Effect of medium milieu on sperm penetration and pronuclear formation of bovine oocytes matured in vitro

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

Download Full-text

In vitro fertilization in inbred BALB/c mice I: isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation

Zygote ◽

10.1017/s0967199408004607 ◽

2008 ◽

Vol 16 (3) ◽

pp. 249-257 ◽

Cited By ~ 5

Author(s):

Seiji Kito ◽

Yuki Ohta

Keyword(s):

Calcium Concentration ◽

Detrimental Effect ◽

Choline Chloride ◽

Positive Control ◽

Basic Medium ◽

Vitro Fertilization ◽

Medium Osmolarity ◽

Sperm Penetration ◽

Pronuclear Formation

SummaryTo optimize IVF conditions for BALB/c mice, which are known to have poor in vitro fertilizability, the requirements for sperm–ova interaction were studied by use of modified simplex optimization medium (mKSOM) as a basic medium. Modified human tubal fluid (mHTF) was used for sperm preincubation and acted as a positive control. When the two media were compared, neither capacitation nor fertilization was supported in mKSOM. Increasing the calcium concentration in mKSOM to 5 mM or more during sperm: ova coincubation improved zona penetration but not male pronuclear (MPN) formation to the same level as those cells incubated in mHTF. When medium osmolarity was varied from 230–305 mOsmol by NaCl at 5 mM CaCl2, MPN formation improved at 280 mOsmol or higher osmolarity to the same level as that found when using mHTF. When NaCl equivalent to 25–75 mOsmol was substituted with trehalose, no significant reduction in fertilization was observed. Substitution of NaCl equivalent to 75 mOsmol with other osmotic reagents (sucrose, choline chloride and sorbitol) resulted in similar levels of fertilization as found with mHTF, except for sorbitol, which reduced fertilization significantly caused by its detrimental effect on sperm viability. At isotonic osmolarity (305 mOsmol), maximum fertilization was observed at 5 mM CaCl2; lower or higher concentrations of CaCl2 resulted in reduced fertilization. Calcium and osmolarity, therefore, are important for sperm : ova interaction in BALB/c mice and the increases in calcium to 5 mM and osmolarity to 305 mOsmol are optimal for BALB/c sperm to penetrate through the zona and to form MPN.

Download Full-text

376 EFFECT OF SPERM TREATMENT ON THE ABILITY TO ACTIVATE OOCYTES AFTER ICSI IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab376 ◽

2007 ◽

Vol 19 (1) ◽

pp. 303

Author(s):

M. Nakai ◽

N. Kashiwazaki ◽

N. Maedomari ◽

M. Ozawa ◽

J. Noguchi ◽

...

Keyword(s):

Freeze Drying ◽

Oocyte Activation ◽

Sham Injection ◽

Oocyte Meiosis ◽

Freeze Dried ◽

Sperm Penetration ◽

Sperm Factor ◽

Pre Treatment ◽

Pronuclear Formation

During fertilization, sperm penetration (gamete membrane fusion and exposure of sperm cytoplasm) allows oocyte activation (resumption of oocyte meiosis, pronuclear formation, etc.) by inducing an elevation of the intracellular free Ca2+ concentration. So a spermatozoon ought to be able to fully activate an oocyte. However, in pig ICSI oocytes, although a spermatozoon is injected successfully into ooplasm, complete activation is deficient in some of the oocytes. A variety of sperm pre-treatments before ICSI have been reported; however, there is a possibility that the treatment affects the ability to activate oocytes after the injection. We examined the effect of sperm treatments (freezing, freeze-drying, and sonication) on the ability to activate oocytes. Ejaculated boar semen was centrifuged (10 min, 600g) and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze 1991 Reprod. Domest. Anim. (Suppl. 1), 231–253). The sperm were then treated with or without sonication for 10 s (fresh whole and sonicated sperm, respectively). The freezing of sperm was carried out as was described (Kikuchi et al. 1998 Theriogenology 50, 615–623). Frozen–thawed spermatozoa were then treated with or without sonication (frozen–thawed sonicated and whole sperm, respectively). The fresh whole and sonicated sperm were subjected to a freeze-drying system and the sperm were then re-hydrated (freeze-dried whole and sonicated sperm, respectively). A whole sperm or 1 or 3 sonicated sperm heads were then injected into in vitro-matured oocytes, as described previously (Nakai et al. 2003 Biol. Reprod. 68, 1003–1008; 2006 Reproduction 131, 603–611). Sham injection was also performed. No artificial stimulation was added to the injected oocytes. The oocytes with more than one pronucleus(i) at 10 h after the injection were defined as being activated. As shown in Table 1, the rates of activated oocytes after injection of one sonicated head or sham injection were significantly lower than those of the oocytes injected with whole sperm or 3 sonicated sperm heads in each sperm source (P < 0.05 by ANOVA and Duncan's multiple range test). Furthermore, the rates of activated oocytes for each injection category were not different among the 3 sperm sources. These results suggest that sonication before ICSI may reduce the quantity of activation-inducing sperm factor. It is also suggested that sperm pre-treatment such as freezing or freeze-drying does not affect the ability for oocyte activation. Table 1. Effect of sperm treatment on oocyte activation after ICSI

Download Full-text

87 EFFECT OF CRYOPROTECTANT CONCENTRATION IN THE VITRIFICATION SOLUTION ON THE ZONA PELLUCIDA HARDENING AND SPERMATOZOA PENETRATION OF BOVINE OOCYTES

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab87 ◽

2011 ◽

Vol 23 (1) ◽

pp. 149 ◽

Cited By ~ 1

Author(s):

A. Quiñones Martorello ◽

G. Rios ◽

A. Cano ◽

R. H. Alberio

Keyword(s):

Zona Pellucida ◽

Calcium Influx ◽

Oocyte Activation ◽

High Concentration ◽

Hoechst 33342 ◽

Polar Bodies ◽

Dmso Concentration ◽

Sperm Penetration ◽

Bovine Oocytes

In the murine model, it has been shown that the high concentration of cryoprotectants required for vitrification can activate the oocytes through a process mediated by calcium influx. This activation induces the zona pellucida (ZP) hardening and affects the sperm penetration. This study aimed to evaluate the effect of exposure of bovine oocytes to the vitrification solutions (VS1 and VS2) in calcium-free medium with 3 concentrations of etilenglycol (EG) and dimetylsulfoxide (DMSO) on the oocyte activation. Cumulus oocyte complexes (COC) were matured in vitro (22 h), partially denuded through pipetting in medium with hyaluronidase, and subject to four treatments: T1, untreated (control); T2, exposed to 20% EG+0% DMSO (VS1) and then 40% EG+0% DMSO (VS2); T3, 10% EG+10% DMSO (VS1) and then 20% EG+20% DMSO (VS2); and T4, 0% EG+20% DMSO (VS1) and then 0% EG+40% DMSO (VS2). The contact with each VS was 3 min and 30 s, respectively. After this, the COC were matured up to 24 h. In Expt. 1, COC were denuded and placed in a solution of pronase E in PBS (1 mg mL–1) to determine the number of oocytes with ZP digested after 9 min of exposure to the enzyme. In Expt. 2, COC were fertilized in TALP medium with 50 mg mL–1 heparin and 1 million mL–1 sperm. After 12 h, COC were denuded and stained with bisbenzimide (Hoechst 33342) and examined under epi-fluorescence. The number of oocytes indicating spermatic penetration was determined by presence of intact sperm heads, spermatic pro-nucleus, or 2 polar bodies. Data were analysed by the PROC GENMOD (SAS; see Table 1). In Expt. 1, there were no differences in the percentage of oocytes without ZP after pronase treatment in groups T1, T2, and T3. The T4 group had the lowest percentage of digestion, and T3 was not different from T4. In Expt. 2 there were no differences in the percentage of sperm penetration between T2, T3, and T4. All treatments had lower values than T1. In conclusion, bovine oocytes undergo hardening of the ZP when put in contact with the cryoprotectants, and this effect was significantly increased with the use of DMSO. Moreover, there was a decrease in sperm penetration in all treated groups, indicating that the natural blocking of polyspermy depends not only on the hardening of the ZP, but another process that could act at the plasma membrane. It is possible that cryoprotectants, regardless of their concentration, may trigger this early block through a mechanism that would be independent of calcium. Table 1.Effect of EG and DMSO concentration in the VS on the ZP hardening and sperm penetration of bovine oocytes exposed to these solutions Acknowledgment: the National Research Agency through the grant PICT 2007/1205.

Download Full-text

320 EFFECTS OF DIFFERENT ACTIVATION REGIMENS ON PRONUCLEAR FORMATION AND PRE-IMPLANTATION DEVELOPMENTAL COMPETENCE OF IN VITRO-MATURED BOVINE OOCYTES AFTER INTRACYTOPLASMIC SPERM INJECTION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab320 ◽

2015 ◽

Vol 27 (1) ◽

pp. 249

Author(s):

M. E. Arias ◽

R. Sanchez ◽

R. Felmer

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Standard Procedure ◽

Chemical Activation ◽

Developmental Rate ◽

Fertilization Rate ◽

Developmental Competence ◽

Assisted Reproductive Technique ◽

Pronuclear Formation ◽

Bovine Oocytes

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique that has been used with considerable success in humans; however, in the bovine species the efficiency of this technique is far from optimal. The objective of the present study was to evaluate the effect of 4 chemical activation treatments, 6-dimethylaminopurine (DMAP), cycloheximide (CHX), anisomycin (ANY), and ethanol (EtOH) on the pronuclear formation and embryo development of bovine embryos generated by ICSI. Cumulus-oocyte complexes were aspirated from abattoir ovaries, selected, and matured in 400-µL drops of standard TCM-199 maturation medium for 22 h at 38.5°C and 5% CO2. The ICSI was performed by a standard procedure. Injected oocytes were randomly distributed and activated by 5 µM ionomycin for 5 min (Io) followed by i) 5 µg mL–1 CHX for 5 h (Io/CHX), ii) 3 h window followed by a second Io treatment plus 1.9 mM DMAP for 4 h (2Io/DMAP), iii) 1 µg mL–1 ANY for 5 h (Io/ANY), and iv) 3 h window followed by 7% ethanol (Io/EtoH). Embryos were cultured in 50-µL drops of KSOM medium under mineral oil at 38.5°C and 5% CO2, 5% O2, and 90% N2. Cleavage was recorded at 72 h and blastocyst rate at 192 h. Pronuclear formation analysis was carried out at 18 hpa with Hoechst staining. An oocyte was considered fertilized when 2 polar bodies and 1 female and 1 male pronucleus (or a decondensed sperm head) could be observed. The data were transformed to arcsine, analysed by ANOVA, and means were compared using Tukey's test with Statgraphics Plus 2 Software. Results with a total of 431 injected oocytes (114, 104, 101, and 112 for DMAP, CHX, ANY, and EtOH, respectively) showed differences in cleavage (P < 0.01) in DMAP, CHX, and ANY treatments (86, 72, and 78%, respectively), relative to EtOH (12%). Similarly, the rate of blastocysts/injected oocyte at 192 h was higher with DMAP, CHX, and ANY (41, 20, and 32%, respectively), relative to EtOH (4%). Sham-injected oocytes showed cleavage and blastocyst rates of 67, 43, 68, and 12% and 32, 11, 19, and 5%, for DMAP, CHX, ANY, and EtOH, respectively. Despite the higher developmental rate observed with DMAP, pronuclear formation assessment revealed that fertilization rate was higher in CHX (87%) and ANY (75%) treatments relative to DMAP (35%). In conclusion, the results of the present study show that activation of bovine oocytes after ICSI is more efficient with DMAP and ANY, compared with CHX and EtOH.Provision of ovaries by our local slaughterhouse (Frigorifico Temuco, Chile) and funding support from FONDECYT 1120241 CONICYT, Chile, are gratefully acknowledged.

Download Full-text

137 Assessment of fertilizing ability of Merino ram semen cold stored up to 48h by heterologous IVF of bovine oocytes

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab137 ◽

2019 ◽

Vol 31 (1) ◽

pp. 194 ◽

Cited By ~ 1

Author(s):

D. A. Galarza ◽

M. Ladrón de Guevara ◽

P. Beltrán-Breña ◽

M. J. Sánchez-Calabuig ◽

A. López-Sebastián ◽

...

Keyword(s):

Semen Analysis ◽

Skim Milk ◽

Semen Parameters ◽

Computer Assisted ◽

Cleavage Rate ◽

Straight Line ◽

Fertilizing Ability ◽

Pronuclear Formation ◽

Bovine Oocytes

The use of cold-stored ram semen has been applied in sheep AI programs, because it preserves its fertilizing ability similar to fresh. Besides, the heterologous IVF has been successfully employed to assess semen fertilizing ability in several species. Hence, we aimed to evaluate the fertilizing ability of ram semen cold stored up to 48h at 5°C by assessing heterologous IVF using bovine oocytes. Fifteen pools of 3 normospermic Merino ram (2-7 years) ejaculates were collected using artificial vagina, diluted to 200×106 spermatozoa mL−1 with ultra-heat-treatment-based extender (skim milk-6% egg yolk) and cold stored up to 48h. In vitro matured zona-intact bovine oocytes were subjected to heterologous IVF using fresh semen (FS, n=707), semen cold stored to 24h (CS24, n=832) or semen cold stored to 48h (CS48, n=611). In parallel, homologous IVF (control, n=1356) and parthenogenesis (parth control non-fertilized oocytes, n=334) were performed. Ram non-selected and selected (BoviPure, Nidacon International, Mölndal, Sweden) semen parameters were evaluated by computer-assisted semen analysis. Sperm-oocyte interaction was assessed at 2.5h post-insemination (hpi) by evaluating the number of bound spermatozoa, whereas penetration and polyspermy were evaluated after 12 hpi. Presumptive zygotes were fixed and stained with Hoechst 33342 at 18, 20, 22, 24 and 26 hpi to assess pronuclear formation using phase contrast and confocal microscopy. Cleavage rate was evaluated in all groups at 48 hpi. Data obtained from 5 replicates were analysed using one-way ANOVA. Data was expressed as mean±standard error of the mean. In terms of sperm storage time, non-selected semen showed a significant decrease (P<0.05) for CS24 and CS48 compared with FS on progressive motility [SPM (%): 52.30±4.1 and 36.9±5.5v. 71.3±1.6] and straight-line velocity (mm s−1: 132.2±6.1 and 109.7±6.3v. 176.7±4.3), respectively. However, selected semen showed a decrease (P<0.05) only for CS48 when compared with CS24 or FS on SPM (35.6±3.9v. 56.1±6.91 and 59.3±2.6) and straight-line velocity (83.5±4.4v. 105.3±6.5 and 110±2.0), respectively. No differences were observed between heterologous IVF groups in all parameters evaluated. Homologous IVF showed a higher percentage of penetration only when compared with heterologous FS group (44.4±6.8v.12.5±4.5%; P<0.01). The polyspermy was higher in heterologous CS24 group when compared with homologous IVF (11.4±3.4v. 3.8±2.2; P<0.05). The homologous IVF group, as expected, showed the higher percentage of pronuclear formation at 18 hpi compared with heterologous IVF with FS (67.3±5.8v. 35.2±5.6%), CS24 (72.1±4.5 v. 37.2±5.7%) and CS48 (63.0±6.0 v. 27.0±5.6%), respectively (P<0.001). Likewise, cleavage rate was higher in homologous group compared with heterologous IVF and parthenogenetic groups for FS (78.3±2.6.8v. 46.3±3.2 and 7.0±2.3%), CS24 (78.4±2.6 v. 48.3±3.2 and 4.9±2.0%), and CS48 (78.4±3.3 v. 43.3±3.5 and 4.3±1.2%), respectively (P<0.001). In conclusion, Merino ram semen cold stored up to 48h maintains its fertilization ability to the same extend as fresh and can be used for sheep crossbreeding programs.

Download Full-text

Effect of progesterone and/or estradiol-17β on sperm penetration in vitro of bovine oocytes

Theriogenology ◽

10.1016/0093-691x(96)00168-9 ◽

1996 ◽

Vol 46 (3) ◽

pp. 459-469 ◽

Cited By ~ 5

Author(s):

R.C. Chian ◽

P. Blondin ◽

M.A. Sirard

Keyword(s):

Sperm Penetration ◽

Bovine Oocytes ◽

Estradiol 17Β

Download Full-text

Reversible changes in protein phosphorylation during germinal vesicle breakdown and pronuclear formation in bovine oocytes in vitro

Zygote ◽

10.1017/s0967199403002156 ◽

2003 ◽

Vol 11 (2) ◽

pp. 119-129 ◽

Cited By ~ 5

Author(s):

R.C. Chian ◽

J.T. Chung ◽

K. Niwa ◽

M.A. Sirard ◽

B.R. Downey ◽

...

Keyword(s):

Protein Phosphorylation ◽

Gel Electrophoresis ◽

Germinal Vesicle ◽

Two Dimensional Gel Electrophoresis ◽

Germinal Vesicle Breakdown ◽

One Dimensional ◽

Cell Cycle Transition ◽

Pronuclear Formation ◽

Bovine Oocytes

This study examined the event of protein phosphorylation in bovine oocytes during germinal vesicle breakdown (GVBD) and formation of pronuclei following fertilisation in vitro. Immature oocytes were obtained from abattoir materials and cultured in vitro. The oocytes were labelled with [32P]orthophosphate at 3 h intervals from 0 to 12 h following maturation in culture or from 3 to 18 h following insemination. One-dimensional gel electrophoresis indicated that levels of protein phosphorylation are low prior to GVBD. However, the levels of protein phosphorylation at approximately 40 kDa, 27 kDa, 23 kDa and 18 kDa increased substantially following GVBD and then decreased gradually as maturation in culture progressed. In contrast, the levels of protein phosphorylation increased gradually in the oocytes following pronucleus formation. Further, two-dimensional gel electrophoresis indicated that the protein at approximately 18 kDa reversibly changed in the oocytes during maturation and fertilisation. These results indicate that the reversible changes of this phosphoprotein may be related to either cell cycle transition or pronucleus formation during maturation and fertilisation in bovine oocytes.

Download Full-text

44 EFFECT OF CYTOPLAST VOLUME ON FERTILIZATION AND EMBRYO DEVELOPMENT IN PORCINE M-II OOCYTES RECONSTRUCTED WITH KARYOPLASTS AND CYTOPLASTS OBTAINED BY THE 'CENTRI-FUSION' METHOD

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab44 ◽

2008 ◽

Vol 20 (1) ◽

pp. 102

Author(s):

N. Maedomari ◽

K. Kikuchi ◽

M. Fahrudin ◽

N. Nakai ◽

M. Ozawa ◽

...

Keyword(s):

Polar Body ◽

Cell Number ◽

Blastocyst Stage ◽

Developmental Competence ◽

First Polar Body ◽

Developmental Capacity ◽

Sperm Penetration ◽

Pronuclear Formation ◽

And Control

Metaphase-II chromosome transfer (M-II transfer) of oocytes is considered to be one of the advanced procedures to improve fertilization and developmental abilities of oocytes with poor cytoplasmic maturation. The aim of this study was to investigate the developmental capacity after IVF and IVC of porcine oocytes reconstructed from karyoplasts and cytoplasts produced by centri-fusion (Fahrudin et al. 2007 Cloning Stem Cells 9, 216–228). In brief, IVM oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041) with a visible first polar body were centrifuged at 13 000g for 9 min to stratify the cytoplasm. Then the zonae pellucidae were removed with pronase treatment. Zona-free oocytes were layered on a 300-µL discontinuous gradient of Percoll in TCM-HEPES with 5 µg mL–1 of cytochalasin B. After centrifugation at 6000g for 4 s, fragmented cytoplasms with approximately equal volumes were obtained, stained with Hoechst-33342, and classified into cytoplasm with (K; karyoplast) or without (C; cytoplast) chromosomes. One karyoplast was fused with 0, 1, 2, 3, and 4 cytoplasts (K, K + 1C, K + 2C, K + 3C, and K + 4C, respectively) by an electric stimulation with a single DC pulse (1.5 kV cm–1 for 20 µs) and cultured for 1 h. Zona-free oocytes without any reconstruction served as control oocytes. The diameters of the reconstructed and control oocytes were measured. All specimens were fertilized in vitro with frozen–thawed boar sperm, and cultured using the well of the well (WOW) system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256–264). Their fertilization status and developmental competence were examined. Data were analyzed by ANOVA followed by Duncan's multiple range tests. The diameter differed significantly among K to K + 4C oocytes (75.0–127.1 µm; P < 0.05), whereas the diameter of K + 2C oocytes was similar to that of the control oocytes (110.5 µm). Regardless of the cytoplast volume, sperm penetration rates (73.1–93.8%) for K to K + 4C oocytes were not significantly different compared to control oocytes (78.0%). Male pronuclear formation rates of K to K + 4C oocytes (92.3–97.1%) were also not different significantly different compared to control oocytes (96.6%). However, monospermy rates of K oocytes was significantly higher (61.6%; P < 0.05) than those of the reconstructed (K + 1C to K + 4C; 18.2–34.9%) and control oocytes (32.9%). The blastocyst formation rates in K, K + 1C, K + 2C, and K + 3C groups (0.0–9.8%; P < 0.05) were significantly lower than those in the control and K + 4C groups (17.8% and 15.3%, respectively; P < 0.05). The total cell numbers per blastocyst in K + 1C and K + 2C groups (7.5 and 8.3 cells, respectively) were significantly lower than in the control, K + 3C, and K + 4C groups (15.3–26.2 cells; P < 0.05). These results suggest that the cytoplast volume of porcine M-II transferred oocytes, produced by reconstruction from a karyoplast and cytoplast(s) and centri-fusion, is important for their ability to develop to the blastocyst stage and influences cell number.

Download Full-text