231 EFFECT OF DIFFERENT EQUINE CHORIONIC GONADOTROPIN CONCENTRATIONS ON IN VITRO-PRODUCED BOVINE EMBRYOS

In vitro maturation (IVM) is one of the most challenging steps in the in vitro production of bovine embryos. The IVM medium must provide the necessary conditions for the occurrence of nuclear and cytoplasmic maturation, close to the physiological conditions. The pituitary gonadotropins are essential components for generating competent oocytes; however, whether these hormones (pituitary, placental, or both) are essential and which concentrations should be used are still controversial. Our work aimed to compare the effect of different concentrations of the placental gonadotropin (eCG) in the IVM medium on the in vitro-produced bovine blastocysts. Cumulus–oocyte complexes (n = 1341, grades I and II), obtained from ovaries from an abattoir were selected and distributed into six groups: (1) eCG (4 IU mL–1; n = 192); (2) eCG (1.5 IU mL–1; n = 204); (3) eCG (1.3 IU mL–1; n = 203); (4) eCG (0.15 IU mL–1; n = 202); (5) eCG (0.015 IU mL–1; n = 199); (6) control: FSH (0.1 mg mL–1), LH (50 µg mL–1), and 10% of fetal calf serum (FCS; n = 341). Medium from groups 1 to 5 also contained LH (50 µg mL–1) and BSA (6 mg mL–1). The cumulus–oocyte complexes were matured in TCM-199 for 24 h and were IVF (Day 0) in TALP-IVF for 22 to 24 h. Viable spermatozoa were selected by Percoll gradient, and they were evaluated (motility and spermatozoa concentration) to provide the insemination concentration (106 spermatozoa mL–1). Presumptive zygotes were cultured in SOF medium supplemented with FCS (2.5%) and BSA (5 mg mL–1) in an incubator (38.3°C, 5% CO2, and maximum humidity). Embryo development was evaluated in terms of cleavage (Day 3), blastocyst (Day 7), and hatching rates (Day 10). Mean rates were analysed by chi-squared test and ANOVA, and significance was considered when P < 0.05. The results obtained from the different groups are shown in Table 1. Cleavage, blastocyst, and hatching rates were not different among groups. We conclude that, under our experimental conditions, even minimal concentrations of eCG (0.015 IU) may be a viable and effective alternative in the replacement of FSH for the IVM of bovine oocytes. Table 1.Cleavage, blastocyst, and hatching rates of the experimental groups (1, 2, 3, 4, and 5) and control group1 Fellowships and support by CAPES and FAPESP.

Download Full-text

31 Effect of fetal calf serum on production and cryotolerance of in vitro bovine embryos from Ecuadorian Creole heifers

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab31 ◽

2019 ◽

Vol 31 (1) ◽

pp. 141

Author(s):

M. S. Méndez ◽

M. E. Soria ◽

L. R. Galarza ◽

F. P. Perea ◽

D. E. Argudo

Keyword(s):

Calf Serum ◽

Bovine Embryos ◽

In Vitro Production ◽

Sodium Pyruvate ◽

Embryo Production ◽

Maturation Medium ◽

Fetal Bovine ◽

And Control ◽

Vitro Production

In the Ecuadorian Andes there is a Creole bovine biotype whose population is disappearing. In vitro embryo production and cryopreservation is an important biotechnology that allows the conservation of animals threatened with extinction. The objective of this study was to determine the in vitro production and cryopreservation of embryos from creole heifers raised in the highlands of Ecuador. Immature cumulus-oocyte complexes were retrieved by ovum pickup from 10 Creole heifers (OPU) and from abattoir ovaries (control). The experiment was completed within 8 replicates. Cumulus-oocyte complexes were cultured in a maturation medium (TCM-199 supplemented with 10% fetal bovine serum, 100µg mL−1 of sodium pyruvate, 0.75mg mL−1 of l-glutamine, 4µg mL−1 of FSH-p, 100µM cysteamine, and 250µg mL−1 of gentamicin) following IVF (SOF medium supplemented with 10µg mL−1 heparin) and in vitro culture (citrate SOF medium). After denudation (Day 1 after IVF), presumptive embryos from each oocyte source (OPU and control) were split into 2 groups: with (FCS+) and without (FCS−) FCS (2.5%), which was added on Day 5 after IVF. On Day 7, embryos were evaluated, and those with quality 1 were vitrified. After warming, embryo re-expansion at 2h and embryo re-expansion and hatching at 24 and 48h were evaluated. Data were analysed by logistic regression in SAS software (SAS Institute Inc., Cary, NC, USA). Results of embryo rate at Day 7 and rates of vitrified, re-expanded, and hatched embryos are shown in Table 1. Regardless of the oocyte source, the addition of 2.5% FCS decreased embryo re-expansion at 2h and reduced embryo hatching at 48h in the OPU group. In conclusion, FCS did not improve embryo production and adversely affected the cryotolerance of embryos produced in vitro from Ecuadorian creole heifers. Table 1.Production and cryotolerance of in vitro bovine embryos

Download Full-text

49 SUCCESSFUL CRYOPRESERVATION USING LOW ETHYLENE GLYCOL CONCENTRATION FOR IN VITRO-PRODUCED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab49 ◽

2017 ◽

Vol 29 (1) ◽

pp. 132

Author(s):

M. Takayama ◽

S. Sato ◽

Y. Nishimura ◽

K. Imai ◽

O. Dochi

Keyword(s):

Ethylene Glycol ◽

Calf Serum ◽

Bovine Embryos ◽

Embryo Survival ◽

Lower Survival Rate ◽

Treatment Groups ◽

Chi Squared ◽

Hatching Rates

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL−1 FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 106 sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.

Download Full-text

221 EFFECT OF STAGE OF CORPUS LUTEUM DEVELOPMENT ON THE IN VITRO PRODUCTION OF BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab221 ◽

2011 ◽

Vol 23 (1) ◽

pp. 209

Author(s):

S. Miyashita ◽

K. Miyata ◽

C. Tachibana ◽

Y. Inaba ◽

H. Koyama ◽

...

Keyword(s):

Corpus Luteum ◽

Calf Serum ◽

Cumulus Cells ◽

Large Mass ◽

Bovine Embryos ◽

In Vitro Production ◽

Stage Of Development ◽

The Mean ◽

Vitro Production

The objective of this study was to investigate the effect of stage of corpus luteum (CL) development on the in vitro production of bovine embryos. Ovaries were classified according to the expected day of the oestrous cycle based on the morphology of the ovaries. Ovaries with a corpus hemorrhagicum and the remnant of the follicular lumen filled with blood were considered the early luteal stage (Days 2 to 4; Day 0 = day of ovulation, n = 46). Ovaries with a large mass of orange tissue in the CL were classified as the midluteal stage (Days 7 to 10, n = 42). Cumulus–oocyte complexes (COC) were collected by aspiration of 2- to 6-mm follicles. The COC were classified into the following grades: COC with >3 compact layers of cumulus cells and evenly granulated cytoplasm were classified into Grade 1; COC with >3 layers cumulus cells and evenly granulated cytoplasm were classified into Grade 2; COC with partially remaining cumulus cells and abnormal cytoplasm were classified into Grade 3; COC without cumulus cells or those with expanded cumulus cells were classified into Grades 4 and 5, respectively. Grades 1 and 2 COC were in vitro matured for 20 h in TCM-199 supplemented with 5% calf serum and 0.02 mg mL–1 of FSH at 38.5°C under an atmosphere of 5% CO2 in air. Matured COC were inseminated with 5 × 106 sperm for 18 h. Presumptive zygotes were cultured in CR1aa medium supplemented with 5% calf serum at 38.5°C under an atmosphere of 5% O2, 5% CO2, and 90% N2 for 9 days (fertilization = Day 0). The mean number of COC and the proportion of COC classified as Grades 1 and 2 were analysed by ANOVA. Cleavage rates on Day 3 and blastocyst rates on Days 7 to 9 were analysed by a chi-square test. The mean number of recovered oocytes in the early luteal stage (18.7 ± 9.5) was significantly higher (P < 0.05) than the number in the midluteal stage (12.2 ± 5.7). The proportion of Grades 1 and 2 oocytes in the early luteal stage [66.7% (531/789)] was significantly higher (P < 0.01) than that in the midluteal stage [51.6% (252/484)]. The cleavage and blastocyst rates in the early luteal stage [60.9% (181/297) and 32.7% (97/297), respectively] were significantly higher (P < 0.05) than those in the midluteal stage [50.7% (76/150) and 20.7% (31/150) respectively].The present study suggests that the stage of development of the CL in bovine ovaries influences the number of recovered oocytes per ovary and the development of in vitro production of bovine embryos.

Download Full-text

88 EFFECT OF VITRIFICATION PROCEDURE ON SURVIVAL RATE OF BOVINE EMBRYOS PRODUCED IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv23n1ab88 ◽

2011 ◽

Vol 23 (1) ◽

pp. 149

Author(s):

E. Y. Herrera ◽

C. de Frutos ◽

R. Laguna-Barraza ◽

A. Gutierrez-Adan ◽

D. Rizos

Keyword(s):

Survival Rate ◽

Survival Rates ◽

Calf Serum ◽

Basal Medium ◽

Bovine Embryos ◽

Oviduct Fluid ◽

Bovine Oocytes ◽

Cryopreservation Method ◽

Hatching Rates

Vitrification as a cryopreservation method has many advantages compared with slow freezing. Many variables in the vitrification process exists that influence the survival rates of vitrified oocytes and embryos. These include the cryoprotectants (type, concentration, and duration of exposure), the temperature of the vitrification solution at exposure, the device used for vitrification, and the quality and developmental stage of embryos. It is worthwhile to mention that vitrification protocols successfully used in bovine oocytes and embryos have been used also with human oocytes and embryos. Vitrification is relatively simple, requires no freezing equipment, and relies on the placement of the embryos in a very small volume of vitrification medium that must be cooled at extreme rates not obtainable in regular enclosed straws. The aim of the present study was to evaluate the efficiency of 4 different vitrification protocols on the survival rate of in vitro produced (IVP) bovine embryos. Blastocysts were produced by a standard IVP procedure following in vitro maturation, fertilization, and culture in synthetic oviduct fluid supplemented with 5% fetal calf serum (FCS). On Day 7 (Day of IVF = Day 0), a total of 297 blastocysts were vitrified using (i) the open pulled straw (OPS) in 20% DMSO and 20% ethylene glycol (EG) in a basal medium of TCM-199 with HEPES supplemented with 20% FCS; (ii) the modified OPS, in 20% DMSO, 20% EG, and 0.5 M sucrose in a basal medium of phosphate buffer saline (PBS) supplemented with 20% FCS; (iii) the cryoloop, in 15% DMSO, 15% EG, 10 mg mL–1 Ficoll 70, and 0.65 M sucrose in a basal medium of PBS supplemented with 20% FCS; and (iv) in 0.25 straws in 20% glycerol, 20% EG, 0.3 M sucrose, 3% polyethylene glycol, and 0.3 M xylose in a basal medium of PBS. After warming, embryos were placed in culture for additional 24 h. Re-expansion and hatching rates were measured at 2 and 24 h after warming. Data were analysed by 1-way ANOVA. At 2 h post-warming, the re-expansion of blastocysts vitrified with cryoloop was significantly higher compared with OPS, modified OPS, and the 0.25 straw methods (54.08 ± 15.53 v. 10.40 ± 3.00, 22.67 ± 9.20, and 8.82 ± 2.15, respectively; P ≤ 0.028). At 24 h post-warming, only embryos from cryoloop and modified OPS were still alive with a survival rate of embryos vitrified with cryoloop significantly higher than that of those vitrified with modified OPS (48.45 ± 17.56 v. 3.75 ± 3.75, respectively; P ≤ 0.007). Hatching rates at 24 h post-warming were not different between cryoloop and modified OPS groups (5.63 ± 4.40 and 1.25 ± 1.25, respectively). These results clearly demonstrate that embryo cryotolerance is affected by the method used for cryopreservation. Moreover, cryoloop vitrification was found to be more effective than OPS and 0.25 straw methods for the cryopreservation of bovine embryos.

Download Full-text

Culture system and long-term storage of culture media in the in vitro production of bovine embryos

Acta Veterinaria Hungarica ◽

10.1556/avet.59.2011.1.12 ◽

2011 ◽

Vol 59 (1) ◽

pp. 129-139 ◽

Cited By ~ 1

Author(s):

Santiago Varga ◽

Carmen Diez ◽

Lina Fernández ◽

Jenny Álvarez ◽

Adelino Katchicualula ◽

...

Keyword(s):

Embryo Development ◽

Culture System ◽

Culture Media ◽

Calf Serum ◽

Significant Loss ◽

Bovine Embryo ◽

In Vitro Production ◽

Fresh Culture Medium ◽

Hatching Rates

The optimum culture system for in vitro matured and fertilised oocytes still remains to be clarified. Culture media (CM) for mammalian embryos are routinely prepared fresh for use and preserved under refrigeration during one or two weeks. The purposes of this work were (1) to compare the efficiency of a synthetic oviduct fluid (SOF) with two different bovine serum albumin (BSA) concentrations (3 and 8 g/L) for the in vitro production of bovine blastocysts, (2) to test the effect of timing on adding fetal calf serum (FCS) to the SOF, and (3) to evaluate the effects on bovine embryo development of freezing and lyophilisation as procedures for preserving the SOF. Supplementation of SOF with 3 g/L BSA increased Day-7 blastocyst expansion rates (18.3 ± 1.6 vs. 14.4 ± 0.7; P < 0.05), although no differences in hatching rates were found. Addition of FCS to SOFaa (SOF with amino acids) medium supplemented with sodium citrate (SOFaaci) at 48 and at 72 h post-insemination (PI) allowed obtaining higher Day-6 embryo development rates than when FCS was added at 18 or 96 h PI (Day-6 morulae + blastocyst rate: 30.0 ± 1.1, 40.8 ± 1.1, 43.9 ± 2.3 and 39.3 ± 0.5 for FCS addition at 18, 48, 72 and 96 h, respectively). Hatching rates were significantly improved when serum was added at 72 h PI. Finally, both refrigeration and lyophilisation appeared as useful cryopreservation procedures for SOFaaci, although a significant loss of its ability to support embryo development, compared to the control fresh culture medium, was observed.

Download Full-text

171 LASER-ASSISTED ZONA PELLUCIDA HATCHING IN FROZEN - THAWED BOVINE EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab171 ◽

2010 ◽

Vol 22 (1) ◽

pp. 244 ◽

Cited By ~ 1

Author(s):

M. K. Chiasson ◽

J. A. Carter ◽

K. R. Bondioli ◽

R. A. Godke ◽

G. T. Gentry

Keyword(s):

Calf Serum ◽

Pregnancy Rates ◽

Water Bath ◽

Assisted Hatching ◽

Direct Transfer ◽

Pulse Treatment ◽

Bovine Embryos ◽

Hatching Rates

Incomplete zona hatching or failure of the zona to rupture compromises post-transfer embryo viability and conceptus development. Assisted hatching prior to the transfer of frozen-thawed bovine embryos has been proposed as a means to increase recipient pregnancy rates. The objective of this study was to determine if laser-assisted hatching would improve in vivo derived frozen-thawed bovine embryo hatching rates. In Exp. 1, direct-transfer beef cattle embryos were air-thawed for 15 s, placed in a 30°C water bath for 15 s, then held in TALP-HEPES, evaluated for stage and grade (1 = good to 3 = poor) and randomly applied to treatments. Embryos (n = 156) received either 2 or 3 symmetrical rents 40% through the outer zona surface using the XYClone diode laser (Hamilton Thorne, Beverly, MA, USA) at 90% power with a 600 μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were then cultured in vitro in CR1aa supplemented with 10% calf serum at 39°C in 5% CO2 and 5% O2 for 4 d. Embryo hatching rates were 47% for Treatment A and 53% for Treatment B. In Exp. 2, in vivo produced, nonsurgically collected direct-transfer Hereford embryos (n = 64) were utilized. In Exp. 3, in vivo produced nonsurgically collected glycerol frozen Brangus embryos (n= 46) were utilized. Embryos utilized in Exp. 2 and 3 were air-thawed for 15 s, placed in a 30°C water bath for 15 s, and then held in 1 M sucrose for 7 min. Embryos were then held in phosphate-buffered saline with 10% calf serum (Exp. 2) or ViGRO Holding Plus (Bioniche, Pullman, WA, USA) (Exp. 3), evaluated for stage and grade before being randomly assigned to either Treatment A or B. Embryos received either 3 symmetrical rents 40% through the outer zona surface using the XYClone laser at 90% power with a 600-μs pulse (Treatment A) or remained zona intact (Treatment B). Embryos were transferred nonsurgically (1 embryo/female) by the same technician into synchronized mixed breed recipient beef cows on Day 7 of the estrous cycle. Pregnancy status was determined at 35 days and 60 days via ultrasonography. In Exp. 2, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Also, treatment did not affect pregnancy rates at 35 or 60 days (41% and 28% for Treatment A and 44% and 41% for Treatment B, respectively). Likewise, there was no difference in calving rate for recipients confirmed pregnant at 60 days for Treatment A (89%) and Treatment B (77%). In Exp. 3, treatment did not affect 60 day pregnancy rates across embryo grades 1, 2, and 3. Pregnancy rates at 35 and 60 days were not affected by treatment (65% and 65% for Treatment A and 76% and 59% for Treatment B, respectively). Calving rates for those recipients in Exp. 3 were not available at the time of abstract preparation. Based on the data presented herein, it does not appear that laser-assisted hatching with the XYClone laser increases the number of in vivo derived frozen-thawed embryos that hatch following in vitro culture or increase pregnancy rates after transfer to recipient cattle.

Download Full-text

80 EFFECT OF PRE-EQUILIBRATION TIME ON THE SURVIVAL RATE OF IN VITRO-FERTILIZED BOVINE EMBRYOS AFTER VITRIFICATION

Reproduction Fertility and Development ◽

10.1071/rdv20n1ab80 ◽

2008 ◽

Vol 20 (1) ◽

pp. 120 ◽

Cited By ~ 1

Author(s):

H. Koyama ◽

A. H. Sugulle ◽

O. Dochi

Keyword(s):

Calf Serum ◽

Equilibration Time ◽

Bovine Embryos ◽

Chi Square ◽

Chi Square Test ◽

Vitrification Solution ◽

Short Time ◽

Further Development ◽

Hatching Rates

Successful cryopreservation of embryos depends on the pre-equilibration time. This study was designed to compare 2 pre-equilibration times – short (1 min) and long (5 min) – and to evaluate the re-expansion and hatching rates of different stages of embryos using the short pre-equilibration method. Cumulus–oocyte complexes (COCs) from ovaries obtained from a slaughterhouse were matured, fertilized, and cultured in vitro. In Experiment 1, expanded blastocysts between 7 and 9 days of culture were pre-equilibrated for the short time (1 min) in 100 μL of vitrification solution 1 (VS1: containing 7.5% ethylene glycol (EG), 7.5% dimethyl sulfoxide (DMSO), and 20% calf serum (CS) in TCM-199), followed by incubation in 100 μL of vitrification solution 2 (VS2: containing 15% EG, 15% DMSO, 20% CS, and 1 m sucrose (Suc) in TCM-199) for 30 s. Another group of blastocysts was subjected to the long pre-equilibration (5 min) in 100 μL VS1, followed by incubation in 100 μL of VS2 for 30 s. The embryos were placed into Cryotops® (Kitasato Supply Co., Tokyo, Japan) and immediately submerged in liquid nitrogen and kept there for 1 week. Blastocysts were warmed by plunging the Cryotops into 1 m Suc in TCM-199 for 1 min, placed in 0.5 m Suc in TCM-199 for 3 min, and finally placed in CR1aa alone for 5 min before being cultured. In Experiment 2, 8-cell embryos, morulae, and expanded blastocysts were vitrified by the previously described short equilibration method. The re-expansion and hatching rates of embryos were determined as the percentage of vitrified–warmed embryos undergoing further development in the in vitro culture. The data were analyzed by the chi-square test. Results are presented in Table 1. There was no difference between the short and long pre-equilibration times in terms of survival (94.0% v. 94.1%, respectively) and morphological appearance immediately after warming. However, re-expansion of blastocysts (ability to develop further) was slightly higher with the short pre-equilibration than with the long pre-equilibration (90.0% v. 85.9%, respectively). In Experiment 2, there were no differences in embryo re-expansion, but the hatching rates of 8-cell embryos were lower than those of morulae, blastocysts, and controls. In conclusion, the results of this study indicate that the length of pre-equilibration time prior to vitrification does not influence embryo re-expansion, and that bovine morulae and blastocysts can be vitrified with equal success. We also conclude that insufficient permeation of cryoprotectants may occur in 8-cell embryos. Table 1. Survival and hatching rates of vitrified bovine embryos after warming

Download Full-text

In vitro production of bovine embryos using flow-cytometrically sexed sperm

Archives Animal Breeding ◽

10.5194/aab-49-133-2006 ◽

2006 ◽

Vol 49 (2) ◽

pp. 133-140 ◽

Cited By ~ 1

Author(s):

L. Kątska-Książkiewicz ◽

B. Ryńska ◽

M. Bochenek ◽

J. Opiela ◽

J. Jurkiewicz

Keyword(s):

Bovine Embryos ◽

In Vitro Production ◽

Blastocyst Development ◽

Vitro Fertilization ◽

Bovine Spermatozoa ◽

Embryo Cleavage ◽

And Control ◽

Vitro Production ◽

Sperm Freezing

Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P<0.0001), sperm sexing (P<0.0001) and sperm freezing (P<0.01). Blastocysts development was affected by sperm freezing (P<0.04) and sperm sexing (P<0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.

Download Full-text

VOLUME DE MEIO E NÚMERO DE OÓCITOS PARA FECUNDACÃO NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS

Archives of Veterinary Science ◽

10.5380/avs.v9i2.4066 ◽

2004 ◽

Vol 9 (2) ◽

Author(s):

D.S. BRUM ◽

F.G. LEIVAS ◽

M.R. NOEBAUER ◽

M.L. BERNARDI ◽

C.A.M. SILVA ◽

...

Keyword(s):

In Vitro Maturation ◽

Hatching Rate ◽

Bovine Embryos ◽

In Vitro Production ◽

Oviduct Fluid ◽

Vitro Production ◽

Hatching Rates

A origem dos Complexos Cumulus-Oócitos (CCO) bovinos e os diferentes protocolos entre equipes geram variações nos índices de produção in vitro (PIV). O processo pode ser influenciado pelo número de CCO e volume das gotas de maturação, fecundação e cultivo. Neste estudo, avaliou-se a densidade de CCO e o volume de meio de fecundação sobre a PIV com 672 oócitos bovinos. A maturação in vitro (MIV) foi conduzida com 20 CCO em 200µL de TCM-199+rFSHh+10% de soro de vaca em estro (SVE), por 24h, em estufa a 39ºC, 5% CO2 em ar e umidade saturada. Na fecundação (Dia 0 =D0) utilizou-se 5, 10 ou 20 CCO e 2 volumes de meio (1:5 e 1:10), num fatorial 3x2, em 6 repetições. O sêmen foi selecionado por migração ascendente e para a fertilização (1x106 espermatozóides/mL) os gametas foram incubados em Fert-Talp, por 18h. O cultivo foi em grupos de 20 zigotos, em 200µL de fluído sintético do oviduto (SOF) aaci+5%SVE, por 8 dias e as avaliações em D2 (clivagem), D7 (Blastocistos), D9 (Blastocistos expandidos, em eclosão e eclodidos). As taxas de clivagem (80, 85 e 87%), embriões em D9 (23, 27 e 28%) e de eclosão (42, 37 e 48%) foram semelhantes (P>0,05) para 5, 10 ou 20 CCO, respectivamente. Não houve diferença (P>0,05) na clivagem (83 e 85%), embriões em D9 (24 e 28%) e eclosão (41 e 44%) entre as proporções 1:5 e 1:10µL. A produção de blastocistos e taxa de eclosão em D9 não são influenciados pelo número de CCO, nem pela proporção de CCO por volume de meio na fecundação. Volume of medium and number of oocytes for fertilization in the in vitro production of bovine embryos Abstract The origin of the bovine Cumulus Oocytes Complexes (COC) and the different protocols among teems are responsible for the variation rates of the in vitro production (IVP). The process is also influenced by COC number and by the volume of the drop in the maturation, fertilization and culture. In this study, the effect of bovine COC density per drop medium and the fertilization medium volume were evaluated during the bovine IVP using 672 COC. The in vitro maturation was performed with 20 COC in 200µL of TCM-199+rFSHh+10% estrus cow serum (ECS), for 24h in an incubator with 5%CO2 in air, saturated humidity, at 39oC. For the fertilization (Day 0 = D0) 5, 10 or 20 COC and 2 volumes ratio of medium (1:5 and 1:10) were used in a factorial outline 3x2, in 6 replications. Thawed semen was prepared by a swim up process and for the fertilization (1x106 spermatozoa/ ml) the gametes were maintained in Fert-Talp for 18h. Groups of twenty presumed zygotes were cultured in 200µL synthetic oviduct fluid (SOFaaci)+5% ECS, for 8 days. The evaluations were carried out in D2 (cleavage), D7 (blastocysts) and D9 (expanded and hatched blastocyts). The rates of cleavage (80, 85 and 87%), embryos in D9/ D2 (23, 27 and 28%) and hatched blastocstys in D9/D7 (42, 37 and 48%) were similar (P>0.05) for 5, 10 and 20 COC, respectively. No difference was observed in cleavage (83 and 85%), embryos in D9 (24 and 28%) and hatching rates (41 and 44%) for 1:5 and 1:10µL of fertilization medium. Blastocysts production and the hatching rate in D9 were neither influenced by the COC number, nor by the proportion of COC per medium volume used during fertilization.

Download Full-text