Comparison of culture media for their effects on development of caprine IVF embryos using fresh and cryopreserved semen

2015 ◽  
Author(s):  
Ajay Pratap Singh ◽  
Dharmendra Kumar ◽  
Gopalakrishna R ◽  
Rakesh Ranjan ◽  
Saurabh K Pandey ◽  
...  
Keyword(s):  
Culture Media ◽  
Developmental Patterns ◽  
Fertilization Rates ◽  
Developmental Rates ◽  
Fresh Semen

The aim of the present study was to compare the effects of two culture media on in vitro fertilized embryo produced by fresh and cryopreserved buck semen respectively. The in vitro fertilized embryos from both semen sources were cultured separately in the two media groups viz., Research Vitro Cleave-BSA (RVCL-BSA) and RVCL-Blast-BSA media. Under RVCL–BSA medium, the fresh semen produced higher rates of cleavage (68.94±1.86 vs 65.62±2.34) and significantly higher (P 0.05) morula (27.89±1.18 vs 23.84±2.23), blastocysts (15.26±0.67 vs 13.07±1.17) and blastomeric counts (232.65±14.26 vs 218.34 ± 10.26) than cryopreserved semen. The similar developmental patterns were exhibited in RVCL-Blast-BSA medium in which the fresh semen showed significantly higher (P?0.05) rates of morula (38.94 ± 2.37 vs 31.87±2.10), blastocysts (29.89 ± 2.02 vs 25.31 ± 1.78) and blastomeric count (272.45 ± 16.54 vs 248.85 ± 14.38). This study concludes that the RVCL-Blast-BSA medium produced significantly higher (P?0.05) embryonic developmental rates in comparison to RVCL– BSA medium irrespective of semen sources and the fresh semen afforded better fertilization rates than cryopreserved semen in both culture groups.

Assessment of binder of sperm protein 1 (BSP1) and heparin effects on in vitro capacitation and fertilization of bovine ejaculated and epididymal sperm

Zygote ◽  
2020 ◽  
Vol 28 (6) ◽  
pp. 489-494
Author(s):  
Paula Rodríguez-Villamil ◽  
Daiane Mentz ◽  
Felipe Ledur Ongaratto ◽  
Luis Henrique Aguiar ◽  
Jose Luiz Rodrigues ◽  
...  
Keyword(s):  
Control Group ◽  
Sperm Capacitation ◽  
Epididymal Sperm ◽  
Sperm Protein ◽  
Fertilization Rates ◽  
Bovine Sperm ◽  
Development Rates ◽  
Developmental Rates

SummaryThe present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen–thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

98 EXPERIMENTAL TRANSFER OF BOVINE IVF-DERIVED 32-CELL STAGE EMBRYOS INTO THE UTERUS: ENVIRONMENTAL EFFECTS ON DEVELOPMENTAL CHARACTERISTICS

2016 ◽  
Vol 28 (2) ◽  
pp. 179
Author(s):  
M. Hoelker ◽  
D. Salilew-Wondim ◽  
F. Rings ◽  
D. Tesfaye ◽  
K. Schellander
Keyword(s):  
Culture Media ◽  
Uterine Cavity ◽  
Blastocyst Stage ◽  
Considerable Proportion ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage ◽  
Developmental Rates ◽  
Uterine Environment

Usually, in vitro-produced bovine embryos are cultured in vitro in static culture systems for 7 to 9 days in media composed according the oviducal fluid although it is well accepted that around Day 4.5–5 the bovine embryo enters the uterine cavity, providing environmental conditions different from the oviduct. Therefore, one has to raise the question whether changing culture media properties after Day 5 of culture could have beneficial effects on early development of bovine embryos. To answer that question, we transferred bovine IVF derived 32-cell stage embryos into the uterine cavity of synchronized recipients. All embryos had been matured and fertilized under routine standard conditions and were cultured in synthetic oviducal fluid supplemented with essential and nonessential amino acids (SOFaa) supplemented with either 0.3% fatty acid free bovine serum albumin (BSAfaf/Uterus) or 10% serum (serum/uterus) at 38.5°C, 5% O2, and 5% CO2 in humidified air prior transfer into the uterine environment, allowing further development to the blastocyst stage within the physiological environment prior recollection at Day 7 by routine uterine flushing followed by comparison with statically in vitro-developed embryos cultured in media supplemented with serum (serum/serum group) or BSAfaf (BSAfaf/BSAfaf group). All in all, a total of 1031 in vitro-derived 32-cell stage embryos were transferred to 21 synchronized Simmental recipient heifers. Of these, a total of 680 embryos (66%) could be recollected at Day 7. Embryos of the serum/serum group reached a higher blastocyst rate compared with embryos of the BSAfaf/BSAfaf group (68% v. 41%; P < 0.05, ANOVA, Tukey test), whereas the developmental rate to the blastocyst stage did not differ after 9 days of in vitro culture, indicating higher developmental kinetics of bovine 32-cell stage embryos when culture media is supplemented with serum. Moreover, embryos of the serum/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos of the serum/serum group (12.9% v. 68.4%). Likewise, embryos in the BSAfaf/uterus group reached significantly lower developmental rates to the blastocyst stage until Day 7 compared with embryos in the BSAfaf/BSAfaf group (16.0% v. 40.1%). When allowed to develop for additional 48h in vitro, developmental rates to the blastocyst stage at Day 9 were still higher in BSAfaf/BSAfaf treatment compared with the BSAfaf/uterus treatment (91.4% v. 74.4%) and the serum/serum treatment compared with the serum/uterus treatment (92.5% v. 56.0%). Taken together, the results of our study demonstrate that uterine transfer of bovine 32-cell stage embryos results in reduction of developmental kinetics as well as lower developmental rates compared with embryos statically cultured in vitro. That might indicate, that a considerable proportion of bovine 32-cell stage embryos might not be able to adapt to the uterine environment.

Liquid storage of ram semen in the absence or presence of some antioxidants

1996 ◽  
Vol 8 (6) ◽  
pp. 1013 ◽  
Author(s):  
WM Maxwell ◽  
T Stojanov
Keyword(s):  
Superoxide Dismutase ◽  
Glutathione Peroxidase ◽  
Intrauterine Insemination ◽  
Fertilization Rates ◽  
Liquid Storage ◽  
Semen Storage ◽  
Acrosome Integrity ◽  
Improvement In Survival ◽  
Fresh Semen

The antioxidants superoxide dismutase (SOD), catalase (CAT), cytochrome c (CHc) and glutathione peroxidase (GP) were added at various concentrations to Tris-glucose-yolk diluent (TGY), and their effects on motility, acrosome integrity and fertility of ram spermatozoa were assessed after extension and liquid storage. All the antioxidants improved the motility and acrosome integrity of spermatozoa, and a combination of SOD and CAT had an additive effect on the survival of spermatozoa stored at 5 degrees C but not at 25 degrees C. There was a linear improvement in survival of spermatozoa with increasing dose of antioxidants except for CAT for which doses higher than 200 U mL-1 were toxic. The proportion of oocytes fertilized in vitro declined with time of semen storage (P < 0.001), and was better for semen diluted with TGY containing SOD or CAT than TGY without antioxidants when stored for 7 days (116/246, 47% v. 25/79, 32%; P < 0.05) but not for 14 days (23/174, 13% v. 8/66, 12%). Fertilization rates were unaffected by the presence or absence in the diluent of CHc or GP. The proportions of ewes with fertilized ova and of recovered ova fertilized were better after insemination with semen diluted in TGY containing SOD and CAT than TGY without antioxidants when stored for 14 days (9/18, 50% and 20/40, 50% v. 2/13, 15% and 5/32, 16%; P < 0.05) but not for 7 days (9/20, 45% and 16/48, 33% v. 8/16, 50% and 24/41, 59%). Pregnancy rates were better after intrauterine insemination of ewes with fresh semen than stored semen (11/18, 61% v. 21/75, 28%; P < 0.01), and with semen stored in TGY containing SOD and CAT than in TGY without antioxidants (15/37, 41% v. 6/38, 16%; P < 0.05).

scholarly journals 316 SUPPLEMENTAL CYSTEINE PRESENCE DURING THE DECONDENSATION OF SPERM CHROMATIN IMPROVES FERTILIZATION AND BLASTOCYST FORMATION AFTER INTRACYTOPLASMIC SPERM INJECTION IN PIGS

2005 ◽  
Vol 17 (2) ◽  
pp. 308
Author(s):  
M. Katayama ◽  
T. Cantley ◽  
A. Rieke ◽  
B. Day
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Morphological Changes ◽  
Formation Rate ◽  
Sperm Chromatin ◽  
Blastocyst Formation ◽  
Fertilization Rates ◽  
Duration Time

The effect of a cysteine supplement in culture media for oocytes matured in vitro after intracytoplasmic sperm injection (ICSI) on fertilization and embryo development were examined. In the first experiment, sperm injected oocytes were cultured in NCSU23 (control) or NCSU23 supplemented with 0.57–3.71 mM cysteine (0.57–3.71 Cys) for 12 h after ICSI, and then fixed to observe pronuclear formation. In the second experiment, to examine the appropriate duration time of cysteine supplement to support fertilization, sperm-injected oocytes were transferred into NCSU23 following culture in NCSU23 supplemented with 1.71 mM cysteine for 1, 2, 3, 4, 5, 6, or 9 h after ICSI, and then fixed at 12 h. At the same time, morphological changes of sperm heads in oocytes cultured in NCSU23 (1.71 Cys) were observed. In the third experiment, to examine the developmental ability of ICSI embryos fertilized in NCSU23 (1.71 Cys), sperm injected oocytes were cultured under the following conditions for a total of 168 h; NCSU23 (control), NCSU23 (1.71 Cys) for 3 h followed by transfer into NCSU23 (1.71 Cys-3 h), NCSU23 (1.71 Cys) for 12 h followed by transfer in NCSU23 (1.71 Cys-12 h), or NCSU23 (1.71 Cys) (1.71 Cys). Data were pooled from at least five replicates. Values in each replicate were analyzed using one-way ANOVA. Significance of differences was assessed by Student's t-test. Culture with several concentrations of cysteine for 12 h showed that 1.71–3.71 Cys significantly (P < 0.05) increased fertilization rates above controls or 0.57 Cys (56–60%, 35%, or 48%, respectively). Culture for several duration times with 1.71 Cys showed that fertilization rates increased as the duration time increased to 3 h which was significantly (P < 0.05) higher than controls (68% and 34%, respectively), and culture times of greater than 3 h did not increase fertilization rates (58–68%). At 3 h, 59% of oocytes cultured in NCSU23 (1.71 Cys) had decondensed sperm heads and 16% of those had enlarged sperm heads. At 6 h, 50% of oocytes cultured in NCSU23 (1.71 Cys) had male pronuclei. Blastocyst formation rate in 1.71 Cys-3 h was 29% which was higher than for controls (20%). On the other hand, 1.71 Cys-12 h cultures showed low blastocyst formation rates, and continuous culture in NCSU23 (1.71 Cys) for 168 h (1.71 Cys) significantly (P < 0.05) decreased blastocyst rates (16% and 7%, respectively). We found that the supplement of 1.71 mM cysteine to NCSU23 for culture of oocytes after ICSI improved fertilization rates. However, the presence of 1.71 mM cysteine for 12 h or longer after ICSI had adverse effects on embryo development. Since 1.71 mM cysteine supplement for 3 h after ICSI improved blastocyst formation with the same fertilization rates as when supplemented for 12 h, the presence of cysteine only during the decondensation of sperm chromatin was found to be associated with the improvement of fertilization and also the promotion of blastocyst formation.

191 ALLEVIATION OF THE TWO-CELL BLOCK OF ICR, ddY, AND AKR MOUSE EMBRYOS BY BOVINE SERUM ALBUMIN

2007 ◽  
Vol 19 (1) ◽  
pp. 212
Author(s):  
K. Ono ◽  
R. Ohishi ◽  
H. Imai ◽  
M. Yamada
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Bovine Serum ◽  
Culture Media ◽  
Mouse Embryos ◽  
Control Group ◽  
Cell Block ◽  
Cell Stage ◽  
Developmental Rates

Bovine serum albumin (BSA) is an embryotrophic macromolecule used in embryo culture media and improves embryo development in vitro. However, when 1-cell embryos from some strains of mouse were cultured in traditional medium, even with BSA, developmental arrest occurred at the 2-cell stage, termed '2-cell block'. The developmental block is known to be alleviated by adding EDTA to the medium for ICR and ddY strains, and deleting phosphate from the medium for the AKR strain. Recently, our preliminary experiments revealed that the 2-cell block is relieved by adding deionized BSA (d-BSA) to the medium for the ICR strain. Thus, in the present study, we investigated whether d-BSA could rescue the embryos from ICR, ddY, and AKR strains from the 2-cell block. Fertilized 1-cell embryos were collected 20 h post-hCG from superovulated ICR, ddY, and AKR females (8-week-old) that had been mated with the ICR strain of males. Stock solutions (15%) of commercially available fraction V BSA, ovalbumin (ova), and γ-globulin (γG) were deionized over a mixed-bed ion adsorption resin. Embryos were cultured in EDTA-depleted KSOM medium with or without these deionized or non-deionized proteins at 37�C under 5% CO2 in air for 4 days. Experiments were done in at least 3 replicates, and the statistical analyses of the data were done by ANOVA and Fisher&apos;s PLDS test. To observe the distribution of BSA in the embryos from the 1-cell to the blastocyst stages, immunofluorescence study was performed using anti-BSA antibody with a laser confocal microscope. The developmental rates to the 4-cell stage of 1-cell embryos cultured in medium without (control group) or with BSA at 0.3% in ICR and 0.6% in ddY and AKR (BSA group) were very low (ICR: 10% (4/38) and 37% (17/47); ddY: 9% (7/73) and 23% (9/37); AKR: 0% (0/60) and 0% (18/30), respectively). However, when embryos were cultured with d-BSA at 0.3% in ICR and 0.6% in ddY and AKR, the rates to the 4-cell stage significantly increased (ICR: 91% (51/56), ddY: 82% (61/76), AKR: 82% (50/60) vs. control group or BSA group: P &lt; 0.05), and development to the blastocyst stage was observed (ICR: 79% (44/56), ddY: 65% (47/76), AKR: 63% (38/60)). When ICR embryos were cultured with 0.3% deionized-ova or deionized-�G, no significant increase was observed in developmental rates to the 4-cell stage (25% (10/40) and 24% (10/42), respectively). We next examined the critical culture period for the beneficial effects of d-BSA and intracellular distribution of BSA using ddY mouse embryos. It was found that exposure to d-BSA during the late 1-cell (24 h post-hCG) and early 2-cell stages (42 h post-hCG) promoted the development beyond the 2-cell stage. The distribution of BSA in the cytoplasm of embryos at any stage was observed. Interestingly, BSA localized in the nuclei of embryos during the late 1-cell and early 2-cell stages. In conclusion, our results suggest that BSA itself has a potential to remove the 2-cell block in ICR, ddY, and AKR strains. In addition, nuclear localization of BSA may play a key role in regulating the development beyond the 2-cell stage in the mouse embryos.

291 EFFECTS OF ANTIOXIDANT AND CULTURE MEDIUM ON THE DEVELOPMENT OF PORCINE IN VITRO-MATURED - IN VITRO-FERTILIZED EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 261
Author(s):  
C. Choe ◽  
D.-S. Son ◽  
S.-H. Choi ◽  
S.-R. Cho ◽  
H.-J. Kim ◽  
...  
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Culture Conditions ◽  
Free Oxygen Radicals ◽  
Blastocyst Formation ◽  
Free Oxygen ◽  
Statistical Analysis System ◽  
Developmental Rates ◽  
Analysis System

Most cells cultured in vitro are exposed to the risk of injury by free oxygen radicals (FOR). However, some of FOR-induced injury could be reduced by the antioxidants and culture medium used for in vitro embryos. This study was undertaken to examine the effects of the antioxidant and culture medium on the development of porcine in vitro-matured–in vitro-fertilized embryos. In Experiment 1, we treated the porcine oocytes in NCSU23 medium with various concentrations of β-mercaptoethanol (β-ME) to determine the effective concentration of antioxidants during IVM of porcine oocytes. In Experiment 2, we tested different culture media to find the proper culture conditions for in vitro porcine embryos. The porcine oocytes that were matured in NCSU23 medium and then fertilized in mTBM medium were cultured in NCSU23 or porcine zygote medium-5. All steps (maturation, fertilization, and development) were carried out in vitro. Differences were analyzed among treatments using the general linear model (GLM) procedure in the Statistical Analysis System (SAS Institute, Inc., Cary, NC, USA). The results were summarized as follows. Various concentrations of β-ME showed different developmental rates in porcine embryos. The rates of blastocyst formation at Day 7 after IVF were 9.2 � 1.8 (n = 65), 10.0 � 4.2 (n = 80), 17.5 � 1.1 (n = 63), 20.7 � 1.7 (n = 82), and 14.6 � 1.4 (n = 82) in oocytes treated with β-ME at 0, 10, 25, 50, and 100 �M during IVM, respectively. Of the concentrations of β-ME tested, 50 �M β-ME markedly increased the rates of blastocyst formation at Day 7 (P &lt; 0.05). The rates of blastocyst formation at Day 7 in the NCSU23 and PZM-5 culture media of porcine IVF-derived embryos were 18.8 � 2.6 (n = 96) and 15.6 � 7.1 (n = 77), respectively. The developmental rates were slightly increased in NCSU23, compared with those in PZM-5, but there were no significant differences (P &lt; 0.05) between the NCSU23 and PZM-5 media. In conclusion, these results suggest that the addition of 50 �M β-ME in the IVM medium can improve developmental the rates of porcine embryos in vitro.

234 NEW CULTURE MEDIA AFFECTS BLASTOCYST DEVELOPMENT AND GENE EXPRESSION LEVELS IN IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 206
Author(s):  
J. M. K. Nielsen ◽  
C. Wrenzycki ◽  
P. Hyttel ◽  
F. Poppicht ◽  
L. Strøbech
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Culture Media ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Developmental Rates ◽  
Advanced Development ◽  
One Way Anova ◽  
Gene Expression Levels

The purpose was to examine effects of different media for bovine in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on blastocyst rates, morpho-kinetics, and relative abundance of mRNA of 8 genes associated with critical processes and developmental competence in the embryo. Abattoir-derived cumulus-oocyte complexes (COC) were in vitro matured (IVM) in either TCM199 [+0.5% BSA and gonadotropins (Suigonan Vet 150 I.E. mL–1)] or in a novel commercially available media (Bo-IVM), and a total of 1196 presumptive zygotes, from 4 replicates, were submitted to in vitro culture (IVC) and cultured in either SOF (+0.5% BSA) or in a novel commercially available media (Bo-IVC). Blastocyst rates and morpho-kinetics were assessed on Day 8 after fertilization. The high-quality blastocysts from each group were analysed by RT-qPCR, on single blastocysts using earlier verified primers, for BAX, BCL2L1, DNMT3A, FASN, G6PD, HSPA1A, SLC2A1, and SLC2A3. Data on blastocyst rates were analysed for statistical differences using a linear regression model, using a binary reproach and general estimating equations. One-way ANOVA was used to detect differences in the relative abundance of mRNA between groups, whereas differences between maturation and culture media were analysed by a 2-way ANOVA. Blastocyst rates in the Bo-IVM/Bo-IVC (37%), TCM199/Bo-IVC (33%), Bo-IVM/SOF (26%), and TCM199/SOF (28%) groups were significantly different from each other (P < 0.0001). Specifically, the Bo-IVM/Bo-IVC group differed significantly from both SOF-cultured groups (P < 0.01). Subjectively, this group also had embryos of the highest quality and most advanced development. Significantly increased levels of mRNA transcripts were found for embryos cultured in Bo-IVC for all genes (P < 0.05) except BCL2L1. In conclusion, the developmental rates and gene expression of in vitro-produced bovine blastocysts were affected by the use of different culture media. Increased blastocyst rates, apparently superior embryo quality, and more abundant gene expression were achieved when blastocysts were cultured in Bo-IVC culture media compared with SOF.The project was supported by The Danish Council for Strategic Research.

scholarly journals Superoxide dismutase affects the viability of thawed European mouflon (Ovis g. musimon) semen and the heterologous fertilization using both IVF and intracytoplasmatic sperm injection

2003 ◽  
Vol 15 (1) ◽  
pp. 19 ◽  
Author(s):  
Fiammetta Berlinguer ◽  
Sergio Ledda ◽  
Irma Rosati ◽  
Luisa Bogliolo ◽  
Giovanni Leoni ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Culture Media ◽  
Fluorescein Isothiocyanate ◽  
Spermatozoa Motility ◽  
Culture Systems ◽  
Fertilization Rates ◽  
Frozen Semen ◽  
Intracytoplasmatic Sperm Injection ◽  
Acrosome Integrity

This study evaluated the effects of superoxide dismutase (SOD) on viability and acrosome integrity of European mouflon spermatozoa after cryopreservation and on the fertilization rates of sheep oocytes after IVF or intracytoplasmatic sperm injection (ICSI). Frozen semen was thawed and washed with synthetic oviduct fluid supplemented with 0.6% bovine serum albumin. After centrifugation, the spermatozoa pellet was split into two culture systems: (i) without SOD; and (ii) in the presence of 1500 IU mL−1 SOD. Sperm viability and acrosome integrity were evaluated simultaneously, immediately after thawing and after 3, 6 and 9 h of culture (5% CO2, 39°C, 90% humidity), by incubating sperm with propidium iodide and fluorescein isothiocyanate-labelled Pisum sativum agglutinin. At the same time, sperm were assessed for motility using a standard scoring system (independent operators’ observation of sperm) that graded degree of motility (i.e. 1 = immotile to 10 = maximum motility, as observed at the moment of thawing). For IVF, frozen–thawed semen derived from the two culture systems was placed in culture together with in vitro-matured sheep oocytes. For ICSI, semen derived from the same culture systems as that for IVF was used, and incubated for 1 h under standard conditions. The results showed a marked difference (P < 0.01) between the percentages of live spermatozoa in medium with SOD and those obtained in medium alone, after 3, 6 and 9 h of culture. The percentages of intact acrosome spermatozoa were higher in medium with SOD after 6 h (P = 0.05) of culture. Spermatozoa motility decreased significantly in SOD containing medium at 3 and 6 h of culture compared with motility in control medium. Fertilization rates were significantly lower in medium with SOD than in medium alone, whereas in the ICSI system fertilization rates were significantly higher in the presence of SOD. The results indicate that the addition of SOD to the culture media enhances the viability rates and the acrosome integrity of cryopreserved mouflon spermatozoa.

121 DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS CULTURED IN SEQUENTIAL OR CONTINUOUS MEDIA

2008 ◽  
Vol 20 (1) ◽  
pp. 141
Author(s):  
L. S. Amorim ◽  
D. J. Walker ◽  
G. E. Seidel Jr
Keyword(s):  
Culture Media ◽  
Cumulus Cells ◽  
Similar Efficacy ◽  
Defined Medium ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Developmental Rates ◽  
Effects Of Media

Slaughtered bovine females have different characteristics including age, nutritional status, breed, and management system, all of which may affect the results obtained in in vitro embryo production. Another key consideration is that early embryos move from the oviduct to a slightly different environment in the uterus, which has led to development of sequential embryo culture media (e.g. Lane M et al. 2003 Theriogenology 60, 407–419). However, the benefits and importance of using sequential media are not fully known. Therefore, the aim of the present study was to compare developmental rates of oocytes obtained from slaughterhouse-derived ovaries from cows or heifers after culture in sequential media (CDM-1, CDM-2) or in a continuous medium (C-CDM). The experiment was a 3 × 2 × 2 factorial design [bulls (A, B, or C), source (cows or heifers), and medium (sequential or continuous)]. Cumulus–oocyte complexes were aspirated, within 5 h of slaughter, from 3- to 8-mm ovarian follicles of cows (1482 oocytes) and fattened heifers usually fed melengesterol acetate (2818 oocytes). Embryos were produced in vitro as described by De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596, with slight modifications. Presumptive zygotes were vortexed to remove cumulus cells and cultured for 2.5 d in C-CDM (CDM supplemented with 5.0 mm L-lactate, essential and nonessential amino acids, and 0.5% FAF-BSA, or in CDM-1 (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Devel. 18, 585–596) at 39°C in a humidified incubator under 5% CO2, 5% O2, and 90% N2. Cleavage was assessed after 2.5 d; 2- to 6-cell embryos were considered as cleaved, but were not cultured further. Embryos at the 7- to 8-cell stage were cultured for an additional 4.5 d in fresh C-CDM or CDM-2. The percentage blastocysts per oocyte was assessed after 7 and 8 days of culture. Data were arcsin-transformed and evaluated by ANOVA. There was a significant interaction between bull and ovary source for both 8-cell embryos and cleavage rate (P < 0.05); however, this interaction was no longer significant for blastocysts. No other interactions were significant nor a source of ovaries. Culturing embryos in CDM-C refreshed after cleavage evaluation (continuous) or culturing embryos in CDM-1 early and CDM-2 after cleavage evaluation (sequential) resulted in similar cleavage and blastocyst rates (Table 1). We conclude that bovine embryos can be produced using a single chemically defined medium (+BSA) with similar efficacy as a system using 2 sequential media. Table 1. Effects of media on embryonic development (mean ± SE)

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection

1997 ◽  
Vol 9 (7) ◽  
pp. 665 ◽  
Author(s):  
M. C. Gómez ◽  
J. W. Catt ◽  
L. Gillan ◽  
G. Evans ◽  
W. M. C. Maxwell
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Acrosome Reaction ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Frozen Semen ◽  
Acrosome Integrity ◽  
And Control ◽  
Fresh Semen ◽  
Percoll Centrifugation

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitrofertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30°C or 39°C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30°C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.

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