POST-THAW VIABILITY OF EUROPEAN BISON (BISON BONASUS) SEMEN FROZEN WITH EXTENDERS CONTAINING EGG YOLK OR LIPIDS OF PLANT ORIGIN AND EXAMINED WITH A HETEROLOGOUS IN VITRO FERTILIZATION ASSAY

2006 ◽  
Vol 37 (2) ◽  
pp. 116-125 ◽  
Author(s):  
S. S. Pérez-Garnelo ◽  
M. Oter ◽  
C. Borque ◽  
C. Talavera ◽  
M. Delclaux ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Egg Yolk ◽  
Bison Bison ◽  
European Bison ◽  
Plant Origin ◽  
Bison Bonasus ◽  
Vitro Fertilization

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.

scholarly journals 289EFFECT OF FERTILIZATION TIME OF PIG OOCYTES MATURED IN-VITRO BY BOAR SPERM STORED AT 4°C

2004 ◽  
Vol 16 (2) ◽  
pp. 264
Author(s):  
Y.J. Yi ◽  
M.Y. Kim ◽  
Y.J. Chang ◽  
D.I. Jin ◽  
C.S. Park
Keyword(s):  
In Vitro Fertilization ◽  
Room Temperature ◽  
Sperm Concentration ◽  
Egg Yolk ◽  
Penicillin G ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Boar Sperm ◽  
Sperm Penetration

The use of boar sperm stored at 4°C may be a useful tool for in vitro production of pig embryos. Therefore, this study was undertaken to investigate the effect of fertilization time of pig oocytes matured in-vitro by boar sperm. The sperm-rich fraction (30–60mL) was slowly cooled to room temperature (20–23°C) by 2h after collection. Semen was transferred into 15-mL tubes, centrifuged at room temperature for 10min at 800g, and the supernatant solution was poured off. The concentrated sperm was resuspended with 5mL of the LEN (11.0g lactose hydrate, 20mL egg yolk, 0.05g N-acetyl-D-glucosamine and 100mL distilled water) diluent to provide 1.0×109 spermmL−1 at room temperature. The resuspended semen was cooled in a refrigerator to 4°C. The medium used for oocyte maturation was TCM-199 supplemented with 26.19mM sodium bicarbonate, 0.9mM sodium pyruvate, 10μgmL−1 insulin, 2μgmL−1 vitamin B12, 25mM HEPES, 10μgmL−1 bovine apotransferrin, 150μM cysteamine, 10IUmL−1 PMSG, 10IUmL−1 hCG, 10ngmL−1 EGF, 0.4% BSA, 75μgmL−1 sodium penicillin G, 50μgmL−1 streptomycin sulfate and 10% pFF. After about 22h of maturation, oocytes were cultured without cysteamine and hormones for 22h at 38.5°C, 5% CO2 in air. Oocytes were inseminated with boar sperm stored at 4°C for 2 days after collection. Oocytes were coincubated for 1, 3, 6 and 9h in 500μL TBM fertilization media with 1×106mL−1 sperm concentration. Thereafter, oocytes were transferred into 500μL NCSU-23 culture medium containing 0.4% BSA for further culture of 6, 48 and 144h, fixed and stained for the evaluation of fertilization parameters and developmental ability. Data were analysed by ANOVA and Duncan’s multiple range test using the SAS program. The rates of sperm penetration and male pronuclear formation were higher in the fertilization times of 6 and 9h than in those of 1 and 3h. The percentage of polyspermic oocytes was highest in fertilization time of 9h compared with other incubation times. The rates of cleaved oocytes were higher in the fertilization times of 6 and 9h (85.0 and 84.6%) compared with those of 1 and 3h (61.1 and 76.8%). The percentage of blastocyst formation from the cleaved oocytes was highest in the fertilization time of 6h (33.6%) than in that of 1, 3 and 9h (11.4, 23.0 and 29.6%). Mean cell numbers per blastocyst were 32.9±3.3, 27.6±2.7, 26.3±2.2 and 24.4±1.8 in the fertilization times of 6, 9, 3 and 1h, respectively. In conclusion, we found out that boar sperm stored at 4°C could be used for in vitro fertilization of pig oocytes matured in vitro. Also, we recommend the coincubation time of 6h in 500μL TBM fertilization medium with 1×106mL−1 sperm concentration for in vitro fertilization of pig oocytes matured in vitro.

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.

131 EFFECT OF BREED AND FROZEN - THAWED RAM SEMEN ON IN VITRO FERTILIZATION AND OVINE EMBRYONIC DEVELOPMENT

2011 ◽  
Vol 23 (1) ◽  
pp. 170 ◽  
Author(s):  
K. C. Lehloenya ◽  
N. Mahoete ◽  
J. P. C. Greyling ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Developmental Stages ◽  
Germplasm Conservation ◽  
Egg Yolk ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Significant Difference

Ovine embryonic development was evaluated 8 days following in vitro fertilization, after using fresh or frozen–thawed Merino and indigenous (Pedi and Zulu) sheep semen. Semen used was collected twice weekly over a 3-month period with the aid of an electro-ejaculator. Following collection, semen samples were evaluated and semen with acceptable sperm motility and a percentage live sperm of 60% diluted with an egg yolk-based extender (Egg-Yolk Citrate). Semen samples were cryopreserved in straws with a programmable freezer to –130°C and then plunged into liquid nitrogen (–196°C) until used for IVF. Fresh and frozen–thawed semen was used to fertilize the matured oocytes in vitro. A total of 791 oocytes were fertilized using fresh semen and 802 oocytes fertilized using frozen–thawed semen. No significant differences were recorded between the fresh and frozen–thawed semen regarding the embryonic developmental stages. The performance of fresh and frozen–thawed semen followed the same trend, with the cleavage rate gradually declining with the progression in time and the embryonic developmental stage. The lowest developmental rate recorded was the occurrence of blastocyst formation, ranging between 0.4 ± 0.4% and 2.6 ± 1.0%. Regarding breed, no significant difference was observed from cleavage to the 2- to 4-cell stages. The use of fresh and frozen–thawed Zulu semen resulted in a significantly (P < 0.05) higher percentage of 8-cell development compared with the Pedi semen. However, the 8-cell embryonic stage recorded with the use of the Zulu ram semen (fresh and frozen–thawed), did not differ significantly from that of the Merino breed. No significant difference between the breeds regarding blastocyst formation was recorded. The overall cleavage rate, 2- to 4-cell, and blastocyst embryonic developmental stages following the use of fresh and frozen–thawed semen from the different rams were generally lower than those recorded by other researchers. The low blastocyst rates obtained warrant more research regarding the in vitro embryo production technique in order to improve the ovine blastocyst formation rate. The study was funded by the University of the Free State and conducted at the Germplasm Conservation and Reproduction Biotechnologies ARC.

365 COMPARISON OF IN VITRO FERTILIZING CAPACITY OF FROZEN - THAWED SEX-SORTED AND SEX-SORTED FROZEN - THAWED BULL SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 298 ◽  
Author(s):  
V. Malcolm ◽  
M. Marfil ◽  
M. Calvi ◽  
F. Rigali ◽  
M. Pugliese ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Egg Yolk ◽  
Reproductive Technologies ◽  
Development Rate ◽  
Vitro Fertilization ◽  
Final Treatment ◽  
Frozen Semen ◽  
Sexed Semen

Sperm sexing has become a world-wide technology, now available in many countries. The method has been incorporated into many reproductive technologies such as embryo production (Zhang et al. 2003 Theriogenology 60, 1657–1663), but sex-sorting is limited when bulls are located far from sorters or when only frozen semen is available. Previous studies on sexing frozen–thawed spermatozoa have been done in rams, which resulted in retention of the spermatozoan functional capacities (Hollinshead et al. 2004 Reproduction 127, 557–568). In vitro characteristics were assessed in bulls after sexing of thawed sperm (Hollinshead et al. 2004 Theriogenology 62, 958–968); however, the fertilizing capacity of frozen–thawed sex-sorted (FTSS) spermatozoa was not tested. The aim of the present study was to compare cleavage and embryo development rate among frozen–thawed (FT), sex-sorted frozen–thawed (SSFT), and FTSS bull spermatozoa. For FT, sperm were diluted to a final concentration of 60 × 106 sperm/mL, packaged in 0.5-mL straws, and frozen. In SSFT, spermatozoa were sex-sorted by flow cytometry following Schenk protocols (1999 Theriogenology 52, 1375–1391). Three × 106 spermatozoa were packaged into 0.25-mL straws and frozen. The final treatment (FTSS) consisted of thawing 6 to 10 frozen straws of 4 different bulls containing an average of 25 × 106 spermatozoa and centrifuging at 600g for 15 min at 21°C to extract cryodiluent. Spermatozoa were diluted and stained with Hoechst 33342 (stain concentration of 112.5 µM, the same used for SSFT treatment) following Schenk sexed-semen protocols (1999), sex-sorted by a flow cytometer, and collected in Tris-base extender containing 20% egg yolk. For each ejaculate frozen–thawed, SSFT and FTSS spermatozoa were prepared for oocyte in vitro fertilization. Also, semen from a bull routinely used as a control in the laboratory was added for a better comparison of results. Oocytes from a slaughterhouse were processed following standard in vitro fertilization procedures (Ferré 2002 Theriogenology 57, 664) 4 times for each bull, and comparison was made between treatments. Results were analyzed by ANOVA. No significant differences were observed among bulls (data not shown) (P &gt; 0.05). Although embryo development rate was statistically different between sexed and non-sexed groups (P &lt; 0.05), results showed that frozen–thawed bull spermatozoa can be sex-sorted and used for in vitro fertilization with comparable developmental rates comparable to those when frozen sexed semen is used (Table 1). This opens a new commercial window for cases where pre-selected sexed embryos from bulls that are not in AI centers are desired, also giving an opportunity for dead bulls. Nevertheless, since a large number of straws are necessary, further studies must be carried out to make this procedure more efficient and economically profitable. Table 1. Results of cleavage and embryo development rates between spermatozoa treatments

Improved fertilization rate in an in vitro fertilization program by egg yolk-treated sperm

1992 ◽  
Vol 58 (1) ◽  
pp. 197-198 ◽  
Author(s):  
Yona Barak ◽  
Ami Amit ◽  
Joseph B. Lessing ◽  
Gedalia Paz ◽  
Zwi T. Homonnai ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Egg Yolk ◽  
Fertilization Rate ◽  
Fertilization Program ◽  
Vitro Fertilization

scholarly journals “Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm’’

Cryobiology ◽  
2019 ◽  
Vol 91 ◽  
pp. 40-52 ◽  
Author(s):  
Halil Ozancan Arslan ◽  
Carolina Herrera ◽  
Eleni Malama ◽  
Mathias Siuda ◽  
Claus Leiding ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Egg Yolk ◽  
Fertilization Rate ◽  
Vitro Fertilization ◽  
Bull Sperm

scholarly journals Effect of Caffeine on Motility of Epididymis Spermatozoa of Bali Bull in Slaughterhouse Cibinong

2020 ◽  
Vol 4 (2) ◽  
pp. 44-47
Author(s):  
Oktora Dwi Putranti ◽  
Lovita Adriani ◽  
S. Soeparna ◽  
Tita Damayanti Lestari
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Motility ◽  
Recovery Rate ◽  
Egg Yolk ◽  
Computer Assisted ◽  
Vitro Fertilization ◽  
Randomized Design ◽  
Straight Line ◽  
Completely Randomized Design

Abattoir is the place to get meat but also a source of potential genetic sperm. Sperm from a slaughterhouse has low motility. Sperm motility can be improved by adding caffeine to the thinner before being used for fertilization. Caffeine is an alkaloid compound that can increase energy through a cAMP cycle. The method used is the testis of 12 cows Bali taken from a slaughterhouse Cibinong and do frozen sperm. Frozen sperm is analyzed using a computer-assisted sperm Analyzed (CASA) who had been treated caffeine 0, 2, 4, and 6 mg/ml. Fertility frozen epididymis sperm was tested using in vitro fertilization. Results were analyzed using a completely randomized design (CRD) with four treatments unidirectional pattern three repetitions. The results showed that the treatment with the addition of caffeine to the thinner of the yolk tris egg yolk epididymis sperm, there was no difference in motility, recovery rate, Curvilinear velocity (VCL), average path velocity (VAP), and straight-line velocity (VSL).

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