scholarly journals 106EVALUATION OF ADDITION OF REDUCED GLUTATHIONE TO COOLING MEDIUM ON IN VITRO FERTILITY AND ACROSOME REACTION IN BOAR SPERMATOZOA

2004 ◽  
Vol 16 (2) ◽  
pp. 175
Author(s):  
C. Matás ◽  
J. Gadea ◽  
F. García-Vázquez ◽  
J.C. Gardón ◽  
S. Cánovas
Keyword(s):  
In Vitro Fertilization ◽  
Acrosome Reaction ◽  
Reduced Glutathione ◽  
Membrane Integrity ◽  
Calcium Ionophore ◽  
Egg Yolk ◽  
Ros Generation ◽  
Vitro Fertilization ◽  
Cooling Medium

The process of cooling to 5°C prior to freezing produces physical and chemical stress on the sperm membrane associated with oxidative stress and reactive oxygen species (ROS) generation that reduces sperm viability and fertilizing ability. The addition of antioxidants to cooling medium could prevent the formation of ROS and improve the seminal parameters. The aim of these experiments was to investigate the effects of addition of reduced glutathione (GSH) to cooling extenders on (1) plasma membrane integrity, (2) acrosome reaction induction by ionophore A 23187 or progesterone, and (3) in vitro fertilization. Ejaculate-rich fractions from three mature pietrain boars were diluted in Beltsville Thaw Solution (BTS) extender and cooled to 15°C over 2h (group C). Thereafter, sperm were centrifuged and diluted in lactose/egg-yolk extender with 0mM (group 0), 1mM (group 1) or 5mM (group 5) of GSH, cooled to 5°C over 2h. The acrosome reaction was then induced by 1μM calcium ionophore or 10μM progesterone in TALP medium and incubated in 5% CO2, 38.5°C for 30 or 45min, respectively. Membrane integrity was evaluated by propidium iodide, and acrosomal status was monitored by means of FITC-labeled peanut agglutinin. Finally, in vitro fertilization was performed with these four spermatozoa groups as described previously (Matás et al. 2003 Reproduction 125, 133–141). ANOVA analysis revealed that the addition of GSH had no effect on the membrane integrity (ranged 58.8 to 66.9) or acrosome reaction induction (ranged 24.3 to 28.2, and 55.7 to 41.4 for progesterone and calcium ionophore, respectively). However, the results of the penetration assay revealed that the cooling affected the penetration rate and the number of sperm per oocyte (Table 1), and this assay is better than the others to predict changes in the spermatozoa functionality (Gadea J and Matás C 2000 Theriogenology 54, 1343–1357). In conclusion, the cooling process affects the in vitro fertilization, but the addition of GSH to the medium did not influence the parameters studied. Supported by AGL2000-0485-CO2-01. Table 1 Homologous in vitro penetration

297 BIRTH OF LIVE RATS THROUGH IN VITRO FERTILIZATION USING CRYOPRESERVED SPERMATOZOA: SPERM FERTILITY IMPROVED BY FREEZING AT - 150°C

2006 ◽  
Vol 18 (2) ◽  
pp. 256
Author(s):  
Y. Seita ◽  
Y. Okuda ◽  
A. Takizawa ◽  
S. Hisamatu ◽  
T. Inomata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Plasma Membrane Integrity ◽  
Freezing Temperatures

We previously reported that damages to spermatozoa by cold shock can be avoided by cooling slowly at 0.5�C/min to 5.0�C (Seita et al. 2005 Reprod. Fertil. Dev. 17, 277-278). The objective of the present study was to develop an in vitro fertilization (IVF) system with frozen-thawed rat spermatozoa for more efficient reproduction of live offspring. We examined the effect of freezing temperatures (cooling 5.0�C to pre-plunging) on post-thaw sperm motility, plasma membrane integrity, and fertility in vitro. Epididymal spermatozoa of Wistar rats were collected in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM (Nova Animal Sales, Inc., Scituate, MA, USA) at room temperature. Samples were loaded into 0.25-mL straws and cooled to 5.0�C at 0.5�C/min in a programmable freezer. Next, the samples were exposed to liquid nitrogen (LN) vapor at various freezing temperatures (-120�C, -150�C or -180�C) above the LN level for 15 min and then plunged into LN. Straws were thawed in a 37�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL in a droplet of 200 �L of R1ECM and then pre-incubated for 5 h. Ovulated oocytes were introduced into the droplet and co-cultured for 10 h. The oocytes were denuded, fixed, and/or examined for two pronuclei (2PN) formation microscopically. The denuded oocytes, which were fertilized with spermatozoa frozen at -150�C and were microscopically confirmed to have 2PN formation, were transferred to pseudo-pregnant recipient females. IVF was also performed by the same method using fresh spermatozoa as the control. Differences in the sperm motility and plasma membrane integrity were analyzed by ANOVA, and the IVF data were analyzed by chi-square test. At 2 h after thawing the motility of spermatozoa frozen at -150�C was significantly higher than that of spermatozoa frozen at -180�C (19.8% and 11.1%; P < 0.05), although the sperm plasma membrane integrity was not significantly different among different freezing temperatures, -120�C, -150�C, and -180�C (18.2%, 23.5%, and 17.9%; P > 0.05). The percentage of oocytes with 2PN was not significantly different between the -150�C frozen and the control (fresh spermatozoa) groups [59% (131/221) and 62% (155/251); P > 0.05], although that of frozen spermatozoa at -120�C and -180�C [20% (38/188) and 23% (35/153)] were significantly lower than that of frozen spermatozoa at -150�C (P < 0.05). A total of 168 putative fertilized zygotes with 2PN were transferred to eleven recipients, and 87 live young were born. In conclusion, our results indicated that post-thaw motility of cryopreserved rat spermatozoa was improved by using a suitable cooling protocol, and the IVF system used in the present study would effectively produce offspring from the cryopreserved epididymal rat spermatozoa. To our knowledge, this procedure is the first successful production of live offspring from cryopreserved rat spermatozoa through in vitro fertilization.

Dynamics of human sperm acrosome reaction: relation with in vitro fertilization

1991 ◽  
Vol 55 (5) ◽  
pp. 994-999 ◽  
Author(s):  
Patrick Fénichel ◽  
Michèle Donzeau ◽  
Dariush Farahifar ◽  
Bernard Basteris ◽  
Noël Ayraud ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Acrosome Reaction ◽  
Human Sperm ◽  
Vitro Fertilization ◽  
Sperm Acrosome Reaction ◽  
Sperm Acrosome

279 LIVE RAT OFFSPRING FROM CRYOPRESERVED EJACULATED SPERMATOZOA THROUGH IN VITRO FERTILIZATION

2006 ◽  
Vol 18 (2) ◽  
pp. 247
Author(s):  
N. Kashiwazaki ◽  
Y. Seita ◽  
M. Shino ◽  
S. Hisamatsu ◽  
T. Inomata
Keyword(s):  
In Vitro Fertilization ◽  
Egg Yolk ◽  
Fertilization Rate ◽  
Efficient Production ◽  
Reliable System ◽  
Vitro Fertilization ◽  
Laboratory Rats ◽  
Pregnant Females ◽  
Cervical Dislocation

We have previously reported successful cryopreservation of epididymal rat spermatozoa (Nakatsukasa et al. 2001 Reproduction 122, 463). However, the procedure for cryopreservation of rat spermatozoa has a disadvantage; a male has to be euthanized for collection of spermatozoa from its epididymides. Obtaining ejaculated spermatozoa repeatedly from the same male could be useful for cryopreservation of invaluable spermatozoa which carry mutations including transgenes. The objective of the present study was to develop a reliable system for cryopreservation of ejaculated rat spermatozoa and efficient production of offspring from the cryopreserved spermatozoa. Matured Wistar females were mated with three males of the same strain, and killed by cervical dislocation after formation of vaginal plugs. The uteri of mated females were excised and flushed with freezing medium containing 23.0% egg yolk, 8.0% lactose, and 0.7% Equex STM to recover ejaculated spermatozoa. The semen samples were loaded into 0.25-mL straws. The straws were cooled to 5.0�C at 0.5�C/min in a programmable freezer and then exposed to liquid nitrogen (LN) vapor at 4 cm (-150�C) above the LN level for 15 min. The straws were then plunged into LN and stored for at least a week. The straws were thawed in a 37.0�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL into a 200-�L droplet of R1ECM and then pre-incubated for 5 h. Ovulated oocytes collected from superovulated females were introduced into the droplet and co-cultured for 10 h for in vitro fertilization (IVF). The oocytes were denuded and examined for the presence of two pronuclei (2PN) microscopically. The denuded oocytes with 2PN were transferred into the oviducts of pseudo-pregnant females. The rates of sperm motility at recovery, post-thaw, and the initiation of IVF (after pre-incubation) were 57 � 6%, 24 � 5%, and 18 � 3%, respectively. After co-culture, 46 (14%) of the total 329 co-cultured oocytes were confirmed to contain 2PN. A total of the 44 putative zygotes were transferred to five recipients, and a total of 21 live young (48%) were born from all of the transferred recipients. We were able to produce zygotes and offspring derived from cryopreserved ejaculated spermatozoa of all three males used in the present study. In conclusion, the cryopreservation system for ejaculated rat spermatozoa used in the present study is a workable protocol for banking of valuable genetic resources of laboratory rats. Further studies on the IVF procedure with cryopreserved ejaculated spermatozoa in the rat are needed to improve the fertilization rate.

250 DISTRIBUTION OF HISTONE macroH2A1 IN BOVINE PRE-IMPLANTATION EMBRYOS DERIVED FROM PARTHENOGENETIC ACTIVATION AND IN VITRO FERTILIZATION

2006 ◽  
Vol 18 (2) ◽  
pp. 232
Author(s):  
C. Kim ◽  
Y. Ma ◽  
C.-C. Chang ◽  
T. Rasmussen ◽  
X. Yang ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Laser Scanning ◽  
Expression Patterns ◽  
Combined Treatment ◽  
Calcium Ionophore ◽  
Ionophore A23187 ◽  
Bovine Embryos ◽  
Vitro Fertilization ◽  
Stage Of Development

It is known that heterochromatin is characterized by the presence of the histone variant of macroH2A1. MacroH2A1 is a core variant histone with a hybrid structure consisting of a domain that resembles a full-length histone H2A1 followed by a large nonhistone domain. We have previously studied the dynamic changes of macroH2A1 accumulation during the pre-implantation developmental period in the mouse. In the present study, we investigated the distribution of microH2A1 in bovine metaphase II oocytes and pre-implantation embryos at 2-, 4-, 8-, 16-cell, and morula stages as well as blastocysts harvested at Days 8, 9, 10, 11, 12, and 13 following activation and in vitro fertilization (IVF). To generate parthenotes, denuded and in vitro-matured oocytes were activated using a combined treatment of calcium ionophore A23187, cycloheximide (CHX), and 6-dimethylaminopurine (6-DMAP). Five oocytes and pre-implantation embryos at each stage of development were used to follow the development expression pattern of microH2A1 by immunocytochemistry. The cross-reactivity of the primary antibody against mouse microH2A1 was verified by Western blot analysis with bovine fibroblasts. Another staining control included immunostaining with antibody against histone molecules. The stained embryos were observed by laser-scanning confocal microscopy and epiflourescence microscopy. No microH2A1 stain was observed in bovine oocytes or pre-implantation embryos up to the expanded blastocyst stages. In the IVF group, the macroH2A1 was first found in elongated blastocysts (Day 11) after hatching. We observed different expression patterns of macroH2A1 in activated vs. IVF bovine embryos. In the parthenote group, we failed to find robust expression even when embryos were cultured for 13 days. Moreover, the pattern of macroH2A1 expression in bovine embryos was different fromn that in the mouse, in which the onset of macroH2A1 expression occurred by the 16-cell morula stage. These results suggest species differences in the establishment of epigenetic signals. This work was supported by grants from USDA to X. Y. and X. C. T.

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