ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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256Artificial Insemination in Farm AnimalsThe induction of IFN- expression appeared genetically determined because IFN- secretionhas been evidenced from hatched blastocysts completely produced in vitro (Hernández-Ledezma et al. 1992, 1993; Stojkovic et al. 1995). Therefore, the uterine environment is notnecessary for the induction of IFN- expression but it could play an essential role in thecontrol of IFN- secretion. Paternal genotype was a significant determinant of the embryo’sability to develop the blastocyst stage and of subsequent IFN- secretion (Kubish et al.2001a). Besides, there is some evidence that the presence of other blastocysts could increaseIFN- secretion (Larson and Kubish 1999).Generally, the evaluation of the development potential of embryos today still depends onsubjective morphological examination (Lindner and Wright, 1983; Buttler and Biggers 1989;Shamsuddin et al. 1992, Massip et al. 1995). However, the development stage and quality ofthe embryos significantly influence the IFN- secretion. Some authors have proposed thatthe produced amount of IFN- could be a useful objective indicator of embryo quality(Hernandez-Ledezma et al. 1992, 1993). But others, considering that the age of blastocystsformation in vitro exhibits significant effects on the IFN- production, have suggested anegative relationship between early IFN- production and blastocysts competence forembryonic development (Kubish et al. 1998, 2004).The aim of this study was therefore to compare the IFN- secretion after hatching in bovineblastocysts of good homogeneous quality wether they are produced in vivo or completely invitro.2. Materials and methods2.1 Production of in vitro bovine blastocysts2.1.1 In vitro maturation of oocytesUnless otherwise indicated, all chemicals in this study were purchased from Sigma-Aldrich,(Saint Quentin. Fallavier, France). Ovaries from Prim Holstein cows were quickly collectedafter death in a local slaughter-house and transferred to the laboratory into a saline solutionwith 0.9% (w/v) NaCl at approximately 35°C. The largest interval between animal killingand oocyte conditioning was 3 hours. Cumulus-oocyte complexes (COCs) were recoveredby aspiration of follicles of 2-8 mm in diameter using a 18 gauge needle under vacuumpressure of approximately 50 mm Hg. The COCs were collected into Hepes-buffered tissueculture medium 199 (TCM 199 ref. M-7528) supplemented with 0.4% (w/v) BSA (CohnFraction V, ref. 9647). Before in vitro maturation, COCs were assessed morphologically: onlythose which displaid a compact and non-atretic cumulus oophorus-corona radiata with anoocyte exhibiting homogeneous cytoplasm were chosen for further in vitro culture. SelectedCOCs were washed thoroughly in TCM 199 (ref. M-4530) plus 10% (v/v) fetal calf serum(FCS, Life Technologies, Inc., Grand Island, NY USA). The maturation of about 60 COCsbatches were achieved in 500µl TCM199 (ref. M-4530) supplemented with 10% FCS, and10ng per ml EGF (ref. E-4127) with 100 µg/ml gentamicin (ref. G-1264), for 24 h at 39°Cunder humidified 5% CO 2 in air.2.1.2 In vitro fertilizationSpermatozoa were prepared from frozen-thawed semen of a sole bull (Prim Holstein breed,electronic number: 44 13 835058) that had been characterized as suitable for in vitrofertilization in our laboratory. The contents of two 0.25 ml straws (each containingapproximately 10 6 spermatozoa per ml) were layered upon a Percoll discontinuous gradient
Relationship between IFN- Production byBovines Embryos Derived Ex Vivo and Completely Produced In Vitro 257(Hasler et al. 1995). Motile spermatozoa were collected after centrifugation at approximately700g for 30 min, at room temperature. Then they washed in Hepes-buffered Tyrode’salbumin lactate pyruvate medium (Talp) (Parrish et al. 1986) and pelleted by centrifugationat approximately 200g for 10 min at room temperature. Meanwhile, COCs were transferredto another four-wells dish containing 250µl of in vitro fertilization Talp supplemented with0.01 mmol/l heparin (ref. H-3149), 0.2 mmol/l penicillamine (ref. P-4875), 0.1 mmol/lhypotaurine, 0.1 mmol/l epinephrine (ref. E-4250) and 6mg/ml fatty acid-free BSA. (ref. A-8806) Insemination was performed to get the final concentration of 2 x 10 6 spermatozoa/ml tofertilize the oocytes. Plates were incubated 5% CO 2 in humidified air at 39°C.2.1.3 In vitro cultureAfter 20 hours, the cumulus cells were removed from the presumptive zygotes byintermittent gentle shaking for 2 min. Presumptive zygotes were then washed 4 times insynthetic oviduct fluid medium SOF (Tervit et al. 1972) according to Holm et al. (1999)containing 0.7 mM Na-pyruvate, 4.2 mM Na-lactate, 2.8 mM myo-inositol, 0.2 mMglutamine, 0.3 mM citrate, 30 ml/l essential amino acids mixture, 10 ml/l non-essentialamino acids, 50 µg/ml gentamycin and 10% (v/v) fetal calf serum (SOF-FCS). All mediawere passed through a 0.2 µm membrane filter and were equilibrated overnight in anincubator at 39°C in 5% O 2 in humidified air. Presumptive zygotes were cultured in groupsof 10-12 in 20µl droplets of SOF-FCS, culture was performed in a humidified atmosphere of5% CO 2 , 5% O 2 and 90% N 2 at 39 C. Cleavage was examined once between days 2 and 3,embryo development rate was evaluated on day 7 post insemination. On day 7, excellentand good quality blastocysts of grade 1 (according to the International Embryo TransferSociety standards-IETS, Stringfellow and Seidel, 1990) were selected for this study.2.2 Production of in vivo bovine blastocystsThe oestrous cycles of french Prim Holstein donor cows were synchronised with an implantof 3mg Norgestomet-3.8 mg valerate oestradiol (Crestar <strong>IN</strong>TERVET S.A.- Holland) for 9days. The oestrous reference was observed 48-68 hours after removing the implant. Thecows were superovulated 8 to 12 days after oestrous with p-FSH (Stimufol Merial SAS -France). A total dose of 450µg p-FSH was administered (8 i.m. injections with decreasingdoses for 4 days) and an analogue of PGF 2 (500 µg i.m.; Cloprostenol Estrumate ScheringPlough <strong>Vet</strong>. S.A.) was injected at the moment of the 5th p-FSH injection. Two artificialinseminations (IA) were performed 12 and 24 hours after starting oestrous (day 0 ). Thefrozen-thawed semen for IA of embryos donor cows was the same that used for theproduction of in vitro bovine blastocysts. Generally, the in vivo embryos have a smallchronological delay in their development in comparison with the in vitro produced embryos,because of the lack of precision in the moment of fertilization. For this reason the cows werecollected at day seven and a half after IA. Embryos were recovered by uterine flushing withPBS (phosphate buffer saline) supplemented with 0.04% (w/v) BSA fraction V (sameprevious reference). Then embryos were washed 4 times in PBS supplemented with 0.4%(w/v) BSA fraction V. The viability of embryos was estimated according to IETS. Onlyexcellent and good quality blastocysts grade I were selected for this study.2.3 Determination of IFN- antiviral activityEmbryo culture media were assayed for antiviral activity by use of a cytopathic effecttitration assay that used a bovine kidney cell line designated Madin and Darby Bovine
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ARTIFICIAL INSEMINATION IN FARM ANIMALS - Phenix-Vet

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