124 CO-CULTURE WITH BOVINE INTACT CUMULUS - OOCYTE COMPLEXES DURING IN VITRO FERTILIZATION OF BUFFALO DENUDED OOCYTES COMPLETELY RESTORES THEIR FERTILIZING AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 167
Author(s):  
M. De Blasi ◽  
M. Rubessa ◽  
L. Boccia ◽  
S. Di Francesco ◽  
M. V. Suárez Novoa ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Control ◽  
Developmental Competence ◽  
Control Group ◽  
Chi Square ◽  
Oocyte Vitrification ◽  
Vitro Fertilization ◽  
Chi Square Test

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.

202 EFFECT OF OSTEOPONTIN ON IN VITRO EMBRYO DEVELOPMENT IN CATTLE

2008 ◽  
Vol 20 (1) ◽  
pp. 180 ◽  
Author(s):  
B. Gasparrini ◽  
E. Monaco ◽  
L. Boccia ◽  
A. De Rosa ◽  
L. Attanasio ◽  
...  
Keyword(s):  
Embryo Development ◽  
Bovine Milk ◽  
Cumulus Cells ◽  
Control Group ◽  
Single Chain ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Oviduct Fluid

Osteopontin (OPN) is an acidic single-chain phosphorylated glycoprotein found both in the oviduct fluid (ODF) and oviductal epithelium in cattle, which is believed to facilitate fertilization. It was recently reported that addition of a rabbit polyclonal immunoglobulin G antibody against purified bovine milk OPN with sperm oocytes, bovine oocytes, or both decreased (P < 0.05) fertilization compared with the in vitro-fertilized control (Goncalves et al. 2007 Theriogenology 67, 468–74). The aim of the present work was to determine the effect of in vitro addition of OPN to the fertilization medium on both cleavage and postfertilization embryo development in cattle. In the first experiment, in vitro-matured oocytes were fertilized in modified TALP medium in the presence of 0.0, 0.1, 1.0, or 10 µg mL–1 of OPN. In a second experiment, matured oocytes were in vitro-fertilized in modified TALP medium in the presence of 0.0, 10, 20, or 40 µg mL–1 of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull previously tested for IVF. After 20 to 22 h of coincubation at 39�C, 5% in CO2 in air, presumptive zygotes were vortexed to remove cumulus cells, washed, and transferred, 30 to 50 per well, into 400 µL of SOF modified medium. Zygotes were incubated in a humidified mixture of 5% CO2, 7% O2, and 88% N2 in air at a temperature of 39�C. On Day 7 of development (Day 0 = day of insemination), cleavage and development rates into transferable embryos (TE)–tight morulae (TM) and blastocysts (Bl) of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by a chi-square test. In experiment 1, numerically higher percentages of TM–Bl (29.5, 29.5, 30.5, and 37.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.25) and Bl (28.6, 27.5, 29.1, and 36.7, respectively, in the control group and in the groups with 0.1, 1, and 10 µg mL–1 of OPN; P = 0.24) were observed with 10 µg mL–1 of OPN. In experiment 2, significantly more cleavage (80.0 v. 71.3%; P < 0.05) and higher percentages of TM–Bl (44.6 v. 34.5%; P < 0.05) and Bl (39.2 v. 30.6%; P = 0.06) were observed with 10 µg mL–1 of OPN v. the control. Combined analysis from both experiments showed an overall effect of 10 µg mL–1 of OPN v. the control in the percentages of TM–Bl and Bl (respectively, 41.1 v. 33.3% and 37.7 v. 30.5%; P < 0.05). These results indicate that it is possible to improve the efficiency of bovine in vitro embryo production by adding the oviductal protein OPN.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

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.

135 SHORT-TERM CRYOPRESERVATION OF VITRIFIED MOUSE MORULAE AT - 79°C

2007 ◽  
Vol 19 (1) ◽  
pp. 185
Author(s):  
Y. Takagi ◽  
M. Shimizu ◽  
M. Morimura ◽  
S. Yokomizo ◽  
K. Hara ◽  
...  
Keyword(s):  
Liquid Nitrogen ◽  
Blastocyst Stage ◽  
Control Group ◽  
Embryo Viability ◽  
Chi Square ◽  
Significant Difference ◽  
Morula Stage ◽  
Chi Square Test ◽  
Student’S T

Embryos of various species are successfully vitrified and cryopreserved in liquid nitrogen (&lt;−150°C). Like the preservation of frozen somatic cells cooled by dry ice (−79°C), the cryopreservation of embryos at −79°C is useful for a reduction in the shipping costs. The purpose of this study was to evaluate the effect of the cryopreservation period at −79°C on the in vitro embryo viability of vitrified mouse morulae after thawing. Morula-stage mouse embryos were collected from superovulated ICR donors 70 h after hCG injection. The embryos were exposed first to 5% DMSO + 5% ethylene glycol (EG) in Dulbecco's PBS + 20% FCS (mPBS) for 2 min, and then equilibrated for 20–30 s in a vitrification solution composed of 10% DMSO + 10% EG + 0.6 M sucrose in mPBS. The embryos were loaded onto cryoloops (Lane et al. 1999 Nat. Biotech. 17, 1234–1236) and plunged directly into liquid nitrogen. The cryoloops were placed in 1.2-mL cryotubes and stored in a −79°C freezer for 1–7 days. The embryos were warmed by passing through 4 dilution media and rinsed with mWM culture medium. They were then cultured at 37°C in 5% CO2 for 44 h. Non-cryopreserved embryos and embryos cryopreserved in liquid nitrogen served as controls. Data were analyzed by the chi-square test and the Student's t-test. Results are shown in Table 1. There was no significant difference (P &gt; 0.01) in the developmental abilities to the blastocyst stage of the vitrified embryos that were cryopreserved at −79°C for 1 day, 3 days, and 5 days, the embryos cryopreserved in liquid nitrogen, and the non-vitrified control. The blastocyst rate of embryos was significantly lower (P &lt; 0.01) for the Day 7 group than for the control group. The cell numbers of blastocysts were significantly lower (P &lt; 0.01) for the Day 1, Day 3, Day 5, and Day 7 groups than for the control group. This study suggests that vitrified mouse morulae can be successfully cryopreserved at −79°C for 5 days. Table 1. Effect of the cryopreservation period on the viability of vitrified mouse morulae preserved at −79°C

205 EFFECT OF OSTEOPONTIN ON CLEAVAGE AND BLASTOCYST RATES IN BUFFALO SPECIES (BUBALUS BUBALIS)

2009 ◽  
Vol 21 (1) ◽  
pp. 200
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Rubessa ◽  
G. Campanile ◽  
R. Di Palo ◽  
...  
Keyword(s):  
Embryo Development ◽  
Cumulus Cells ◽  
Bubalus Bubalis ◽  
The Other ◽  
Control Group ◽  
Single Chain ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization

It was previously reported that osteopontin (OPN), an acidic single-chain phosphorylated glycoprotein found in the oviductal fluid in cattle (Gabler C et al. 2003 Reproduction 126, 721–729), is able to facilitate fertilization in this species (Gasparrini B et al. 2008 Reprod. Fertil. Dev. 20(Suppl. I), 180 abst). The present study aimed to investigate whether the addition of OPN to the fertilization medium would affect both cleavage and postfertilization embryo development in the buffalo. To assess the influence of OPN on cleavage and blastocyst rates, in vitro-matured oocytes were fertilized in modified Tyrode’s albumin lactate pyruvate medium (Lu KH et al. 1987 Vet. Rec. 121, 259–260) supplemented with penicillamine, hypotaurine, and heparin, in the presence of 0.0 (n = 258), 0.1 (n = 263), 1 (n = 261), and 10 μg mL–1 (n = 264) of OPN. In vitro fertilization was carried out with frozen–thawed spermatozoa from a bull already tested for IVF. After 20 to 22 h of co-incubation at 38.5°C and 5% CO2 in air, putative zygotes were gently pipetted to remove cumulus cells, washed, and transferred, 10 per droplet, into 20 μL of SOF medium including essential and nonessential amino acids and BSA (Tervit HR et al. 1972 J. Reprod. Fertil. 30(3), 493–497), in a controlled gas atmosphere consisting of 5% CO2, 7% O2, and 88% N2, in humidified air, at 38.5°C. The culture medium was changed on Day 5 (Day 0 = day of insemination), when cleavage rate was assessed and embryos were moved into fresh medium for an additional 2 days. On Day 7, development rates into blastocysts of superior quality were recorded. Differences in the percentages of both cleavage and blastocyst rates among groups were analyzed by chi-square test. Significantly higher cleavage rates (59.3, 70.3, 71.6, and 42.4%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. Likewise, higher blastocyst rate percentages (17.4, 27.4, 29.9, and 9.5%, respectively, in the control group and in the groups with 0.1, 1, and 10 μg mL–1 of OPN; P < 0.01) were observed in the groups with 0.1 and 1 μg mL–1 of OPN compared with the other groups. In conclusion, these results showed that addition of low concentrations of OPN in the fertilization medium improved both cleavage and postfertilization embryo development in the buffalo, whereas the higher concentration resulted in impaired late-stage embryo development.

scholarly journals 3D Liquid Marble Microbioreactors Support In Vitro Maturation of Prepubertal Ovine Oocytes and Affect Expression of Oocyte-Specific Factors

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1101
Author(s):  
Daniela Bebbere ◽  
Stefano Mario Nieddu ◽  
Federica Ariu ◽  
Davide Piras ◽  
Sergio Ledda
Keyword(s):  
Mammalian Species ◽  
3D Culture ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
Beneficial Effects ◽  
Fumed Silica Nanoparticles ◽  
Liquid Marble

In vitro oocyte maturation (IVM) is a well-established technique. Despite the high IVM rates obtained in most mammalian species, the developmental competence of IVM oocytes is suboptimal. The aim of this work was to evaluate the potential beneficial effects of a liquid marble microbioreactor (LM) as a 3D culture system to mature in vitro prepubertal ovine oocytes, as models of oocytes with intrinsic low competence. Cumulus–oocyte complexes of prepubertal sheep ovaries were in vitro matured in a LM system with hydrophobic fumed-silica-nanoparticles (LM group) or in standard conditions (4W control group). We evaluated: (a) maturation and (b) developmental rates following in vitro fertilization (IVF) and embryo culture; (c) expression of a panel of genes. LM and 4W groups showed similar IVM and IVF rates, while in vitro development to blastocyst stage approached significance (4W: 14.1% vs. LM: 28.3%; p = 0.066). The expression of GDF9, of enzymes involved in DNA methylation reprogramming and of the subcortical maternal complex was affected by the IVM system, while no difference was observed in terms of cell-stress-response. LM microbioreactors provide a suitable microenvironment to induce prepubertal sheep oocyte IVM and should be considered to enhance the developmental competence of oocytes with reduced potential also in other species, including humans.

Effect of cyanocobalamin on oocyte maturation, in vitro fertilization, and embryo development in mice

Zygote ◽  
2020 ◽  
pp. 1-8
Author(s):  
Tamana Rostami ◽  
Fardin Fathi ◽  
Vahideh Assadollahi ◽  
Javad Hosseini ◽  
Mohamad Bagher Khadem Erfan ◽  
...  
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Cumulus Cells ◽  
Treated Group ◽  
Blastocyst Stage ◽  
Control Group ◽  
Vitro Fertilization ◽  
Maturation In Vitro

Summary The aim of this study was to investigate the effect of cyanocobalamin supplementation on in vitro maturation (IVM), in vitro fertilization (IVF), and subsequent embryonic development competence to the blastocyst stage, and in vitro development of mouse 2-cell embryos. Cumulus cells were prepared from mouse cumulus–oocyte complexes (COCs) and incubated for 24 h in an in vitro culture (IVC) medium that contained different concentrations of cyanocobalamin (100, 200, 300 or 500 pM). We collected 2-cell embryos from superovulated NMRI mice and cultured them in the same concentrations of cyanocobalamin (100, 200, 300 or 500 pM). After 42 h of IVM, we observed significantly increased oocyte maturation in the 200 pM cyanocobalamin-treated group compared with the control group (P < 0.0001). Mature oocytes cultured in 200 pM cyanocobalamin were fertilized and cultured in IVC medium with cyanocobalamin (100, 200, 300 or 500 pM) during early embryogenesis. The matured oocytes that were cultured in 200 pM cyanocobalamin had significantly higher 2-cell development rates compared with the control oocytes (P < 0.01). Embryos obtained from in vitro mature oocytes and in vivo fertilized oocytes that were cultured in 200 pM cyanocobalamin had significantly greater frequencies of development to the blastocyst stage and a significant reduction in 2-cell blocked and degenerated embryos compared with the control embryos (P < 0.0001). Embryos derived from oocytes fertilized in vivo with 200 pM cyanocobalamin had a higher percentage of blastocyst embryos compared with those derived from matured oocytes cultured in vitro (P < 0.0001). These finding demonstrated that the effects of cyanocobalamin on oocyte maturation, fertilization, and embryo development in mice depend on the concentration used in IVC medium.

scholarly journals Abilities of cumulus and granulosa cells to enhance the developmental competence of bovine oocytes during in vitro maturation period are promoted by midkine; a possible implication of its apoptosis suppressing effects

Reproduction ◽  
2006 ◽  
Vol 132 (4) ◽  
pp. 549-557 ◽  
Author(s):  
S Ikeda ◽  
K Saeki ◽  
H Imai ◽  
M Yamada
Keyword(s):  
Dna Fragmentation ◽  
Granulosa Cells ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Blastocyst Development ◽  
Bovine Oocytes

We previously reported that when midkine (MK), a heparin-binding growth differentiation factor was used inin vitromaturation (IVM) culture of bovine cumulus-enclosed oocytes (CEOs), their developmental competence to the blastocyst stage afterin vitrofertilization (IVF) was enhanced and the effect of MK might be mediated by its action upon mural granulosa cells and cumulus cells that closely surround the oocyte. In the present study, when denuded oocytes (DOs) were matured in IVM medium with or without MK (200 ng/ml) in the presence or absence of isolated cumulus cell masses and subjected to IVF, the enhancing effects of MK on the developmental competence of DOs to the blastocyst stage after IVF were exerted only in the presence of cumulus cells. In addition, we prepared the conditioned media of granulosa cells cultured with or without 200 ng MK/ml (CMMK+ or CMMK− respectively) and examined their effects on the IVM of DOs in terms of their developmental competence to the blastocyst stage after IVF. The supplementation of CMMK+ into IVM medium at 40% (v/v) significantly enhanced the blastocyst development compared with the no additive control and the CMMK− supplemented groups. Furthermore, the effects of MK during IVM of bovine CEOs on the cumulus cell apoptosis were investigated. CEOs were cultured up to 24 h in IVM medium without (control) or with 200 ng MK/ml. The genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect the apoptotic internucleosomal DNA fragmentation. DNA fragmentation was scarcely detected at the start of IVM, whereas it increased time-dependently as the IVM culture progressed. The degree of the fragmentation was significantly lower in the MK-treatment group compared with the control group at 18 and 24 h of IVM. The apoptosis-suppressing effect of MK on cumulus cells was further confirmedin situby using TUNEL on CEOs. In conclusion, data from the present study further confirmed that MK enhances the developmental competence of bovine oocytes via cumulus and granulosa cells. It was also demonstrated that MK suppresses the apoptosis that occurs in cumulus cells during the period of IVM of bovine CEOs. The putative soluble factor(s) from cumulus cells was suggested from the experiment using CMMK+ . MK may promote the production of such factors in part by its anti-apoptotic effects on cumulus cells.

scholarly journals 252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF

2005 ◽  
Vol 17 (2) ◽  
pp. 276 ◽  
Author(s):  
J. Pryor ◽  
S. Romo ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Sperm Concentration ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Before And After ◽  
Chi Square Test ◽  
Group 2 ◽  
Live Sperm ◽  
Sperm Separation

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.

P–219 mtDNA content in bovine cumulus cells does not predict oocytés developmental competence

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Á Martíne. Moro ◽  
I Lamas-Toranzo ◽  
L González-Brusi ◽  
A Pérez-Gómez ◽  
P Bermejo-Álvarez
Keyword(s):  
Cumulus Cell ◽  
Cumulus Cells ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Developmental Competence ◽  
Success Rates ◽  
Developmental Potential ◽  
Mtdna Sequence

Abstract Study question Does cumulus cell mtDNA content correlate with oocyte developmental potential in the bovine model? Summary answer The relative amount of mtDNA content did not vary significantly in oocytes showing different developmental outcomes following IVF What is known already Cumulus cells are closely connected to the oocyte through transzonal projections, serving essential metabolic functions during folliculogenesis. These oocyte-supporting cells are removed and discarded prior to ICSI, thereby constituting an interesting biological material on which to perform molecular analysis aimed to predict oocyte developmental competence. Previous studies have positively associated oocytés mtDNA content with developmental potential in both animal models and women. However, it remains debatable whether mtDNA content in cumulus cells could be used as a proxy to infer oocyte developmental potential. Study design, size, duration Bovine cumulus cells were allocated into three groups according to the developmental potential of the oocyte: 1) oocytes developing to blastocysts following IVF (Bl+Cl+), 2) oocytes cleaving following IVF but arresting their development prior to the blastocyst stage (Bl-Cl+), and 3) oocytes not cleaving following IVF (Bl-Cl-). Relative mtDNA content was analysed in 40 samples/group, each composed by the cumulus cells from one cumulus-oocyte complex (COC). Participants/materials, setting, methods Bovine cumulus-oocyte complexes were obtained from slaughtered cattle and individually matured in vitro (IVM). Following IVM, cumulus cells were removed by hyaluronidase treatment, pelleted, snap frozen in liquid nitrogen and stored at –80 ºC until analysis. Cumulus-free oocytes were fertilized and cultured in vitro individually and development was recorded for each oocyte. Relative mtDNA abundance was determined by qPCR, amplifying a mtDNA sequence (COX1) and a chromosomal sequence (PPIA). Statistical differences were tested by ANOVA. Main results and the role of chance Relative mtDNA abundance did not differ significantly (ANOVA p &gt; 0.05) between the three groups exhibiting different developmental potential (1±0.06 vs. 1.19±0.05 vs. 1.11±0.05, for Bl+Cl+ vs. Bl-Cl+ vs. Bl-Cl-, mean±s.e.m.). Limitations, reasons for caution Experiments were conducted in the bovine model. Although bovine folliculogenesis, monoovulatory ovulation and early embryo development exhibit considerable similarities with that of humans, caution should be taken when extrapolating these data to humans. Wider implications of the findings: The use of molecular markers for oocyte developmental potential in cumulus cells could be used to enhance success rates following single-embryo transfer. Unfortunately, mtDNA in cumulus cells was not found to be a good proxy for oocyte quality. Trial registration number Not applicable

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