Sperm capacitation combined with removal of the sperm acrosome and plasma membrane enhances paternal nucleus remodelling and early development of bovine androgenetic embryos

2013 ◽  
Vol 25 (4) ◽  
pp. 624 ◽  
Author(s):  
Yao Xiao ◽  
Hualin Zhang ◽  
Sibtain Ahmad ◽  
Liya Bai ◽  
Xiaomin Wang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Early Development ◽  
Developmental Competence ◽  
Sperm Capacitation ◽  
Paternal Genome ◽  
Morula Stage ◽  
Chromatin Integrity ◽  
Androgenetic Embryos ◽  
Sperm Acrosome ◽  
Sperm Decondensation

The androgenetic embryo is a useful model for functional analysis of the paternal genome during embryogenesis. However, few studies have focused on the factors involved in the suppressed developmental competence of such embryos or why sperm cloning-derived androgenetic embryos fail to develop beyond the morula stage in large domestic animals. To overcome this developmental failure, we tried to improve sperm decondensation, as well as to enhance embryonic development by sperm capacitation and removal of the acrosome and plasma membrane before injection of the spermatozoa. Before injection of the spermatozoa, we quantified the effects of sperm capacitation combined with sperm pretreatment on the acrosome and plasma membrane status. We also evaluated sperm decondensation potential, sperm viability and chromatin integrity. Immunostaining data showed that the sperm acrosome and plasma membrane could be more efficiently removed after capacitation. Dithiothreitol-induced sperm decondensation potential was improved with capacitation and removal of the acrosome and plasma membrane. Although most spermatozoa lost viability after pretreatment, their chromatin remained integrated. The patterns of paternal chromatin remodelling within uncleaved androgenetic embryos and the nucleus morphology of cleaved embryos indicated that capacitation combined with membrane disruption could make injected spermatozoa decondense synchronously not only with each other, but also with the developmental pace of the ooplasm. We successfully produced androgenetic blastocysts, and efficiency increased with sperm pretreatment. In conclusion, sperm decondensation and the early development of androgenetic embryos were enhanced with sperm capacitation and removal of the acrosome and plasma membrane prior to sperm injection.

scholarly journals Epidermal growth factor alleviates the negative impact of urea on frozen-thawed bovine sperm, but the subsequent developmental competence is compromised

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Rasoul Kowsar ◽  
Shahrzad Ronasi ◽  
Nima Sadeghi ◽  
Khaled Sadeghi ◽  
Akio Miyamoto
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Dna Fragmentation ◽  
Acrosome Reaction ◽  
Developmental Competence ◽  
Ros Production ◽  
Sperm Capacitation ◽  
Bovine Sperm ◽  
Epidermal Growth ◽  
High Level

AbstractUpon insemination, sperm cells are exposed to components of the female reproductive tract (FRT) fluids, such as urea and epidermal growth factor (EGF). It has been shown that both urea and EGF use EGF receptor signaling and produce reactive oxygen species (ROS) that are required at certain levels for sperm capacitation and acrosome reaction. We therefore hypothesized that during bovine sperm capacitation, a high level of urea and EGF could interfere with sperm function through overproduction of ROS. High-level urea (40 mg/dl urea is equal to 18.8 mg/dl of blood urea nitrogen) significantly increased ROS production and TUNEL-positive sperm (sperm DNA fragmentation, sDF) percentage, but decreased HOS test score, progressive motility, acrosome reaction and capacitation. The EGF reversed the negative effects of urea on all sperm parameters, with the exception of ROS production and DNA fragmentation, which were higher in urea-EGF-incubated sperm than in control-sperm. The developmental competence of oocytes inseminated with urea-EGF-incubated sperm was significantly reduced compared to the control. A close association of ROS production or sDF with 0-pronuclear and sperm non-capacitation rates was found in the network analysis. In conclusion, EGF enhanced urea-reduced sperm motility; however, it failed to reduce urea-increased sperm ROS or sDF levels and to enhance subsequent oocyte competence. The data suggests that any study to improve sperm quality should be followed by a follow-up assessment of the fertilization outcome.

The early development of haploid and aneuploid parthenogenetic embryos

Development ◽  
1975 ◽  
Vol 34 (3) ◽  
pp. 645-655
Author(s):  
Matthew H. Kaufman ◽  
Leo Sachs
Keyword(s):  
Early Development ◽  
Polar Body ◽  
Chromosome Counts ◽  
Cell Stage ◽  
Haploid Chromosome Number ◽  
Stage Of Development ◽  
Morula Stage ◽  
Second Polar Body ◽  
Similar Development ◽  
Parthenogenetic Embryos

The early development of parthenogenetically activated oocytes has been studied in C57BL × CBA-T6T6 (F1T6) translocation heterozygote mice and C57BL × CBA-LAC (F1LAC) mice. All F1T6 oocytes had either a quadrivalent or a univalent-trivalent configuration at meiosis I; no such chromosome configurations were observed in the F1LAC oocytes. At ovulation 36·5 % of the F1T6 oocytes had 19 or 21 chromosomes, whereas 97 % of the F1LAC had the normal haploid chromosome number of 20. After parthenogenetic activation, chromosome counts at metaphase of the first cleavage mitosis were made of the eggs with a single pronucleus following extrusion of the second polar body. These activated eggs had similar frequencies of 19, 20 and 21 chromosomes as had the oocytes at ovulation. The activated 1-cell eggs were transferred to the oviducts of pseudopregnant recipients and the embryos recovered 3 days later. At this stage of development, most of the F1T6 embryos with 19 chromosomes were no longer found, but the frequency of 21-chromosome embryos was similar to the frequency of 21-chromosome oocytes and activated eggs. There was a similar mean number of cells in the embryos with 20 and 21 chromosomes. The results indicate that nearly all the embryos with 19 chromosomes failed to develop, probably beyond the 2-cell stage, whereas oocytes with 21 chromosomes had a similar development to oocytes with 20 chromosomes up to the morula stage.

scholarly journals 131DIFFERENTIAL DNA METHYLATION CHANGES OF THE REPETITIVE SEQUENCES DURING PREIMPLANTATION DEVELOPMENT IN THE MOUSE

2004 ◽  
Vol 16 (2) ◽  
pp. 188
Author(s):  
S.-H. Kim ◽  
Y.-K. Kang ◽  
D.-B. Koo ◽  
M.-J. Kang ◽  
K.-K. Lee ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Repetitive Sequences ◽  
Dna Demethylation ◽  
Preimplantation Development ◽  
Mouse Development ◽  
Cell Stage ◽  
Paternal Genome ◽  
Satellite Sequences ◽  
Centromeric Satellite ◽  
Morula Stage

DNA demethylation as an epigenetic change is a unique event genome-wide, occurring at preimplantation and germ cell stages during mouse development. The paternal genome after fertilization is demethylated first, referred to active demethylation, followed by demethylation of the maternal genome during preimplantation development in the mouse. To examine methylation changes in the early mouse embryo, methylation states of various genomic regions such as intracisternal A-particle (IAP), early retrotransposon (Etn) and centromeric satellite sequences were determined by means of a bisulfite sequencing method. For methylation analysis, genomic DNA was first isolated from each developmental stage of embryo (about 300 cells in total), respectively, and exposed to 1.9M sodium bisulfite overnight. Targeted DNA sequences were amplified from bisulfite-treated genomic DNAs by PCR, cloned into pGEM T-easy vector and sequenced. Results indicated that IAP sequences maintained high levels of methylation until the morula stage and were demethylated in blastocysts. In contrast to the IAP sequences, methylation states of Etn elements were remarkably erased after fertilization, completely demethylated at the 8-cell stage and then remethylated at the morula stage. Centromeric satellite sequences showed low methylation states throughout all preimplantation stages of embryos, indicating that the satellite sequences are substantially demethylated in both paternal and maternal genomes. The results suggest that differential epifenetic changes among the repetitive sequences may be responsible for peculiar chromatin structure of respective genomic loci and/or may regulate gene expression during preimplantation development in the mouse.

62 REPROGRAMMING EVENTS AND DEVELOPMENTAL COMPETENCE OF RHESUS MONKEY EMBRYOS PRODUCED BY SOMATIC CELL NUCLEAR TRANSFER

2006 ◽  
Vol 18 (2) ◽  
pp. 139 ◽  
Author(s):  
S. Mitalipov ◽  
Q. Zhou ◽  
J. Byrne ◽  
W.-Z. Ji ◽  
D. Wolf
Keyword(s):  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Developmental Competence ◽  
Lamin A ◽  
Cell Nuclei ◽  
Blastocyst Development ◽  
Morula Stage

Successful reprogramming of somatic cell nuclei after nuclear transfer requires active remodeling by factors present in the nonactivated cytoplast. High levels of maturation promoting factor (MPF) activity are associated with this remodeling process which includes nuclear envelope breakdown (NEBD), premature chromosome condensation (PCC), and spindle formation. In this study, we examined the extent of nuclear remodeling in monkey somatic cell nuclear transfer (SCNT) embryos by monitoring the dynamics of lamin A/C appearance, as detected immunocytochemically, following fusion of donor cells with recipient cytoplasts. In the control, intracytoplasmic sperm injection (ICSI) fertilized embryos, lamin A/C was readily detected at the pronuclear stage but disappeared in early cleaving embryos only to reappear by the morula stage in association with the activation of the embryonic genome. We initially documented lack or incomplete NEBD and PCC in SCNT embryos in the form of retention of lamin A/C signal emanating from the donor nucleus. This observation was consistent with premature cytoplast activation due to the manipulation procedures. SCNT embryos produced by this approach typically arrested at the morula stage. Significant modifications in nuclear transfer protocols were then employed. Optimization of procedures resulted in robust NEBD and PCC, as indicated by loss of lamin A/C signal from the donor cell. Also, significant improvement of SCNT embryo development in vitro was observed, with a markedly improved blastocyst formation rate (21%). Several different fetal and adult somatic cell types screened as nuclear donors supported blastocyst development. SCNT blastocysts displayed a pattern of Oct-4 expression similar to that of sperm fertilized counterparts, indicative of efficient nuclear reprogramming. However, no pregnancies were established following a preliminary trial of 8 embryo transfers with 48 cloned embryos. Nevertheless, our results represent a breakthrough in efforts to produce cloned monkeys and should provide the resources required for the derivation of embryonic stem cells from SCNT blastocysts.

251 COMPARATIVE ANALYSIS OF GENE EXPRESSION PATTERNS IN PORCINE PRE-IMPLANTATION EMBRYOS DERIVED FROM DIFFERENT ORIGINS

2006 ◽  
Vol 18 (2) ◽  
pp. 233
Author(s):  
J.-G. Kim ◽  
H.-F. Jin ◽  
B.-K. Mohana ◽  
H.-J. Song ◽  
Y.-J. Jeong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Expression Patterns ◽  
Rna Isolation ◽  
Developmental Competence ◽  
Histone H2a ◽  
Cell Stage ◽  
Real Time Quantitative Pcr ◽  
Morula Stage ◽  
Different Origins

The amount of information gathered on the kinetics and quantitative profile of gene expression in nuclear transferred (NT) pre-implantation embryos is still scarce and limited to a handful of genes in pig. In the present study, we compared the relative abundance (RA) of six development-related genes of pre-implantation embryos from different origins. Cumulus-oocyte complexes (COCs) were matured, fertilized and cultured by the method of Abeydeera et al. (2000 Theriogenology 54, 787-797). Parthenogenetic (PA) and NT embryos were produced as described by Kim et al. (2005 Mol. Rep. Dev. 70, 308-313). Sets of 10 embryos each at 4-cell, 8-16-cell, morula, and Day 7 blastocyst stages were used to extract total RNA for analyzing the expression pattern of Bax (pro-apoptotic), Bcl-xl (anti-apoptotic), Oct-4 (pluripotent transcription), Stat3 (cytoplasmic transcription), IFN-tau (implantation) and VEGF (vasculogenesis) genes (three replicates) with real-time quantitative PCR (LightCycler�; Roche Diagnostics, Mannheim, Germany) following RNA isolation (with Dynabeads� mRNA Direct" kit; Dynal, Oslo, Norway) and cDNA amplification (Oligo (dT)12-18 primer and Superscript" III cDNA synthesis kit; Invitrogen, Carlsbad, CA, USA). The expression of each gene was normalized to Histone H2A expression. Statistically significant (P < 0.05) differences in gene expression were analyzed by ANOVA. Oct-4, Stat3, Bax, and Bcl-xl were expressed at the 4-cell stage. However, IFN-tau and VEGF were expressed at the morula stage. The expression patterns of all genes throughout the embryonic development in all types of embryos were similar. However, the relative abundance (RA) of Bax in the 8-16 cell stage of NT and PA was significantly (P < 0.05) increased as compared to that in IVP counterparts. The RA of Oct-4 and Bcl-xl did not differ throughout all stages in NT and PA embryos. There were no significant differences in all types of embryos with respect to the RA of Oct-4 and Bcl-xl with exception of the blastocysts in NT (significantly decreased, P < 0.01). The RA of Stat3 was significantly (P < 0.05) increased in the 8-16 cell stage (NT and PA) and in blastocysts (NT). Similarly, the RA of IFN-tau was significantly (P < 0.05) increased in NT blastocysts. The RA of VEGF was not significantly different in all stages and types of embryos except NT blastocysts (decreased expression, P < 0.01). These results suggest that expression patterns of Bax, Bcl-xl, Oct-4, Stat3, IFN-tau, and VEGF can be used as markers to assess the developmental competence of porcine nuclear transfer embryos. This work was supported by Grant No. 1000520040020000 from Biogreen 21, Republic of Korea.

348 CO-CULTURE OF PORCINE OOCYTES - CUMULUS COMPLEXES DERIVED FROM SMALL FOLLICLE WITH CUMULUS-CELL MASSES FROM MIDDLE FOLLICLE IMPROVES MEIOTIC MATURATION OF THE OOCYTES

2010 ◽  
Vol 22 (1) ◽  
pp. 330
Author(s):  
R. Matsunaga ◽  
H. Funahashi
Keyword(s):  
Early Development ◽  
Cumulus Cell ◽  
Developmental Competence ◽  
Meiotic Maturation ◽  
Protein Profiles ◽  
Blastocyst Formation ◽  
Maturation Rate ◽  
Sperm Penetration ◽  
Functional Peptide ◽  
Pronuclear Formation

It is known that maturation rate of oocytes derived from small follicles (SF) is lower than that of oocytes from middle follicles (MF). Since it has been reported that cumulus cells have important role during oocytes maturation, the ability of SF oocytes to complete the meiotic maturation may be affected by additional cumulus-cell mass. The present study was undertaken to examine the effects of co-culture of oocyte-cumulus complexes (OCCs) derived from SF with additional cumulus-cell masses on in vitro maturation and developmental competence of the oocytes. OCCs were aspirated from small (SF; 1-2 mm in diameter) or middle follicles (MF; 3-6 mm in diameter) of prepuberal ovaries. OCCs were cultured in porcine oocyte medium (POM; Research Institute for the Functional Peptide, Yamagata, Japan) supplemented with gonadotropins and dbcAMP for a first 20-h period and then in gonadotropin-free and dbcAMP-free POM for another 24 h. Culture medium was collected after the first 20-h culture and the end of IVM, and analyzed for the protein profiles. Following IVM, some oocytes were co-incubated with spermatozoa in a drop of modified Medium199 containing 0.4% BSA and 5 mM caffeine for 8 h and then incubated in PZM5 (Research Institute for the Functional Peptide, Yamagata, Japan) for 6 days. Sperm penetration, cleavage, and the early development of the oocytes were examined before culture in PZM5 or Day 2 and Day 6 of culture, respectively. OCCs derived from SF were co-cultured with cumulus-cell masses derived from SF or MF during IVM (SFO-SFC and SFO-MFC groups, respectively). Some OCCs derived from SF or MF were cultured for IVM without additional cumulus-cell masses (SFO and MFO, respectively). After culture, meiotic maturation of the oocytes was examined. To analyze the developmental competence of oocytes of SF, MF, and SFO-MFC groups, sperm penetration, pronuclear formation, cleavage, and blastocyst formation were examined. Protein profiles in the IVM media were examined by 10% SDS-PAGE. Statistical analysis was performed by ANOVA with a Bonferroni-Dunn post hoc test (significance, P ≤ 0.05). After culture for IVM, the diameters of SFO and SFO-MFC were not different from that of MFO (113.3-114.5 μm). The maturation rate of SFO-MFC oocytes (75.5 ± 6.2%) was higher than SFO (52.2 ± 2.8%) and comparable with the rate of MFO oocytes (83.2 ± 6.3%), while there was not significant difference between the mature rate of SFO+SFC oocytes (63.6 ± 4.0%) and SFO oocytes. There were no significant differences between groups in sperm penetration, pronuclear formation, and cleavage. Blastocyst formation of SF oocytes was not improved by co-culture with MF cumulus-cell masses. Certain band was detected only in MF medium of collected at 20 h (24.5 kD). From these results, we conclude that secretions from cumulus-cell masses derived from MF well improve the meiotic progress of oocytes derived from SF, but not the early development following IVF.

75 EFFECT OF PHENAZINE ETHOSULFATE ON PORCINE BLASTOCYST DEVELOPMENT, APOPTOSIS, AND CRYOTOLERANCE AFTER OPEN PULLED STRAW VITRIFICATION

2008 ◽  
Vol 20 (1) ◽  
pp. 118
Author(s):  
B. Gajda ◽  
Z. Smorag ◽  
M. Bryla
Keyword(s):  
Cell Number ◽  
Developmental Competence ◽  
Control Group ◽  
Tunel Staining ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Morula Stage ◽  
Phenazine Ethosulfate ◽  
And Control

It is possible to improve the success of cryopreservation of in vitro-produced bovine embryos by modifying the embryos with the metabolic regulator phenazine ethosulfate (PES) (Seidel 2006 Theriogenology 65, 228–235). The PES treatment increased glucose matabolism, tended to increase the pentose phosphate pathway flux of glucose, and clearly reduced accumulation of lipids in cultured bovine embryos (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607). It is known that porcine embryos have a considerably high content of lipids, and the success rates of their cryopreservation appear to be highly correlated with cytoplasmic lipid content. In our preliminary study, we observed that supplementation of NCSU-23 medium with PES has a positive effect on efficiency of pig blastocysts of good quality (Gajda et al.. 2007 Acta Biochim. Pol. 54(Suppl 1), 52 abst). In the present study, the effects of PES on pig blastocyst development, apoptosis, and survival after vitrification were investigated. In Exp. 1, porcine zygotes obtained from superovulated gilts were cultured in NCSU-23 medium supplemented with 0 (control), 0.025, 0.05, or 0.075 µm PES. The culture was performed at 39�C, with 5% CO2 in air, for 96–120 h. Embryo quality criteria were developmental competence (cleavage, morula stage, and blastocyst stage), cell number per blastocyst, and the degree of apoptosis as assessed by TUNEL staining. In Exp. 2, expanded blastocysts cultured with 0.025 µm PES were vitrified in a ethylene glycol and dimethyl sulfoxide mixture using open pulled straw (OPS) technology (Vajta et al. 1997 Acta Vet. Scand. 38, 349–352). After thawing, the blastocysts were cultured in vitro for re-expansion or transferred to synchronized recipients. Data were analyzed by chi-square test. There was a difference between the 0.025 µm PES-treated and the control group in percentage of cleaved embryos (99.0 and 91.4%, respectively; P < 0.05), between all experimental groups and control in percentage of morula stage (90.7, 87.8, 83.8, and 80.0%, respectively), and between 0.025 and 0.05 µm PES-treated and control in percentage of blastocyst rates (70.0, 75.5, and 65.7%, respectively). The number of cells and percentage of TUNEL-positive nuclei per blastocyst were lower in the PES-treated than in the control group. The survival rate of blastocysts after vitrification and thawing was enhanced in the presence of PES compared to that in the PES-free group (45.2 and 38.9%, respectively; P < 0.05). After transfer of 56 expanded blastocysts cultured with PES and vitrified into 3 recipients, two gilts were confirmed pregnant at 35 days of gestation. In conclusion, a higher blastocyst percentage with a low incidence of apoptosis was obtained in the presence of PES compared to control. These blastocysts also had an increased ability to survive cryopreservation.

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&lt;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&lt;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&lt;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&gt;0.05). No differences were found in blastocyst diameter between the Ca1x (220.4µm) and Ca2x (251.2µm) groups (P&gt;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&gt;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.

99 In Vitro Development of Alpaca Embryos Obtained by Bisection

2018 ◽  
Vol 30 (1) ◽  
pp. 189
Author(s):  
L. Landeo ◽  
R. S. Molina ◽  
M. E. Zuñiga ◽  
T. R. Gastelu ◽  
C. Sotacuro ◽  
...  
Keyword(s):  
Amino Acids ◽  
Streptomyces Griseus ◽  
Petri Dish ◽  
Cumulus Cells ◽  
Essential Amino Acids ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Morula Stage

The objective of this study was to evaluate the in vitro developmental competence of alpaca embryos bisected at different embryonic stages. Gametes were obtained from ovaries and testes collected from a local abattoir. Cumulus-oocyte complexes (COC) were recovered (n = 120) by aspiration of ovarian follicles using a 5-mL syringe with an 18-gauge needle. Then, COC with at least 3 layers of cumulus cells and a homogeneous cytoplasm were matured in TCM-199 supplemented with 10% FCS, FSH (0.02 IU [JM1] [P2] [P3]), and 0.01 mg mL−1 oestradiol 17β [JM4] for 26 h at 38.5°C and 5% CO2 in air. After in vitro maturation, COC were placed in a 30-mL Petri dish containing FERT-TALP solution for 30 min. Then, epididymal alpaca spermatozoa (3 × 106 mL−1) were added to the dish and co-incubated with the COC for 20 h at 38.5°C and 5% CO2 in air. Motile epididymal sperm were selected by swim-up method centrifuged for 15 min at 350 × g in 2 mL of SPERM-TALP supplemented with 6 mg mL−1 of fatty-acid-free BSA. Sperm pellet was extended and culture in 5% CO2 in air at 38.5°C for 45 min. Thirty-three viable embryos at different stages [2-cells (n = 6), 8-cells (n = 15), and morulae (n = 12)] were bisected into approximately equal halves using a micro-surgical blade. The embryos were previously treated with 2 mg mL−1 of protease from Streptomyces griseus (P 8811, Sigma, St. Louis, MO, USA) for 2 min to remove the zona pellucida. After bisection, the demi-embryos were cultivated in in vitro culture (IVC) medium containing 0.036 mg mL−1 sodium pyruvate, 0.146 mg mL−1 l-glutamine, 1% essential amino acids, 0.5% nonessential amino acids, and supplemented with 10% FCS using the well-of-the-well system. The demi-embryos were incubated for 7 days (changing the media every 48 h) in 5% CO2 in air at 38.5°C. Additional embryos (n = 60) were obtained using the same conditions described above and used as a control group (unmanipulated). We obtained 66 demi-embryos [2-cells (n = 12), 8-cells (n = 30), and morulae (n = 24)] after bisection that were considered for IVC. From 12 demi-embryos bisected at 2-cell and 30 bisected at 8-cell stages, 3 (25%) and 30 (100%) reached the morula stage respectively. However, they did not develop any further. Interestingly, 18 demi-embryos bisected in morula reached the blastocyst stage (80%). For unmanipulated embryos, we obtained 42% (25/60), 35% (21/60), 32% (19/60), and 28% (17/60) of cleavage, morulae, and blastocyst and hatched blastocyst rates, respectively. In conclusion, alpaca embryos bisected at earlier stages (less than 8-cell) are not suitable to produce blastocysts. The earliest stage to produce blastocyst from bisected alpaca embryos is the morula stage.

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