| 1. |
- Alminana, C, et al.
(författare)
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Adjustments in IVF system for individual boars: Value of additives and time of sperm-oocyte co-incubation
- 2005
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 64:8, s. 1783-1796
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Tidskriftsartikel (refereegranskat)abstract
- In vitro fertilization (IVF) in pigs is still considered sub-optimal, due to the variable occurrence of polyspermy, variability mainly related to sperm differences. The present study was conducted in an attempt to increase the efficiency of the in vitro production of porcine embryos by optimizing the in vitro fertilization (IVF) protocol for individual males, with regard to the composition of the fertilization medium (experiments 1 and 2) and the length of gamete co-incubation time (experiment 3). A total of 5943 COCs were in vitro matured (IVM) and inseminated with frozen-thawed spermatozoa froth 2 boars (A and B). Experiment 1 determined the effect of additives caffeine (2 mM), hyaluronic acid (HA; [0.5 mg/mL]) and adenosine (10 mu M), alone or in combination, to the IVF-medium during sperm-oocyte co-incubation. Experiment 2 tested the addition of various HA (0, 0.5, 1.0 and 1.5 mg/ml) and adenosine (0, 10, 20 and 40 mu M) concentrations in the fertilization medium; while experiment 3 investigated the effect of two periods of sperm-oocyte co-incubation (10 thin or 6h). In the case of 10 min sperm-oocyte co-incubation, oocytes with attaching spermatozoa were further cultured in IVF-medium containing no spermatozoa until the 6 h of insemination was completed. Presumptive zygotes were cultured in embryo culture medium for 1215 h to assess fertilization parameters. In experiment 1, only caffeine significantly influenced the outcome of fertilization, albeit being a clearly boar-dependent effect. In experiment 2, similar boar differences were seen for HA supplementation while presence of exogenous adenosine did not influence fertilization parameters in either boar. The results of experiment 3 demonstrated that a short co-incubation time significantly (P less than 0.001) increased penetration rate and mean number of spermatozoa per oocyte (74.9 +/- 3.9% versus 62.7 +/- 3.9% and 1.5 +/- 3.2 versus 1.3 +/- 3.5 for 10 min or 6 h, respectively), but reduced monospermy (P less than 0.001, 57.9 +/- 2.5% versus 70.0 +/- 2.8%) when boar A was used. However, such effects were not seen with boar B, in which sperm-oocyte co-incubation time did not affect the efficiency of fertilization. In view of the present results, a preliminary screening for each individual male is required to select optimal conditions for IVF. (c) 2005 Elsevier Inc. All rights reserved.
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| 2. |
- Cuello, C., et al.
(författare)
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Superfine open pulled straws vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation
- 2010
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Ingår i: Reproduction, Fertility and Development. - : CSIRO Publishing. - 1031-3613 .- 1448-5990. ; 22:5, s. 808-817
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Tidskriftsartikel (refereegranskat)abstract
- The present study investigated the in vitro development of and cytoskeletal disruption suffered by in vivo-derived porcine blastocysts subjected to superfine open pulled straws (SOPS) vitrification. Blastocysts were either untreated prior to SOPS vitrification or were subjected to one of the following three pretreatment protocols: (1) centrifugation (12 min, 13 000g); (2) 25 min equilibration with 7.5 mu g mL(-1) cytochalasin B; or (3) equilibration with cytochalasin B followed by centrifugation. After 24 h culture, fresh (n = 32) and vitrified-warmed (n = 188) blastocysts were evaluated by stereomicroscopy, with survival and hatching rates recorded. Some blastocysts were stained with 4,6-diamidino2- phenylindole and processed for cytoskeletal evaluation. Three cytoskeletal patterns were identified: Grade I, intact cytoskeleton; Grade II, gross maintenance of integrity, but with some clumps of actin within the cytoplasm; and Grade III, a highly disrupted cytoskeleton. There were no differences in the survival, hatching and cell death rats, total cell number or cytoskeletal integrity between the different vitrification groups. Cell death was greater for vitrified blastocysts than for fresh blastocysts (3.6 +/- 0.4% v. 0.4 +/- 0.7%, respectively; P less than 0.05) and the percentage of blastocysts with a Grade I cytoskeletal pattern was lower for vitrified compared with fresh blastocysts (60.8% v. 92%, respectively; P less than 0.05). The vitrified-warmed blastocysts that hatched during culture exhibited a Grade I cytoskeletal pattern. In conclusion, successful SOPS vitrification of porcine blastocysts does not require pretreatment with cytochalasin B and/or centrifugation.
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| 3. |
- Cuello, C., et al.
(författare)
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Vitrification of in vitro cultured porcine two-to-four cell embryos
- 2007
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 68:2, s. 258-264
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Tidskriftsartikel (refereegranskat)abstract
- The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master (R) technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows (n = 11) on day 2 (DO = onset of estrus). Some embryos (N = 63) were vitrified within 3 It after collection, warmed and cultured for 120 h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96 It in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24 h (Group VB; N = 65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N = 70) but were cultured in vitro for 120 h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6 +/- 0.7% and 3.2 +/- 0.5%, respectively). The survival and hatching rates of VB embryos (75.0 +/- 0.69% and 33.6 +/- 0.13%) were lower (p less than 0.001) than those obtained with control embryos (89.1 +/- 0.8% and 47.5 +/- 0.12%). Hatched VB embryos had a lower (p less than 0.01) total cell number than hatched control embryos (70.3 +/- 4.5 versus 90.6 +/- 3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master (R). (c) 2007 Elsevier Inc. All rights reserved.
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| 4. |
- Sanchez-Osorio, J., et al.
(författare)
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In vitro postwarming viability of vitrified porcine embryos: Effect of cryostorage length
- 2010
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Ingår i: Theriogenology. - : Elsevier. - 0093-691X .- 1879-3231. ; 74:3, s. 486-490
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Tidskriftsartikel (refereegranskat)abstract
- Porcine embryos, which had been vitrified and stored in liquid nitrogen for up to three yr, were retrospectively analyzed to evaluate the influence of duration of storage on their in vitro viability post-warming. All embryos were vitrified (OPS or SOPS) and warmed (three-step or direct warming) using procedures that resulted in the same in vitro survival, hatching rates, and numbers of cells. Therefore, embryo data obtained using the different procedures were pooled according to their developmental stage as morulae (n = 571) or blastocysts (n = 797) and to the length of their storage in liquid nitrogen: a) 1-9 d; b) 10-30 d; c) 31-90 d; d) 1-3 yr. Non-vitrified embryos of corresponding developmental stages were used as a fresh control group (n = 280). Survival and hatching rates were evaluated after in vitro culture to assess embryo viability. The total number of cells was counted in the resulting viable blastocysts as an indicator of quality. A total of 1,648 fresh and vitrified embryos were analyzed. In vitro survival and hatching rates, but not the number of cells, differed significantly between vitrified morulae and their fresh counterparts irrespective of the duration of cryostorage. Length of storage in liquid nitrogen (LN(2)) did not influence in vitro viability among different groups of vitrified/warmed morulae nor embryos at the blastocyst stage. In conclusion, duration of storage in LN2 has no effect on the post-warming viability of porcine embryos vitrified at morula or blastocyst stage. (C) 2010 Elsevier Inc. All rights reserved.
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| 5. |
- Spjuth, Linda, et al.
(författare)
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Pre-pubertal Di(2-ethylhexyl) phthalate (DEHP) exposure of young boars did not affect sperm In vitro penetration capacity of homologous oocytes post-puberty
- 2007
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Ingår i: Archives of andrology. - : Taylor and Francis. - 0148-5016 .- 1521-0375. ; 53:3, s. 141-147
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Tidskriftsartikel (refereegranskat)abstract
- Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinylchloride (PVC) products (e.g., plastic bags and medical equipment), has been reported to have toxic effects on animal reproduction and is considered an environmental hazard based, mostly, on rodent studies. However, the doses used in these studies are often considerably higher than that presumed in human exposure. In the present study we used young boars as model animals to assess the effects of pre-pubertal DEHP exposure on the ability of spermatozoa to penetrate homologous oocytes in vitro. Eight pairs of cross-bred male boar siblings were used. One brother in each pair became, at random, the test animal exposed to DEHP per os, three times a week, from 3 to 7 weeks of age while the other acted as the control, i.e., placebo-exposed. Semen was collected and frozen between 8 and 9 months of age and stored until spermatozoa were evaluated for their ability to in vitro penetrate in vitro-matured homologous oocytes post-thaw. Both the penetration rate and the number of spermatozoa per oocyte were considered within expected ranges for frozen boar semen of good quality. Penetration rate did not significantly differ (p greater than 0.05) between the groups with DEHP-exposed: 50%; control: 59%, which could be owing to a large variation between boars, and between replicates. The number of spermatozoa in the ooplasm was low and similar (p greater than 0.05) between the groups with DEHP-exposed: 1.5 and the control: 1.7. Under the conditions of the present experiment, pre-pubertal exposure to DEHP does not seem to cause a deleterious effect on the in vitro fertilizing ability of frozen spermatozoa post-puberty.
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