scholarly journals 320 USE OF SYNTHETIC HYALURONAN OR POLYVINYLPYRROLIDONE FOR INTRACYTOPLASMIC SPERM INJECTION INTO MOUSE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 310
Author(s):  
P.N. Moreira ◽  
J. De la Fuente ◽  
A.T. Palasz ◽  
A. Gutiérrez-Adán

The use of polyvinylpyrrolidane (PVP) in intracytoplasmic sperm injection (ICSI) seems to be exclusively related to its surfactant and colloidal properties. In contrast to PVP, which can be toxic to mouse embryos, hyaluronan (HA) is a biological compound. In addition to its colloidal property, HA plays an important biochemical role in cell proliferation and migration and can be found intracellularly in the cleaving stage of mouse, sheep and primate embryos (Hunter RHF 1994 Mol. Reprod. Dev. 39, 176–181). We expect that the viscoelastic properties of HA in combination with its physiological functions may benefit the ICSI procedure. Oocytes at MII stage were collected from CD-1 mice 14 h after hCG injection (h-pi) and were kept at 37°C in KSOM medium for 30 min before ICSI. Semen used for injection was frozen by direct plunge into liquid nitrogen in M2 medium without cryoprotectants. Samples were thawed at 25°C in the air and mixed (1:5) with M2 medium containing either 10% PVP; 360000 MW (w/v; Sigma, St. Louis, MO, USA) or 60% (v/v) synthetic HA (s-HA; MAP-5; Bioniche Inc, Belleville, ON Canada) with comparable viscosity. Injections were performed at 25°C using a mercury-containing pipette attached to a piezo impact unit (Prime Tech, Ibaraki, Japan). A total of 239 oocytes (115 PVP and 124 s-HA) were injected in groups of ten in four replicates. Individual sperm heads decapitated by the freeze/thaw procedure were injected into oocytes and kept for 15 min at 25°C. Oocytes that survived ICSI were placed in 35 μL drops of KSOM medium (∼15 zygotes per drop) under paraffin oil at 37°C and 5% CO2 in humidified air. Cleavage and developmental rates were recorded at 24, 48, and 96 h after oocyte injection. Embryos which developed to the blastocyst stage were transferred to pseudo-pregnant females mated with vasectomized males. At Day 13, recipient mice were sacrificed and the number of implantations and fetuses were recorded. Data were compared between groups by Chi-square analysis. Significantly (P < 0.05) more embryos survived ICSI in PVP (74%) than in s-HA group (56%), which was primarily related to sperm adhesiveness to the injection pipette. However, there were no differences in developmental rates at any stage of in vitro embryo culture between groups (2 cell, 93 vs. 100%; 4–8 cell, 100 vs. 100%; blastocyst, 44 vs 50%) for PVP and s-HA, respectively. Significant differences (P < 0.05) between groups were observed in embryo implantation rates. When ICSI was performed with s-HA, 29 out of 35 blastocysts (83%) transferred to synchronized recipients were implanted, which was accomplished only by 19 of the 35 from the PVP group (54%). However, there was no difference between groups in the number of fetuses detected (8 (23%) vs. 9 (26%) for PVP and s-HA, respectively). The use of s-HA for mouse ICSI can be a valuable alternative to PVP. Hyaluronan may show further benefit if sperm adhesiveness to the micropipette can be eliminated, and may be superior to PVP if embryo implantation rates in the s-HA group can be sustained. The authors would like to thank Bioniche, Inc., Belleville, ON, Canada for donating MAP-5.

2012 ◽  
Vol 24 (1) ◽  
pp. 214
Author(s):  
J. Jarazo ◽  
A. Gambini ◽  
A. De Stefano ◽  
L. Muredas ◽  
J. G. Oriol ◽  
...  

Intracytoplasmic sperm injection (ICSI) is an alternative method for producing in vitro-fertilized embryos in horses. Some authors have suggested that using the piezo drill to inject the spermatozoon is required to obtain acceptable blastocyst rates after ICSI. In order to avoid the use of this equipment, the aim of our study was to evaluate 4 different chemical activation protocols and their effect on embryo development. Cumulus–oocyte complexes were recovered from ovaries of slaughtered mares. The maturation medium was DMEM/F12 supplemented with 10% fetal bovine serum (FBS), 1 μL mL–1 of insulin-transferrin-selenium, 1 mM sodium pyruvate, 100 mM cysteamine and 0.1 mg mL–1 of FSH at 39°C in a humidified atmosphere of 6.5% CO2 in air for 24 h. The ICSI was carried out in 20-μL droplets of TALP-HEPES with a 9-μm pipette, using frozen-thawed spermatozoa from 1 stallion. Spermatozoa were held separate in 100-μL droplets of Modified Whittens. Motile spermatozoa were aspirated and transferred to a 5-μL drop of 7% (v/v) polyvinylpyrrolidone, where 1 sperm was immobilized by swiping the injection pipette across its tail; then, the sperm was injected into the oocyte. All injected oocytes were subjected to 8.7 μM ionomycin for 4 min, followed by 1 of 3 further activation treatments: (1) 4-h culture in 1 mM 6-DMAP and 10 mg mL–1 of cycloheximide, starting 3 h after ionomycin; (2) 5-h culture in 10 mg mL–1 of cycloheximide, starting 10 min after ionomycin; (3) An extra incubation with 5 mM ionomycin for 4 min, starting 3 h after ionomycin. Some injected oocytes were left without a further activation protocol (group 4). After activation, injected oocytes were cultured in 100-μL droplets of DMEM/F12 with 5% of FBS at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. Cleavage (48 h after activation) and blastocyst formation (7–8 days) of all experimental groups were assessed. Culture medium was renewed on Day 3 with fresh DMEM/F12 with 5% of FBS. At Day 9, the zona pellucida of some blastocysts was removed and the blastocysts were maintained in culture until Day 15. Blastocyst growth was determined every 24 h. Statistical differences (using chi-square analysis) were observed in cleavage with treatments 1 and 3 when compared to the other groups (1: 30/52, 58%; 2: 8/40, 20%; 3: 9/25, 36%; and 4: 10/38, 26%). There was no difference on blastocyst rates based on injected oocytes (1: 5/52, 9.6%; 2: 2/40, 5%; 3: 1/25, 4%; and 4: 2/38, 5.3%). On Day 7, blastocyst quality did not differ among treatments and on Day 15, blastocysts from groups 3 and 4 reached 1130 μm and 4300 μm, respectively. Despite the difference observed in cleavage, this work suggests that equine blastocysts could be obtained with all of the activation protocols, without the use of the piezo drill. Further studies are required to assess the effect of chemical activation on in vivo development of produced blastocysts to confirm that they are not parthenogenetic. We are grateful to Mr. Willem Melchior, La Vanguardia Polo Club for some financial support and encouragement to undertake this project.


2014 ◽  
Vol 26 (1) ◽  
pp. 223
Author(s):  
N. C. Canel ◽  
R. J. Bevacqua ◽  
M. I. Hiriart ◽  
D. F. Salamone

TM-intracytoplasmic sperm injection (ICSI) was demonstrated to be an effective technique for the production of transgenic animals. However, this method has not been widely applied for transgenesis in cattle, because of the low embryo developmental rates. This problem may be related to the incomplete sperm decondensation and subsequent pronuclei formation that occurs in cattle after ICSI (Malcuit et al. 2006 Reprod. Fertil. Dev. 18, 39–51). Delgado et al. showed that pretreatment with heparin-sodium salt combined with reduced glutathione (Hep-GSH) could improve bull sperm decondensation (2001 Archives of Andrology 47, 47–58). The objective of this work was to test the use of pretreated sperm with Hep-GSH for TM-ICSI, because an improvement of male pronucleus formation could cause an increase on the frequency of exogenous DNA integration. To this aim, cumulus-oocyte complexes were collected from slaughtered cow ovaries and in vitro matured for 21 h. Frozen sperm from a bull that was previously determined to produce low developmental rates post ICSI and IVF was used. It was thawed and washed twice by centrifuging at 390 × g for 10 min. After that, sperm were incubated with Tris medium supplemented with 80 μM Hep and 15 mM GSH for 20 h. After washing, semen was co-incubated with 50 ng μL–1 of pCX-EGFP plasmid for 5 min on ice and used for ICSI (Hep-GSH ICSI group). An ICSI control group was injected with semen not treated with Hep-GSH. Sham controls were injected with 50 ng μL–1 of pCX-EGFP. Haploid and diploid parthenogenetic controls were also included (Haplo PA and Diplo PA groups). Oocytes were activated by a 4 min exposure to 5 μM ionomycin, placed on TCM-199 for 3 h, and treated with 1.9 mM DMAP for 3 h; Diplo PA were immediately exposed to DMAP after ionomycin treatment. Embryos were cultured in SOF medium. Cleavage and blastocyst rates were evaluated on Days 2 and 7 post ICSI, respectively. Expression of egfp was assayed at Day 4 and at the blastocyst stage. Results: Hep-GSH ICSI group showed higher cleavage rates than ICSI control (68.5%, n = 89 v. 35%, n = 60), and lower than Sham, Diplo PA, and Haplo Pa groups (94% n = 50, 95.1% n = 61, and 85.1% n = 47, respectively; Fisher's exact test, P ≤ 0.05). Although blastocyst rates from ICSI groups did not differ from Haplo PA (21.2%) and Sham groups (8%), Hep-GSH ICSI produced higher rates than ICSI control (19.1 v. 5%). The higher blastocyst rates were observed for Diplo PA (47.5%; P ≤ 0.05). Transgene expression levels at Day 4 were higher for both Hep-GSH ICSI and ICSI control than for Sham control (24.7 and 11.7% v. 0%, respectively; P ≤ 0.05). Rates of egfp expressing blastocysts/injected oocytes were significantly higher for Hep-GSH ICSI than for ICSI and Sham control groups (13.5 v. 1.7 and 0%, respectively; P ≤ 0.05). Conclusions: Pretreatment of bull sperm with Hep-GSH can increase blastocyst rates after ICSI, even when low quality semen is used. Additionally, the employment of Hep-GSH treatment increased rates of transgene expressing blastocysts. It could be a useful strategy for massively implementing TM-ICSI in bovine, for the production of transgenic animals.


2004 ◽  
Vol 16 (2) ◽  
pp. 258
Author(s):  
H. Irving-Rodgers ◽  
S. Morris ◽  
R. Collett ◽  
K. Catanzariti ◽  
T. Peura ◽  
...  

Oocytes from small, non-FSH-dependent follicles are associated with reduced developmental competence following in vitro embryo production (IVP) compared to oocytes from larger follicles. It has been suggested that, for small follicles, oocytes derived from atretic follicles are more developmentally competent than those from healthy follicles (Blondin P and Sirard MA, 1995 Mol. Reprod. Dev. 41, 54–62). Little is known of the characteristics of small follicles that support developmentally competent oocytes. Here we examine the development to blastocyst stage of oocytes collected from histologically-assessed bovine 2–5mm follicles. Ovaries were obtained at a local abattoir;; 4 follicles were dissected from each ovary and oocytes were recovered. A section of each follicle wall was taken and fixed in 2.5% glutaraldehyde for histological assessment of the follicle and characterization of the morphology of the follicular basal lamina by electron microscopy (Irving-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228). Oocytes recovered from follicles underwent IVP utilizing a novel single IVP system. Oocytes were matured for 24h (10μL per COC) in TCM199, supplemented with FSH, hCG, FCS, cysteamine and pyruvate. Mature oocytes were inseminated with 1×106 sperm mL−1 for an additional 24h using Bovine Fertilization Medium (10μL per COC;; Cook, Australia). Following insemination, putative zygotes were stripped of remaining cells and placed within individual micro-wells prepared in 1% agar in Bovine Early Cleavage Medium, Cook, Australia. The agar (350μL) was prepared within wells of a 4-well plate and small plugs of agar were removed to form micro-wells. The agar was over-laid with 450μL of Early Cleavage Medium and 250μL mineral oil, and equilibrated overnight before putative zygotes were placed individually within micro-wells. Culture was performed under 7% O2, 6% CO2, and 87% N2 at 39°C. On Day 5 following insemination, fetal calf serum (final concentration 10% v/v) was added to facilitate blastocyst development. Blastocyst formation was assessed on Day 8. A total of 211 oocytes were cultured and 69% were from healthy follicles;; 67 oocytes (32%) had developed to the blastocyst stage by Day 8. Forty-three percent of oocytes recovered from atretic follicles (28/65) had developed to the blastocyst stage by Day 8, as compared to only 27% (39/146) oocytes recovered from healthy follicles, this difference was significant (P&lt;0.05, chi-square analysis). Seventy-eight percent (14/18) of oocytes from healthy follicles with additional follicular basal lamina material (Irging-Rodgers HF and Rodgers RJ, 2000 J. Reprod. Fert. 118, 221–228) failed to develop, whereas only 44% (4/9) of oocytes from healthy follicles with a normal basal lamina failed to develop (P&gt;0.08). The present study finds a direct association between the follicle morphology and oocyte maturational potential within non-FSH dependent follicles, revealing that high levels of development (&gt;40%) can be obtained from atretic follicles. Furthermore, differences between healthy follicles may also contribute to developmental variation.


2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  

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.


2007 ◽  
Vol 19 (1) ◽  
pp. 173 ◽  
Author(s):  
J. Braun ◽  
C. Otzdorff ◽  
T. Tsujioka ◽  
S. Hochi

The effects of slow freezing or vitrification as well as exposure to the cryoprotective media without cooling and warming of in vitro-matured domestic cat oocytes on the in vitro development to the blastocyst stage was investigated. Cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 3 mg mL−1 BSA, 1 µg mL−1 estradiol, 0.1 IU mL−1 FSH, and 0.0063 IU mL−1 LH. Denuded oocytes with a detectable first polar body were inseminated with 2 × 106 cells mL−1 cauda epididymal spermatozoa for 22 h in TALP solution. Presumptive zygotes were cultured in modified SOF medium at 38.5°C in 5% CO2 in air. For slow freezing, oocytes were equilibrated for 20 min at ambient temperatures in PBS with 20% FCS containing either 1.5 M ethylene glycol (EG) + 0.2 M sucrose or 1.5 M EG + 0.2 M trehalose. Oocytes were loaded into 0.25-mL straws, cooled to −7°C at 2°C min, held for 5 min, seeded, cooled down to −30°C at 0.3°C min, and finally plunged into liquid nitrogen. The straws were thawed for 5 s at room temperature and for 30 s in a waterbath at 30°C. Oocytes were washed 3 times before insemination. In vitro-matured oocytes were exposed to the cryoprotective media for 30 min before they were inseminated and then they were cultured for 7 days. For vitrification (Hochi et al. 2004 Theriogenology 61, 267–275), a minimum-volume cooling procedure using Cryotop (Kitazato Supply Co., Tokyo, Japan) as a cryodevice was applied. No blastocysts could be obtained after slow freezing with a cryoprotective medium containing 0.2 M sucrose. Simple exposure to the same freezing medium after in vitro maturation without cryopreservation resulted in a blastocyst rate of 7.9% (control oocytes, 10.7%; not significant (NS); chi-square analysis). Use of trehalose as an extracellular cryoprotectant resulted in the harvest of one blastocyst (0.6%) after slow freezing. Exposure to the same cryoprotective medium resulted in a blastocyst rate of 10.0% (fresh control, 10.9%; NS). After exposure of in vitro-matured oocytes to the vitrification solution, a blastocyst rate of 16.0% was observed (8/50), which was not statistically different from the blastocyst rate in fresh control oocytes (16.3%; 15/92). No blastocysts could be obtained after vitrification (0/64). The results (Table 1) demonstrate that there is no obvious toxic effect of the cryoprotectants employed here for slow freezing or vitrification on the in vitro-matured oocytes, but the developmental potential of cryopreserved oocytes to the blastocyst stage is severely impaired. Table 1. Effect of slow freezing or exposure to freezing medium of matured cat oocytes on the development to the blastocyst stage in vitro


2006 ◽  
Vol 18 (2) ◽  
pp. 203
Author(s):  
C. Ponsart ◽  
H. Quinton ◽  
A. Rohou ◽  
J. Kelhembo ◽  
G. Bourgoin ◽  
...  

Previous studies have shown that the time between flushing and freezing of bovine embryos can influence pregnancy rates (PRs) following embryo transfer (ET). The aim of this study was to determine which time components can influence ET results. Time components between flushing of a superovulated donor and freezing of the collected embryos were investigated under field conditions. Embryos were frozen in 1.5 M ethylene glycol (EG) for direct transfer. During January 2003, ET technicians (EmbryoTop, Rennes cedex, France) recorded systematically times corresponding to each step comprising the time spent in vitro (TIV) from 153 recovery sessions (RS) with freezing: end of flushing, beginning and end of search of embryos, start of equilibration in EG, beginning and end of straw loading, introduction to −7°C in the freezer, and seeding. Numbers of donor cows and ET technicians doing the freezing (n = 5) were noted for each RS. Embryo (stage, quality) and recipient (breed, parity) characteristics were also noted. A total of 548 frozen embryos were transferred and PRs were assessed. Variability of time components was investigated (Bourgoin et al. 2004 Reprod. Fertil. Dev. 16, 207). The influence of time components and other variation factors was tested on PRs (t-tests and chi-square analysis). The TIV averaged 210 ± 80 min and did not influence PR (≤4 h = 51.9% (n = 393) vs. >4 h = 55.5% (n = 155); P > 0.05), as well as duration of flushing (32 ± 8 min), interval between end of flushing and search (31 ± 27 min), duration of search (45 ± 25 min) and interval between end of search and beginning of freezing (101 ± 63 min). Only significant factors were kept for further analysis. The effects of recipient parity, number of donor cows per RS, and interval between introduction of straw to −7°C, and seeding were tested in a multivariate logistic model. PR varied strongly with parity of recipient (+25% in heifers vs. cows; P = 0.001). PRs were higher when the interval between straw introduction in the freezer and seeding lasted at least 5 min (2–4 min = 48.0% (n = 254) vs. 5–8 min = 57.1% (n = 294); P = 0.009). Time and operator effects were confounded. Overall PR results for the two technicians who used mostly 2–4 min intervals averaged 47% (operator values = 35.6, 48.9, and 54.5) whereas PRs were 54.9 and 60.5% for those waiting 5 min or more before inducing seeding (n = 2). PRs were higher when at least two donor cows were collected per RS (1 donor cow = 49% (n = 259) vs. ≥2 donor cows = 56.4% (n = 289); P = 0.003). This was not in agreement with previous observations in fresh embryos (Bourgoin et al. 2004). However, the number of donor cows strongly influenced the number of viable embryos per RS (1 donor cow = 11 ± 5 vs. ≥2 donor cows = 18 ± 8.5; P < 0.05) and could permit the choice of more embryos to be frozen. These results show that good PR may be achieved with a delay of several hours between flushing and freezing, when heifers are used as recipients. Moreover, confirmed from higher numbers of operators, these data show that it is better to wait at least 5 min to achieve equilibration of the embryo before seeding.


2006 ◽  
Vol 18 (2) ◽  
pp. 195
Author(s):  
D. Rizos ◽  
B. Pintado ◽  
J. de la Fuente ◽  
P. Lonergan ◽  
A. Gutierrez-Adan

It is well known that modification of the post-fertilization culture environment of mammalian pre-attachment embryos can affect blastocyst quality, manifested in terms of morphology, cryotolerance, and relative abundance of certain gene transcripts. Culture of in vitro-produced bovine zygotes in the ewe oviduct leads to the development of blastocysts of a quality similar to those derived totally in vitro (Rizos et al. 2002 Biol. Reprod. 66, 589-595). However, such a system has disadvantages from a practical and animal welfare point of view. The isolated mouse oviduct (IMO) culture system is a potential alternative and has been successfully used in the in vitro culture of mouse, rat, hamster, and pig embryos from the one-cell stage to the morula/blastocyst stage. The aim of this study was to examine (1) the development of bovine zygotes in the IMO maintained in two different media (SOF and KSOM) in organ culture, and (2) the quality of the resultant blastocysts assessed in terms of the relative abundance of transcripts for several genes that have been previously implicated in embryo quality. Mouse oviducts were isolated from adult Swiss females (CD1, Harlan) the day after mating with an intact male. Approximately 10-15 presumptive bovine zygotes, produced by in vitro oocyte maturation and fertilization, were transferred to the ampullae of the isolated oviducts and were cultured in Transwell plates (Costar, Corning, NY, USA) over 1.1 mL of culture medium (SOF, n = 241 or KSOM, n = 320) at 39�C in an atmosphere of 5% CO2 in air at maximum humidity. A control group of embryos was cultured in droplets (25 �L) of the same culture medium and conditions in parallel (SOF, n = 278, KSOM, n = 225). Five replicates (=days of bovine ovary collection) were carried out. Following 6 days of culture, embryos were recovered from the oviducts/culture drops and blastocysts were snap-frozen in liquid nitrogen. Quantification of all gene transcripts was carried out by real time quantitative RT-PCR. Data on embryo development were analyzed by chi-square analysis and differences in transcript abundance by ANOVA. Culture in the IMO did not affect the proportion of zygotes developing to the blastocyst stage compared to the respective control droplets (SOF: 21.0 vs. 21.9%; KSOM: 22.0 vs. 22.2%). Culture in the IMO in SOF resulted in an increase (P d 0.05) in the abundance of transcripts for Oct-4 and SOX and reduced abundance of Glut-1, Na/K transporter, Cx43, and survivin, compared to control embryos. In contrast, culture in the IMO in KSOM resulted in increased abundance of transcripts for Glut-1, Cx43, Oct-4, and survivin and a reduced expression of Na/K transporter and SOX. Transcripts for G6PDH, IFN, and E-Cad were unaffected by culture environment. In conclusion, culture in the IMO leads to alterations in the relative abundance of transcripts that have been previously associated with embryo quality following culture in the ewe oviduct. However, the effect is dependent on the basal medium used.


2006 ◽  
Vol 18 (2) ◽  
pp. 187
Author(s):  
J. De la Fuente ◽  
A. Gutiérrez-Adán ◽  
P. Beltrán Breña ◽  
S. S. Pérez-Garnelo ◽  
A. T. Palasz

It is assumed that, contrary to phosphate buffers, zwitterionic buffers are neutral. However, zwitterionic buffers containing hydroxymethyl or hydroxyethyl residues may interact with OH-groups in the media and produce formaldehyde (Shiraishi et al. 1993 Free Radic. Res. Commun. 19, 315-321). Also, it was shown that three zwitterionic buffers tested in this study interact with DNA (Stellwagen et al. 2000 Anal. Biochem. 287, 167-175). Our objective was to evaluate the effect of the following buffers: TES (T), MOPS (M), HEPES (H) (pKa values at 20�C: 7.2-7.5), and PBS on in vitro development and morphology of bovine embryos. Zwitterionic buffers and PBS were prepared at a concentration of 10 mM in TALP medium and the final pH was adjusted to 7.2. Bovine follicular fluid was aspirated from abattoir-derived ovaries and evenly divided into four tubes. Collected oocytes (five replicates) from each tube were processed separately through the entire IVM, IVF, and IVC procedures using washing medium buffered with: PBS (n = 490), Group 1; H (n = 438), Group 2; M (n = 440), Group 3; and T (n = 394), Group 4. All buffers contained 4 mg/mL BSA. Oocytes were matured in TCM-199 + 10% FCS and 10 ng/mL of epidermal growth factor and fertilized in Fert-TALP containing 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, 6 mg/mL BSA-FAF, and 10 �g/mL heparin with 1 � 106 spermatozoa/mL. After 24 h, oocytes-sperm co-incubation presumptive zygotes were cultured in SOFaa medium with 8 mg/mL BSA at 39�C under paraffin oil and 5% CO2 in humidified air. Cumulus-oocyte complexes and zygotes were held in designated buffers ?16 min before oocyte maturation, ~7 min after IVM and before IVF, and ~18 min after IVF and before culture. The total time of oocyte/embryo exposure to each buffer was ?41 min. Embryo development was recorded on Days 4, 7, 8, and 9. A total of ten, Day 8 blastocysts were taken randomly from each treatment and fixed in 4% paraformaldehyde for total and apoptotic cells counts, and five blastocysts from each replicate and treatment were frozen for later mRNA analysis. Apoptosis were determined by TUNEL, using commercial In situ Cell Death Detection Kit (Roche Diagnostic, SL, Barcelono, Spain). Embryo development among groups was compared by chi-square analysis. The cleavage rates were not different among the groups: PBS, 70.8%; H, 76.5%; M, 77.5% and T, 73.6%. The number of embryos that developed to d8 cells at Day 4 was higher in M, 36.2%, and PBS, 37.6%, than in H, 30.6%, and T, 29.7%, but was not significantly different. However, more (P < 0.05) blastocysts developed at Days 7, 8, and 9 in H and M than in PBS and T groups (21.9% and 22.9% vs. 16.9% and 14.9%, respectively). No difference was found between groups in total cell number (98.8 � 7, PBS; 111.8 � 11.9, M; 106.8 � 12.9, H; and 104.3 � 9.7, T) and the number of apoptotic cells (9.2 � 1.0, P; 9.2 � 0.8, M; 12.9 � 1.8, H; and 9.7 � 0.9, T). Based on the results of this study, we conclude that within our protocol choice of buffer may affect embryo developmental rates but not morphology.


2006 ◽  
Vol 18 (2) ◽  
pp. 137
Author(s):  
A. Lucas-Hahn ◽  
E. Lemme ◽  
K.-G. Hadeler ◽  
H.-G. Sander ◽  
H. Niemann

The reproductive performance of cloned cattle was investigated by assessing the efficiency of transvaginal ultrasound-guided ovum pickup (OPU) and embryo production in vitro. Fetal fibroblasts from the endangered species, German Blackpied Cattle, had been used for nuclear transfer to produce three live cloned offspring (Lucas-Hahn et al. 2002 Theriogenology 57, 433). In the three cloned animals at 12–20 months of age, OPU was performed once per week and the total number of collected oocytes was recorded. In the case of Blondie, the procedure was terminated due to too small ovaries associated with insufficient function. Oocytes suitable for IVF were matured in vitro for 24 h and fertilized in vitro with the semen of a fertile bull. Oocytes derived from abbatoir ovaries were processed in parallel as controls. Embryos were in vitro-cultured in SOFaaBSA medium. Cleavage and developmental rates up to the morula/blastocyst stage were recorded in all groups. Statistical significance was tested using ANOVA and the Student-Newman-Keuls test. The results are presented in Table 1. Embryos from clones had lower cleavage and blastocyst rates compared to those derived from abattoir oocytes. However, results may have been confounded by potential OPU effects. Some of the blastocysts produced from Blacky (n = 5) and Paula (n = 2) were transferred to recipients. Two pregnancies resulted from the Paula transfers. The two male calves were delivered normally. After the completion of this experiment, all three cloned animals were artificially inseminated, became pregnant, delivered healthy calves, and are pregnant again at present. Further studies are needed to explore the fertility of cattle derived from somatic cloning. Table 1. OPU and in vitro embryo production in cloned cattle


2010 ◽  
Vol 22 (1) ◽  
pp. 304
Author(s):  
L. M. C. Pegoraro ◽  
M. N. Dode ◽  
C. F. Weissheimer ◽  
F. G. Leivas ◽  
A. Vieira ◽  
...  

Bovine in vitro production systems are one of the most used assisted reproductive technique. However, this technique has some limitations especially in Bos taurus breeds, because of the low percentage of viable blastocysts produced (around 40% of oocytes inseminated) and higher cryosensitivity due to higher lipid content. Growth hormone (GH) can be a promising additive to increase in vitro embryo production. The aim of this study was to evaluate the embryo developmental rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) and ultrastructural features in Bos taurus embryos produced in SOFaa medium with or without GH. Cumulus oocyte complexes (COC) were recovered from slaughterhouse-derived ovaries (Angus Red crosses) and by ovum pick-up (OPU) from Jersey donors. After IVM and IVF, the presumptive zygotes were allocated in the SOFaa medium without (control) or with addition of GH (100 ng mL-1), for culture at 39°C in an atmosphere of 5% CO2. The cleavage and viable blastocyst rates were recorded 2 and 8 days after initiation of IVF, respectively. The results were compared by chi-square analysis. Similar (P > 0.05) cleavage rates were found in different culture medium and bovine breeds (61 v. 63% for Jersey control and Jersey GH; 71 v. 72% for cross-breed control and cross-breed GH). The development rates (blastocyst/oocytes cleaved and blastocyst/oocytes inseminated) did not differ in culture medium with or without GH within breeds (35 v. 30% for Jersey control and GH; 52 v. 56% for cross-breed control and GH; 21 v. 20% for Jersey control and GH; 36 v. 41% for cross-breed control and GH, respectively; P > 0.05). However, when breeds were compared, higher development rates were observed in cross-breed obtained from slaughterhouses than Jersey donors obtained by OPU (35 v. 52% for Jersey v. cross breed control; 30 v. 56% for Jersey v. cross-breed GH; 21 v. 36% for Jersey v. cross-breed control; 20 v. 41% for Jersey v. cross-breed GH. P < 0.001). The analyses of ultrastructure demonstrated no difference in the lipid proportion and organelle distribution of embryos produced with or without GH. We concluded that GH addition to SOFaa medium did not increase developmental rates for cross-breed or Jersey IVP embryos.


Sign in / Sign up

Export Citation Format

Share Document