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.

scholarly journals 253 COMPARISON OF THREE DIFFERENT IVF PROTOCOLS FOR BOVINE OOCYTES

2005 ◽  
Vol 17 (2) ◽  
pp. 277
Author(s):  
S. Romo ◽  
J. Pryor ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Embryo Culture ◽  
Culture Media ◽  
Sperm Concentration ◽  
Vitro Fertilization ◽  
Group 3 ◽  
Group 2 ◽  
The Given ◽  
Sperm Separation ◽  
Group 1

Recently, the development of commercially available defined media and sperm centrifugation gradients has offered new possibilities for increasing the efficiency of commercial in vitro fertilization (IVF) systems. The objective of this study was to compare three different IVF protocols using two different separation gradients, two fertilization media, and two embryo culture media, as follows: Group 1. sperm separation (SS): Percoll (Sigma, St. Louis, MO, USA), fertilization medium (FM): TALP-Fert (TFM), embryo culture media (ECM): G1/G2 (version 3, Vitrolife, Englewood, CO, USA). Group 2. SS: Percoll, FM: Bovine vitro Fert (Cook, Brisbane, Australia), ECM: Bovine vitro Blast/Bovine vitro Cleave (Cook); and Group 3. SS: EquiPure (Nidacon, Spectrum Technologies, Healdsburg, CA, USA), FM: TFM, ECM: G1/G2. Oocytes were obtained from slaughterhouse ovaries and matured in vitro (Looney et al. 1994 Theriogenology 41, 67). IVF was conducted using frozen/thawed semen from one bull. Semen was separated by centrifugation at 700g for 30 min in the given density gradients; Percoll was used in a 45% to 90% gradient. Sperm viability after separation was assessed by fast-green/eosin stain (Sigma). IVF was carried out in 0.5 mL of the given fertilization medium supplemented with PHE1 and heparin (10 μg/mL), in humidified 5% CO2 in air atmosphere at 38.7°C. Final sperm concentration in the IVF wells was 1 × 106/mL. In Experiment 1, a total of 368 oocytes (2 replicates) were fixed and stained (Hoechst 33342, Sigma) 24 h post-IVF to assess sperm penetration (Group 1, n = 128, Group 2, n = 108, Group 3, n = 132). In Experiment 2, a total of 400 embryos (2 replicates) were cultured in 0.5 mL of the given culture medium under mineral oil in a 5% O2, 5% CO2, 90% N2 atmosphere at 38.7°C with high humidity for 112 h before fixation and staining. Embryos in Groups 1 (n = 129) and 3 (n = 139) and Group 2 (n = 132) were changed to G2 and Cleave media, respectively, at 84 h. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 218 vs. 383 × 106 for EquiPure; P < 0.05), but resulted in higher total motility (60% vs. 41%, respectively; P < 0.05) and higher viability (93% vs. 70%, respectively; P < 0.05) of separated sperm. In Experiment 1, rates of normal fertilization were significantly lower for Group 3 (58%) than for Groups 1 and 2 (74% and 77%, respectively, P < 0.05). In Experiment 2, rates of development to <8, 9 to 16, and >16 cells at 112 h were not significantly different among groups (43, 48, and 46% for Group 1; 22, 18, and 31% for Group 2; and 35, 34, and 23% for Group 3, respectively; P > 0.1). These results indicate that the commercial separation medium, EquiPure, may be associated with lowered sperm motility, viability, and fertilization rates when compared to a standard medium (Percoll) for bovine sperm separation. Commercial fertilization and embryo culture media (Bovine vitro Fert, Cleave, and Blast) provided equivalent embryo development to that currently in use by our laboratory (TFM, G1/G2).

212 EFFECT OF CONSECUTIVE SUPERSTIMULATORY TREATMENT-INDUCED FOLLICULAR WAVE SYNCHRONIZATION TO OPTIMIZE OOCYTE RETRIEVAL AND EMBRYO PRODUCTION BY OVUM PICKUP AND IN VITRO FERTILIZATION IN COWS

2011 ◽  
Vol 23 (1) ◽  
pp. 205
Author(s):  
K. Imai ◽  
M. Ohtake ◽  
Y. Aikawa ◽  
S. Sugimura ◽  
M. Hirayama ◽  
...  
Keyword(s):  
Development Program ◽  
Oocyte Retrieval ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Follicular Wave ◽  
The Third ◽  
The Mean ◽  
Student’S T

We previously reported that superstimulatory (SS) treatment-induced follicular wave synchronization after ovum pickup (OPU) was effective in enhancing the quality of obtained oocytes and blastocysts derived from in vitro maturation (IVM) and fertilization (IVF; Imai et al. 2010 Reprod. Fertil. Dev. 22, 296). The present study was designed to examine the efficiency of embryo production by 4 sessions of OPU-IVF using a series of the SS treatment-induced follicular wave synchronizations. For the SS protocols, 3 consecutive SS (3CSS) and 2 separated SS (2SSS) were used. In the 3CSS group, the first OPU was performed on random days of the oestrous cycle (Day 0) and all follicles larger than 2 mm in diameter were aspirated. On Day 5, follicles larger than 8 mm in diameter were aspirated and a CIDR (InterAg, Hamilton, New Zealand) was inserted. The cows then received 20 armour units of FSH (Kawasaki-Seiyaku, Kawasaki, Japan) in twice-daily decreasing doses by IM injection from Day 7 to 10. Cloprostenol (PGF; 0.75 mg, Fujita-Pharm, Tokyo, Japan) was administered on the morning of Day 9. The second OPU was performed 48 h after PGF administration on Day 11; the CIDR was removed from the cows just before OPU. After the second OPU, donors were treated consecutively with the SS protocol mentioned above for the third and fourth OPU sessions. In the 2SSS group, donors received 2 sets of the SS treatment mentioned above, with an interval of 11 days between the second and the third OPU session. Four OPU sessions were performed every 11 days on all cows. In this study, 8 Japanese Black cows were divided into the 3CSS and 2SSS groups, and the treatment for each group was reversed after a 65-day interval as crossover trials. After OPU, Grade 1 and 2 oocytes were used for IVM and IVF, and putative zygotes were cultured as described by (Imai et al. 2006 J. Reprod. Dev. 52, S19–S29 suppl.). A part of the zygotes were cultured in a micro-well system. Data were analysed by Student’s t-test and chi-square test. There were differences (P < 0.05) in the mean (±SD) number of follicles, collected oocytes, and cultured oocytes in the 3CSS (35.0 ± 8.6 and 24.4 ± 11.2, respectively) and 2SSS (30.8 ± 10.5 and 20.2 ± 9.0, respectively) groups. There were no differences in mean percentage of blastocyst formation and Grade 1 blastocyst rates between the 3CSS (38.5 and 55.8%, respectively) and 2SSS (34.8 and 54.8%, respectively) groups. However, the mean number of blastocysts produced per OPU session was significantly (P < 0.05) higher in the 3CSS group (8.1 ± 6.3) compared with the 2SSS group (5.8 ± 4.4). These results indicate that a series of 3 consecutive SS treatments had greater efficiency in producing OPU-IVF embryos. This work was supported in part by the Research and Development Program for New Bio-industry Initiatives.

scholarly journals 266IN VITRO-DERIVED EMBRYO PRODUCTION WITH SEXED AND UNSEXED SEMEN FROM DIFFERENT BULLS

2004 ◽  
Vol 16 (2) ◽  
pp. 253 ◽  
Author(s):  
L. Ferré ◽  
C. Ohlrichs ◽  
D. Faber
Keyword(s):  
In Vitro Fertilization ◽  
Embryo Development ◽  
Calf Serum ◽  
Sperm Concentration ◽  
Reproductive Technologies ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Sexed Semen ◽  
Chi Square Analysis

The production of pre-sex-selected calves by in vitro fertilization (IVF), using sexed semen, does show some benefits due to the small quantity of sperms needed for the process as compared to other reproductive technologies. The objective of this study was to determine differences among bulls and sperm concentrations in embryo development with sexed and unsexed semen. Follicles ranging from 2 to 6mm in diameter were aspirated from slaughterhouse ovaries. COC were selected and matured in groups of maximum of 30 in 1.8mL of TCM-199, supplemented with 10% fetal calf serum, 0.01UmL−1 bFSH, 0.01UmL−1 bLH and 10μLmL−1 penicillin-streptomycin for 24h at 38.5°C. Fertilization (Day 0) was carried out in micro-drops (50μL) with TALP-FERT medium containing PHE (3μgmL−1 penicillamine, 11μgmL−1 hypotaurine and 0.18μgmL−1 epinephrine), 10μLmL−1 non-essential amino acid and 2μgmL−1 heparin. Frozen/thawed sexed (female) and non-sexed sperms from five bulls were selected in a discontinuous percoll gradient. Sperm concentration was 1×106 for non-sexed semen and 1×106 or 2×106 for sexed semen. After 18–20h, presumptive zygotes were denuded and cultured in groups of 10 in 50-μL micro-drops of SOF citrate with 5% FCS (Holm P et al., 1999 Theriogenology 52, 683–700) under paraffin oil in a 5% O2, 5% CO2, 90% N2 atmosphere with high humidity. On Day 7, blastocysts (BL) were morphologically evaluated and recorded. Results are shown in Table 1. Data was compared by chi-square analysis. Sexed frozen bovine sperm can be used successfully in IVF systems. More research needs to be done to optimize and standardize bovine in vitro fertilization with sexed semen. Table 1 Results of comparisons between bulls, sperm concentrations, cleavage and embryo development

scholarly journals Factors Related to the Number of Antral Follicles on InVitro Fertilization (IVF)

2016 ◽  
pp. 75
Author(s):  
Fransiskus C Raharja ◽  
Ketut Suwiyoga ◽  
IPG Wardhiana
Keyword(s):  
Prevalence Ratio ◽  
Cross Sectional Study ◽  
Chi Square ◽  
Cross Sectional ◽  
Vitro Fertilization ◽  
Antral Follicles ◽  
Chi Square Test ◽  
The Mean ◽  
Infertile Patients

Objective: To determine factors which are related to the number of antral follicles on infertile patients. Method: This cross sectional study was conducted in In-Vitro Fertilization (IVF) clinic of Graha Tunjung, Sanglah hospital, Bali. All fertile patients following the IVF program were calculated the number of antral follicles in both ovarian using transgene USG. This sample was recruited by random sampling from April 1st, 2001 to April 30th, 2011. We analyzed the data using Chi square test through SPSS for Windows 17.0 version. Result: Of 102 samples, the mean of patients’ age was 32.9% (SD 4.6) years old. From 72 patients (70.6%) experienced above 3 years of infertile period, the primary infertile was on 69 patients (67.7%). There was a relationship between patients’ age and the number of antral follicles significantly (prevalence ratio (PR) 1.41; 95% CI 1.11- 1.79). Meanwhile, the number of antral follicles and type of infertile (PR 1.02; 95% CI 0.76-1.37) also infertile period (PR 0.95; 95% CI 0.72-1.27) were not associated significantly. Conclusion: Patients’ age has an association with the number of antral follicles on IVF. [Indones J Obstet Gynecol 2016; 4-2: 75-77] Keywords: age, infertile, infertile period, number of antral follicles and type of infertile

scholarly journals Predictive Factors For Three Pronuclear Zygote In Ivf Cycle

KnE Medicine ◽  
2016 ◽  
Vol 1 (1) ◽  
Author(s):  
Budi Wiweko
Keyword(s):  
Strong Predictor ◽  
Multivariate Logistic Regression Analysis ◽  
Controlled Ovarian Hyperstimulation ◽  
Categorical Variables ◽  
Continuous Variables ◽  
Chi Square ◽  
Preimplantation Genetic Testing ◽  
Vitro Fertilization ◽  
Chi Square Test

<p>Introduction: Preimplantation Genetic Testing is one of the methods to screen genetic defects in embryos created through In Vitro Fertilization. In developing country this technique is still new and expensive method. Thus, knowing several factors in predicting the occurrence of three-pronuclear zygote can help much.</p><p>Method: 472 cycles in 449 subjects who underwent controlled ovarian hyperstimulation in IVF cycles between January 2013 and August 2014 were included in the study. Categorical variables were compared using Chi Square test and continuous variables were analyzed using Independent t-test, and statistically significant was considered when p &lt; 0.05. Multivariate logistic regression analysis was performed in order to correlate clinical variables and the occurrence of three-pronuclear zygote (3pn). </p><p>Results: There were 38 3pn was identified in this study (8.05%). No correlation was found between age of the women, number of mature follicles, which are less than six with the incidence of 3pn. However, level of Anti Mullerian Hormones (AMH) found to be the strongest predictor with the incidence of 3PN (p &lt; 0,01, RR 2.5, CI95% 1,55; 4.16).</p><p>Conclusion: level of AMH is known to be strong predictor of thee pronuclear zygote after IVF cycle.</p>

scholarly journals Collection, freezing of Ban epididymal sperm and evaluation of in vitro fertility after thawing

2021 ◽  
Vol 18 (4) ◽  
pp. 625-632
Author(s):  
Nguyen Viet Linh ◽  
Nguyen Thi Nhung
Keyword(s):  
In Vitro Fertilization ◽  
Sperm Concentration ◽  
Epididymal Sperm ◽  
Freezing And Thawing ◽  
Vitro Fertilization ◽  
Mountainous Regions ◽  
Before And After ◽  
Thawing Processes ◽  
Motility Of Sperm

Many researches on characteristics of porcine ejaculated semen on popular commercialized breeds collecting at breeding centers or households with pig breeding services have been carried out in Vietnam. However, very few investigation had been dealt with epididymal sperms, especially the ones from boar of Ban, a popular Vietnam native mini-pig breed which is usually freely raised in far rural and mountainous regions in some provinces of Northern Vietnam. In the present study, we surveyed on criteria of epididymal sperms from 4 Ban boars at collection, after freezing and thawing processes, and tested their fertility by an in vitro fertilization and embryo culture experiment. Approximate volume of sperm collected from a Ban boar did not differ (3-4 mL), with sperm concentration from 6.4 x 109 to 11.3 x 109 sperm/mL. Motility varied from 8.7 to 27.0%, whereas vitality was from 58.0 to 85.6%. After freezing and thawing, the motility of sperm slightly decreased to values of 6.3 to 25.7%, and viability significantly decreased to values of 41.3 to 79.6%. No difference was found between rates of abnormal morphology before and after freezing and thawing (10.6 to 31.0% and 12.0 to 32.0%, respectively). A test by in vitro fertilization with Landrace oocytes revealed that two sperm lots had acceptable in vitro fertility with rates of blastocyst formation from 14.4 to 18.8%. In conclusion, a study on collection, freezing and fertility testing of epididymal sperm collected from Ban boars has been carried out. The results of the present study could contribute necessary information as well as standardized sperm lots as important materials to further research on in vitro fertilization of Ban in Vietnam.

scholarly journals Evaluation of equine corneal endothelium after exposure to 0.05% brilliant blue - an in vitro study

2019 ◽  
Vol 71 (4) ◽  
pp. 1158-1164
Author(s):  
M.C.C. Andrade ◽  
T.M. Moreno ◽  
M.S. Muccillo ◽  
J.A.T. Pigatto ◽  
E.V. Camilo
Keyword(s):  
Corneal Endothelium ◽  
In Vitro Study ◽  
Brilliant Blue ◽  
Control Group ◽  
Chi Square ◽  
Uniform Size ◽  
Chi Square Test ◽  
Group 2 ◽  
Scanning Electron

ABSTRACT The aim of this study was to evaluate the immediate effects of 0.05% brilliant blue on corneal endothelium of horses. Thirty-eight corneas of 19 horses, male or female, of different ages were studied. Corneas were randomly divided into two groups. Group 1: Corneal endothelium was covered with 0.3mL of brilliant blue 0.05% for 60 seconds followed by rinsing with a balanced salt solution. Group 2: Corneal endothelium was covered with BSS for 60 seconds. The corneas were excised with an 8mm trephine and prepared to analyze posterior endothelial surface using a light microscope (24 corneas) and a scanning electron microscope (14 corneas). The equine posterior corneal endothelium surface observed by optical microscopy and scanning electron microscopy revealed a continuous layer of polygonal cells of uniform size and shape in both the control and treatment groups. Due to non-normal residuals at ANOVA mean comparison, a generalized linear model was utilized at 5% level of significance. The chi-square test stated that treatment and control group were not different statistically. The 0.05% brilliant blue did not cause damage to equine corneal endothelium.

188 EFFECT OF TRANSFER OF FROZEN-THAWED IVF EMBRYOS ON PREGNANCY IN REPEAT-BREEDER INSEMINATED OR NON-INSEMINATED HOLSTEIN CATTLE

2006 ◽  
Vol 18 (2) ◽  
pp. 202 ◽  
Author(s):  
O. Dochi ◽  
M. Tanisawa ◽  
S. Goda ◽  
H. Koyama
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Calf Serum ◽  
Pregnancy Rates ◽  
Holstein Cattle ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Chi Square Test ◽  
Repeat Breeder

Repeat-breeding is one of the important factors that affect dairy management. The objective of this study was to investigate the effect of transfer of frozen–thawed IVF embryos on pregnancy in repeat-breeder Holstein cattle. Cumulus–oocyte complexes (COCs) were collected by aspiration of 2–1-mm follicles from ovaries obtained at a local abattoir. COCs were matured for 20 h in TCM-199 supplemented with 5% calf serum (CS) and 0.02 mg/mL of FSH at 38.5°C under a 5% CO2 atmosphere in air. Matured oocytes were inseminated with spermatozoa of 5 × 106/mL in BO solution (Brackett and Oliphant 1975 Biol. Reprod. 12, 260–274) containing 10 mM hypotaurine and 4 units/mL heparin. After 18 h of gamete co-culture, presumptive zygotes were cultured in CR1aa (Rosenkrans et al. 1991 Theriogenology 35, 266) supplemented with 5% CS for 8 days at 38.5°C under 5% CO2, 5% O2, 90% N2 atmosphere in air. After in vitro fertilization, Day 7 and Day 8 blastocysts were frozen in 1.5 M ethylene glycol (EG) in Dulbecco's PBS (DPBS) supplemented with 0.1 M sucrose and 20% CS. Embryos were transferred into a freezing medium, loaded into 0.25-mL straws, and allowed to stand for 15–20 min for equilibration. The straws were then plunged into a −7°C methanol bath of a programmable freezer for 1 min, seeded at −7°C, maintained at −7°C for 15 min, cooled to −30°C at the rate of −0.3°C/min, and then plunged into liquid nitrogen. Recipient animals (43 heifers, 131 cows) included those that did not conceive after being artificially inseminated (AI) 3 to 15 times. The frozen–thawed IVF embryos were directly transferred to the recipient animals 7 days after estrus or AI. Pregnancy rates were analyzed by chi-square test. The results are presented in Table 1. There were no significant differences in the pregnancy rates between treatments. However, a slightly higher pregnancy rate was achieved by embryo transfer after AI. These results suggest that embryo transfer may increase the pregnancy rate in repeat-breeder Holstein cattle. Table 1. Pregnancy rates after transfer of IVF frozen–thawed embryos in repeat-breeder Holstein cattle

184 EFFECTS OF X-RAY ELECTROMAGNETIC RADIATION ON BOVINE OOCYTE DEVELOPMENT

2010 ◽  
Vol 22 (1) ◽  
pp. 250
Author(s):  
W. E. Snyder ◽  
J. T. Aaltonen ◽  
H. D. Sigal ◽  
N. M. Loskutoff
Keyword(s):  
Electromagnetic Radiation ◽  
High Energy ◽  
X Rays ◽  
Embryo Production ◽  
Chi Square ◽  
X Ray ◽  
Chi Square Test ◽  
Bovine Oocytes ◽  
Experimental Group

This study examined the effects of X-rays on bovine oocytes used for in vitro embryo production. In recent years, airport security has become more stringent and the use of X-ray screening may be required for all items, which could be problematic when transporting oocytes. Ionizing radiation such as X-rays are high-energy particles that can penetrate into the nucleus of a cell and cause single and double-strand breaks in the DNA chain. Oocytes exposed to X-ray radiation may have DNA damage affecting maturation, fertilization, and embryonic development. Bovine ovaries obtained from a local abattoir were used to collect immature oocytes by follicular aspiration. The oocytes were divided into a control group and an experimental group then placed in maturation medium. The oocytes in the experimental group were placed in a hospital X-ray machine with a single exposure of 100 mA, 120 kVp for 0.3 s. This exposure challenged the oocytes with a comparable, calculated amount of mrems equivalent to a single screening from an airport X-ray machine (30 mrems). After X-ray exposure, the experimental group was cultured with the control at 37°C and 5% CO2. After 18 h, cryopreserved bovine sperm were thawed and processed using BoviPure (Nidacon International, Mölndal, Sweden) density gradient centrifugation and the oocytes were inseminated with 1 × 106 sperm in fertilization medium at 37°C and 5% CO2. After 24 h, both groups were transferred to 50-μL droplets of G1 medium (Vitrolife, Göteborg, Sweden) at 37°C and 6% CO2 and both were transferred into G2 medium after 72 h. Development of the oocytes was scored after an additional 72 h. Results were based on 2 criteria: the percent cleaved (presumptive fertilization) and the percent cleaved that developed to morula and blastocyst stages. The results indicated no significant difference in the percentages of oocytes cleaving after insemination for the control and experimental groups (P = 0.403, chi-square test). However, there was a significant increase (P = 0.037, chi-square test) in the percentages of morulae and blastocysts developing in the control (non-irradiated) v. experimental (X-rayed) groups. The outcome of this study cautions on the potential damage caused to bovine oocytes used for embryo production in vitro when exposed to X-ray electromagnetic radiation during routine air transport screening operations. Table 1.

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.

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