98 Assessing Endangered Felid Puma concolor Sperm Fertility by In Vitro Fertilization with Domestic Cat Oocytes

2018 ◽  
Vol 30 (1) ◽  
pp. 188
Author(s):  
M. Duque ◽  
A. Sestelo ◽  
D. F. Salamone
Keyword(s):  
Puma Concolor ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Suspension ◽  
Morula Stage ◽  
Cryopreserved Sperm

The Puma concolor population has been decreasing during the last 30 years. Semen cryopreservation of this species has been accomplished successfully and offers the possibility of preserving endangered species. We previously showed that fertilizing capability of wild felid spermatozoa can be evaluated using intracytoplasmic sperm injection (ICSI) with in vitro-matured domestic cat oocytes (Moro et al. 2014 Reprod. Domest. Anim. 49, 693-700). Due to the lack of homologous oocytes, we evaluated the capability of the Puma concolor sperm to induce domestic cat oocyte fertilization and subsequent pre-implantation embryo development. In the present study, cryopreserved sperm obtained by electroejaculation from five different males were used for IVF of in vitro-matured (IVM) domestic cat oocytes. Straws were thawed by exposing them to air for 10 s and then immersing in a 37°C water bath for 30 s. The contents of the straws were poured into a sterile 1.5-mL microtube pre-warmed to 37°C. The sperm suspension was diluted (1:3 v/v) by the slow (drop-by-drop) addition of a modified Tyrode’s solution. For IVF, IVM oocytes (n = 370) were co-incubated with 0.5 × 105 motile spermatozoa mL−1 in an atmosphere of 21% O2 in air at 38.5°C for 18 to 20 h. Presumptive zygotes were cultured in vitro in 50-μL drops of modified Tyrode’s medium on 6.5% CO2 in air at 38.5°C. Cleavage was determined at 48 h post-fertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Results (mean ± SEM) showed a high cleavage rate (179/370, 49.0 ± 4.0%), and a high development to morula stage (137/370, 34.4 ± 7.2%), and to blastocyst stage (94/370, 23.4 ± 4.7%) for all males. These results indicated that Puma concolor spermatozoa can induce domestic cat oocyte activation and development to blastocyst stage in similar rates to domestic cat homologous IVF: IVM oocytes (n = 291), cleavage rate (199/291, 67.1 ± 6.1%), development to morula stage (144/291, 47.8 ± 4.9%), and to blastocyst stage (86/291, 30.1 ± 1.6%). In conclusion, we demonstrated that domestic cat oocyte can be used to evaluated cryopreserve sperm samples from another felid species.

Births of kittens produced by intracytoplasmic sperm injection of domestic cat oocytes matured in vitro

2000 ◽  
Vol 12 (8) ◽  
pp. 423 ◽  
Author(s):  
M. C. Gómez ◽  
C. E. Pope ◽  
R. Harris ◽  
A. Davis ◽  
S. Mikota ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Oocyte Activation ◽  
Morula Stage ◽  
Cytoplasmic Maturation ◽  
Vitro Development

In Experiment 1, cleavage frequency and in vitrodevelopment of domestic cat embryos produced after in vitro maturation of oocytes obtained from ovaries after ovariohysterectomy (in vivo) with that of oocytes retrieved from follicle-stimulating hormone-treated donors at 24 h after administration of luteinizing hormone (in vivo) and fertilization by intracytoplasmic sperm injection (ICSI) or IVF were compared. In each group presumptive zygotes were assessed for cleavage on IVC Days 1 and 4 and for development to blastocysts on IVC Day 7. In vitro matured oocytes had lower frequencies of meiotic maturation (59.2% v. 66.5%), cleavage at Day 1 (41.4% v. 64.9%) and development to the morula stage at Day 4 (65.8% v. 87.9%) than did in vivo matured oocytes, after ICSI and IVF. Development to the blastocyst stage was lower in in vitro matured oocytes (19.0%) than in vivo matured oocytes (29.5%) after ICSI. In Experiment 2, we evaluated the capacity of sperm injected oocytes without a visible polar body to undergo cleavage and in vitro development. More in vivo matured than in vitro matured oocytes underwent cleavage at Day 1 (46.6% v. 12.6%) and developed to the morula stage by Day 4 (66.7% v. 46.1%), but no blastocysts were obtained at Day 7 in either group. In Experiment 3, we evaluated the in vivo viability of domestic cat embryos derived from ICSI of in vitro matured oocytes. Morula stage embryos were transferred to 18 domestic cat recipients either on Day 4 or 5 after oocyte recovery. A total of 3 domestic cat recipients were pregnant after transfer to recipients on Day 5. Two pregnant cats delivered two normal and healthy live male kittens on Day 68 of gestation and the remaining cat delivered a male kitten on Day 62 that died during the last two days of gestation. These results demonstrate that: (1) inadequate cytoplasmic maturation of in vitro matured domestic cat oocytes is the main cause of deficient oocyte activation; (2) the injection of oocytes without a visible polar body is a useful technique to evaluate oocyte cytoplasmic maturation; and (3) blastocysts obtained after ICSI of in vitro matured oocytes are viable and not a result of parthenogenesis.

105 Functionality evaluation of two extenders for Leopardus geoffroyi sperm cryopreservation by interspecific IVF with domestic cat oocytes

2019 ◽  
Vol 31 (1) ◽  
pp. 178
Author(s):  
A. J. Sestelo ◽  
M. D. Rodriguez ◽  
N. Gañan ◽  
D. F. Salamone ◽  
L. Barañao ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Freezing Process ◽  
Sperm Cryopreservation ◽  
Step Method ◽  
Cleavage Rate ◽  
Sperm Suspension ◽  
Acrosome Integrity

Even though knowledge in sperm cryopreservation of endangered felids advanced in recent years, very little is known about suitable protocols to cryopreserve sperm from Leopardus geoffroyi (LG). In the present study, sperm obtained by electroejaculation from 5 different males were cryopreserved in either a Tes-Tris- or a lactose-based diluent (Gañan et al. 2009 Theriogenology 72, 341-352) with modifications in the freezing process using a one-step method: straws were placed horizontally on a metal rack, 4cm above the surface of liquid nitrogen in a styrofoam box, and kept for 10min before plunging them in LN. The objectives were to (1) compare in vitro motility and acrosome status of LG sperm cryopreserved in both extenders and (2) test functionality of LG sperm cryopreserved in both extenders through their ability to fertilize mature domestic cat oocytes. Straws were thawed by exposing them to air for 10s and then immersing them in a water bath at 37°C for 30s. The contents of the straws were poured into a sterile 1.5-mL microtube prewarmed to 37°C. The sperm suspension was diluted (1:3 vol/vol) by the slow (drop by drop) addition of a modified Tyrode’s solution. Sperm parameters, percentage of motile spermatozoa, and quality of motility were assessed and sperm motility index (SMI) was calculated as follows: [% motile sperm+(quality×20)]/2. Acrosome integrity was assessed by staining with Coomassie brilliant blue. For IVF, in vitro-matured domestic cat oocytes (n=238 Tes-Tris, n=239 lactose) were co-incubated with 0.5×105 motile spermatozoa/mL under 5% CO2 in air at 38.5°C for 18-20h (Pope et al. 2006 Methods Mol. Biol. 254, 227-244). Presumptive zygotes were cultured in vitro in 50-µL drops of modified Tyrode’s medium at 38.5°C in 5% CO2, 5% O2, 90% N2 atmosphere. Cleavage was assessed 48h postfertilization, and 5% FBS was added at Day 5 of in vitro culture. Blastocyst stage was evaluated at Day 8. Data was analysed by Fisher’s exact test using GraphPad Prism 6.0 (GraphPad Inc., San Diego, CA, USA), significant at P<0.05. Results, mean (±standard error of the means), showed that SMI and acrosome integrity (pre- and post-thawing) were similar for both extenders: prethawed (SMI=56±3.3v. 59±5.5; acrosome integrity=88±3.0% v. 90±2.0%), and post-thawed (SMI=46±5.0v. 44±7.0; acrosome integrity=57±7.5% v. 68±2.4%) Tes-Tris v. lactose, respectively. For IVF, results showed a high cleavage rate in both groups (117/238, 49% v. 117/239, 49%), and a high development to morula (96/238, 40% v. 94/239, 39%) and to the blastocyst stage (61/238, 26% v. 51/239, 21%) for all males Tes-Tris v. lactose, respectively. There were no significant differences between groups at any development stage. In conclusion, we found that both extenders can be used to cryopreserve LG sperm maintaining functional conditions and that fertilizing capacity can be tested using in vitro-matured domestic cat oocytes.

280 PORCINE BLASTOCYSTS DERIVED FROM IN VITRO-MATURED OOCYTES INJECTED INTRACYTOPLASMICALLY WITH SPERM FROM TESTICULAR TISSUE XENOGRAFTED INTO NUDE MICE

2006 ◽  
Vol 18 (2) ◽  
pp. 247 ◽  
Author(s):  
K. Kikuchi ◽  
M. Nakai ◽  
N. Kashiwazaki ◽  
M. Ozawa ◽  
N. Maedomari ◽  
...  
Keyword(s):  
Nude Mice ◽  
New Technology ◽  
Testicular Tissue ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Male Gametes ◽  
Sperm Suspension

The utilization of spermatogonia from testicular tissue after xenografting into immuno-deficient mice should lead to new insights for the conservation of male gametes. However, successful embryo production using sperm cells from xenografted testicular tissues has been limited to rhesus monkeys (Honaramooz et al. 2004 Biol. Reprod. 70, 1500-1503). In the present study, the objective was to establish this new technology for pig conservation in combination with intracytoplasmic sperm injection. Testes were obtained from male piglets 6 to 15 days old, in which most of the germ cells were gonocytes; these were minced into pieces of approximately 1.5 � 1.5 � 1.5 mm. Approximately 20 fragments were transplanted under the back skin of castrated nude mice 5 to 8 weeks old. The testicular grafts were recovered between 125 and 192 days after xenografting, minced in Dulbecco's phosphate-buffered saline, and centrifuged several times, to serve as a sperm suspension. In vitro maturation of the recipient oocytes (Kikuchi et al. 2002 Biol. Reprod. 66, 1033-1041) and injection with an intact spermatozoon, followed by electrical stimulation at 1 h post-injection (Nakai et al. 2003 Biol. Reprod. 68, 1003-1008), were carried out. The putative zygotes were cultured in vitro for 6 days (Kikuchi et al. 2002), and were then fixed, stained, and assessed for embryonic development and quality. From a total of 27 mice that were xenografted with testicular tissues, spermatids and spermatozoa were obtained in 19 of the mice (70.4%). Most of the spermatozoa were matured morphologically, showing faint motility after release into the collection medium. From a total of 253 oocytes (four replications) that were injected with sperm, 63 (24.9 � 7.1%) oocytes developed to the blastocyst stage. The average total cell number was 41.9 � 3.9. These values are comparable to those in in vitro fertilization by frozen-thawed spermatozoa, resulting in developmental ability to piglets after embryo transfer (25.3% and 48.7 cells; Kikuchi et al. 2002). These results suggest the possibility of embryo production using porcine spermatozoa that are differentiated from gonocytes within the xenografts.

17 Effect of the zona-free aggregation on the developmental competence of kodkod (Leopardus guigna) embryos generated by interspecies somatic cell nuclear transfer

2019 ◽  
Vol 31 (1) ◽  
pp. 134
Author(s):  
D. Veraguas ◽  
C. Aguilera ◽  
D. Echeverry ◽  
D. Saez-Ruiz ◽  
F. O. Castro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Somatic Cell ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Relative Expression ◽  
Standard Curve ◽  
Morula Stage ◽  
Leopardus Guigna

The kodkod is considered a vulnerable species by the International Union for Conservation of Nature. Phylogenetically, the kodkod is classified in the Leopardus genus, which has only 36 chromosome pairs compared with the domestic cat, which has 38. The proposed hypothesis was that domestic cat oocytes are capable of reprogramming somatic cells from kodkod after interspecies somatic cell NT (SCNT), allowing the in vitro embryo development up to blastocyst stage. Five experimental groups were made based on the technology and culture system: (1) cat embryos generated by IVF (IVF), (2) cat embryos generated by SCNT (Ca1x), (3) aggregated cat embryos generated by SCNT (Ca2x), (4) kodkod embryos generated by interspecies SCNT (K1x), and (5) aggregated kodkod embryos generated by interspecies SCNT (K2x). Interspecies SCNT was performed using a zona-free method. Reconstructed embryos were activated by 2 electrical pulses of 140 kV cm−1 for 40 µs and then incubated for 5h in 10μg mL−1 of cycloheximide and 5μg mL−1 of cytochalasin B. Embryos were cultured in SOF media using the well of the well system in a 5% O2, 5% CO2, and 90% N2 atmosphere at 38.5°C for 8 days. The morulae and blastocysts rates were estimated, and diameter of cloned blastocysts was measured. The relative expression of OCT4, SOX2, and NANOG was evaluated in blastocysts by RT-qPCR using the standard curve method; SDHA was used for normalization. The Kruskal-Wallis test was used to evaluate the developmental parameters and gene expression. The t-test was used to evaluate blastocyst diameter. Statistical differences were considered at P<0.05. The number of replicates was IVF=10, Ca1x=8, Ca2x=6, K1x=3, and K2x=8. The morulae rate was lower when clone embryos were cultured individually (IVF=97/153, 63.4%; Ca2x=28/51, 54.9%; K2x=63/110, 57.3%; Ca1x=48/126, 38.1%; K1x=22/87, 25.3%; P<0.05). In the domestic cat, blastocysts rate was higher in IVF (58/153, 37.9%) and Ca2x (28/51, 29.4%) groups than in the Ca1x group (21/126, 16.7%; P<0.05). No blastocysts were generated in the K1x group (0/87), whereas 5.5% of blastocysts were obtained from the K2x (6/110; 5.5%); this was not statistically different compared with the K1x group (P>0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P>0.05). However, the diameter of the blastocysts from the K2x group (172.8µm) tended to be lower than that of the blastocysts from the Ca2x group (P=0.05). Regarding gene expression, only 1 of the 6 kodkod blastocysts expressed OCT4, and none expressed SOX2 and NANOG. On the other hand, the relative expression of OCT4 tended to decrease in blastocysts from the Ca1x and Ca2x groups compared with the IVF group (P=0.09), but no differences were found in the expression of SOX2 and NANOG among groups (P>0.05). In conclusion, after interspecies SCNT, domestic cat oocytes support the development of kodkod embryos until the morula stage. However, the embryo aggregation did not significantly improve the blastocyst rate and gene expression.

356 BLASTOCYST DEVELOPMENT FROM DOMESTIC CAT OOCYTES INJECTED WITH DEHYDRATED SPERMATOZOA

2006 ◽  
Vol 18 (2) ◽  
pp. 285
Author(s):  
A. Moisan ◽  
S. Leibo ◽  
J. Lynn ◽  
M. Gómez ◽  
C. Pope ◽  
...  
Keyword(s):  
Room Temperature ◽  
Calcium Ionophore ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Sperm Suspension ◽  
Glass Slides ◽  
Freeze Dried ◽  
Artificial Vagina

Live offspring have been produced by intracytoplasmic sperm injection (ICSI) of dehydrated spermatozoa into mouse and rabbit oocytes (Wakayama and Yanagimachi 1998 Nature Biotechnol. 16, 639; Liu et al. 2004 Biol. Reprod. 70, 1776). The objective of the present study was to determine the capability of dehydrated domestic cat spermatozoa to fertilize oocytes after ICSI. Spermatozoa were collected from three toms by ejaculation into an artificial vagina. Freshly collected samples were layered under 1 mL of Tyrode's salt solution supplemented with HEPES, BSA, glutamine, pyruvate, lactate, and 50 mM ethyleneglyeotetraacetic acid (EGTA) pH = 8.2-8.4, and allowed to swim-up during a 12-min incubation at 38�C. From the upper 600 �L of the sperm suspension, four 100-�L aliquots were collected and placed into 2-mL glass ampoules, plunged into liquid nitrogen (LN2), freeze dried at -43�C to -45�C and 44 to 76 � 10-3 mbar pressure, and stored at 4�C. Four to twelve 10-�L aliquots of the same suspension were placed onto glass slides, air-dried for 30 min, and stored in a desiccator at room temperature. Domestic cat oocytes were collected from minced ovaries and allowed to mature in vitro for 22 to 24 h. After maturation, oocytes were either fertilized in vitro (IVF; n = 36), or sperm injected (ICSI) with fresh or refrigerated (n = 74), freeze-dried (n = 45), or air-dried (n = 45) spermatozoa. After ICSI or IVF, oocytes were cultured in a three-step-sequential medium (G�mez et al. 2004 Cloning Stem Cells 6, 247) for up to 8 days. Cleavage and development to the blastocyst stage was assessed on Days 2 and 8 of culture, respectively. Cleavage rates after IVF (56%), ICSI with freeze-dried (60%), or ICSI with fresh spermatozoa (59%) were higher than those obtained after ICSI with air-dried spermatozoa (35%; P < 0.05). Blastocyst development after ICSI treatments was obtained only with fresh spermatozoa (9%), and was lower than that obtained after IVF (25%; P < 0.05). Recently, one hatching blastocyst was produced when oocytes (n = 18) were exposed to calcium ionophore 2 h after ICSI with freeze-dried sperm. This is the first report that domestic cat embryos can be produced in vitro by injecting oocytes with dried spermatozoa.

230 ACTIVATION OF IN VITRO-MATURED BOVINE OOCYTES WITH IONOMYCIN AND ROSCOVITINE AFTER INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 194
Author(s):  
C. B. Fernandes ◽  
L. G. Devito ◽  
L. R. Martins ◽  
T. S. Rascado ◽  
F. C. Landim-Alvarenga
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Kinase Inhibitor ◽  
Polar Body ◽  
Sperm Concentration ◽  
Oocyte Activation ◽  
Cleavage Rate ◽  
Sperm Suspension ◽  
Artificial Activation ◽  
Bovine Oocytes

In all mammalian species studied so far, fertilization induces oocyte activation necessary for pronuclear formation, syngamy, and the beginning of embryonic cleavage. The aim of this experiment was to evaluate the effectiveness of a protocol for artificial activation for bovine oocytes using ionomycin and roscovitine either in combination with intracytoplasmic sperm injection (ICSI) or alone. In this study, ionomycin was used to facilitate the increase of intracellular calcium, due to the release of calcium from intracellular stores. This compound was used in conjuction with roscovitine, a specific cdc2 kinase inhibitor. The success of the treatment was compared with that of oocytes fertilized by IVF. Three replicates were carried out using bovine oocytes harvested from slaughterhouse ovaries. In vitro-matured oocytes were cultured in TCM-199 plus 10% FCS, pyruvate, estradiol, hCG, and gentamicin at 39�C in an atmosphere of 5% of CO2 in air for 20 h. After in vitro maturation, oocytes were divided into 3 groups. For parthenogenetic activation, 100 oocytes were stripped of cumulus cells and placed in H-MEM plus 10% FCS and 5 µm ionomycin for 8 min, maintained in H-MEM plus 10% FCS, 66 mm roscovitine and 7.5 mg mL–1 cytochalasin B for 6 h, and placed into culture. In the ICSI group, oocytes were denuded and transferred to 5-µL H-MEM plus 20% FCS drops. Only MII oocytes were microinjected. The sperm drop was prepared with a mixture of 4 µL polyvinylpyrrolidone (PVP) and 1 µL of the sperm suspension produced by Percoll gradient. For injection, a single normal mobile sperm was aspirated with the tail first. A single oocyte was fixed by holding the pipette to position the polar body at the 6 or 12 o'clock position. The injection pipette was pushed through the zona pellucida and the oolema and the spermatozoan was released into the cytoplasm. After ICSI, the oocytes were subjected to the same activation protocol described earlier and cultured. For IVF, sperm was prepared by swim-up and 100 oocytes were fertilized in Fert-Talp for 18 h (sperm concentration: 1 � 106). All oocytes were cultured in HTF:BME plus 0.6% BSA, 10% FCS, 0.01% myoinositol, and gentamycin at 39�C in an atmosphere of 5% of CO2 in air for 72 h. Cleavage was evaluated visually and the embryos were stained with Hoechst 33342 for estimation of nuclei numbers. The data were analyzed by ANOVA, followed by the Tukey test (P < 0.05). The results showed a cleavage rate of 76% for the IVF group, 57% for the ICSI group, and 51% for the parthenogenic group. The artificial activation proposed was efficient in inducing oocyte activation and cleavage; however, the rates obtained were significantly lower then the ones observed after IVF. Injection of a viable sperm into the oocyte through ICSI did not improve the cleavage rate after activation. This result indicates that the membrane fusion and/or sperm interaction with the oocyte during fertilization is important for the physiological modifications that result in oocyte cleavage in bovine.

scholarly journals Development to Blastocyst Stage of Pig Oocytes Matured, Fertilized and Electroactivated In Vitro

2002 ◽  
Vol 45 (6) ◽  
pp. 547-556
Author(s):  
N. R. Mtango ◽  
M. D. Varisanga ◽  
D. Y. Juan ◽  
P. Wongrisekeao ◽  
T. Suzuki
Keyword(s):  
In Vitro Maturation ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Significant Difference ◽  
Activation Rates ◽  
Oviduct Fluid

Abstract. This study was designed 1) to determine the effectiveness of two in vitro maturation (IVM) media (tissue culture medium [TCM] and modified synthetic oviduct fluid supplemented with amino acids [mSOFaa]), 2) to compare the effects of two in vitro fertilization (IVF) media (modified Tris-buffered medium [mTBM] and mSOFaa) on the developmental competence of pig oocytes, and 3) to test the activation ability of IVM pig oocytes matured in TCM or mSOFaa, electroactivated and cultured in mSOFaa. The nuclear maturation rates were similar between IVM media (91.0 % vs. 89.0 %). A similar result was obtained when the activation rates were 54.2 % in TCM and 56.0 % in mSOFaa, and the blastocyst rates were 7.9 % and 6.1 %, respectively. There was no significant difference between mSOFaa and mTBM in the percentage of embryos with two pronuclei 33.2 % vs. 13.8 % or polypronuclei 5.3 % vs. 13.4 %. The cleavage rate was the same in both media. The medium mSOFaa gave a significantly higher (P< 0.05) blastocyst rate than mTBM (12.7 % vs. 3.9 %). We concluded that mSOFaa can enhance in vitro maturation, fertilization and culture of pig oocytes.

28 EFFECT OF TRICHOSTATIN A ON IN VITRO EMBRYO DEVELOPMENT OF INTERSPECIES NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED FROM CAT DONOR NUCLEI AND BOVINE CYTOPLASM

2013 ◽  
Vol 25 (1) ◽  
pp. 161 ◽  
Author(s):  
M. Wittayarat ◽  
Z. Namula ◽  
V. V. Luu ◽  
L. T. K. Do ◽  
Y. Sato ◽  
...  
Keyword(s):  
Nuclear Transfer ◽  
Trichostatin A ◽  
Blastocyst Stage ◽  
Domestic Cat ◽  
Cell Stage ◽  
Histone Deacetylase Inhibitor Trichostatin ◽  
Invaluable Tool ◽  
Morula Stage ◽  
Bovine Oocytes

Interspecies somatic cell nuclear transfer (iSCNT) is an invaluable tool for studying nucleus-cytoplasm interactions and may provide an alternative for cloning endangered animals whose oocytes are difficult to obtain. The developmental ability of iSCNT embryos decreases with increases in taxonomic distance between the donor and recipient species. The development of cat-bovine iSCNT embryos is reportedly blocked at the 8-cell stage (Thongphakdee et al. 2008 J. Reprod. Dev. 54, 142–147). Abnormal epigenetic reprogramming, such as DNA methylation or histone modifications, may cause low iSCNT efficiencies. The present study was conducted to evaluate the effect of the histone deacetylase inhibitor trichostatin A (TSA), previously used to enhance nuclear reprogramming following SCNT, on the developmental ability of cat iSCNT embryos using bovine oocytes matured in vitro. The matured bovine oocyte was enucleated by the glass needle and the domestic cat fetal fibroblast used as the donor nuclei was then placed into the perivitelline space adjacent to the plasma membrane of the oocyte. Couplets with bovine ooplasm were fused and activated simultaneously with a single DC pulse of 2.3 kV cm–1 for 30 µs, respectively, using an electro cell fusion generator followed by cycloheximide treatment. Reconstructed cat-bovine embryos were treated with 0, 25, 50, and 100 nM concentrations of TSA for 24 h following fusion. The percentages of embryos cleaved and embryos developed to the blastocyst stage were subjected to arc sin transformation before ANOVA. The TSA treatment at 50 nM contributed significantly higher rates of cleavage and blastocyst formation (n = 139; 84.3 and 4.6%, respectively) compared with untreated embryos (n = 187; 63.8 and 0%, respectively) and embryos treated with 100 nM TSA (n = 172; 71.4 and 0%, respectively; P < 0.05). Development to the morula stage of iSCNT embryos was observed in the TSA treatment groups, whereas no embryos developed beyond the 16-cell stage in the untreated group. In conclusion, our results indicate that TSA treatment for 24 h following fusion improves the development of iSCNT embryos. Specifically, 50 nM TSA treatment provides a beneficial effect on cleavage and development to the blastocyst stage of cat iSCNT embryos using bovine oocytes matured in vitro as recipients and domestic cat fibroblasts as donor nuclei.

scholarly journals Effect of sperm preparation method on in vitro fertilization in pigs

Reproduction ◽  
2003 ◽  
pp. 133-141 ◽  
Author(s):  
C Matas ◽  
P Coy ◽  
R Romar ◽  
M Marco ◽  
J Gadea ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blastocyst Formation ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Preparation ◽  
Male Pronucleus ◽  
Penetration Time

This study was designed to determine the effect of different sperm preparation treatments before IVF on the acrosome reaction, oocyte penetration time, early embryo development and timing of female and male pronucleus formation. Pooled sperm-rich fractions were (i) washed in PBS, (ii) left unwashed, or (iii) layered in a Percoll gradient. In Expt 1, the proportion of acrosome-reacted spermatozoa, determined by staining with fluorescein isothyocyanate-labelled peanut agglutinin lectin and propidium iodide, was highest after treatment with Percoll (P < 0.001). In Expt 2, oocytes matured in vitro were co-cultured with spermatozoa for 2, 4 or 6 h. Attached spermatozoa were then removed and the oocytes were cultured in fresh IVF medium for 16 h. Both sperm treatment and co-culture time were found to affect penetrability and monospermy rates (P < 0.001); spermatozoa treated with Percoll showed fastest oocyte penetration and highest penetrability. In Expt 3, matured oocytes were co-incubated with spermatozoa pretreated by the three above mentioned procedures (i, ii, iii) for 2, 6 and 2 h respectively. Putative zygotes were then washed and transferred to medium NCSU-23 until the blastocyst stage. In this experiment, sperm treatment had a significant effect on the cleavage rate (P < 0.001) and rate of blastocyst formation (P < 0.05); the group treated with Percoll showed the highest rate of blastocyst formation. Finally, in Expt 4, timing of female and male pronucleus formation for each sperm treatment was determined 4, 6 and 8 h after insemination. The time of female and male pronucleus formation was affected by the sperm treatment and was faster for the Percoll group (P < 0.05). The findings of the present study indicate that treatment with Percoll yields the best results in this in vitro pig embryo production system.

In vitro maturation, in vitro fertilization and embryonic development of canine oocytes

Zygote ◽  
2007 ◽  
Vol 15 (4) ◽  
pp. 347-353 ◽  
Author(s):  
S.R. Lee ◽  
B.S. Kim ◽  
J-W. Kim ◽  
M.O. Kim ◽  
S.H. Kim ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Fertilization Rate ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Nuclear Maturation ◽  
Heparin Treatment ◽  
Maturation In Vitro ◽  
Maturation Rate ◽  
Morula Stage

SummaryIn this study we have investigated the efficiency of in vitro maturation (IVM) as a basic way to study the development of canine oocytes after in vitro fertilization (IVF). We decided, therefore, to perform two-part experiments. Firstly, experiment I compared the effects of TCM199 without fetal bovine serum (FBS) with TCM199 supplemented with 5% FBS on the in vitro nuclear maturation rate of canine oocytes. For the efficiency of meiotic development to the metaphase II (MII) stage, we found that 4.7% (4/64) of all oocytes grown in TCM199 without FBS developed to the MII stage compared with only 1.7% (1/59) of those grown in TCM199 with 5% FBS for 48 h. Therefore, FBS did not increase in vitro nuclear maturation. In experiment II, the cleavage rate of canine oocytes used for IVF was investigated following heparin treatment. Canine oocytes were fertilized in four groups: Fert–TALP medium without heparin (Fert I) or Fert–TALP medium supplemented with 10, 20 or 30 µg/ml heparin (Fert II, Fert III, Fert IV, respectively). Oocytes that were grown for 24 h in Fert I following fertilization showed the highest rate of all of the groups, 6.5% (5/77) and developed to the early morula stage. Markedly, the oocytes cultured in Fert I for 24 h following insemination had a higher rate of embryonic development than other groups. We can assert that, unlike findings in other mammals, heparin treatment in canine IVF does not increase the efficiency of the fertilization rate and is therefore not an important factor.

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