Bovine Immunodeficiency Virus in Relation to Embryos Fertilized In Vitro

The association of bovine immunodeficiency virus (BIV) with embryos derived by in vitro fertilization from oocytes of experimentally infected heifers or oocytes/embryos exposed to the virus in vitro was investigated. Using a nested-PCR assay, proviral DNA of BIV was not detected in follicular fluid or in embryos derived from BIV-infected donors. In vitro exposure of oocytes to BIV during maturation or insemination with BIV-infected semen resulted in zona pellucida-intact embryos testing negative for BIV provirus. However, exposure of zona pellucida-free day-7 embryos to the virus resulted in a positive BIV assay for 28% of the batches of embryos, suggesting that the zona pellucida has a role in protecting against BIV infection. The presence of BIV in the IVF system had no apparent effect on the development of bovine embryos to the blastocyst stage.     

Embryo transfer in relation to bovine leukemia virus control and eradication

Two hundred and seven, zona pellucida-intact bovine embryos were collected from bovine leukemia virus-infected donors, washed, and transferred to uninfected recipients: 111 of these embryos were sired by bovine leukemia virus-infected bulls. Fifty live calves were obtained from the 57 pregnancies resulting from the transfers. None of the recipients or calves developed antibodies to bovine leukemia virus. Nine zona-intact ova, 12 zona-intact morulae and 15 hatched blastocysts, exposed “in vitro” to bovine leukemia virus, washed and then tested for bovine leukemia virus were negative. Twenty-seven, zona-intact embryos and 14 hatched embryos were similarly exposed and washed prior to being transferred in groups to two uninfected recipients: no pregnancies resulted, nor did the recipients develop antibodies to bovine leukemia virus up to 120 days posttransfer. The conclusion from these and other bovine leukemia virus studies is that zona-intact embryos can be transferred from bovine leukemia virus-infected donors, including those bred by bovine leukemia virus-infected bulls, without risk of transmitting bovine leukemia virus, providing that they are properly washed prior to transfer.     

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.     

An attempt to render oocytes and embryos free from the porcine circovirus type 2 after experimental in vitro exposure

The purpose of this study was to investigate the possibility of rendering oocytes and embryos free of porcine circovirus type 2 (PCV2). Groups of cumulus oocytes complexes, cumulus free oocytes, and embryos 3 to 5 d post breeding were exposed to PCV2 (10(5) TCID50/mL) prior to disinfection by washing and different combinations of enzymatic treatments. The study suggests that under the in vitro conditions used, standard washing procedures with, or without, trypsin or incorporating pronase or hyaluronidase and DNase/RNase in the treatment was not effective in rendering oocytes and embryos free from PCV2 nucleic acid. Since the virus is noncytopathic in cell culture and for embryonic cells, it appears that there is a possibility of introducing viral contamination through oocytes collected from infected pigs into the in vitro fertilization system with subsequent potential of producing in vitro fertilized embryos associated with PCV2.     

Failure to Remove Bovine Diarrhea Virus (BVDV) from Bull Semen by Swim up and other Separatory Sperm Techniques Associated with in vitro Fertilization

Semen samples, collected from bulls pesistently infected with bovine viral diarrhea virus (BVDV) and containing BVDV (titer 10⁵ - 10⁶TCID₅₀/ml), were subjected to sperm separation procedures (washing, swim up, Percoll gradient, glass wool filtration, glass beads filtration) that are commonly used prior to in vitro fertilization (IVF) to determine if these procedures would yield spermatozoa free from BVDV. The final sperm pellets from frozen and fresh ejaculates were tested for the presence of BVDV by the immunoperoxidase technique; all tests were positive for BVDV in the range of 10³ - 10⁴TCID₅₀/ml). The study shows that when semen containing BVDV at the level of 10⁵ - 10⁶TCID₅₀/ml) is used for IVF, the virus is not completely removed by any of the simple physical methods commonly used to prepare sperm for IVF. Inhalt: Feldversuch das bovine Diarrhoe Virus (BVDV) aus Bullensperma durch Swim up oder andere Trennverfahren in Verbindung mit der In vitro Fertilisation zu entfernen Spermaproben, die von Bullen gewonnen wurden, welche dauerhaft mit dem Virus der bovinen Virus-Diarrhoe (BVDV) infiziert waren und einen Titer zwischen 10⁵ - 10⁶TCID₅₀/ml enthielten, wurden verschiedenen Trennverfahren unterzogen (Waschen, swim up, Percoll gradient, Glaswollenfiltration, Glaskugelfiltration). Diese Verfahren werden üblicherweise für die Vorbereitung zur In vitro Befruchtung verwendet, und es sollte geprüft werden, ob diese Verfahren auch das Sperma von dem BVD-Virus befreien können. Das endgültige Spermapellet von gefrorenen/aufgetauten und frischen Ejakulaten wurde in Gegenwart des BVD-Virus und mit Hilfe der immunoperoxidase Technik getestet. Alle Testergebnisse waren positiv für BVD-Virus in einem Bereich von 10³ - 10⁴ TCID₅₀/ml. Die Studie zeigt, daß Sperma, wenn es 10⁵-10⁶TCID₅₀/ml des Virus enhält, nicht vollständig mit den für die IVF üblichen, einfachen physikalischen Methoden von dem Virus befeit werden kann.     

Effect of trypsin in semen on in vivo fertilization and early embryonic development in superovulated heifers

The effect of trypsin on the fertilizing capacity of bull semen was investigated as part of the evaluation of the addition of trypsin to semen as a method for destroying or inactivating infectious agents. Parts of the ejaculates from four bulls were treated with 0.3% trypsin solution. Both the treated and untreated aliquots of semen were frozen, thawed and used for the artificial insemination of superovulated heifers. Two hundred and thirty ova and embryos were collected from 22 heifers on day 7 after oestrus (insemination). One hundred and ten out of 164 (67%) embryos and ova from 15 heifers inseminated with trypsin-treated semen were classified as of transferable quality compared to 46 out of 66 (70%) in the control group of 7 heifers (p>0.05). There was no difference in the proportion of fertilized ova or degenerated embryos resulting from the control or trypsin-treated samples of frozen-thawed semen, which is consistent with results obtained previously using fresh semen.