Use of cryopreserved spermatozoa for caprine in vitro fertilization (IVF).

Proceedings Annual Conference of the International Embryo Transfer Society, Nice Acropolis, Nice, France, Jan., 1997

Effects of EGTA and cytochalasin-B during freezing and vitrification of immature and mature bovine and rhesus monkey (M. mulatta) oocytes.

Cryopreservation procedures have been shown to be extremely disruptive to the plasma membrane and intracellular organization of mammalian oocyte. Cytochalasin-B (Cyt-B) or EGT A may stabilize oocyte cytoskeletal elements prior to Cryopreservation and make the plasma membrane less rigid and more elastic to avoid injury during the osmotic stresses of freezing. Experiments were conducted to assess the morphological viability and developmental potential of bovine and rhesus monkey oocytes roter exposure to Cyt-B or EGTA prior to freezing and vitrification. ln the bovine experiments: The methods of oocyte isolation, in vitro maturation (IVM), in vitro fertilization (IVF) , and embryonic development (IVC) were as described elsewhere (Biol Reprod. 55:333-339, 1996). AIl data were analyzed by ANOV A and Chi-square using "StatMost" for Windows. ln an initial experiment, a group of ilnmature (GV) oocytes and oocytes roter IVM (ova) were randomly selected and pretreated for 5 min with EGTA (O.IM) or Cyt-B (Img/ml) without cryoprotectant (CPA), and another group were equilibrated for 15 min with the CPA (1.5 M ethylene glycol mixed with 0.5 M glycerol in PHS) roter pretreatment with EGTA or Cyt-B. AIl oocytes were diluted and/or washed and immediately used for IVM and/or IVF and IVC. No significant differences in cleavage (C) development and blastocyst (B) hatching among treatments in the GV oocytes group. Percentages of early C, morula (M), B, and expanded B (EB) development ranged 72-82%, 53-65%, 40-47% and 25-35% respectively. Ova treated with EGTA without CPA produced significantly (P<.05) higher C (31/41,75%), M (48.8%), B (40%) and EB (24.4%) than with Cyt-B. ln the slow freezing experiment, a total of 105 GV oocytes and 155 ova were exposed to (a) zero (b EGTA, or ~) Cyt-B before equilibration with CPA + 0.5 sucrose in PHS plus 15% Fetal bovine serum (PBS/FBS). Gametes were loaded into sterile cryovials, cooled at 2°Chnin to -7°C, seeded, cooled at 0.3°Chnin to -32°C, then plunged in LN2. They were thawed.in air (10 sec), then wann water (35°C), and diluted out of CPA in 3 steps (1, 0.5, 0.2 M sucrose). Three replicated showed that although many of the frozen-thawed oocytes/ova survived as judged by morphology (range 70-80%), their developmental potential was severely retarded. C ranged between 0% to 14.3%. ln the vitrification experiment, a total of 135 GV oocytes and 90 ova were grouped as above and vitrified in straws containing a mixture of 3.25M glycerol and 4.5M ethylene glycol plus 0.5 sucrose in PBS/FBS. Here, higher (P<.05) morphological survival rates (98%) foIlowed pretreatment with EGT A. However, the developmental potential was very low among aIl groups ranging between 0% to 17.7%. ln the monkey experiments: The methods of oocyte isolation, IVM and IVF was as described ( Theriogenology 43:362, 1995). A total of 72 GV oocytes and 42 ova were subjected to slow rate freezing and vitrification in groups as described for cow oocytes above. Pretreatment of both GV oocytes or ova with EGTA or Cyt-B prior to freezing or vitrification resulted in 77.6% morphological survival which was significantly (P<.OI) higher than for the control (50%). No differen?s in fertilization and development were detected mnong treatment. Per?ntages of C to 4-?Il stage ranged from 50-67%. Results suggest prefreeze treatment with EGTA and/or Cyt-B may be useful in enhancement of oocyte/ovum cryopreservation.Proceedings Annual Conference of the International Embryo Transfer Society, Nice Acropolis, Nice, France, Jan., 1997

Improvement in bovine embryo production in vitro by glutathione-containing culture media

Bovine oocytes were matured, fertilized, and cultured (TCM 199 with serum and co-culture) in vitro (IVMFC) with addition, during different phases of the procedure, of antioxidants: superoxide dismutase (SOD) and reduced glutathione (GSH). The addition of SOD (1,500 or 3,000 IU/ml) did not improve proportions of oocytes undergoing cleavage or the development of embryos to morula and blastocyst stages. The cleavage rates were significantly lower than in the control group (CTR 57.5%) when SOD was present during the insemination interval (IVF) or throughout the entire procedure (IVMFC). Thus when the lower concentration was present for IVF and IVMFC, 35.1% and 36.4% of inseminated oocytes cleaved (P < 0.01 compared to CTR) and cleavage results with the higher concentration during IVF and IVMFC were 38.5% and 29.2% (P < 0.025 and P < 0.001 compared to CTR, respectively). Significant improvements in proportions of oocytes undergoing cleavage (84.5% vs. 57.0%, P < 0.001) and morula/blastocyst development (33.3% vs. 13.9%, P < 0.005) were achieved when GSH (1 mM) was added to the culture medium. In a defined medium for culture (mSOF and BSA) the presence of SOD (3,000 IU/ml) was ineffective, but in a defined medium supplemented with GSH (1 mM) at day 6 postinsemination (i.e., when 90% of developing embryos were in 8-16 cell stages), development to the morula and blastocyst stages was supported for 35.5% of cultured oocytes (P < 0.005 compared to 19.2% for CTR). These data suggest that bovine embryos are sensitive to oxidative stress and that medium supplementation with the radical scavenger glutathione can improve embryo development in vitro

Vitrification of human embryos subjected to blastomere biopsy for preimplantation genetic screening produces higher survival and pregnancy rates than slow freezing. J Assist Reprod Genet,

Abstract Purpose Cryopreservation of blastocysts, especially those subjected to the trauma due to blastomere biopsy for the purposes of pre-implantation genetic screening (PGS), requires significant optimization. Laboratory and clinical outcomes were compared to determine the effect of two different cryopreservation techniques on the development of human pre-implantation embryos that underwent blastomere biopsy and blastocoel drainage prior to cryopreservation. Design Retrospective clinical study. Patient(s) Women who requested cryotransfer of supernumerary blastocysts were analyzed by FISH. Results The main outcome measures were post-thaw survival (SR), pregnancy (PR), and implantation (IR). The SR of slowly frozen blastocysts was 83% compared to 97% for vitrified blastocysts. In 160 cases where biopsied embryos were cryotransferred, the results for slowly frozen versus vitrified blastocysts were: SR (71% vs. 95%), PR (23% vs. 37%), and IR (26% vs. 36%, P&lt;0.05), respectively. Conclusion The results revealed that vitrified blastocysts provided higher SR, PR and IR as compared to slowly frozen counterparts

Adverse effect of cake collapse on the functional integrity of freeze-dried bull spermatozoa

Under optimal freeze-drying conditions, solutions exhibit a cake-like porous structure. However, if the solution temperature is higher than the glass transition temperature of the maximally freeze-concentrated phase (Tg′) during drying phase, the glassy matrix undergoes viscous flow, resulting in cake collapse. The purpose of the present study was to investigate the effect of cake collapse on the integrity of freeze-dried bull spermatozoa. In a preliminary experiment, factors affecting the Tg′ of conventional EGTA buffer (consisting of Tris–HCl, EGTA and NaCl) were investigated in order to establish the main experimental protocol because EGTA buffer Tg′ was too low (−45.0 °C) to suppress collapse. Modification of the EGTA buffer composition by complete removal of NaCl and addition of trehalose (mEGTA buffer) resulted in an increase of Tg′ up to −27.7 °C. In the main experiment, blastocyst yields after ooplasmic injection of freeze-dried sperm preserved in collapsed cakes (drying temperature: 0 or −15 °C) were significantly lower than those of sperm preserved in non-collapsed cake (drying temperature: −30 °C). In conclusion, freeze-dried cake collapse may be undesirable for maintaining sperm functions to support embryonic development, and can be inhibited by controlling both Tg′ of freeze-drying buffer and temperature during the drying phase.ArticleCRYOBIOLOGY. 68(3):354-360 (2014)journal articl

Aneuploidy Detection in Pigs Using Comparative Genomic Hybridization: From the Oocytes to Blastocysts

Data on the frequency of aneuploidy in farm animals are lacking and there is the need for a reliable technique which is capable of detecting all chromosomes simultaneously in a single cell. With the employment of comparative genomic hybridization coupled with the whole genome amplification technique, this study brings new information regarding the aneuploidy of individual chromosomes in pigs. Focus is directed on in vivo porcine blastocysts and late morulas, 4.7% of which were found to carry chromosomal abnormality. Further, ploidy abnormalities were examined using FISH in a sample of porcine embryos. True polyploidy was relatively rare (1.6%), whilst mixoploidy was presented in 46.8% of embryos, however it was restricted to only a small number of cells per embryo. The combined data indicates that aneuploidy is not a prevalent cause of embryo mortality in pigs

In vitro maturation and fertilization of prepubertal and pubertal black Bengal goat oocytes

Oocytes retrieval, in vitro maturation (IVM) and fertilization (IVF) efficiency are inevitable steps towards in vitro production of embryos. In the present study, these parameters were investigated in the ovaries of prepubertal (n = 31) and pubertal (n = 61) black Bengal goats obtained from a slaughterhouse. Nuclear maturation was evaluated upon aspiration and following IVM in TCM-199 (Earle's salt with L-glutamine and sodium bicarbonate) for 27 h at 39℃ under 5% CO2 in humidified air. The oocytes retrieval and efficiency (mean ± SD) per prepubertal and pubertal goats were 5.2 ± 0.6 and 6.8 ± 0.6, and 77.3 ± 0.1% and 80.5 ± 0.6%, respectively. Anaphase I - telophase I stages differed significantly (7.3 ± 0.8 vs. 2.6 ± 0.2, p < 0.05) between the two groups of goats. After IVM, the percentages of metaphase II were significantly higher (66.3 vs. 60.3, p < 0.05) in pubertal goats than in their prepubertal counterparts. The percentages of normal in vitro fertilization (IVF) in Fert-Tyrode's albumin lactate pyruvate of pubertal goat oocytes did not differ between Percoll and swim-up sperm separation methods (36.7 ± 0.9% vs. 32.7 ± 1.3%, p > 0.05). Furthermore, sperm capacitation by heparin alone or in combination with ionomycin did not lead to a significant increase in the normal fertilization rate (34.8 ± 1.7 vs. 32.2 ± 1.5%, respectively) in the oocytes of pubertal goats. In conclusion, the ovaries of pubertal black Bengal goats obtained from the slaughterhouse could be used for in vitro embryo production. However, further optimization of the IVM and IVF techniques are necessary for satisfactory in vitro embryo production

Successful Long-Term Preservation of Rat Sperm by Freeze-Drying

Background: Freeze-drying sperm has been developed as a new preservation method where liquid nitrogen is no longer necessary. An advantage of freeze-drying sperm is that it can be stored at 4uC and transported at room temperature. Although the successful freeze-drying of sperm has been reported in a number of animals, the possibility of long-term preservation using this method has not yet been studied. Methodology/Principal Findings: Offspring were obtained from oocytes fertilized with rat epididymal sperm freeze-dried using a solution containing 10 mM Tris and 1 mM EDTA adjusted to pH 8.0. Tolerance of testicular sperm to freeze-drying was increased by pre-treatment with diamide. Offspring with normal fertility were obtained from oocytes fertilized with freeze-dried epididymal sperm stored at 4uC for 5 years. Conclusions and Significance: Sperm with –SS – cross-linking in the thiol-disulfide of their protamine were highly tolerant to freeze-drying, and the fertility of freeze-dried sperm was maintained for 5 years without deterioration. This is the first report to demonstrate the successful freeze-drying of sperm using a new and simple method for long-term preservation

Single-cell whole-genome amplification technique impacts the accuracy of SNP microarray-based genotyping and copy number analyses

Methods of comprehensive microarray-based aneuploidy screening in single cells are rapidly emerging. Whole-genome amplification (WGA) remains a critical component for these methods to be successful. A number of commercially available WGA kits have been independently utilized in previous single-cell microarray studies. However, direct comparison of their performance on single cells has not been conducted. The present study demonstrates that among previously published methods, a single-cell GenomePlex WGA protocol provides the best combination of speed and accuracy for single nucleotide polymorphism microarray-based copy number (CN) analysis when compared with a REPLI-g- or GenomiPhi-based protocol. Alternatively, for applications that do not have constraints on turnaround time and that are directed at accurate genotyping rather than CN assignments, a REPLI-g-based protocol may provide the best solution