243 IN VITRO PRODUCTION OF LLAMA EMBRYOS BY IVF OR ICSI

2007 ◽  
Vol 19 (1) ◽  
pp. 237
Author(s):  
P. A. Conde ◽  
C. Herrera ◽  
M. G. Chaves ◽  
S. M. Giuliano ◽  
A. Director ◽  
...  
Keyword(s):  
Defined Medium ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Chi Square ◽  
Vitro Fertilization ◽  
South American Camelids ◽  
Lama Glama ◽  
Initial Description

Interest in South American camelids has increased in the last few years. Assisted reproduction techniques, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) with epididymal spermatozoa, have shown poor results in these species (Tibary et al. 2005 Theriogenology 64, 618–638). The aim of the present study was to compare the efficacy of IVF vs. ICSI for in vitro embryo production in llama (Lama glama) using oocytes collected from ovarian follicles of different sizes. A total of 193 oocytes were collected from 223 follicles aspirated from 21 adult females by flank laparotomy after ovarian stimulation. Before aspiration, the diameter of each follicle was measured, and oocytes from each female were randomly assigned to either IVF or ICSI treatment. Semen samples were collected by electroejaculation and incubated in 25% (v/v) collagenase solution at 37°C to reduce viscosity. For IVF, spermatozoa were either non-treated or treated with heparin, penicillamine, and hypotaurine as capacitating agents. For ICSI, some oocytes were activated immediately after sperm injection with 5 µM of ionomycin for 10 min and 2 mM of 6-DMAP for 3 h, while others were subjected only to sperm injection. Spermatozoa used for ICSI were not treated with capacitating agents. All presumptive zygotes were cultured in SOFaas for 8 days. The percentage of total oocytes and mature (MII) oocytes recovered from follicles <7 mm and >7 mm in diameter were compared in each female. The proportion of oocytes inseminated via IVF and ICSI that cleaved and developed to the blastocyst stage was compared. The proportion of total oocytes, MII-oocytes, cleaved embryos, and blastocysts were compared between treatments by chi-square and Fisher's tests. The percentages of total (77/100; 77%) and MII (9/31; 29%) oocytes collected from <7 mm follicles were significantly lower than those of total (116/126; 92%) and MII (43/55; 78%) oocytes collected from >7 mm follicles (P < 0.01). The highest cleavage rates were observed in oocytes collected from follicles >7 mm in diameter and fertilized by IVF with (56%) or without (50%) capacitating agents; these rates were significantly different from those of the other treatments (P < 0.05, Table 1). Further studies will determine if the present results can be obtained with a higher number of oocytes. The results of the present study provide the first demonstration that Lama glama embryos can be produced in vitro using fresh semen. In addition, we have provided the initial description of blastocyst development after culture of ICSI-derived embryos in a defined medium. Table 1.Cleavage and blastocyst formation in different-sized llama oocytes inseminated via IVF or ICSI

scholarly journals 144 EFFECT OF ANTRAL FOLLICLE COUNT IN BEEF HEIFERS ON IN VITRO FERTILIZATION/PRODUCTION

2017 ◽  
Vol 29 (1) ◽  
pp. 180
Author(s):  
C. C. Chase ◽  
R. A. Cushman ◽  
A. K. McNeel ◽  
E. C. Wright-Johnson ◽  
G. A. Perry ◽  
...  
Keyword(s):  
Antral Follicle ◽  
Antral Follicle Count ◽  
Blastocyst Stage ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
University Of Florida ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Vitro Production

Our objective has been to compare the IVF and in vitro production (IVP) of embryos from low and high antral follicle count (AFC) heifers. This is the fourth year of the study with years 1 to 3 reported individually. For this report, we add data for the fourth year and present a combined analysis (years 1 to 4) for the first time. Each year, AFC was determined on ~120 Angus heifers using transrectal ultrasonography. Ten heifers with the lowest AFC and 10 heifers with the highest AFC and all with evidence of oestrous cyclicity were synchronized with two 5-mL injections of PGF2α 11 days apart. Half were harvested on Day 5 to 6 and half on Day 15 to 16 of the oestrous cycle. The IVF procedure was slightly modified each year. For year 4, the IVF procedure included protocols for semi-defined media and was as described (IVP Protocol, P. J. Hansen’s Laboratory, University of Florida). Cumulus-oocyte complexes (COC) from follicles less than 8 mm in diameter were cultured in maturation medium (5% CO2; 38.5°C) for 24 h. Matured COC were fertilized using thawed frozen semen from a bull that was purified using isolate. Motile spermatozoa were added to COC in fertilization medium at a final concentration of 1 × 106 spermatozoa per mL. About 24 h later, presumptive zygotes were placed in micro drops of development medium under oil, and cultured (5% CO2; 5% O2; balance N2; 38.5°C). On Day 3 and 8 after fertilization, cleavage and blastocyst development rates, respectively, were assessed. Data were analysed using the MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) and the model included the effects of year (1 to 4), group (high or low AFC), and their interaction. The year × group interaction was not significant (P > 0.10). Low AFC heifers, compared with high AFC heifers, had fewer numbers of COC (P < 0.0001; 12.8 ± 1.83 v. 31.9 ± 1.86), fewer numbers of COC that cleaved (P < 0.0001; 8.0 ± 1.38 v. 21.6 ± 1.40), and fewer numbers of COC that developed to the blastocyst stage (P < 0.0001; 1.7 ± 0.58 v. 5.7 ± 0.58). Year affected the numbers of COC that cleaved (P < 0.003) and the numbers of COC that developed to the blastocyst stage (P < 0.0001). Year also influenced the percentage of COC that cleaved (P < 0.0002) and the percentage of COC that developed to blastocysts (P < 0.0001). Group (AFC) did not influence (P > 0.19) the percentage of COC that cleaved (61.2 ± 2.83 v. 66.4 ± 2.83%, for low v. high AFC, respectively). Low AFC heifers had a lower (P < 0.002) percentage of COC that developed to blastocysts (10.3 ± 1.52%) than high AFC heifers (17.6 ± 1.52%). These results indicate that high AFC heifers, compared to low AFC heifers, have more COC recovered, more COC cleaved, and more COC developed to the blastocyst stage. The percentage of COC cleaved did not differ between AFC groups; however, the percentage of COC that developed to the blastocyst stage was greater for high than low AFC heifers. This suggests a potential advantage in maternal to embryonic transition for high compared with low AFC heifers.

247 EFFECTS OF CYSTEINE IN IVM MEDIA ON IN VITRO MATURATION UNDER LOW OXYGEN TENSION, IN VITRO FERTILIZATION, AND IN VITRO CULTURE OF PORCINE OOCYTES

2008 ◽  
Vol 20 (1) ◽  
pp. 203
Author(s):  
N. V. Linh ◽  
D. N. Q. Thanh ◽  
M. Ozawa ◽  
B. X. Nguyen ◽  
K. Kikuchi ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Blastocyst Stage ◽  
Low Oxygen ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Low Oxygen Tension ◽  
High O2 ◽  
Group 1

Cysteine is considered to promote male pronuclear (MPN) formation in porcine through oocyte glutathione (GSH) synthesis (Yoshida et al. 1993 Biol. Reprod. 49, 89–94). The GSH has an important role in providing cells with a redox state and in acting to protect cells from toxic effects of oxidative damage (Meister et al. 1976 AM Rev. Biochem. 45, 559–604). However, such previous investigations were carried out under high O2 tension (20% O2) incubation conditions. Here we simply study IVM-IVF-IVC competence of porcine oocytes matured in IVM media supplemented with cysteine of different concentrations under low oxygen tension (5% O2). Cumulus–oocyte complexes (COCs) from prepubertal gilts were collected, matured, and fertilized in vitro according to Kikuchi et al. (2000 Biol. Reprod. 66, 1033–1041). COCs were cultured in IVM medium supplemented with 0 (Group 1; control), 0.05 (Group 2), 0.1 (Group 3), 0.2 (Group 4), and 0.6 mm (Group 5) cysteine under low oxygen tension. Nuclear maturation of oocytes, fertilization status, and number of cells in resultant embryos were assessed with orcein staining; also, the GSH content of IVM oocytes was measured by the method described by Ozawa et al. (2002 Reproduction 124, 683–689). Maturation rates of Groups 1–5 were 68.2 � 3.2, 70.6 � 7.7, 69.7 � 15.9, 75.9 � 7.7, and 68.8 � 8.0%, respectively, indicating no difference in maturation competence among the groups (P > 0.05 by ANOVA). The rates of sperm penetration, MPN formation (95.9 � 2.4, 100 � 0, 92.8 � 4.7, 94.0 � 4.1, and 92.4 � 2.7%, respectively), monospermy, and even blastocyst rates after 6 days of IVC were not different among the groups (P > 0.05 by ANOVA). Moreover, the cell numbers of blastomeres in blastocysts (38.68 � 3.5, 40.1 � 3.1, 37.5 � 3.0, 36.2 � 3.3, and 43.8 � 4.0, respectively) were uniformly the same among the groups (P > 0.05 by ANOVA). However, GSH content of IVM oocytes increased significantly (P < 0.05 by ANOVA) as the concentration of cysteine increased (12.2 � 0.6, 14 � 0.8, 15.1 � 0.5, 16.4 � 0.4, and 16.4 � 0.5 pmol/oocyte, respectively). The GSH level of oocytes in Group 1 (control) seems to be higher than that reported by Aberydeera et al. (1998 Biol. Reprod. 58, 213–218), who matured porcine oocytes under high O2 tension. This may reflect the effect of low O2 tension and explain the same developmental rate to the blastocyst stage as that of oocytes matured in the media supplemented with cysteine in this study. In conclusion, an addition of 0.05–0.6 mm cysteine during IVM, under 5% O2 tension, of porcine oocytes significantly increased intracellular GSH synthesis according to its concentration. However, it had no promoting effects on nuclear maturation, fertilization, male pronucleus formation, and subsequent embryonic development to the blastocyst stage. Thus, O2 tension during IVM of oocytes is suggested to be important for the in vitro production of porcine blastocysts.

scholarly journals In vitro production of cattle blastocysts in chemically defined medium with or without insulin supplementation

1996 ◽  
Vol 5 (5) ◽  
pp. 509-514 ◽  
Author(s):  
Kristiina Bredbacka ◽  
Peter Bredbacka
Keyword(s):  
Bovine Serum Albumin ◽  
Culture Medium ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Chemically Defined Medium ◽  
In Vitro Production ◽  
Cell Numbers ◽  
Vitro Production ◽  
Bovine Oocytes

In this study we evaluated the use of a chemically defined medium in the production of blastocysts from bovine oocytes fertilized in vitro. As culture medium we used CRI-PVP, a modification of CRlaa medium with bovine serum albumin replaced by polyvinylpyrrolidone. After 168 h of culture (192 h after insemination) 8.7%, 10.5 and 12.8% of the cleaved embryos developed to the blastocyst stage in the presence of 0, 2 or 200 nM insulin, respectively. The supplementation of 200 nM insulin tended to increase cell numbers in morulae and blastocysts (P=0.10). It is concluded that CRI-PVP can be used as a chemically defined medium in the production of blastocysts from bovine 1-cell embryos. However, further modifications are needed, and the insulin concentrations used may be below the optimum for blastocyst production.

222 EFFECT OF THE PRESENCE OR ABSENCE OF PERCOLL CENTRIFUGATION; PENICILLAMINE, HYPOTAURINE, AND EPINEPHRINE; AND HEPARIN ON IN VITRO PRODUCTION OF BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 259 ◽  
Author(s):  
J. F. Hasler ◽  
J. E. Stokes
Keyword(s):  
Percoll Gradient ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Rat Liver Cells ◽  
Lactate Medium ◽  
Bovine Oocytes ◽  
Percoll Centrifugation

Protocols for the production of bovine embryos in vitro routinely include Percoll centrifugation of semen, usually include heparin, and often include penicillamine, hypotaurine, and epinephrine (PHE) in the fertilization media. This study examined the contribution of each of these components to the success of in vitro fertilization of bovine oocytes and subsequent blastocyst development. Bovine oocytes were aspirated from 2- to 10-mm follicles within 5 h after slaughter of cattle at a local abattoir. Groups of 30 to 40 cumulus–oocyte complexes (COC) were matured in 0.5 mL of TCM-199 with 10% FCS, 4 µg mL–1 of FSH, and 6 µg mL–1 of LH (NOBL Laboratories, Sioux Center, IA, USA) for 24 h (39°C, 4% CO2 in air). The COC were then washed and placed in 0.5 mL of modified Tyrode-lactate medium for IVF with various combinations of 2 µg mL–1 of heparin, 20 µM penicillamine, 10 µM hypotaurine, and 1 µM epinephrine. Each group of COC was inseminated with 0.25 × 106 frozen–thawed sperm from a single bull after 30 min of centrifugation with (Exp. 1) or without (Exp. 2) a 45/90% Percoll gradient with sperm TALP. Oocytes were vortexed to remove the cumulus after 18 h and placed in co-culture wells containing a monolayer of buffalo rat liver cells and 0.5 mL of Menezo’s B2 medium supplemented with 10% FCS. On the fourth day of in vitro culture, cleavage was defined as 2 cells or greater and embryos were transferred to fresh co-culture wells. There were 4 replicates in the first experiment and 6 in the second. Data were analysed by ANOVA. In the first experiment, the use of a Percoll gradient during centrifugation for separation of viable sperm from seminal plasma and cryprotectants resulted in significantly higher cleavage and Day 8 blastocyst rates than did the absence of Percoll when PHE and heparin were used together, and both cleavage and blastocyst rates were lower when only PHE or heparin was used separately compared with when both were used together (Table 1). The absence of Percoll, PHE, and heparin resulted in the lowest rates of cleavage and development. In the second experiment, the absence of either PHE or heparin resulted in lower cleavage rates, but not blastocyst rates, compared with the use of both, and the absence of both resulted in the lowest cleavage and blastocyst rates in spite of the use of Percoll. Table 1.Effects of Percoll; penicillamine, hypotaurine, and epinephrine (PHE); and heparin on cleavage and subsequent embryo development per oocyte

scholarly journals In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals

2017 ◽  
Vol 20 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A.E. Abdelkhalek ◽  
Sh.A. Gabr ◽  
W.A. Khalil ◽  
Sh.M. Shamiah ◽  
L. Pan ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Epididymal Spermatozoa ◽  
Vitro Production

Abstract Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

Glucose and glycine synergistically enhance the in vitro development of porcine blastocysts in a chemically defined medium

2012 ◽  
Vol 24 (3) ◽  
pp. 443 ◽  
Author(s):  
Tomomi Mito ◽  
Koji Yoshioka ◽  
Shoko Yamashita ◽  
Chie Suzuki ◽  
Michiko Noguchi ◽  
...  
Keyword(s):  
Culture Medium ◽  
Survival Rates ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Atp Content ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Defined Culture ◽  
Vitro Development

In the present study, the effects of glucose and/or glycine on the in vitro development of Day 5 (Day 0 = IVF) porcine blastocysts were determined. The addition of 2.5–10 mM glucose to the chemically defined culture medium porcine zygote medium (PZM)-5 significantly increased blastocyst survival rates compared with those of blastocysts cultured in the absence of glucose. The addition of 5 and 10 mM glycine to PZM-5 containing 5 mM glucose significantly enhanced the development to hatching and the number of hatched blastocysts compared with no addition of glycine. However, the addition of glycine to PZM-5 with no glucose did not improve blastocyst development. The ATP content of Day 6 blastocysts cultured with glucose was significantly higher than that of blastocysts cultured in the absence of glucose, regardless of glycine supplementation. The diameter and total cell numbers were significantly greater, and the apoptotic index was significantly lower, in Day 6 blastocysts cultured with both glucose and glycine. These results indicate that glucose is an important energy source for the porcine blastocyst and that glucose and glycine act synergistically to enhance development to the hatching and hatched blastocyst stage in vitro.

241 DIBUTYRYL CYCLIC AMP AND EGF-LIKE FACTORS SUPPORT IN VITRO MATURATION OF PORCINE OOCYTES IN A CHEMICALLY DEFINED MEDIUM WITHOUT GONADOTROPINS

2009 ◽  
Vol 21 (1) ◽  
pp. 218
Author(s):  
Y. Akaki ◽  
K. Yoshioka ◽  
H. Funahashi
Keyword(s):  
Cyclic Amp ◽  
In Vitro Maturation ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Chemically Defined Medium ◽  
Dibutyryl Cyclic Amp ◽  
Vitro Fertilization ◽  
Porcine Oocyte

Exposure of porcine oocyte–cumulus complexes (OCC) to gonadotropins induces meiotic resumption, but the details of this mechanism are still unknown. The present study was undertaken to examine combinational effects of EGF-like factors and dibutyryl cyclic AMP (dbcAMP) in a chemically defined medium on in vitro maturation (IVM) of porcine oocytes. The OCC were aspirated from 3- to 6-mm-diameter follicles of prepuberal ovaries and used in the current study. The basic culture medium was a chemically defined medium, Porcine Oocyte Medium (POM; Research Institute for the Functional Peptides, Yamagata, Japan). In the first experiment, various concentrations (0, 10, and 1000 ng mL–1) of EGF-like factors (EGF, amphiregulin, and betacellulin) were added to POM during an entire IVM period (44 h). In the second experiment, to determine the additive effect of EGF-like factors, each EGF-like factor with an effective concentration was combined with the others. In the last experiment, to examine the combined effect with dbcAMP, OCC were exposed to EGF (10 ng mL–1), amphiregulin (1000 ng mL–1), and dbcAMP (1 mm) during the first 20 h of IVM and then the culture was continued in the absence of EGF-like factors and dbcAMP. After culture, in all experiments, meiotic resumption and the progress of oocytes were examined after denuding, fixing, and staining. Statistical analyses was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P < 0.05). In the first experiment, all treatments without supplementation with 10 ng mL–1 amphiregulin increased the incidence of oocytes maturing to the MII phase, as compared with controls (29.1 to 39.3% v. 11.1%, P < 0.05). In the second experiment, combinations with 2 kinds of EGF-like factor slightly (but not significantly) improved the percentage of oocytes at the MII stage (37.7 to 47.4%). In the last experiment, supplementation with 1 mm dbcAMP during the first 20 h of IVM, regardless of the presence of EGF-like factors, significantly increased the incidence of MII oocytes as compared with controls, whereas the incidence was the highest when 1 mm dbcAMP, 10 ng mL–1 EGF, and 1000 ng mL–1 amphiregulin were supplemented (75.5%). When those oocytes were cultured in a chemically defined medium after in vitro fertilization, the developmental competence of oocytes to the blastocyst stage (25.0%) was not different from oocytes matured in the presence of gonadotropins and dbcAMP during the first 20 h of IVM (17.3%). These observations indicate that supplementation of a chemically defined maturation medium with EGF-like factors and dbcAMP during the first 20 h of IVM can support the meiotic progress and developmental competence of porcine oocytes well. Currently, we are examining the developmental competence of those oocytes after embryo transfer. The results will be presented at the meeting. This study was supported by MAFF AgriBio1605.

237 THE EFFECTS OF BULLS AND X-SORTING OF SPERM ON THE ACCURACY OF NONINVASIVE CRITERIA TO PREDICT BLASTOCYST FORMATION OF IN VITRO-PRODUCED BOVINE EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 208
Author(s):  
S. Matoba ◽  
T. Somfai ◽  
T. Nagai ◽  
M. Geshi
Keyword(s):  
Formation Rate ◽  
Calf Serum ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Blastocyst Development ◽  
Chi Square

Previously, an early first cleavage and a second cleavage after IVF with a normal cleavage pattern defined by even blastomeres without fragments or protrusions was found to be a potent marker for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLoS ONE 7, e36627). The aim of this study was to investigate the effects of bulls and X-sorting of sperm on the ability of these simple noninvasive markers to predict the potency of bovine IVF embryos to develop to the blastocyst stage in vitro. Immature oocytes were matured in TCM199 supplemented with 0.02 armour unit mL–1 FSH and 5% calf serum at 38.5°C in 5% CO2 and 95% air for 22 to 23 h. After maturation, oocytes were inseminated with either of non-sorted frozen-thawed sperm from 3 bulls (A–C) or X-sorted sperm of bull A. Putative zygotes were cultured (IVC) in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL–1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2 for 216 h. Embryo kinetics were observed individually by time-lapse cinematography (CCM-1.3Z; Astec, Fukuoka, Japan; Sugimura et al. 2010 Biol. Reprod. 83, 970–978). First and second cleavage kinetics and pattern were categorized according to Sugimura et al. (2012). For each bull, blastocyst development from embryos possessing the following 3 selection markers was compared: (marker 1) the first cleavage within 28 h after IVF, (marker 2) marker 1 combined with 2 even blastomeres without fragments or protrusions, and (marker 3) marker 2 combined with the second cleavage within 50 h after IVF with ≥6 even blastomeres without fragments or protrusions, respectively. Data were analysed by the Yates' corrected chi-square test. A total of 823 oocytes were used in at least 3 replications. When non-sorted sperm was used for IVF, there was not difference (P > 0.05) in total blastocyst formation rates on Day 8 (Day 0 = IVF) among bulls (ranging between 49.5 and 60.8%); however, blastocyst formation rate of embryos generated from X-sorted sperm of bull A (39.5%) was lower (P < 0.05) compared with other groups despite of similar cleavage rates. Embryos having marker 3 criteria developed to the blastocysts stage at significantly higher rates than those having marker 1 criteria in case of non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A (75.9, 87.0, 90.0, and 75.0% v. 59.5, 62.2, 63.6, and 46.3%, respectively). In groups produced from non-sorted sperm of bulls A, B, C, and X-sorted sperm of bull A, blastocyst development rates of embryos with marker 2 criteria (73.7, 75.0, 90.0, and 65.8%, respectively) were higher (P < 0.05) than those of embryos having marker 1 criteria but did not differ significantly from those with marker 3 criteria. Our results reveal that a first cleavage within 28 h after IVF to 2 even blastomeres without fragments or protrusions are potent predictive markers of the developmental competence of bovine embryos to the blastocyst stage regardless of bulls and sperm sorting.Research was partly supported by JSPS KAKENHI (26450388).

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
M. Alomar ◽  
H. Tasiaux ◽  
S. Remacle ◽  
F. George ◽  
D. Paul ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test ◽  
Kinetics Of

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P &lt; 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P &lt; 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P &lt; 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P &lt; 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P &lt; 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

Polyspermic penetration in porcine IVM - IVF systems

2003 ◽  
Vol 15 (3) ◽  
pp. 167 ◽  
Author(s):  
Hiroaki Funahashi
Keyword(s):  
Zona Pellucida ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Final Maturation ◽  
Morphological Differences ◽  
Vitro Production ◽  
Boar Spermatozoa

Although techniques for in vitro production of porcine embryos have proceeded very rapidly during the past decade, polyspermic penetration still remains a persistent obstacle to porcine in vitro fertilization (IVF) systems. Considerable research on in vitro polyspermic penetration in porcine in vitro-matured (IVM) oocytes has been undertaken to try to solve this problem. In the current paper, recent advancements in overcoming the problems of polyspermy in porcine IVF systems are reviewed. Partial induction of the acrosome reaction of boar spermatozoa in IVF media that contain caffeine is likely to be one of the major causes of polyspermy. A reduction in the number of incompletely acrosome-reacted spermatozoa, which can bind tightly to the zona pellucida and mask free sperm receptors of the zona pellucida, could reduce the incidence of polyspermic penetration; however, morphological differences in the reaction of the zona pellucida have been observed between IVM and ovulated oocytes, which suggests that altered zona morphology may be another cause of polyspermic penetration. It has been shown that the developmental ability of polyspermic porcine embryos to the blastocyst stage is similar to that of normal embryos but that developmental competence to term is much lower. To overcome the current problems of polyspermy, it is suggested that future efforts should be focused on controlling boar sperm function and/or sperm–zona binding to achieve the final maturation associated with normal zona modifications of porcine oocytes at fertilization.

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