79 DEVELOPMENT TO TERM OF RAT ZYGOTES DERIVED FROM CRYOPRESERVED MATURE OOCYTES AND SPERM THROUGH INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 120
Author(s):  
N. Kashiwazaki ◽  
D. Sano ◽  
Y. Seita ◽  
S. Sugio ◽  
C. Suzukamo ◽  
...  
Keyword(s):  
Germ Cells ◽  
Intracytoplasmic Sperm Injection ◽  
Wistar Rats ◽  
Room Temperature ◽  
Developmental Competence ◽  
Key Technology ◽  
Experimental Animals ◽  
Epididymal Spermatozoa ◽  
Vitrification Solution ◽  
Physiological Research

The rat, as well as the mouse, is one of the most valuable experimental animals for biomedical and physiological research. There are numerous valuable mutant rats including transgenetic strains. Cryopreservation of rat oocytes and sperm as haploid germ cells is a key technology for banking the genetic resources efficiently. The aim of the present study was to examine survival of vitrified/warmed oocytes and developmental competence of resultant zygotes in the rat. Rats used in the present study were all Wistar rats. Epididymal spermatozoa were frozen as described previously (Seita et al. 2005 Reprod. Fertil. Dev. 18, 256). After thawing, spermatozoa were sonicated to obtain sperm heads for intracytoplasmic sperm injection (ICSI). Oocytes were collected from immature females superovulated with eCG and hCG. Oocytes were equilibrated in 7.5% (v/v) ethylene glycol (EG) + 7.5% (v/v) dimethylsulfoxide (DMSO) + 20% (v/v) FCS in PB1 for 5 min and then transferred into 15.0% EG (v/v) + 15.0% DMSO (v/v) + 20% FCS + 0.5 m sucrose in PB1 (vitrification solution) for 1 min at room temperature (22–24�C). During exposure to the vitrification solution, oocytes were loaded on a Cryotop� (Kitazato Supply Co., Tokyo, Japan). At warming, the film of Cryotop was directly immersed into PB1 containing 0.5 m sucrose and 20% FCS at 37.5�C. The warmed oocytes were washed three times and put into a HEPES-buffered (22 mm) modified R1ECM (310 mOsm) medium. The sperm heads were microinjected intracytoplasmically into the warmed oocytes. Then, presumptive zygotes were transferred surgically into the oviducts of recipient females (Day 0), and Caesarean section of the recipients was performed on Day 22. After vitrification and warming, 245 of 275 (88%) oocytes survived morphologically, 240 of the warmed oocytes were injected, and 156 oocytes (65%) were morphologically normal after the injection. To confirm development to term of zygotes derived from cryopreserved oocytes and sperm, 143 injected oocytes were transferred to 9 recipients, resulting in 3 pregnancies and the generation of one live pup. The results indicate that rat zygotes derived from cryopreserved oocytes and sperm through ICSI can develop to term, and full developmental competence can be preserved in rat oocytes after cryopreservation.

scholarly journals Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection

Reproduction ◽  
2004 ◽  
Vol 127 (2) ◽  
pp. 187-194 ◽  
Author(s):  
Y H Choi ◽  
L B Love ◽  
D D Varner ◽  
K Hinrichs
Keyword(s):  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Culture Media ◽  
Developmental Competence ◽  
Glucose Medium ◽  
Factors Affecting ◽  
Nuclear Maturation ◽  
High Glucose Medium

This study was conducted to evaluate the effect of initial cumulus morphology (expanded or compact) and duration of in vitro maturation (24, 30 or 42 h) on the developmental competence of equine oocytes after intracytoplasmic sperm injection (ICSI). The effect of manipulation temperature (room temperature vs 37 °C) at the time of ICSI and concentration of glucose (0.55 vs 5.5 mM) during embryo culture was also investigated. The nuclear maturation rates of expanded (Ex) oocytes were significantly (P < 0.001) higher than those of compact (Cp) oocytes at all maturation times (61–72 vs 23–25% respectively). Forty-eight hours after ICSI of mature Ex oocytes, the rate of cleavage with normal nuclei was significantly (P < 0.05) higher for oocytes matured for 24 h than for those matured for 30 or 42 h (73 vs 57–59% respectively). For Cp oocytes, the morphologic cleavage rates for oocytes matured for 30 h were significantly higher (P < 0.05) than for those matured for 24 or 42 h (86 vs 55–61% respectively). The overall proportion of embryos having more than four normal nuclei at 48 h culture was significantly higher (P < 0.05) for Cp than for Ex oocytes. Manipulation temperature did not affect development of embryos from Ex or Cp oocytes at 96 h after ICSI. Culture in high-glucose medium significantly increased morphologic cleavage of Cp, but not Ex, oocytes (P < 0.05). Embryos from Cp oocytes had a significantly higher average nucleus number after 96-h culture than did embryos from Ex oocytes. These data indicate that developmental competence differs between Ex and Cp equine oocytes, and is differentially affected by the duration of maturation and by composition of embryo culture media.

scholarly journals Fertility of mice receiving vitrified adult mouse ovaries

Reproduction ◽  
2006 ◽  
Vol 131 (4) ◽  
pp. 681-687 ◽  
Author(s):  
Toshio Hani ◽  
Takanori Tachibe ◽  
Saburo Shingai ◽  
Nobuo Kamada ◽  
Otoya Ueda ◽  
...  
Keyword(s):  
Germ Cells ◽  
Adult Mouse ◽  
Green Fluorescence Protein ◽  
Green Fluorescence ◽  
Experimental Animals ◽  
Immature Mouse ◽  
Estrous Cyclicity ◽  
Fluorescence Protein ◽  
High Degree ◽  
Old Mice

Cryopreservation of the ovaries is a useful technology for preservation of germ cells from experimental animals, because if the female founder is infertile or has mutated mitochondrial DNA, preservation of female germ cells is necessary. Although it is possible to cryopreserve immature mouse ovaries with a high degree of viability by vitrification with a mixture of several cryoprotectants, the viability of cryopreserved adult mouse ovaries is still unknown. Here, we investigated the viability of mouse ovaries at various ages after cryopreservation by vitrification techniques. Donor ovaries were collected from 10-day-, 4-week-, 10-week- and 7-month-old, female, nulliparous, green fluorescence protein (GFP)-transgenic mice and cryopreserved by vitrification. The vitrified-warmed ovaries were orthotopically transplanted to 4- or 10-week-old mice. GFP-positive pups were obtained in all experimental groups. In the 4-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 44%, 9%, 12% and 4% respectively. In the 10-week-old recipients, the percentages of GFP-positive pups among the total pups from recipients transplanted with ovaries of 10-day-, 4-week-, 10-week- and 7-month-old donors were 36%, 16%, 2% and 9% respectively. Furthermore, GFP-positive pups also were obtained from recipients transplanted with ovaries of donors without normal estrous cyclicity. Our results indicate that cryopreservation of mouse ovaries by vitrification is a useful method for the preservation of female germ cells from mice of various ages.

Involvement of Fas/FasL system in apoptotic signaling in testicular germ cells of male Wistar rats injected i.v. with microcystins

Toxicon ◽  
2009 ◽  
Vol 54 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Qian Xiong ◽  
Ping Xie ◽  
Huiying Li ◽  
Le Hao ◽  
Guangyu Li ◽  
...  
Keyword(s):  
Germ Cells ◽  
Wistar Rats ◽  
Apoptotic Signaling ◽  
Male Wistar Rats

Developmental competence of blastocysts produced following intracytoplasmic sperm injection (ICSI)

2002 ◽  
Vol 78 ◽  
pp. S9
Author(s):  
T Rahil ◽  
S.A Brody ◽  
M.T Butler ◽  
M.N Ho ◽  
M.F Rousseau ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Developmental Competence

scholarly journals ASSESSMENT OF IMMUNOMODULATORY POTENTIAL OF AN AYURVEDIC FORMULATION, NIROCIL SYRUP IN WISTAR RATS

2020 ◽  
pp. 154-158
Author(s):  
DINESH DILIP GHADIGAONKAR ◽  
MUKESH B CHAWDA ◽  
KAPIL S THAKUR
Keyword(s):  
Wistar Rats ◽  
Animal Ethics ◽  
Complete Blood Count ◽  
Treated Group ◽  
Control Group ◽  
Differential Leukocyte Count ◽  
Experimental Animals ◽  
Prior Approval ◽  
Hemagglutination Titer ◽  
Immunomodulatory Potential

Objective: This study aims to assess the immunomodulatory potential of an Ayurvedic formulation, Nirocil syrup, in Wistar rats. Methods: The experiments were conducted on Wistar rats with prior approval from the Institutional Animal Ethics Committee. Nirocil syrup was administered for 6 weeks to experimental animals. Parameters such as hemagglutination titer, histopathology of immunological organs, complete blood count, differential leukocyte count, and immunological paw edema were recorded and compared with controlled (untreated) and becozinc treated groups. Results: Nirocil treated group significantly enhanced the antibody titer in comparison to the control group. The results are supported by the increase in blood lymphocyte count and antigenic stimulation in immunological organs (spleen). Nirocil syrup enhanced antibody formation and suppressed the immunological edema in experimental animals. Conclusions: The study concludes that the Ayurvedic formulation Nirocil syrup has immunopotentiating activity.

Prolonged survival of mouse epididymal spermatozoa stored at room temperature

genesis ◽  
2001 ◽  
Vol 31 (4) ◽  
pp. 147-155 ◽  
Author(s):  
Masahiro Sato ◽  
Aki Ishikawa ◽  
Ayako Nagashima ◽  
Toshiteru Watanabe ◽  
Norihiro Tada ◽  
...  
Keyword(s):  
Room Temperature ◽  
Prolonged Survival ◽  
Epididymal Spermatozoa

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

Time-lapse imaging was used to establish the morphokinetics of equine embryo development to the blastocyst stage after invitro oocyte maturation (IVM), intracytoplasmic sperm injection (ICSI) and embryo culture, in oocytes held overnight at room temperature (22–27°C; standard conditions) before IVM. Embryos that developed to the blastocyst stage underwent precleavage cytoplasmic extrusion and cleavage to the 2-, 3- and 4-cell stages significantly earlier than did embryos that arrested in development. We then determined the rate of blastocyst formation after ICSI in oocytes held for 2 days at either 15°C or room temperature before IVM (15-2d and RT-2d treatment groups respectively). The blastocyst development rate was significantly higher in the 15-2d than in the RT-2d group (13% vs 0% respectively). The failure of blastocyst development in the RT-2d group precluded comparison of morphokinetics of blastocyst development between treatments. In any condition examined, development to the blastocyst stage was characterised by earlier cytoplasmic extrusion before cleavage, earlier cleavage to 2- and 4-cell stages and reduced duration at the 2-cell stage compared with non-competent embryos. In conclusion, this study presents morphokinetic parameters predictive of embryo development invitro to the blastocyst stage after ICSI in the horse. We conclude that time-lapse imaging allows increased precision for evaluating effects of different treatments on equine embryo development.

44 PRODUCTION OF LIVE PIGLETS AFTER CRYOPRESERVATION OF IMMATURE PORCINE OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 136
Author(s):  
T. Somfai ◽  
K. Kikuchi ◽  
K. Yoshioka ◽  
F. Tanihara ◽  
H. Kaneko ◽  
...  
Keyword(s):  
Polar Body ◽  
Petri Dish ◽  
Developmental Competence ◽  
Basic Medium ◽  
High Survival ◽  
Blastocyst Development ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Vitrification Solution

Development to term of vitrified porcine follicular oocytes is reported in the present study. Immature cumulus-oocyte complexes (COC) were collected from slaughtered prepubertal gilts and were vitrified according to our method published recently (Somfai et al. 2013 J. Reprod. Dev., in press). Briefly, after pretreatment with 7.5 μg mL–1 of cytochalasin B (CB) for 30 min in modified NCSU-37 (a basic medium, BM) at 38.5°C, groups of 88 to 121 COC were equilibrated in a mixture of 2% ethylene glycol (EG), 2% propylene glycol (PG), and 7.5 μg mL–1 CB for 13 to 15 min. Then, COC were washed in vitrification solution (17.5% EG, 17.5% PG, 5% polyvinyl pyrrolidone, and 0.3 M trehalose in BM) and then dropped with 2 μL of vitrification solution onto the surface of aluminum foil floating on liquid nitrogen (LN2). Microdroplets (each containing 10–25 COC) were transferred into cryotubes. After storage in LN2 for 2 to 4 weeks, the oocytes were warmed by dropping the microdroplets directly into 2.5 mL of warming solution (0.4 M trehalose in BM) kept in a 35-mm Petri dish on a 42°C hotplate for less than 1 min. Then, the warming dish was placed on a 38°C hotplate and COC were consecutively transferred for 1-min periods into BM containing 0.2, 0.1, or 0.05 M trehalose at 38°C. The COC were matured in vitro for 44 h using porcine oocyte medium (POM) supplemented with 10% follicular fluid (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Then, oocytes were denuded, and their live/dead status and nuclear maturation were determined by their morphology and the presence of the first polar body, respectively. To assess their developmental competence, vitrified and non-vitrified (control) oocytes were in vitro fertilized (IVF; Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) and then in vitro cultured in porcine zygote medium-5 (PZM-5; Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213). Blastocyst rates were recorded on Days 5, 6, and 7 of culture (Day 0 = the day of IVF). The experiment was replicated 4 times. Data were analysed with 1-way ANOVA and the Tukey test. The results revealed that 86.4% (364/424) of oocytes survived after vitrification, which was significantly lower (P < 0.05) than that of controls [100% (326/326)]. Live oocytes in vitrified and control groups did not differ statistically in terms of nuclear maturation (63.9 v. 65.3%). Blastocyst rates of surviving vitrified oocytes were significantly lower compared with controls on Days 5 (2.4 v. 12.7%), 6 (4.8 v. 17.6%), and 7 (5.6 v. 18.4%). To test their ability to develop to term, 16 and 27 blastocysts on Day 5 developing from vitrified COC were transferred into 2 recipients. Both recipients became pregnant and farrowed a total of 10 live piglets (4 and 6 piglets, respectively). These data demonstrate that large groups of immature porcine oocytes could be cryopreserved by this method showing high survival and maturation rates. Furthermore, despite a low rate of blastocyst development, transfer of Day-5 blastocysts generated from vitrified oocytes resulted in piglet production for the first time in the world. Partially supported by JSPS and HAS under the Japan-Hungary Research Cooperative Program.

225 DEVELOPMENTAL COMPETENCE AND QUALITY OF PIG EMBRYOS OBTAINED FROM STANDARD IN VITRO FERTILIZATION OR INTRACYTOPLASMIC SPERM INJECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 260 ◽  
Author(s):  
I. Grad-Mandryk ◽  
J. Kosenyuk ◽  
B. Gajda
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Quality Criteria ◽  
Cell Number ◽  
Total Cell ◽  
Developmental Competence ◽  
Tunel Staining ◽  
In Vitro Production ◽  
Total Cell Number ◽  
Vitro Fertilization

In vitro production of porcine embryos is still relatively inefficient. The main reasons for this limited performance are polyspermy after IVF and the poor developmental ability of obtained zygotes. Intracytoplasmic sperm injection (ICSI) is one possible solution to eliminate polyspermy. The aim of this study was to compare the developmental competence of pig zygotes, total cell number, and DNA fragmentation of pig blastocysts derived from IVF or ICSI. Cumulus–oocyte complexes were obtained by aspiration from antral follicles of ovaries collected from slaughtered gilts. The oocytes were then cultured in modified TC-199 medium to metaphase II for 42 h. Semen for IVF was incubated in modified capacitation medium (M199) for 1 h. The sperm fraction (1 × 106 cells mL–1) was introduced into droplets containing oocytes, and then gametes were co-incubated for 4 h in modified TC-199 medium. Intracytoplasmic sperm injection was performed using a mechanical micromanipulator (Research Instruments Limited, Cornwall, UK). Micromanipulation was carried out in modified NCSU-37 medium. The tails of spermatozoa were broken, and then single spermatozoa were aspirated into the injection pipette. The oocyte was fixed by a holding pipette, and the sperm head was then introduced into the oocyte cytoplasm. Presumptive zygotes were cultured in vitro for 144 h in NCSU-23 medium. The embryo quality criteria were developmental competence (morula and blastocyst rates), total cell number per blastocyst, and degree of apoptosis assessed by TUNEL staining. Data were analysed by chi-squared test. The experiment was performed on 136 zygotes (6 replicates) obtained after IVF and 83 zygotes (4 replicates) obtained after ICSI. Percentages of embryos developed to the morula and blastocyst stages were 42.3 ± 6.1 and 28.8 ± 4.7 after IVF, respectively, and 51.7 ± 15.4 and 34.5 ± 18.9 after ICSI, respectively (no differences were observed). Significant differences were noticed in total number of cells per blastocyst between embryos after IVF and ICSI (33.7 ± 5.39 v. 22.8 ± 3.22; P < 0.01). However, there was no difference in the degree of apoptosis between IVF and ICSI embryos (5.14 ± 3.49 and 6.14 ± 4.88, respectively). Our preliminary studies demonstrated a higher proportion of cell numbers in IVF-derived embryos compared with those produced by ICSI, but the developmental competence and degree of apoptosis, as evaluated by the TUNEL method, in both groups were comparable. This study was funded by project N N311 516140 by the NCN, Poland.

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