Fertilisation of cryopreserved sperm and unfertilised quail ovum by intracytoplasmic sperm injection

2016 ◽  
Vol 28 (12) ◽  
pp. 1974 ◽  
Author(s):  
Kyung Soo Kang ◽  
Tae Sub Park ◽  
Deivendran Rengaraj ◽  
Hyung Chul Lee ◽  
Hong Jo Lee ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Development Rate ◽  
Animal Biotechnology ◽  
Animal Cloning ◽  
Female Quail ◽  
Stage 4 ◽  
Conservation Of Genetic Resources ◽  
Inositol 1,4,5 Trisphosphate ◽  
Cryopreserved Sperm

Intracytoplasmic sperm injection (ICSI) is an important technique in animal biotechnology for animal cloning and conservation of genetic resources, but has been a challenge for avian species. In the present study, we investigated the ability of cryopreserved quail spermatozoa to achieve fertilisation and embryo development. Female quail were killed 70–120 min after previous oviposition to collect unfertilised oocytes from the oviduct. Fresh or cryopreserved–thawed spermatozoa were injected into the cytoplasm of unfertilised oocytes, and the manipulated oocytes were incubated in quail surrogate eggshells. Injection of fresh spermatozoa supplemented with inositol 1,4,5-trisphosphate (IP3) resulted in a significantly increased rate of embryo development compared with injection of fresh spermatozoa alone (90% vs 13%, respectively). Although >80% of embryos stopped cell division and development before Hamburger and Hamilton (HH) Stage 3, approximately 15% of embryos from the fresh sperm injection developed to past HH Stage 4, and one embryo survived up to HH Stage 39 (11 days of incubation). In the case of cryopreserved spermatozoa, the embryo development rate was 30% after ICSI, and this increased significantly to 74% with IP3 supplementation. In conclusion, cryopreserved spermatozoa combined with ICSI followed by surrogate eggshell culture can develop quail embryos.

P–084 Microfluidic Sperm Sorting (MFSS) technique versus Physiological Intracytoplasmic Sperm Injection (PICSI) technique in high DNA fragmentation index sperm samples

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
G Kant ◽  
K D Nayar ◽  
H Sharma ◽  
S Gupta ◽  
S Mishra ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Dna Fragmentation ◽  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Development Rate ◽  
Clinical Pregnancy ◽  
Sperm Dna Fragmentation ◽  
Fragmentation Index ◽  
Sperm Dna ◽  
Dna Fragmentation Index

Abstract Study question To evaluate the effectiveness of using Microfluidic Sperm Sorting (MFSS) technique and Physiological Intracytoplasmic Sperm Injection (PICSI) technique in patient with high DNA fragmentation index (DFI) sperm samples. Summary answer Sperm selected by microfluidic sorting are associated with significant increase in day 3 grade A embryo development rate, clinical pregnancy rate over PICSI. What is known already DNA damage is unrecognisable in living sperm prior to insemination and an increased sperm DNA fragmentation index has been associated with lower fertilization rates, impaired embryo development and reduced pregnancy rates. Standard semen processing techniques are associated with centrifugation, which may induce reactive oxygen species and DNA damage. In strategies to minimize sperm DNA fragmentation, Physiological ICSI can relatively reduce sperm DNA fragmentation by 67.9% (Parmegiani et al., 2010) while new technique Microfluidic sperm sorter technique also demonstrate sperm selection with significantly reduced DNA damage. Study design, size, duration A prospective randomised study was conducted from 1st August 2019 to 31st December 2020. Two hundred patients were randomised by computer generated list and divided into 2 groups. Group A (n = 100) , in which sperm were processed by microfluidic sperm sorter (MFSS) while in group B (n = 100), sperm were selected by Physiological Intracytoplasmic Sperm Injection (PICSI) technique and morphologically normal motile sperm were injected by Intracytoplasmic sperm injection (ICSI) technique in all mature oocytes. Participants/materials, setting, methods The study period included all normozoospermic patients with high DNA fragmentation index (>25% ) while oligospermic, asthenozoospermic samples, patients with poor ovarian reserve and advanced age were excluded from the study. All A grade embryos were vitrified and transferred in frozen embryo replacement cycle. Both groups were compared on the basis of fertilisation rate, day 3 grade A embryo development rate , clinical pregnancy rate and miscarriage rate. Main results and the role of chance Cycle characteristics (female age, length of stimulation, gonadotrophin dose, number of oocytes and number of transferred embryos) were similar in both groups. Between the 2 groups, There was a significant increase observed in day 3 grade A embryo development rate (60% vs. 42%, p–0.016) and clinical pregnancy rate (62% vs. 46%, p–0.049), while no statistical significant difference observed in fertilisation rate (82% vs. 78%, p–0.80) and miscarriage rate ( 12% vs. 11%, p- 1). Limitations, reasons for caution: Larger randomised control studies are needed to strengthen these results. Wider implications of the findings: We have demonstrated that sperm sorted by microfluidic helps in selection of sperm with better DNA integrity over Physiological ICSI. Using it in routine practice can help in reducing the negative effect of reactive oxygen species and thus improve pregnancy rate and live birth rate. Trial registration number MCDH/2019/31

Mutations in ZP4 are associated with abnormal zona pellucida and female infertility

2021 ◽  
pp. jclinpath-2020-207170
Author(s):  
Xiaoli Wei ◽  
Youzhu Li ◽  
Qicai Liu ◽  
Wensheng Liu ◽  
Xiaohong Yan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Zona Pellucida ◽  
Intracytoplasmic Sperm Injection ◽  
Treatment Strategies ◽  
Suction Pressure ◽  
Human Female ◽  
Female Reproduction ◽  
Efficient Treatment ◽  
Whole Exome

BackgroundThe zona pellucida (ZP) of human oocytes plays essential protective roles in sperm–egg interactions during fertilisation and embryo development. ZP4-null female rabbits exhibit a thin and irregular ZP, which severely impairs embryo development and fertility. However, the effects of ZP4 defect on human female reproduction remain unknown.Methods and resultsWe performed whole-exome sequencing in 26 female patients with abnormal (thin and irregular) ZP and identified heterozygous variants in ZP4 (OMIM: 613514) from 3 patients (approximately 11%). No ZP4 variant was found in the 30 control women with proven fertility. We constructed ZP4-mutated plasmids and found that the variants reduced the secretion of ZP4 in vitro. Lower suction pressure facilitated egg retrieval, and intracytoplasmic sperm injection (ICSI) was a desirable treatment for ZP4-mutated patients with abnormal ZP.ConclusionsWe identified ZP4 as a novel gene for human abnormal ZP and found that lower suction pressure and ICSI are efficient treatment strategies.

Use of time-lapse imaging to evaluate morphokinetics of in vitro equine blastocyst development after oocyte holding for two days at 15°C versus room temperature before intracytoplasmic sperm injection

2019 ◽  
Vol 31 (12) ◽  
pp. 1862 ◽  
Author(s):  
N. A. Martino ◽  
G. Marzano ◽  
A. Mastrorocco ◽  
G. M. Lacalandra ◽  
L. Vincenti ◽  
...  
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Room Temperature ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Group 13 ◽  
Time Lapse Imaging

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.

208 CLONING AND CHARACTERIZATION OF BUFFALO INTERFERON-TAU AND EFFICACY OF RECOMBINANT BUFFALO INTERFERON-TAU FOR IN VITRO EMBRYO DEVELOPMENT

2015 ◽  
Vol 27 (1) ◽  
pp. 194
Author(s):  
S. Saugandhika ◽  
H. N. Malik ◽  
S. Saini ◽  
V. Sharma ◽  
S. Bag ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Embryo Development ◽  
Large Scale ◽  
Signal Sequence ◽  
Development Rate ◽  
Blastocyst Development ◽  
Interferon Tau ◽  
Significant Difference ◽  
Pregnancy Recognition

Interferon tau (IFN-tau) is known as maternal pregnancy recognition factor in ruminants. IFN-tau not only acts as a signalling molecule of pregnancy recognition but also performs various functions for successful implantation and pregnancy establishment. The aim of the present study was to produce recombinant buffalo interferon-tau (BuIFN-Tau) and observe if it has any effect on in vitro embryo development. The BuIFN-Tau gene was obtained through polymerase chain reaction (PCR) from hatched buffalo blastocysts and was cloned into pJET cloning vector. Screening of the recombinant colonies gave 8 distinct buffalo IFN-tau isoforms, out of which the predominant buffalo IFN-t tau1 isoform (gene bank accession number JX481984), was subcloned into expression vector pET22b without signal sequence. The recombinant plasmid was induced to express the recombinant protein by isopropyl b-D-1-thiogalactopyranoside. Analysis of the products of recombinant BuIFN-tau without signal sequence by SDS–PAGE revealed a new 20-kDa protein coinciding with the molecular weight of IFN-tau as reported earlier in literature. The purified recombinant BuIFN-tau was confirmed by Western blot using anti-HIS antibody and was subjected to three steps of large-scale purification using HIS affinity chromatography, anion exchange chromatography, and gel filtration chromatography. Finally, a relatively pure histidine-tagged recombinant protein, which had a purity of at least 90%, was generated as confirmed through SDS. The concentration of recombinant BuIFN-tau was 1 mg mL–1 by Bradford assay. The purified recombinant BuIFN-tau was used as supplement of the culture medium for IVF early buffalo embryos at the following concentrations: control, 1, 2, and 4 µg mL–1. Sixty oocytes each in 4 groups (with 20 oocytes/drop in three replicates for each group) were used for in vitro maturation. After 24 h, the matured oocytes were incubated with in vitro capacitated sperm cells for 18 h; thereafter, the presumptive zygotes were cultured in IVC medium supplemented with 0, 1, 2, or 4 µg mL–1 of the purified recombinant BuIFN-tau. The experiment was repeated 3 times. The data were analysed using SYSTAT 7.0 (SPSS Inc., Chicago, IL, USA) after arcsin transformation of percentage values. The differences were analysed by one-way ANOVA followed by Fisher's least significant difference test. Out of 3 concentrations of recombinant BuIFN-tau, the 2 µg mL–1 concentration significantly promoted the rate of blastocyst development, 45.55% against 31.1% (control; P < 0.01). Blastocyst development rate for low and high concentrations was 29.97% and 10.18% respectively. It is concluded that the addition of 2 µg mL–1 of recombinant BuIFN-tau enhances the blastocyst development rate in buffalo, and hence there is some evidence that BuIFN-tau has not only a role in maternal recognition of pregnancy but also in embryonic development.

271 EFFECT OF MARE AGE ON OOCYTE MORPHOLOGY AND DEVELOPMENTAL COMPETENCE AFTER INTRACYTOPLASMIC SPERM INJECTION

2008 ◽  
Vol 20 (1) ◽  
pp. 215
Author(s):  
J. L. Altermatt ◽  
T. K. Suh ◽  
J. E. Stokes ◽  
L. F. Campos-Chillon ◽  
E. M. Carnevale
Keyword(s):  
Embryo Development ◽  
Zona Pellucida ◽  
Intracytoplasmic Sperm Injection ◽  
Pregnancy Rates ◽  
Developmental Competence ◽  
Vitelline Membrane ◽  
Computer Assisted ◽  
Developmental Potential ◽  
Oocyte Morphology ◽  
Oocyte Developmental Competence

Reduced fertility in aged mares is associated with delayed early embryo development and lower pregnancy rates, potentially related to oocyte developmental competence. Human oocyte morphology has been associated with developmental potential, although comparative evidence is lacking in the mare. Exogenous FSH may be beneficial in obtaining more oocytes; however, effects on oocyte morphology and competence are unknown. Objectives were to determine if zona pellucida thickness (ZPT), ooplasm volume (OV), and perivitelline space volume (PVSV) were related to mare age or FSH treatment and to cleavage, blastocyst, and pregnancy rates after intracytoplasmic sperm injection (ICSI). Cycles with and without eFSH treatment were alternated; eFSH treatments began in diestrus with a cohort of follicles ≥20 mm. Oocytes were collected by transvaginal aspiration from follicles >30 mm from young (4 to 9 years) and old (>20 years) mares at 20 to 24 h after administration of recombinant eLH. Oocytes were cultured for 18 h in TCM-199 at 38.5�C in 6% CO2 in air. Sperm were injected 40 � 1 h after eLH, using frozen sperm from a single ejaculate. Presumptive zygotes were incubated in Dulbecco's modified Eagle's medium/F12 + 10% fetal calf serum at 38.5�C in 5% CO2, 5%O2, and 90% N2. Cleavage (≥2 cells) was recorded 48 h after ICSI. Blastocysts considered viable (formation before 9 d and good quality) were transferred nonsurgically into recipients 3 to 7 days after ovulation. Only pregnancies of fetuses with heart beats were included. Morphological parameters of oocytes (old, n = 40; young, n = 37) were obtained from photographic images taken at ICSI and analyzed by computer-assisted measurement using digital calipers (Spot Software, Diagnostic Instruments, Inc., Sterling Heights, MI, USA). Zona pellucida thickness was averaged from 2 measurements 90� to 180� apart. Ooplasm volume was calculated (4/3πr3) from the average of 2 diameters of the ooplasm 90� apart; and PVSV was calculated as the difference of the vitelline membrane volume and that of the volume at the inner volume of the ZP calculated as an oblate spheroid (4/3πa2b) from the average of 2 diameters. Zona pellucida thickness, OV, and PVSV were analyzed using 2-way ANOVA for main effects of age and treatment and 3-way ANOVA by adding cleavage as a factor. Zona pellucida thickness was less (P = 0.007) for old compared with young (least squares mean SEM of 11.4 � 0.2 and 12.3 � 0.2 µm, respectively) with no effect on cleavage, blastocyst, or pregnancy rates. Ooplasm volume was not different (P = 0.14) between old and young (309 036 � 5373 and 320 544 � 5639 µm3, respectively) and did not affect cleavage, blastocyst, or pregnancy rates. The PVSV was greater (P = 0.001) in old compared with young (157 505 � 10 853 and 102 161 � 11 388 µm3, respectively) and may be related to the lower cleavage (P = 0.03), blastocyst (P = 0.02), and pregnancy (P = 0.05) rates. Treatment with FSH had no effect (P > 0.1) on morphology or embryo development. In this study, ZPT and PVSV differed with mare age and could be of predictive value for oocyte developmental competence.

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