Prolonged storage of epididymal spermatozoa does not affect their capacity to fertilise in vitro-matured domestic cat (Felis catus) oocytes when using ICSI

2011 ◽  
Vol 23 (6) ◽  
pp. 818 ◽  
Author(s):  
J. Ringleb ◽  
R. Waurich ◽  
G. Wibbelt ◽  
W. J. Streich ◽  
K. Jewgenow
Keyword(s):  
Freeze Drying ◽  
Storage Conditions ◽  
Domestic Cat ◽  
Prolonged Storage ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Fertilisation Rate ◽  
Epididymal Spermatozoa ◽  
The Impact

The impact of different storage conditions of epididymal spermatozoa (including prolonged storage, cryopreservation and freeze-drying) on their fertilisation capacity was tested using intracytoplasmic sperm injection (ICSI). This kind of information is urgently needed when applying assisted reproductive technology to endangered felids in zoos. In particular, the utilisation of epididymal spermatozoa of castrated or deceased felids often requires time-consuming transportation and is therefore susceptible to loss of gamete quality. Sperm cells were stored at 4°C for up to 72 h followed by cryopreservation or freeze-drying. Thawed motile and immotile spermatozoa were used for ICSI and the embryo cleavage rate was assessed 36 h after injection. A significant impact on the fertilisation rate of oocytes could only be detected when using immotile thawed or rehydrated spermatozoa. Cryopreservation or storage at 4°C showed no influence. The simulation of transport conditions using domestic cat spermatozoa revealed that in vitro production of felid embryos with gametes from euthanised individuals is possible if testes are stored cool and arrive at the laboratory within 72 h. An essential prerequisite is the application of ICSI to achieve fertilisation even with single motile spermatozoa. Additional cryopreservation of spermatozoa after transportation is possible and will allow the establishment of a sperm bank for felids.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

scholarly journals Nanoparticles induce genetic, biochemical, and ultrastructure variations in Salvadora persica callus

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Manar S. Fouda ◽  
Mohamed H. Hendawey ◽  
Ghada A. Hegazi ◽  
Hayat M. Sharada ◽  
Nagwa I. El-Arabi ◽  
...  
Keyword(s):  
Folk Medicine ◽  
Antimicrobial Activities ◽  
In Vitro Production ◽  
Salvadora Persica ◽  
Zno Nps ◽  
Active Constituent ◽  
Benzyl Isothiocyanate ◽  
Stress Markers ◽  
The Impact

Abstract Background Salvadora persica is an endangered medicinal plant due to difficulties in its traditional propagation. It is rich in bioactive compounds that possess many pharmaceutical, antimicrobial activities and widely used in folk medicine. The current study aims at in vitro propagation of Salvadora persica and the application of different nanoparticles (NPs) to induce the synthesis of bioactive and secondary metabolites within the plant. The cellular and genetic responses to the application of different NPs were evaluated. Results The impact of nanoparticles NPs (ZnO, SiO2, and Fe3O4) on callus growth of Salvadora persica and the production of its active constituent benzyl isothiocyanate was examined, regarding some oxidative stress markers, antioxidant enzymes, and genetic variabilities. An encouraging impact of 0.5 mg/l ZnO NPs on benzyl isothiocyanate production was shown reaching up to 0.905 mg/g callus fresh weight in comparison to 0.539 mg/g in control callus. This was associated with decreasing hydrogen peroxide content and increasing superoxide dismutase and peroxidase activities. The deposition of the NPs on cellular organelles was detected using a transmission microscope. Fifteen Inter-Simple Sequence Repeats (ISSR) primers detected an overall, 79.1% polymorphism among different treatments. A reduction in genomic DNA template stability (GTS) was made and was more pronounced in higher doses of different NPs. Conclusion This study is a stepping stone in developing a productive protocol for in vitro production of benzyl isothiocyanate from Salvadora persica using NPs as a valuable anticancer compound.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

scholarly journals In vitro fertilizing potential of urethral and epididymal spermatozoa collected from domestic cats (Felis catus)

2017 ◽  
Vol 20 (1) ◽  
pp. 19-24 ◽  
Author(s):  
S. Prochowska ◽  
W. Niżański
Keyword(s):  
Comparative Analysis ◽  
In Vitro Fertilization ◽  
Felis Catus ◽  
Domestic Cats ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Commercial Medium ◽  
Blastocyst Rate ◽  
Epididymal Spermatozoa

Abstract The aim of this study was to provide a comparative analysis of in vitro fertilizing potential of frozen-thawed urethral and epididymal feline spermatozoa. Both types of semen were collected from 7 cats and cryopreserved in liquid nitrogen. To perform in vitro fertilization, both urethral and epididymal samples from the same individual were thawed and spermatozoa were co-incubated with in vitro matured cat oocytes. Obtained embryos were cultured in vitro for 7 days in a commercial medium. Cleavage rate, morula rate and blastocyst rate were calculated. Experiment was run in 10 replicates. The examined parameters showed no significant differences between urethral and epididymal spermatozoa (p>0.05). Cleavage rate and embryo’s development were highly variable between replicates, even for the different sperm samples collected from one individual. There was no significant correlation between fertilizing capacity of two types of spermatozoa collected from the same male. In this study we confirmed that cryopreserved urethral spermatozoa have equally good fertilizing potential as epididymal ones, and both can be successfully used for in vitro fertilization in cats with the use of commercial medium.

137 EFFECT OF POST-IVF DEVELOPMENTAL KINETICS ON SURVIVAL AND QUALITY OF VITRIFIED - WARMED DOMESTIC CAT BLASTOCYSTS

2007 ◽  
Vol 19 (1) ◽  
pp. 186
Author(s):  
T. Tsujioka ◽  
C. Otzdorff ◽  
J. Braun ◽  
S. Hochi
Keyword(s):  
Survival Rates ◽  
Cell Number ◽  
Domestic Cat ◽  
Minimum Volume ◽  
Cleavage Rate ◽  
Exact Probability ◽  
Cell Staining ◽  
Number Of Cells ◽  
Cooling Procedure

A limited number of reports are available for cryopreservation of IVF-derived cat blastocysts (Karja et al. 2006 Theriogenology 65, 415–423). In the present study, the IVF-derived domestic cat blastocysts exhibiting differences in their developmental kinetics were cryopreserved by vitrification. Pre- and post-warm blastocysts were examined for their cell number, and the survival rate was determined by in vitro culture of the post-warm embryos. Cumulus–oocyte complexes, recovered from the ovaries of post-pubertal queens, were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Oocytes were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. Newly formed blastocysts were harvested 6 and 7 days post-IVF and subjected to vitrification (Hochi et al. 2004 Theriogenology 61, 267–275) by a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice. The post-warm blastocysts were cultured for 24 h, and complete re-expansion was considered to be an indicator of survival. Embryos were differentially stained with Hoechst 33342 and propidium iodide to assess the total number of cells and the ICM ratio in the blastocysts. The cleavage rate of oocytes was 47.1% (314/666) and the percentage of oocytes developing to blastocysts on Day 6 and Day 7 was 9.8 and 9.5%, respectively (total blastocyst yield: 19.2%; 128/666). Post-warm in vitro survival rates of blastocysts harvested at Days 6 and 7 were 73.8% (31/42) and 66.7% (18/27), respectively (P = 0.59; exact probability test). There were no significant differences in the total number of cells and the ICM ratio between fresh control and vitrified blastocysts (P ≥ 0.05; ANOVA), although the ICM ratio of surviving Day 7 blastocysts was significantly smaller than that of fresh controls (18.9 vs. 28.9%; P = 0.03), as shown in Table 1. These results indicate that IVF-derived domestic cat blastocysts can survive vitrification by a minimum-volume cooling procedure without a reduction in the parameters studied, as long as the blastocysts are harvested on Day 6. Table 1. Differential cell staining of fresh and vitrified Day 6 and Day 7 cat blastocysts

248 IN VITRO PRODUCTION OF CATTLE × BUFFALO HYBRID EMBRYOS USING BOVINE OOCYTES AND AFRICAN BUFFALO (SYNCERUS CAFFER CAFFER) EPIDIDYMAL SPERM

2007 ◽  
Vol 19 (1) ◽  
pp. 240
Author(s):  
O. D. Owiny ◽  
D. M. Barry ◽  
M. Agaba ◽  
R. A. Godke
Keyword(s):  
Bison Bison ◽  
Abnormal Development ◽  
African Buffalo ◽  
Syncerus Caffer ◽  
Epididymal Sperm ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Bovine Oocytes

Interspecies hybridization of bovids occurs between domestic cattle and at least 3 other species: the American bison (Bison bison), yak (Bos grunniens), and banteng (Bos banteng). Birth of a cattle � buffalo hybrid was reported in Russia, but the report was never authenticated. Such hybrids could be important in improving livestock production and managing diseases that impede production in tropical Africa. We investigated hybridization between cattle and their closest African wild relative, the African buffalo (Syncerus caffer caffer). In an attempt to produce pre-implantation cattle � buffalo hybrid embryos in vitro, matured bovine oocytes were subjected to a standard IVF procedure with either homologous (n = 1166 oocytes) or heterologous (n = 1202 oocytes) buffalo epididymal sperm. After IVF, 67.2% of the oocytes inseminated with homologous sperm cleaved. In contrast, insemination with buffalo sperm resulted in a 4.6% cleavage rate. Cleavage was also slower in hybrids than in cattle embryos. Up to 52.2% of cleaved homologous embryos progressed to the morula stage compared with 12.7% for hybrids. No hybrid embryos developed beyond the 16-cell stage, whereas 40.1% of the cleaved bovine embryos developed to the blastocyst stage. Developmental anomalies such as polyspermy, uneven cleavage, vacuolization, and absence of nuclei in some blastomeres were common in the hybrid embryos. We conclude that interspecies fertilization of cattle oocytes with African buffalo sperm occurs in vitro and that the barrier to hybridization is in the early stages of embryonic development. Also, chromosomal disparity is the likely cause of fertilization abnormalities, abnormal development, and subsequent arrest, impairing the formation of pre-implantation hybrid embryos. Investigation into developmental abnormalities, including reciprocal hybridization and genetic studies of the hybrid embryos, are recommended.

60 IMPROVEMENT IN EFFICIENCY AND QUALITY OF CLONED AND PARTHENOGENETIC PORCINE EMBRYOS BY AMINO ACIDS

2007 ◽  
Vol 19 (1) ◽  
pp. 147
Author(s):  
H. T. Lee ◽  
J. M. Jang ◽  
S. H. Lee ◽  
M. K. Gupta
Keyword(s):  
Amino Acids ◽  
Culture Media ◽  
Cell Number ◽  
Control Group ◽  
In Vitro Production ◽  
Cleavage Rate ◽  
Fetal Fibroblasts ◽  
Blastocyst Rate

In vitro production of cloned porcine embryos by somatic cell nuclear transfer (SCNT) has become routine in several laboratories but the efficiency and quality of the resultant blastocysts remains sub-optimal. Cloned porcine blastocysts show low cell number, high fragmentation rate, and apoptosis which results in lower pregnancy rates upon embryo transfer. Earlier we reported that supplementation of culture media with amino acids benefit pre-implantation embryo development of in vivo- as well as in vitro-fertilized porcine embryos (Koo et al. 1997 Theriogenology 48, 791–802). This study evaluated how exogenous amino acids could affect pre-implantation development and quality of cloned or parthenogenetic porcine embryos. The effects of commercially available amino acids, referred to as Eagle's non-essential amino acids (NEAA), added or not added (control) to NCSU23 medium containing fatty acid-free BSA were studied. Oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro and parthenogenetically activated (PA) or nuclear-transferred with fetal fibroblasts (SCNT), as described earlier (Uhm et al. 2000 Mol. Reprod. Dev. 57, 331–337). At 168 h post-activation, blastocysts were harvested for assessment of embryo quality by TUNEL labeling, Hoechst 33342 staining, and gene expression analysis. Results showed that, in the PA group, the cleavage rate was not affected by the supplementation of NEAA. However, the blastocyst rate was significantly improved when NEAA was present in the medium compared to that of the control group (38.9 ± 0.3 vs. 27.5 ± 0.3%, respectively) throughout the culture period. The supplementation during the pre-compaction period alone gave better results than during the post-compaction period alone (59.5 ± 0.9 vs. 33.4 ± 0.3%, respectively). In the SCNT group, however, both cleavage (73.6 ± 0.2 vs. 64.2 ± 0.4%) and blastocyst rate (18.7 ± 0.2 vs. 13.8 ± 0.3%) were improved by NEAA supplementation. Furthermore, these blastocysts had higher hatching ability (30.0 ± 1.8 vs. 14.6 ± 4.9%) than those of control group (P < 0.05). Supplementation of NEAA also increased the mean nuclei number of PA-derived (76.1 ± 4.9 vs. 66.5 ± 3.3) as well as SCNT-derived (43.1 ± 2.6 vs. 31.8 ± 1.9) blastocysts and reduced the time during which blastocysts formed. TUNEL assay revealed that incidence of nuclear fragmentation and apotosis was reduced by NEAA. Real-time qRT-PCR for Bax and Bcl-XL transcripts revealed that the relative abundance of Bax was reduced while that of Bcl-XL was increased. These effects were more pronounced when NEAA was present during the pre-compaction period alone. Thus, our data suggest that NEAA improves the yield and quality of cloned porcine embryos by enhancing blastocyst expansion and positively modulating the total cell number and apoptosis. These data may have implications for understanding the nutritional needs of cloned porcine embryos produced in vitro and for optimizing the composition of culture media to support their development. This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.

In vitro production of GHB in blood and serum samples under various storage conditions

2012 ◽  
Vol 214 (1-3) ◽  
pp. 113-117 ◽  
Author(s):  
S.W. Zörntlein ◽  
A. Kopp ◽  
J. Becker ◽  
T.J. Kaufmann ◽  
J. Röhrich ◽  
...  
Keyword(s):  
Storage Conditions ◽  
In Vitro Production ◽  
Serum Samples ◽  
Vitro Production

In vitro production (IVP) of domestic cat embryos using a modified IVP bovine system

1999 ◽  
Vol 51 (1) ◽  
pp. 285 ◽  
Author(s):  
P Freistedt ◽  
M.B Stojkovic ◽  
T Bonhoeffer ◽  
J Braun ◽  
E Wolf
Keyword(s):  
Domestic Cat ◽  
In Vitro Production ◽  
Vitro Production

scholarly journals 91IMPACT OF PROPYLENE GLYCOL V. ETHYLENE GLYCOL ON OOCYTE MATURATION, SPINDLE INTEGRITY, FERTILIZATION AND EMBRYO DEVELOPMENT IN VITRO IN THE DOMESTIC CAT

2004 ◽  
Vol 16 (2) ◽  
pp. 167
Author(s):  
P. Comizzoli ◽  
D.E. Wildt ◽  
B.S. Pukazhenthi
Keyword(s):  
Ethylene Glycol ◽  
Propylene Glycol ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Domestic Cat ◽  
Hoechst 33342 ◽  
Immature Oocytes ◽  
Development In Vitro ◽  
The Impact

A thorough characterization of cryoprotectant (CPA) sensitivity is required to formulate a successful cryopreservation protocol for any biomaterial. The aim of this study was to characterize the toxic impact of various CPA types, concentrations, and exposure temperatures on the immature domestic cat oocyte. In Experiment 1, grade I immature oocytes (n=561) were exposed (30min; 25°C or 0°C) to 0M, 0.75M, 1.5M, or 3M of propylene glycol (PrOH) or ethylene glycol (EG) in PBS+20% fetal calf serum (v/v). After exposure, CPA was removed step-wise by subjecting oocytes to decreased CPA concentrations. Oocytes were cultured (30h; 38.5°C, 5% CO2) in IVM medium as reported previously (Wolfe and Wildt 1996 J. Reprod. Fertil. 106, 135–141). Oocytes were then fixed and stained to examine nuclear status (Hoechst 33342) and spindle integrity (FITC-labeled anti-α-tubulin antibodies; Sigma Chemical Co., St. Louis, MO). Experiment 2 was designed on the basis of Experiment 1 results to assess the impact of the spindle abnormalities on subsequent embryo development. Oocytes (n=776) were exposed to CPA conditions yielding optimal nuclear maturation with either high (0.75M or 3.0M PrOH or 1.5M EG at 25°C) or low (1.5M PrOH at 25°C) proportions of abnormal spindle. After IVM, oocytes were inseminated with thawed semen (5×105 motile sperm mL−1 ) in Ham’s F-10 (Irvine Scientific, St-Anna, CA). At 16h post-insemination, oocytes were fixed and stained (Hoechst 33342) to assess IVF success (pronuclear formation) or cultured in vitro for 7 days to assess embryo development. Data were analyzed by ANOVA and Tukey’s multiple comparison test. In Experiment 1, CPA treatment had no effect (NS) on meiotic progression to metaphase I. However, percentage of oocytes reaching metaphase II (MII) was reduced (P<0.05) in 3.0M PrOH at 0°C (29.3±8.3%; mean±SD), 3.0M EG at 25°C (33.7±8.9%), and 0°C (29.4±11.0%) compared to all other conditions examined (range, 52.0% to 62.0%). All CPA treatments also increased (P<0.05) spindle abnormalities at MII (range, 40.3% to 75.9%) compared to control (13.8±8.6%), except 1.5M PrOH at 25°C (20.7±10.1%). None of the CPA treatments in Experiment 2 influenced IVF success (range, 55% to 63%; NS). However, percentage of cleaved embryos was reduced (P<0.05) in 0.75M PrOH (32.1±4.1%), 1.5M EG (33.4±4.0%), and 3.0M PrOH (29.3±3.8%) compared to control (50.1±4.0%) or 1.5M PrOH (50.6±4.9%). Developmental competence (number of blastocysts relative to number of cleaved embryos) also was impaired (P<0.05) in 1.5M EG (16.5±7.4%) and 3.0M PrOH (14.9±7.8%) compared to the other conditions (range, 32.5% to 38.5%), including 1.5 PrOH at 25°C (32.5±7.8%). In conclusion, exposure of immature oocytes to 1.5M PrOH at 25°C does not adversely impact oocyte maturation, MII spindle, fertilization, or embryo development in vitro in the domestic cat.

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