377 EMBRYO PRODUCTION BY OVUM PICKUP-INTRACYTOPLASMIC SPERM INJECTION-IVC IN AN EQUINE OVUM PICKUP PROGRAM USING SEMEN FROM FERTILE AND INFERTILE STALLIONS

2010 ◽  
Vol 22 (1) ◽  
pp. 345 ◽  
Author(s):  
S. Colleoni ◽  
R. Duchi ◽  
G. Lazzari ◽  
C. Galli
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Oocyte Quality ◽  
High Fertility ◽  
Cleavage Rate ◽  
Chi Square ◽  
Female Component ◽  
Chi Square Test

The introduction in equine reproduction of ovum pickup (OPU) combined with intracytoplasmic sperm injection (ICSI), IVC, and embryo transfer, has allowed for the production of offspring from donors and stallions that could not reproduce by conventional techniques. For this reason, we used in our OPU-ICSI-IVC program both fertile stallions and stallions with field records of low or no fertility. Overall, 805 and 584 OPU oocytes were fertilized with sperm from fertile and infertile stallions, respectively. Cleavage rate was statistically lower in the latter group (65.94 v. 59.24%, chi square test; P < 0.05) but embryo development was similar (11.67 v. 8.20% blastocysts/injected oocytes, chi-square test). In order to further investigate the stallion effect on embryo development, we selected 3 stallions with low (A) or no (B, C) fertility in the field and we compared the results of the OPU program with embryo development obtained using oocytes recovered from abattoir ovaries and matured, fertilized, and cultured in vitro as the OPU oocytes. Part of the abattoir oocytes was fertilized with a stallion with known high fertility both in vivo and in vitro (abattoir fertile). Overall, the results (shown in the table) suggest a reduction in the efficiency of stallions A, B, and C compared with to the fertile stallion used as control (10.79, 7.69, and 5.0% v. 17.35%, respectively). For stallions A and B, the efficiency was further reduced in the OPU setting, indicating that the female component can play a role in the overall efficiency of the procedure. In particular, 4 mares out of 8 had a history of no pregnancy and all mares had some rate of inbreeding with the respective stallion used for the ICSI. Instead, the oocytes from the abattoir ovaries were collected in large pools from several mares, representing an average oocyte quality, and the mares were of different breed than the stallions. All data were analyzed by chi-square test and significance was set at P < 0.05. In conclusion, we demonstrated that, for those stallions in which fertility in the field is low or absent, OPU-ICSI-IVP is a suitable choice to obtain embryos, although the efficiency is variable depending not only on the stallion but also on the origin of the oocytes. Table 1.Stallion effect on embryo development of ovum pickup (OPU) and abattoir oocytes This work was supported by Fondazione Cariplo and Regione Lombardia.

Effect of butafosfan supplementation during oocyte maturation on bovine embryo development

Zygote ◽  
2019 ◽  
Vol 27 (05) ◽  
pp. 321-328
Author(s):  
Lucas Teixeira Hax ◽  
Joao Alveiro Alvarado Rincón ◽  
Augusto Schneider ◽  
Lígia Margareth Cantarelli Pegoraro ◽  
Letícia Franco Collares ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Nuclear Maturation

SummaryAround 60–80% of oocytes maturated in vivo reached competence, while the proportion of maturation in vitro is rarely higher than 40%. In this sense, butafosfan has been used in vivo to improve metabolic condition of postpartum cows, and can represent an alternative to increase reproductive efficiency in cows. The aim of this study was to evaluate the addition of increasing doses of butafosfan during oocyte maturation in vitro on the initial embryo development in cattle. In total, 1400 cumulus–oocyte complexes (COCs) were distributed in four groups and maturated according to supplementation with increasing concentrations of butafosfan (0 mg/ml, 0.05 mg/ml, 0.1 mg/ml and 0.2 mg/ml). Then, 20 oocytes per group were collected to evaluate nuclear maturation and gene expression on cumulus cells and oocytes and the remaining oocytes were inseminated and cultured until day 7, when blastocysts were collected for gene expression analysis. A dose-dependent effect of butafosfan was observed, with decrease of cleavage rate and embryo development with higher doses. No difference between groups was observed in maturation rate and expression of genes related to oocyte quality. Our results suggest that butafosfan is prejudicial for oocytes, compromising cleavage and embryo development.

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

scholarly journals Improvement of quality of oocytes collected in the autumn by enhanced removal of impaired follicles from previously heat-stressed cows

Reproduction ◽  
2001 ◽  
pp. 737-744 ◽  
Author(s):  
Z Roth ◽  
A Arav ◽  
A Bor ◽  
Y Zeron ◽  
R Braw-Tal ◽  
...  
Keyword(s):  
Heat Stress ◽  
Embryo Development ◽  
Treated Group ◽  
Oocyte Quality ◽  
Control Group ◽  
Delayed Effect ◽  
Cleavage Rate ◽  
Lactating Cows ◽  
Summer Heat

The fertility of dairy cows decreases during the summer and remains low during the cooler autumn although the animals are no longer under heat stress. The aim of this study was to characterize a delayed effect of summer heat stress on oocyte quality in the autumn and to improve oocyte quality by enhanced removal of follicles damaged during the previous summer. Lactating cows (n = 16) were subjected to heat stress during the summer. In autumn, ovarian follicles (3-7 mm in diameter) were aspirated by an ultrasound-guided procedure during four consecutive oestrous cycles. Follicles were aspirated from control cows on day 4 and from treated cows on days 4, 7, 11 and 15 of each oestrous cycle. All cows received PGF(2alpha) and GnRH injections on days 19 and 21, respectively, and maintained cyclicity, as indicated by plasma progesterone concentrations. On day 4 of each cycle, the oocytes recovered were examined morphologically, matured and activated in vitro, and cultured for 8 days. In cycle 1 (early October) both groups showed low percentages of grade 1 oocytes, cleavage, four- and eight-cell embryos, morulae and parthenogenetic blastocysts. Subsequently, the number of grade 1 oocytes increased earlier (cycle 2) in treated than in control cows (cycle 3; P < 0.05). The cleavage rate in the control group remained relatively low throughout (32-58%), whereas in the treated group it increased from 40% (cycle 1) to 75% (cycles 3 and 4; P < 0.05). The number at each stage of embryo development increased slightly but remained low throughout in the control group, whereas in the treated group significant (P < 0.05) increases of all stages were observed in cycles 3 and 4. The results show a delayed effect of summer heat stress on oocyte quality and embryo development in the autumn. Enhanced removal of the impaired cohort of follicles led to earlier emergence of healthy follicles and high quality oocytes in the autumn.

70 NON-PLATED GRANULOSA AND CUMULUS CELLS AND FIRST PASSAGE FIBROBLASTS AS NUCLEUS DONOR FOR GOAT CLONING

2006 ◽  
Vol 18 (2) ◽  
pp. 143
Author(s):  
D. Salamone ◽  
M. Catala ◽  
A. Gibbons ◽  
F. Pereyra Bonnet ◽  
M. Cueto
Keyword(s):  
Granulosa Cells ◽  
Room Temperature ◽  
Uv Light ◽  
Cumulus Cells ◽  
Cleavage Rate ◽  
First Passage ◽  
Chi Square ◽  
Donor Nucleus ◽  
Chi Square Test

Different types of somatic cells have been used as nucleus donors for cloning. Most of them were previously cultured in vitro as a monolayer through several plate passages. The experiment reported here was conducted to study the potential usages of granulosa and cumulus cells for cloning without previous culture as a monolayer. A first-plate-passage fibroblast was also used. Oocytes were aspirated by laparoscopy from Criolla goats and matured in TCM-199 + 5% FCS at 39°C for 24 h. Matured oocytes were denuded by vortexing for 3 min in TL HEPES with 1 mg/mL bovine testis hyaluronidase. Metaphases were assessed and oocytes were enucleated by visualization with Hoechst 33342 (5 μg/mL) under UV light (<6 s). Granulosa and cumulus cells were also recovered by laparoscopy and maintained in maturation medium in cryotube for 20 h at room temperature or 39°C, respectively. Goat adult ear fibroblasts were cultured for 1 or 2 weeks and used 2 days after confluence. All types of donor cells were transferred to the perivitlline space of enucleated oocytes and fused by an electrical pulse. After 2 h, activation was induced by incubation in TL-HEPES with 5 µM ionomycin for 4 min and 2 mM 6-DMAP for 3 h. The oocytes were then washed with TL-HEPES and cultured in SOF medium and atmosphere of 5% CO2 + 5% O2 + 90% N2. Cleavage (Day 2) and development to blastocysts (Day 6) were recorded and analyzed by chi-square test. The cleavage rate for non-plated granulosa cells was higher than for the other treatment goups; cumulus cells had a lower rate of development to blastocysts (Table 1). These results suggest that granulosa cells collected and maintained for 24 h at room temperature could be used to produce cloned blastocysts. Table 1. Effect of non-plated granulosa and cumulus cells and first passage fibroblasts as donor nucleus oocytes in goat cloning

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

2 SPECIFIC FATTY ACID FOLLOW-UP REVEALS RUMEN-PROTECTED FAT SUPPLEMENTATION EFFECTS ON BOVINE OOCYTE QUALITY AND EMBRYO DEVELOPMENT

2014 ◽  
Vol 26 (1) ◽  
pp. 115 ◽  
Author(s):  
A. F. González-Serrano ◽  
C. R. Ferreira ◽  
V. Pirro ◽  
J. Heinzmann ◽  
K.-G. Hadeler ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Palmitic Acid ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Oocyte Quality ◽  
Lipid Profiles ◽  
Dependent Manner ◽  
Fat Supplementation

Information on how supplementation of high-yield dairy cows with rumen-protected fat affects fertility in cattle herds is scarce. Here, Holstein-Friesian heifers (n = 84) received a supplement consisting of either rumen-protected conjugated linoleic acid (CLA; cis-9,trans-11-CLA and trans-10,cis-12-CLA) or stearic acid 18 : 0 (SA) on top of an isocaloric grass silage diet. Two supplementation doses were used (100 and 200 g d–1). Blood and follicular fluid were collected at the start and end of the supplementation period for analysis of cholesterol, insulin-like growth factor (IGF), and nonesterified fatty acids (NEFA), and for fatty acid profiling. Although cholesterol, IGF, and NEFA levels did not differ among experimental groups, lipid profiles in blood and follicular fluid were affected in a dose-dependent manner by both supplements. After 45 days of supplementation, oocytes were collected by ovum pick-up (OPU). The mRNA relative abundance of target genes (IGF1r, GJA1, FASN, SREBP1, and SCAP) was analysed in single in vitro- (24 h IVM) and in vivo-matured (collected by OPU 20 h after GnRH injection) oocytes and in vitro-produced blastocysts (Day 8) by qPCR (n = 6/group). Lipid profiling of individual oocytes from the CLA-supplemented (n = 37) and the SA-supplemented (n = 50) was performed by desorption electrospray ionization mass spectrometry (DESI-MS). Oocytes from the CLA-supplemented (n = 413) and the SA-supplemented (n = 350) groups were used for assessing maturation and blastocysts development rates. In immature oocytes, CLA supplementation led to an increase of triacylglycerol 52 : 3 [TAG (52 : 3)] and TAG (52 : 2), squalene, palmitic acid 16 : 0, and oleic acid 18 : 1, and decreased abundance of TAG (56 : 3), TAG (50 : 2) and TAG (48 : 1). In vitro-matured oocytes showed different lipid profiles, with increased abundances of TAG (52 : 3), and TAG (52 : 2) as well as phosphatidylinositol 34 : 1 [Plo (34 : 1)], whereas phosphatidylglycerol (34 : 1) [PG (34 : 1)] and palmitic acid 16 : 0 were less abundant in in vitro-matured oocytes. SCAP was significantly down-regulated in in vitro-matured oocytes from supplemented heifers compared with their in vivo-matured counterparts. Maturation (CLA = 74% v. SA = 67%) and blastocyst rates (CLA = 22.4% v. SA = 12.7%) were different among experimental groups. One-way ANOVA and the Tukey-Kramer test were applied for a multiple comparison of means (P-value ≤ 0.05 was considered as statistically significant). In conclusion, we demonstrate here that fatty acid monitoring along different compartments (i.e. blood system, follicular fluid, and intra-oocyte) after rumen-protected fat supplementation of dairy heifer diet reveals nutritional footprints on oocyte quality and embryo development. These results demonstrate the close relationship between nutrition and cattle herd's fertility and, at the same time, support the role of the bovine model for understanding nutritional-dependent fertility impairments.

72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN PERIVITELLINE SPACE MICROINJECTION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
D. M. de Souza ◽  
H. Fernandes ◽  
P. V. Silva ◽  
B. Cazari ◽  
P. D. Moço ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Training Model ◽  
Perivitelline Space ◽  
Chi Square ◽  
Testicular Cells ◽  
Chi Square Test ◽  
Cellular Components

The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the perivitelline space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Financial support was received from FAPESP of Brazil.

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

Fertilizing capacity of epididymal and testicular sperm using intracytoplasmic sperm injection (ICSI)

1995 ◽  
Vol 7 (2) ◽  
pp. 281 ◽  
Author(s):  
SJ Silber ◽  
P Devroey ◽  
H Tournaye ◽  
Steirteghem AC Van
Keyword(s):  
Pregnancy Rate ◽  
Intracytoplasmic Sperm Injection ◽  
Pregnancy Rates ◽  
Obstructive Azoospermia ◽  
Motile Sperm ◽  
Epididymal Sperm ◽  
Testicular Sperm ◽  
Cleavage Rate

For men with uncorrectable obstructive azoospermia, their only hope of fathering a child is microsurgical epididymal sperm aspiration (MESA) combined with in vitro fertilization (IVF). In 1988, proximal epididymal sperm were demonstrated to have better motility than senescent sperm in the distal epididymis, and it was thought that retrieval of motile sperm from the proximal epididymis would yield reliable fertilization and pregnancy rates after conventional IVF. However, the results to date have been poor, and although a minority of patients achieved good fertilization rates with IVF, the vast majority (81%) had consistently poor or no fertilization and the pregnancy rate averaged only 9%. Recently, intracytoplasmic sperm injection (ICSI) has been successfully used to achieve fertilization and pregnancies for patients with extreme oligoasthenozoospermia. ICSI has therefore been applied to cases of obstructive azoospermia and, in this report, 67 MESA-IVF cases are compared with 72 MESA-ICSI cases. The principle that motile sperm from the proximal segments of the epididymis should be used for ICSI was followed, although in the most severe cases in which there was an absence of the epididymis (or absence of sperm in the epididymis), testicular sperm were obtained from macerated testicular biopsies. These sperm only exhibited a weak, twitching motion. In 72 consecutive MESA cases, ICSI resulted in fertilization and normal embryos for transfer in 90% of the cases, with an overall fertilization rate of 46%, a cleavage rate of 68%, and ongoing or delivered pregnancy rates of 46% per transfer and 42% per cycle. The pregnancy and take-home baby rates increased from 9% and 4.5% with IVF to 53% and 42% with ICSI. There were no differences between the results for fresh epididymal, frozen epididymal or testicular sperm, and the number of eggs collected did not affect the outcome. The results were also unaffected by the aetiology of the obstruction such as congenital absence of the vas deferens or failed vasoepididymostomy. The only significant factor which affected the pregnancy rate was female age. It is concluded that although complex mechanisms involving epididymal transport may be beneficial for conventional fertilization of human oocytes (in vivo or in vitro), none of these mechanisms are required for fertilization after ICSI. Given the excellent results with epididymal and testicular sperm, ICSI is obligatory for all future MESA patients. Finally, the use of ICSI with testicular sperm from men with non-obstructive azoospermia is also discussed.

scholarly journals Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen-thawed spermatozoa

Reproduction ◽  
2002 ◽  
pp. 455-465 ◽  
Author(s):  
YH Choi ◽  
CC Love ◽  
LB Love ◽  
DD Varner ◽  
S Brinsko ◽  
...  
Keyword(s):  
Intracytoplasmic Sperm Injection ◽  
Polar Body ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
First Polar Body ◽  
Bovine Oocytes

This study was undertaken to evaluate the development of equine oocytes in vitro and in vivo after intracytoplasmic sperm injection (ICSI) with either fresh or frozen-thawed spermatozoa, without the use of additional activation treatments. Oocytes were collected from ovaries obtained from an abattoir and oocytes classified as having expanded cumulus cells were matured in M199 with 10% fetal bovine serum and 5 microU FSH ml(-1). After 24-26 h of in vitro maturation, oocytes with a first polar body were selected for manipulation. Fresh ejaculated stallion spermatozoa were used for the experiment after swim-up for 20 min in sperm-Tyrode's albumen lactate pyruvate. Frozen-thawed spermatozoa from the same stallion were treated in a similar way. Spermatozoa were immobilized and injected into the oocytes using a Piezo drill. Presumptive zygotes were cultured in G1.2 medium for 20 or 96 h after the injection was administered, or were transferred to the oviducts of recipient mares and recovered 96 h later. In addition, bovine oocytes with first polar bodies were injected with the two types of stallion spermatozoa and fixed 20 h after injection to examine pronuclear formation. Fertilization rate (pronucleus formation and cleavage) at 20 h after injection of spermatozoa was not significantly different between fresh and frozen-thawed sperm groups in either equine or bovine oocytes. Pronucleus formation after injection of spermatozoa into bovine oocytes was significantly higher than that for equine oocytes (P < 0.05). There were no significant differences in cleavage rate or average number of nuclei at 96 h between equine oocytes injected with fresh or frozen-thawed spermatozoa. However, embryos developed in vivo for 96 h had a significantly higher number of nuclei in both sperm treatments compared with those cultured in vitro. These results indicate that good activation rates may be obtained after injection of either fresh or frozen-thawed equine spermatozoa without additional activation treatment. Injection of frozen-thawed equine spermatozoa results in similar embryo development to that obtained with fresh equine spermatozoa. In vitro culture of equine zygotes in G1.2 medium results in a similar cleavage rate but reduced number of cells compared with in vivo culture within the oviduct. Bovine oocytes may be useful as models for assessing sperm function in horses.

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