAP) for 4 h. Out of three different pulse strengths (1.2, 2.1 or 3.3 kV/cm) used, 2.1 kV/cm resulted in the highest blastocyst rate (25.3%). On comparing different chemical agents and electric pulse, highest blastocyst rate was observed for calcium ionophore (CaI) (28.6%) followed by ethanol (25.0%), electric pulse (22.5%) and combined CaI and ethanol treatment (16.7%) although differences among them were not significant. Furthermore, a significantly reduced developmental potential was observed in zona‐free oocytes when compared to zona‐intact ones up to the blastocyst stage (44.3% vs 27.1%). In conclusion, zona‐free buffalo oocytes can be successfully activated for parthenogenetic development using chemical or electrical stimulation. Out of different agents examined, CaI followed by 6‐DMAP resulted in the highest blastocyst rate.     

Oviductal Fluid Proteins Associated with the Bovine Zona Pellucida and the Effect on In Vitro Sperm–Egg Binding,Fertilization and Embryo Development

Studies have demonstrated that oviductal fluid (ODF) proteins associate with eggs of numerous species including the bovine. In this study, the association of three ODF proteins, the bovine oestrus‐associated protein, osteopontin (OPN), lipocalin‐type prostaglandin D synthase (L‐PGDS), with the bovine zona pellucida (ZP) was demonstrated by immunohistochemistry and western blot. The biological function of ODF derived egg‐associated OPN and L‐PGDS in sperm binding, fertilization and embryonic development was also explored. In vitro matured bovine oocytes were pre‐incubated with ODF collected by cannula from cows in oestrus, or ODF with antibodies to OPN, L‐PGDS and bovine serum albumin (BSA). Following incubation, oocytes were inseminated with 1 × 10⁵ frozen‐thawed spermatozoa, and they were evaluated for sperm binding, fertilization and embryonic development in vitro. Pre‐treatment of ODF with antibodies to all of proteins reduced sperm binding to the ZP and fertilization in vitro. Cleavage rates were not significantly different among incubations, but rates of embryo development were significantly decreased. We conclude that antibodies to OPN, L‐PGDS and BSA react with oocytes incubated with ODF and inhibit sperm binding, fertilization and embryonic development in vitro, suggesting a potential role of these proteins in these events.     

Influence of Osteopontin in Bovine Uterine Tube Fluid on Sperm Binding and Fertilization in RCA-1 Lectin-treated Oocytes

This study was conducted to determine the effect of pre‐exposure of oocytes to Ricinus communis (RCA‐1) lectin and osteopontin (OPN) in uterine tube fluid (UTF) on in vitro sperm–egg binding and fertilization. In vitro‐matured bovine oocytes were incubated (39°C, 5% CO₂ in air) for 2 h in the following treatments: (i) 500 μl of fertilization medium (FM); (ii) 250 μl of FM with 0.25 ml of non‐luteal ampullary uterine tube fluid (NLAUTF); (iii) 250 μl of FM with 250 μl of NLAUTF and 4 μl of RCA‐1 lectin; (iv) 250 μl of FM with 250 μl of NLAUTF, a rabbit polyclonal antibody (1:200) against purified bovine milk OPN, and RCA‐1 lectin; (v) 500 μl of FM and RCA‐1 lectin. Following incubation, oocytes were washed, placed in FM with 2 μg heparin, and incubated with 1 × 10⁵ frozen–thawed spermatozoa per 10 oocytes. Oocytes used to assess sperm binding were stained with Hoescht 33342, and the number of sperm bound per zona pellucida counted. The remaining oocytes were fixed in acid alcohol, stained with 1% acetate‐orcein and observed to determine the presence of pronuclei. More sperm bound to the zona pellucida (mean ± SEM) when oocytes were incubated in treatment 3 (59.0 ± 5.5) than in treatments 2 (46.4 ± 5.6), 4 (18.1 ± 5.4), 5 (33.4 ± 5.6) or 1 (32.5 ± 5.6). More oocytes were fertilized when incubated in treatment 3 (91% ± 3.0) than in 2 (84% ± 3.0), 4 (40% ± 3.0), 5 (77% ± 3.0) or 1 (76% ± 3.0). As in previous studies, this study suggests that RCA‐1 lectin enhances binding of UTF‐derived OPN to bovine oocytes, resulting in increased sperm–egg binding and fertilization in vitro and a possible role in fertilization.     

Effect of co‐culture with intact embryos on development of bovine separated blastomeres

To improve embryo development in bovine separated blastomeres, we evaluated applicability of co‐culture with intact embryos. The morphological quality of blastocysts derived from separated blastomeres and rate of blastocyst formation were only slightly increased when the cells were co‐cultured with intact embryos, which did not provide significant differences when statistically analyzed. However, the cell count of inner cell mass (ICM), trophectoderm (TE) and total number of cells in Day 8 blastocysts were significantly higher when the cells were co‐cultured with the intact embryos than those with the cells cultured individually (P < 0.05). Transfer of four monozygotic pairs of blastocysts derived from the cells co‐cultured with intact embryos led to three pregnancies even when the blastomeres were produced by in vitro maturation and in vitro fertilization of oocytes collected by ovum pick‐up from elite cows. These results suggest that co‐culturing with intact embryos may enhance development of bovine separated blastomere.     

Comparison of persistence of seven bovine viruses on bovine embryos following in vitro exposure

The ability of seven cytopathic strains of bovine viruses to adhere to the zona pellucida of six-to-eight day-old bovine embryos were compared. Embryos were exposed to virus by placing them either in virus suspensions or by culturing them on infected bovine turbinate cultures for 18-24 h. After exposure to bovine virus diarrhea virus (BVDV), infectious bovine rhinotracheitis virus (IBV), bluetongue virus (BTV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV), parainfluenza 3 virus (PI3), or bovine enterovirus virus (BEV), the embryos were tested for virus by culture in bovine turbinate cells and by morphological examination using electron microscopy (EM). A special technique to minimize loss of embryos processed for EM was developed. More embryos had viral particles on the surface of the zona pellucida after exposure to 18-24 hour infected cell cultures than did embryos exposed to viral culture suspensions. The most dramatic finding was that BTV adhered in large numbers to the surface of the zona pellucida of exposed embryos. IBRV, PRV, and VSV comprised an intermediate group, with virions occasionally detected on the surface of exposed embryos after 5 washes. Therefore, extensive washing is required. The PI3 and BEV were easily removed from embryo-exposed virus by washing. BVD was difficult to identify morphologically, making assessment by EM unreliable. There was no evidence that any one of the seven viruses penetrated the intact zona pellucida. Using a micromanipulator, 42 embryos were also directly inoculated through the zona pellucida with +/- 50 picoliters of virus inoculum or medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Serum-Free Culture Media on in vitro Development of Domestic Cat Embryos Following In Vitro Maturation and Fertilization

This study was conducted to determine the adequate medium for a serum‐free culture system of domestic cat embryos produced by in vitro maturation (IVM) and fertilization (IVF). Cumulusoocyte complexes recovered from cat ovaries were matured in vitro for 24 h, and then inseminated in vitro for 12 h. After insemination, the oocytes were cultured in five media [Ham's F10, Waymouth 752/1 (Waymouth), TCM199, modified Earle's balanced salt solution (MK‐1) and CR1aa], each of which contained 0.4% bovine serum albumin. There were no significant differences among the rates of fertilization of oocytes cultured in five media following IVF. The rate of oocytes/embryos developed to at least the morula stage was significantly lower (p < 0.05) in Waymouth than in MK‐1, TCM199 and CR1aa. Moreover, none of the embryos cultured in Ham's F10 and Waymouth developed to the blastocyst stage. There were no differences among the rates of development to the blastocyst stage of oocytes/embryos cultured in MK‐1, TCM199 and CR1aa. These results indicate that the type of serum‐free medium has a major impact on in vitro development of domestic cat embryos derived from IVM/IVF, and MK‐1, TCM199 and CR1aa media are suitable for in vitro culture of cat embryos in a serum‐free culture system.     

Assessment of DNA Damage during In Vitro Development of Buffalo (Bubalus bubalis) Embryos: Effect of Cysteamine

Comet assay was used in the present study to examine DNA damage to buffalo oocytes and embryos during in vitro culture. Embryos were produced in vitro from oocytes obtained from slaughterhouse ovaries in presence of cysteamine (IVM and IVC media supplemented with 50 and 100 μm , respectively) or in its absence (controls). Compared to controls, cysteamine supplementation increased (p < 0.01) cleavage rate and proportion of oocytes that developed to 8‐ to 16‐cell stage. The incidence of DNA damage was lower (p < 0.01) in cysteamine group than that in controls at 8‐ to 16‐ (19.3 ± 4.24 vs 72.0 ± 5.22%) but not in 2‐cell stage embryos (11.7 ± 5.63 vs 20.8 ± 5.49%) or in mature oocytes (5.3 ± 3.43 vs 10.3 ± 4.73%). The tail length, which indicates magnitude of DNA damage, was shorter (p < 0.01) in cysteamine group than in controls in mature oocytes (25.5 ± 0.5 vs 36.0 ± 0.71 pixels) and 8‐ to 16‐cell stage (49.2 ± 1.64 vs 152.7 ± 1.28 pixels) but not in 2‐cell stage embryos (36.3 ± 1.54 vs 36.4 ± 0.75 pixels). Also, exposure of oocytes/embryos to UV radiation or H₂O₂ caused extensive DNA damage. In conclusion, these results suggest that oocytes/embryos suffer from DNA damage during progress of in vitro culture, which can be partly ameliorated by cysteamine supplementation of culture media.     

Evidence of bovine immunodeficiency virus (BIV) infection: serological survey in Argentina

This is the first report of serological evidence for bovine immunodeficiency virus (BIV) infection in Argentina. The analysis was performed in 589 dairy bovine sera samples, applying indirect enzyme-linked immunosorbent assay (I-ELISA) using a synthetic antigen (transmembrane peptide, TM) and Immunofluorescent assay (IFA). In this study, 9 dairy herds from 4 Argentinian provinces were evaluated and 12% of the animals tested positive for BIV. Seven of the 9 herds tested were BIV seropositive and the percentage of BIV seropositive animals in the herds ranged from 2% to 42%. Direct detection of BIV provirus applying nested PCR was not conclusive. Antibody detection against bovine leukemia virus (BLV) in all sera was also performed applying immunodiffusion (ID) assay and 59% resulted seropositive. Statistical analysis of the results was carried out and possible evidence of association between BIV and BLV infection was considered. Future studies should be performed including local field isolates strains of BIV.     

Interaction of a Green Recombinant Bovine Herpesvirus 4 with In Vitro-produced Bovine Embryos

The objective of the present study was to assess whether bovine herpesvirus 4 (BHV-4) is able to infect in vitro-produced bovine embryos. A green recombinant BHV-4 (BHV-4EGFPTK), obtained by insertion of an EGFP gene into the TK locus of BHV-4, was used. The presence of this marker protein made it possible easily to detect infected cells under physiological conditions, without harmful manipulation of the cells or the addition of exogenous substrates, so that the spread of the virus could be followed in real time. Zona pellucida intact (ZP-I) and zona pellucida open (ZP-O) blastocytes were exposed to 10⁶ TCID₅₀ viral particles and infection was monitored by fluorescent microscopy for 48 h. Expression of EGFP and degeneration of embryonic cells was observed in three of the 18 ZP-O embryos, but in none of the ZP-I embryos. It was concluded from this preliminary study that BHV-4 has only a low ability to infect in vitro-produced bovine embryos, depending on the absence of ZP, the amount of virus present and the stage of embryonic development. However, embryonic stem cells could be transduced by BHV-4EGFPTK just after differentiation, as shown by expression of EGFP.     

Synergistic Effect of Porcine Follicular Fluid and Dibutyryl Cyclic Adenosine Monophosphate on Development of Parthenogenetically Activated Oocytes from Pre‐Pubertal Gilts

This study investigated the effect of porcine follicular fluid (PFF) and dibutyryl cyclic adenosine monophosphate (dbcAMP) during in vitro maturation (IVM) of porcine oocytes on meiotic maturation, fertilization and embryo development, and compared the effect of supplementing the embryo culture media with PFF or foetal bovine serum (FBS) on embryo development. Oocytes from pre‐pubertal gilts were IVM for 44 h, and parthenogenetically activated or in vitro‐fertilized. Embryos were cultured in porcine zygote medium (PZM3) for 7 days. Cleavage and blastocyst rates were evaluated at 48 h and 7 days of culture. The supplementation of the IVM medium with 25% PFF and 1 mm dbcAMP for the first 22 h resulted in more (p < 0.05) embryos developing to the blastocyst stage as compared with the inclusion of dbcAMP alone. The dbcAMP + PFF combination increased (p < 0.05) the average number of nuclei per blastocyst as compared with either of these components alone or in its absence. A synergistic effect of dbcAMP + PFF during IVM was also reflected in the capacity of oocytes to regulate sperm penetration and prevent polyspermy, as twice as many oocytes from the control group were penetrated by more than one sperm as compared with those matured in the presence of both dbcAMP and PFF. The supplementation of PZM3 with 10% FBS from days 5 to 7 of culture significantly improved the total cell quantity in embryos derived either from control or dbcAMP + PFF matured oocytes. There was no effect on the total cell quantity when FBS was replaced by the same concentration of PFF. These studies showed that dbcAMP, PFF and FBS can improve both the quantity (57.3% vs 41.5%) and quality (74.8 vs 33.3 nuclei) of porcine blastocysts derived from oocytes recovered of pre‐pubertal gilts.     

Systemic Immunosuppression by Methylprednisolone and Pregnancy Rates in Goats Undergoing the Transfer of Cloned Embryos

The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona‐free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre‐treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona‐free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo‐ vs in vitro‐matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona‐free and zona‐intact goat cloned embryos.     

The effects of ceftiofur sodium (Naxcel) on bovine oocyte and preimplantation embryonic development assessed by in vitro embryo production techniques

Ceftiofur sodium is a third-generation cephalosporin antibiotic with broad spectrum bactericidal activity against Gram-positive and Gram-negative bacteria including the beta-lactamase producing strains. In this study, we use in vitro techniques to examine the effects of low and high levels of ceftiofur sodium on the development of bovine oocytes/embryos during oocyte maturation, oocyte fertilization and embryo culture. A total of 8590 oocytes was used in six independent experiments, each in a randomized complete block design. Each replication within each experiment consisted of oocytes from the same abattoir collection of ovaries. There was no difference in embryo development when oocytes were exposed to ceftiofur sodium during oocyte maturation or fertilization at low (10 and 50 μg/mL) or high (100 and 200 μg/mL) concentrations. However, when fertilized oocytes were exposed to concentrations 50 μg/mL during culture, ceftiofur sodium significantly retarded embryo development (e.g. the numbers of ova developing to the morula and blastocyst stages were reduced, and a large proportion of embryos were blocked at the 8-cell stage). We conclude that ceftiofur sodium does not appear to have detrimental effects on oocyte maturation and fertilization. However, long term exposure to high dosages of ceftiofur sodium during post-fertilization culture adversely affects embryo development in vitro.     

Developmental Kinetics of Pig Embryos by Parthenogenetic Activation or by Handmade Cloning

The developmental kinetics of pig embryos produced by parthenogenetic activation without (PAZF) or with (PAZI) zona pellucida or by handmade cloning (HMC) was compared by time‐lapse videography. After cumulus cell removal, the matured oocytes were either left zona intact (PAZI) or were made zona free by pronase digestion (PAZF) before they were activated (PA). Other matured oocytes were used for HMC based on foetal fibroblast cells. On Day 0 (day of PA or reconstruction), the embryos were cultured for 7 days in vitro in our time‐lapse system. Pictures were taken every 30 min, and afterwards, each cell cycle was identified for each embryo to be analysed. Results showed that the PA embryos (both PAZF and PAZI) had shorter first cell cycle compared with HMC (17.4. 17.8 vs 23.6 h), but had a longer time length from four cell to morula stages (57.9, 53.8 vs 44.9 h). However, at the second cell cycle, PAZF embryos needed shorter time, while PAZI embryos had similar time length as HMC embryos, and both were longer than PAZF (23.4, 24.8 vs 14.6 h). Both PAZF and PAZI embryos used similar time to reach the blastocyst stage, and this was later than HMC embryos. In addition, when all of these embryos were grouped into viable (developed to blastocysts) and non‐viable (not developed to blastocysts), the only difference in the time length was observed on the first cell cycle (18.6 vs 24.5 h), but not on the later cell cycles. In conclusion, our results not only give detailed information regarding the time schedule of in vitro‐handled pig embryos, but also indicate that the first cell cycle could be used as a selecting marker for embryo viability. However, to evaluate the effect of the produced techniques, the whole time schedule of the pre‐implantation developmental kinetics should be observed.