312 EXPOSURE OF SPERMATOZOA TO SOLUBILIZED EXTRACTS OF THE OVIDUCTAL EPITHELIUM APICAL PLASMA MEMBRANE ENHANCES FERTILIZATION IN PORCINE IN VITRO FERTILIZATION

2007 ◽  
Vol 19 (1) ◽  
pp. 272
Author(s):  
N. Satake ◽  
A. K. Alhaider ◽  
W. V. Holt ◽  
P. F. Watson
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Plasma Membranes ◽  
Fertilization Rate ◽  
Apical Plasma Membrane ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Fertilization Rates

In vitro production (IVP) of porcine embryos is currently suboptimal compared with IVP in species such as mice and cattle. In vitro fertilization (IVF) usually involves the co-culture of oocytes and spermatozoa in a medium droplet. Oocyte quality is the focus of many studies. In vivo, the quality of spermatozoa is as important as the oocyte, and females have many mechanisms to select the highest quality spermatozoa for their oocytes. Oviductal proteins have been shown to affect sperm motility of subpopulations within an ejaculate. The present study was carried out to investigate normal and polyspermic fertilization rates of spermatozoa exposed to oviductal epithelial apical plasma membrane (APM) proteins, a mixture of peripheral proteins extracted by 1 M NaCl from isolated oviductal apical plasma membranes, prior to co-culture with oocytes in IVF. Porcine oocytes were aspirated from ovaries and grade I quality oocytes (cumulus–oocyte complexes with a spherical shape, visible nucleus, even-density cytoplasm, and multiple layers of cumulus cells) were selected and matured for 48 h in TCM-199 supplemented with LH (0.5 �g mL-1), FSH (0.5 �g mL-1), and EGF (10 ng mL-1). Ejaculates were washed through a Percoll gradient to obtain a concentrated pellet. Spermatozoa were diluted in capacitation–fertilization medium in the presence or absence of APM proteins (100 �g mL-1), incubated for 10 min, and then co-cultured with oocytes for 6 h in modified Tween medium B with milk powder medium (Abeydeera and Day 1997 Theriogenology 48, 537–544) supplemented with BSA (0.4%) and sodium bicarbonate (5 mM). Presumptive zygotes were cultured in NCSU23 medium for a further 48 h. The oocytes/zygotes were then fixed and stained with propidium iodide for evaluation by confocal microscopy for fertilization and cleavage (n = 1235 oocytes). Fertilization rates were compared between treatments in a chi-squared test using the Mantel-Haenszel approach. The overall fertilization rate was significantly higher (78 vs. 86%) when spermatozoa were incubated in the presence of APM proteins (P < 0.05), and in the group of fertilized oocytes, polyspermic fertilization (47 vs. 21%) was significantly reduced when spermatozoa were exposed to APM proteins (P < 0.01). However, cleavage rates were not different. These results suggest that exposure of spermatozoa to APM proteins prior to IVF increases the fertilization rate and decreases the incidence of polyspermic penetration.

173 COMPARISON OF FERTILIZATION RATES OF EQUINE OOCYTES USING IN VITRO FERTILIZATION, PERIVITELLINE AND INTRACYTOPLASMIC SPERM INJECTIONS

2012 ◽  
Vol 24 (1) ◽  
pp. 198
Author(s):  
D. R. Sessions ◽  
J. K. Graham ◽  
E. M. Carnevale
Keyword(s):  
In Vitro Fertilization ◽  
Normal Embryo ◽  
Fertilization In Vitro ◽  
Perivitelline Space ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Chi Square Analysis ◽  
Bovine Oocytes

In vitro fertilization in horses has had limited and inconsistent success for oocytes matured in vivo or in vitro. However, oocytes transferred to the oviducts of inseminated recipient mares are consistently fertilized and result in normal embryo development. Potentially, equine sperm are unable to penetrate the zona pellucida (ZP) in vitro, which could be a result of failure of capacitation or hyperactivation. We hypothesised that ZP of equine oocytes impairs fertilization in vitro and bypassing the ZP would be beneficial for fertilization rates. Oocytes were retrieved using transvaginal follicle aspirations from oestrous mares 22 to 24 h after administration of a gonadotropin-releasing hormone analog (deslorelin, 1.1 mg). After culture for 20 h in TCM-199 with Earle's salts and 10% FCS, oocytes were assigned to 1 of 3 groups: 1) IVF, equine (n = 9) and bovine (n = 80) cumulus–oocyte complexes co-incubated with sperm; 2) PVI, sperm injections into the perivitelline space of equine (n = 13) and bovine (n = 11) oocytes; and 3) ICSI, intracytoplasmic sperm injections of equine oocytes (n = 13). Fresh sperm from 2 stallions was used. For IVF, sperm were treated with 20 μM dilauroyl phosphatidylcholine (PC12) to induce capacitation but not the acrosome reaction; sperm for PVI and ICSI were treated with 40 μM PC12 to capacitate and acrosome-reacted sperm. For IVF, each equine oocyte was co-incubated with 8 to 11 bovine oocytes and 250 000 sperm in fertilization media (De La Torre-Sanchez et al. 2006 Reprod. Fertil. Dev. 18, 597–607) for 18 h. For PVI, 10 to 15 sperm were injected into the perivitelline space of each equine and bovine oocyte and for ICSI, 1 sperm was injected into the cytoplasm of each equine oocyte. Oocytes were then placed in embryo culture medium (DMEM-F12 and 10% FCS) for 24 h before examination for cleavage. Cleavage rates for each group were compared using chi-square analysis. After IVF or PVI, no equine oocytes cleaved; in comparison, 11 of 13 (85%) cleaved in the ICSI group. However, of the bovine oocytes, 10 of 80 (12%) cleaved after IVF and 1 of 11 (9%) cleaved after PVI with equine sperm. An additional 6 PVI injections with equine (n = 6) oocytes and sperm were stained with Hoechst 33324 to determine sperm localization within the oocytes. All injected sperm remained in the perivitelline space and did not appear to fuse with the oolemma or enter the ooplasm. The results of this study suggest that the equine sperm prepared with PC12 were unable to penetrate the oolemma. In our experiment, sperm treated with PC12 resulted in high fertilization rates when placed directly into the ooplasm during ICSI; however, the sperm were unable to penetrate the ZP or oolemma. Additional research is needed to determine the cause of penetration failure and whether this is due to inadequate sperm treatments or a missing oviducal component in our in vitro systems. Funding for this project was provided by the benefactors for the Preservation of Equine Genetics and the Cecil and Irene Hylton Foundation.

161 IN VITRO FERTILIZATION OF DROMEDARY CAMEL (CAMELUS DROMEDARIUS) OOCYTES WITH EPIDIDYMAL SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 192 ◽  
Author(s):  
A. R. Moawad ◽  
G. M. Darwish ◽  
M. R. Badr ◽  
A. B. El-Wishy
Keyword(s):  
In Vitro Fertilization ◽  
Calcium Ionophore ◽  
Fertilization Rate ◽  
Dromedary Camel ◽  
Vitro Fertilization ◽  
Fertilization Rates ◽  
Significant Difference ◽  
Epididymal Spermatozoa ◽  
Normal Fertilization

Various techniques such as AI and ET have been reported to improve reproductive efficiency and genetic potential in camelids. In vitro fertilization and the development of IVP embryos are considered an alternative for genetic improvement in this species. This study investigated the effects of different sperm cell concentrations (1, 2, 3 and 4 × 106 sperm mL–1), different capacitating materials (5 mM caffeine, 10 μg mL–1 of heparin, 10 mg mL–1 of theophylline, 1 mM calcium ionophore A23178 and 10 μg of heparin + 5 mM caffeine), post-slaughter epididymal flushing time and fertilization media supplements (Fert-TALP + 6 mg mL–1 of BSA and Fert-TALP + 3 mg mL–1 of polyvinylpyrrolidone ) on fertilization rates and subsequent development of dromedary camel oocytes. Cumulus–oocyte complexes obtained at slaughter were matured in vitro in TCM-199 for 36 h at 39°C in a humidified atmosphere of 5% CO2. For IVF, spermatozoa were collected from epididymides of slaughtered male camels at 1 to 2 h post-slaughter or after 24 h of epididymal storage at 4°C. The spermatozoa were then prepared for IVF by the swim-up technique. Following sperm capacitation, oocytes and spermatozoa were co-incubated for 18 h. Oocytes were then stained using aceto-orcein for evaluation of fertilization events. Presumptive zygotes were cultured in vitro in TCM-199 medium supplemented with 5% FCS for 9 days at 39°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At least 3 replicates were performed for each experimental group. Data were analysed by chi-square test. Fertilization rates were 55.5, 62.5, 62.7 and 47.2% in oocytes inseminated with 1, 2, 3, or 4 × 106 sperm mL–1, respectively. Normal fertilization rate (oocytes with 2 pronuclei) was higher (P =  0.06) in oocytes inseminated with 2 × 106 sperm mL–1 (29.7%) than in those inseminated by 4 × 106 sperm mL–1 (11.1%). Treatment of epididymal spermatozoa with 5 mM caffeine significantly increased (P ≤ 0.05) fertilization rate (61.9%) compared with calcium ionophore A23178 (32.4%). These values were not significantly different from other groups (38.5, 54.1 and 50.0% in heparin, theophylline and heparin + caffeine, respectively). Normal fertilization was highest (25.4%) in oocytes inseminated with caffeine-treated spermatozoa. Insemination of oocytes in Fert-TALP medium containing BSA resulted in a higher fertilization rate (21.4%) compared with oocytes in polyvinylpyrrolidone-supplemented medium (5.7%; P =  0.06). Storage of camel epididymides at 4°C for 24 h did not affect fertilization rates. Cleavage rate (48 h post-insemination) was higher in oocytes fertilized with caffeine-treated spermatozoa than in oocytes in the theophylline group (26.8 vs 10.5%; P =  0.08). No significant difference was observed in the frequency of blastocyst development (5 days post-insemination) between the 2 groups (5.4 vs 2.6%); based on the number of cleaved oocytes, the same proportions of blastocyst embryos were reported (20.0 and 25.0%). Taken together, these results suggest that dromedary camel oocytes can be matured, fertilized and subsequently developed in vitro with high developmental potential. Epididymal spermatozoa at a concentration of 2 × 106 sperm mL–1 prepared in a medium containing caffeine as a capacitating agent can be used effectively in IVF of camel oocytes.

scholarly journals Production of viable bovine embryos by intracytoplasmic sperm injection of oocytes harvested from slaughtered old cows

2021 ◽  
Vol 26 (1) ◽  
pp. 7-14
Author(s):  
Mihai Cenariu ◽  
Mihai Borzan ◽  
Sorin Dan ◽  
Remus Chiorean ◽  
Emoke Pall
Keyword(s):  
In Vitro Fertilization ◽  
Intracytoplasmic Sperm Injection ◽  
Fertilization Rate ◽  
Fertilization Success ◽  
Vitro Fertilization ◽  
Bull Semen ◽  
Intracytoplasmic Injection ◽  
Lower Fertility

Abstract: (1) Background: Intracytoplasmic sperm injection (ICSI) is currently used to increase fertilization success by avoiding several oocyte or sperm deficiencies that would normally prevent conception after in vivo fertilization or classical in vitro fertilization. This paper aimed at improving the in vitro fertilization protocol of bovine oocytes, harvested from old cows after slaughtering, using intracytoplasmic sperm injection; (2) Methods: Oocytes were harvested by puncture of follicles from ovaries obtained from slaughtered old cows, followed by aspiration. Out of the 127 cumulus-oocyte complexes that were harvested, 84 (66.14%) were declared suitable for cultivation, after morphological evaluation. Following oocyte maturation for 22 hours, 77 cumulus-oocyte complexes were morphologically intact and could undergo the steps required for intracytoplasmic injection of spermatozoa. Frozen-thawed bull semen was used for ICSI and the 77 fertilized oocytes were kept for 24 hours in an atmosphere enriched with 5% CO2.; (3) Results: Fertilized oocytes transformed into 46 zygotes (fertilization rate of 59.74%), while after 168 h of cultivation 38 transferable compact morulae or early blastocysts were obtained; (4) Conclusions: Intracytoplasmic sperm injection can represent a viable alternative to classical IVF, when oocytes or sperm with lower fertility are used.

scholarly journals 202FERTILIZING ABILITY OF EPIDIDYMAL CAT SPERMATOZOA AFTER CRYOPRESERVATION OR STORAGE AT 4°C.

2004 ◽  
Vol 16 (2) ◽  
pp. 222 ◽  
Author(s):  
L. Bogliolo ◽  
P. Bonelli ◽  
L. Madau ◽  
M.T. Zedda ◽  
C. Santucciu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Genetic Material ◽  
Membrane Integrity ◽  
Long Distance ◽  
Vitro Fertilization ◽  
Plasma Membrane Integrity ◽  
Fertilization Rates ◽  
Epididymal Spermatozoa

The possibility of harvesting cat epididymal spermatozoa from excised testis represents a potentially important tool for preserving valuable genetic material from males that die unexpectedly. The purpose of this study was to evaluate plasma membrane integrity, acrosomal status and in vitro fertilizing capability of epididymal cat sperm after short- and long-term storage at 4°C and after cryopreservation. Spermatozoa were collected by flushing the cauda epididymis and the vasa deferentia removed from adult cats undergoing orchiectomy. Spermatozoa were split into three aliquots: A-fresh, B-frozen, and C-cooled. The A portion was immediately tested on the day of collection (control), the B portion was frozen in pellets in Test Yolk Buffer (TYB) with 8% glycerol, while the C portion was extended in TYB and stored at 4°C for 1 day, 2 days or 7 days. Cat oocytes recovered from minced ovaries were matured for 24h in TCM 199+1UI/mL hCG+0.5UI/mL FSH+0.3% BSA at 38.5°C in 5% CO2. In vitro fertilization (IVF) of in vitro-matured (IVM) oocytes was performed using spermatozoa from portions A, B or C in modified Tyrode’s solution supplemented with 0.6% BSA at 38.5°C in 5% in air. Before IVF, aliquots of sperm samples from the three portions were stained simultaneously with fluorescein isothiocyanate-labelled Pisum Sativum agglutinin (FITC-PSA) and propidium iodide to evaluate the percentage of plasma membrane-intact spermatozoa with acrosomes present. At least 200 spermatozoa were counted in duplicate for each sample. After 24h of incubation, to evaluate fertilization rate, the oocytes were stained with aceto-lacmoid. Oocytes with two pronuclei, presumably male and female, were classified as fertilized. The percentages of spermatozoa maintaining plasma membrane integrity and intact acrosomes after 1 day or 2 days of storage at 4°C were 75% and 65%, respectively, and were similar to that of fresh epididymal spermatozoa (78%). However, the percentages of spermatozoa with intact plasma membranes that had intact acrosomes after 7 days of storage at 4°C (52%) or after cryopreservation (48%) were significantly lower (P<0.01, ANOVA) than those of samples stored for 1 or 2 days at 4°C. As shown in the Table, fertilization rates of oocytes inseminated with fresh spermatozoa and spermatozoa stored for 1 or 2 days were similar. In contrast, the fertilization rates of oocytes inseminated with spermatozoa that had been cryopreserved or stored for 7 days were significantly lower than those obtained with spermatozoa used immediately after collection or storage for 1 day. From our results, we suggest that storage of epididymal spermatozoa at 4°C may be a potentially useful method for temporary storage of spermatozoa from endangered cats, and may allow more efficient use and long-distance transport of genetically important germplasm. This work was supported by MIUR (ex 40%). Table 1 In vitro fertilization of in vitro-matured cat oocytes with fresh, stored at 4°C and cryopreserved epididymal cat spermatozoa

scholarly journals Effect of Fertility Blend® Administration on the Oocytes Quality and Embryonic Development using assisted Reproductive Technology in Mice

2017 ◽  
Vol 11 (1) ◽  
pp. 67-71
Author(s):  
S. Al-dujaily ◽  
Salah M. Al-Chalabi ◽  
N. Khadem ◽  
M. Abdul-mageed ◽  
Mo Abdul lateef
Keyword(s):  
In Vitro Fertilization ◽  
Embryonic Development ◽  
Ovarian Function ◽  
Treated Group ◽  
Fertilization Rate ◽  
Control Group ◽  
Vitro Fertilization ◽  
Female Mice

Female Fertility Blend ® (FFB) is one of new nutritional supplement that used to enhance the fertility status in women. This supplement containing vitamins, minerals, enzymes, amino acids, all may improve the oocytes quality and ovarian function; at the same time protect oocytes from free radicals damage. The aim of the study is to examine the in vivo effect of FFB on oocyte quality, and in vitro fertilization rate (IVFR), and embryonic development (ED) at early cleavage stages using the mice as a model for human being. Therefore, two groups of mature female mice were involved (20 mouse each). The treated group is daily orally administrated by 3.4mg/kg /body weight from FFB for 10 days and the other groups (the control) were treated with FFB- free distilled water only for the same period. Oocytes were collected and an epididymal sperms from mature fertilized male mice are obtained and in vitro fertilization (IVF) is done. Following 24 and 48hrs from IVF, the FR and ED rate are recorded. This results showed a significant (P<0.05) differences in fertilization rate and embryonic development when treating the female mice with FFB compared to control group. It is concluded that the FFB treatment has a great improvement in oocytes maturation and in vitro fertilization and embryonic development status.

scholarly journals A Brief Incubation of Cumulus-Enclosed Mouse Eggs in a Calcium-Free Medium Containing a High Concentration of Calcium-Chelator Markedly Improves Preimplantation Development

2020 ◽  
Vol 17 (10) ◽  
pp. 3505
Author(s):  
Valeria Merico ◽  
Silvia Garagna ◽  
Maurizio Zuccotti
Keyword(s):  
In Vitro Fertilization ◽  
Mammalian Species ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Preimplantation Development ◽  
High Concentration ◽  
Vitro Fertilization ◽  
Positive Effects

The presence of cumulus cells (CCs) surrounding ovulated eggs is beneficial to in vitro fertilization and preimplantation development outcomes in several mammalian species. In the mouse, this contribution has a negligible effect on the fertilization rate; however, it is not yet clear whether it has positive effects on preimplantation development. Here, we compared the rates of in vitro fertilization and preimplantation development of ovulated B6C3F1 CC-enclosed vs. CC-free eggs, the latter obtained either after a 5 min treatment in M2 medium containing hyaluronidase or after 5–25 min in M2 medium supplemented with 34.2 mM EDTA (M2-EDTA). We found that, although the maintenance of CCs around ovulated eggs does not increment their developmental rate to blastocyst, the quality of the latter is significantly enhanced. Most importantly, for the first time, we describe a further quantitative and qualitative improvement, on preimplantation development, when CC-enclosed eggs are isolated from the oviducts in M2-EDTA and left in this medium for a total of 5 min prior to sperm insemination. Altogether, our results establish an important advancement in mouse IVF procedures that would be now interesting to test on other mammalian species.

scholarly journals In Vitro Fertilization of In Vitro and In Vivo Matured Oocytes from Prepubertal Meishan Gilts

1990 ◽  
Vol 61 (11) ◽  
pp. 1011-1016
Author(s):  
Takashi MIYANO ◽  
Kiyoshi YOSHIKAWA ◽  
Seishiro KATO ◽  
Hiroshi HARAYAMA ◽  
Iwao NANJO ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Vitro Fertilization

scholarly journals Efficiency Comparison Between Dry And Humid Cultures For Clinical Outcome of The In Vitro Fertilization-Embryo Transfer

2021 ◽  
Author(s):  
Weihai Xu ◽  
Lin Zhang ◽  
Ling Zhang ◽  
Shishi Li ◽  
Jing Shu
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Transfer ◽  
Implantation Rate ◽  
Fertilization Rate ◽  
Cleavage Rate ◽  
High Quality ◽  
Vitro Fertilization ◽  
Efficiency Comparison ◽  
Ivf Et

Abstract Background: In this study, we compared the in vitro embryo development, embryo transfer outcome and the offspring outcome in the in vitro fertilization-embryo transfer (IVF-ET) between dry culture (DC) and humid culture (HC). Methods: Our study was divided into two parts. Firstly, we determined the fertilization rate, cleavage rate and high-quality embryo rate from 21 cycles in the DC group (N=262 oocytes) and HC group (N=263 oocytes). Secondly, we determined the embryo transfer outcome and the offspring outcome in DC group (N=184 cycles) and HC group (N=136 cycles). Results: Compared with the HC group, significant increase was observed in the high-quality embryo rate (66.1.2% vs. 55.3%, p=0.037) and implantation rate (49.8% vs. 40.6%, p=0.027) in the DC group. No statistical differences were observed in the pregnant outcome and birth defect of the offspring (p>0.05). Compared with HC, DC was associated with a higher high-quality embryo rate and a higher implantation rate after embryo transfer. Conclusions: No statistical differences were noticed in the offspring conditions between the two culture modes. Taken together, DC may serve as a promising method for IVF-ET.

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