Detection of ovine lentivirus in the cumulus cells, but not in the oocytes or follicular fluid, of naturally infected sheep.
ABSTRACT The aim of this study was to examine the Maedi-Visna virus (MVV) infection status of oocytes, cumulus cells, and follicular fluid taken from 140 ewes from breeding flocks. MVV proviral-DNA and MVV RNA were detected using nested-PCR and RT-PCR MVV gene amplification, respectively in the gag gene. Nested-PCR analysis for MVV proviral-DNA was positive in peripheral blood mononuclear cells in 37.1% (52/140) of ewes and in 44.6% (125/280) of ovarian cortex samples. The examination of samples taken from ovarian follicles demonstrated that 8/280 batches of cumulus cells contained MVV proviral-DNA, whereas none of the 280 batches of oocytes taken from the same ovaries and whose cumulus cells has been removed, was found to be PCR positive. This was confirmed by RT-PCR analysis showing no MVV-viral RNA detection in all batches of oocytes without cumulus cells (0/280) and follicular fluid samples taken from the last 88 ovaries (0/88). The purity of the oocyte fraction and the efficacy of cumulus cell removal from oocytes was proved by absence of granulosa cell-specific mRNA in all batches of oocytes lacking the cumulus cells, using RT-PCR. This is the first demonstration that ewe cumulus cells harbor MVV genome and despite being in contact with these infected-cumulus cells, the oocytes and follicular fluid remain free from infection. In addition, the enzymatic and mechanical procedures we used to remove infected-cumulus cells surrounding the oocytes, are effective to generate MVV free-oocytes from MVV-infected ewes.
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ABSTRACT: Introduction of bovine viral diarrhea virus (BVDV) with cumulus-oocyte-complexes (COCs) from the abattoir is a concern in the production of bovine embryos in vitro. Further, International Embryo Transfer Society (IETS) guidelines for washing and trypsin treatment of in-vivo-derived bovine embryos ensure freedom from a variety of pathogens, but these procedures appear to be less effective when applied to IVF embryos. In this study, COCs were exposed to virus prior to IVM, IVF and IVC. Then, virus isolations from cumulus cells and washed or trypsin-treated nonfertile and degenerated ova were evaluated as quality controls for IVF embryo production. The effect of BVDV on rates of cleavage and development was also examined. All media were analyzed prior to the study for anti-BVDV antibody. Two approximately equal groups of COCs from abattoir-origin ovaries were washed and incubated for 1 h in minimum essential medium (MEM) with 10% equine serum. One group was incubated in 10(7) cell culture infective doses (50% endpoint) of BVDV for 1 h, while the other was incubated without virus. Subsequently, the groups were processed separately with cumulus cells, which were present throughout IVM, IVF and IVC. Cleavage was evaluated at 4 d and development to morulae and blastocysts at 7 d of IVC. After IVC, groups of nonfertile and degenerated ova or morulae and blastocysts were washed or trypsin-treated, sonicated and assayed for virus. Cumulus cells collected at 4 and 7 d were also assayed for virus. Anti-BVDV antibody was found in serum used in IVM and IVC but not in other media. A total of 1,656 unexposed COCs was used to produce 1,284 cleaved embryos (78%), 960 embryos > or = 5 cells (58%), and 194 morulae and blastocysts (12%). A total of 1,820 virus-exposed COCs was used to produce 1,350 cleaved embryos (74%), 987 embryos > or = 5 cells (54%), and 161 morulae and blastocysts (9%). Rates of cleavage (P = 0.021), cleavage to > or = 5 cells (P = 0.026) and development to morula and blastocyst (P = 0.005) were lower in the virus-exposed group (Chi-square test for heterogeneity). No virus was isolated from any samples from the unexposed group. For the exposed group, virus was always isolated from 4- and 7-d cumulus cells, from all washed nonfertile and degenerated ova (n = 40) and morulae and blastocysts (n = 57) and from all trypsin-treated nonfertile and degenerated ova (n = 80) and morulae and blastocysts (n = 91). Thus, virus persisted in the system despite the presence of neutralizing antibody in IVM and IVC media, and both washing and trypsin treatment were ineffective for removal of the virus. Presence of virus in 4- and 7-d cumulus cells as well as in nonfertile and degenerated ova were good indicators of virus being associated with morulae and blastocysts.Theriogenology 02/2000; 53(3):827-39. · 2.08 Impact Factor
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ABSTRACT: This study was designed to test the efficiency of recently developed vitrification technology followed by microscope-free thawing and transfer of sheep embryos. In a first set of experiments, in vivo derived embryos at the morula to blastocyst stage were frozen in an automated freezer in ethylene glycol, and after thawing and removal of cryoprotectants, were transferred to recipient ewes according to a standard protocol (control group). A second group of embryos were loaded into open-pulled straws (OPS) and plunged into liquid nitrogen after exposure at room temperature to the media: 10% glycerol (G) for 5 min, 10% G+20% ethylene glycol (EG) for 5 min, 25% G+25% EG for 30s; or 10% EG+10% DMSO for 3 min, 20% EG+20% DMSO+0.3M trehalose for 30s. The OPS were thawed by plunging into tubes containing 0.5M trehalose. After this rapid thawing, the embryos were directly transferred using OPS as the catheter for the transplantation process. In a second set of experiments, in vivo derived and in vitro produced expanded blastocysts were vitrified in OPS and then transferred as described above. The lambing rates recorded (59% for the conventionally cryopreserved in vivo derived embryos, 56% for the vitrified in vivo derived embryos, and 20% for the vitrified in vitro produced embryos), suggest the suitability of the vitrification technique for the transfer of embryos obtained both in vivo and in vitro. This simple technology gives rise to a high embryo survival rate and will no doubt have applications in rearing sheep or other small ruminants.Theriogenology 04/2003; 59(5-6):1209-18. · 2.08 Impact Factor
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ABSTRACT: The limited knowledge on the regulation of oocyte formation, the different steps of folliculogenesis and the required conditions for oocytes to undergo proper growth, differentiation and maturation are major causes of the failure in obtaining viable offspring from in vitro cultured early oocytes from domestic animals and humans. This review highlights the factors that at present are known to be involved in the formation of mammalian oocytes and their growth, differentiation and maturation within ovarian follicles.Theriogenology 05/2005; 63(6):1717-51. · 2.08 Impact Factor