scholarly journals Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production

2012 ◽  
Vol 64 (3) ◽  
pp. 525-532 ◽  
Author(s):  
A.C Lucio ◽  
M.V Resende ◽  
J.A. Dernowseck-Meirelles ◽  
A.P. Perini ◽  
L.Z Oliveira ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Density Gradient ◽  
Staining Method ◽  
Control Group ◽  
Bovine Embryo ◽  
Sperm Viability ◽  
Selection Methods ◽  
Selection Procedures ◽  
Specific Sequences

The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y- specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.

scholarly journals Effect of insulin–transferrin–selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development

Zygote ◽  
2016 ◽  
Vol 24 (6) ◽  
pp. 890-899 ◽  
Author(s):  
A.L.S. Guimarães ◽  
S.A. Pereira ◽  
M. N. Diógenes ◽  
M.A.N. Dode
Keyword(s):  
Ascorbic Acid ◽  
Cell Number ◽  
Total Cell ◽  
Control Group ◽  
Bovine Embryo ◽  
Total Cell Number ◽  
Embryo Production ◽  
Embryo Size ◽  
Blastocyst Rate

SummaryThe aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

scholarly journals 136 EVIDENCE OF A DIRECT EFFECT OF P4 ON IVF-DERIVED BOVINE 8-CELL EMBRYOS

2005 ◽  
Vol 17 (2) ◽  
pp. 219 ◽  
Author(s):  
C.E. Ferguson ◽  
T.R. Davidson ◽  
M.R.B. Mello ◽  
A.S. Lima ◽  
D.J. Kesler ◽  
...  
Keyword(s):  
Embryo Development ◽  
Direct Effect ◽  
Blastocyst Stage ◽  
Control Group ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Direct Role ◽  
Treatment Groups ◽  
And Control

There has been much debate over a direct role for progesterone (P4) in early bovine embryo development. While previous attempts to supplement bovine embryos in vitro with P4 produced results that vary and are often contradictory, this may be a response of administering P4 at inappropriate times. Therefore, the objective of these experiments was to determine if P4 could exert a direct effect on developing IVF-derived bovine embryos when administered at an appropriate time of embryo development. In Exp. I, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 168); (2) vehicle, CR1aa + ETOH (0.01%) (n = 170); and (3) P4, CR1aa + ETOH + P4 (20 ng/mL in 50-μL droplet) (n = 173). In Exp. II, IVF-derived bovine 8-cell embryos were randomly allotted to treatments: (1) control, CR1aa medium (n = 160); (2) vehicle, CR1aa + DMSO (0.01%) (n = 180); and (3) P4, CR1aa + DMSO (0.01%) + P4 (20 ng/mL in 50-μL droplet) (n = 170). All embryos were evaluated on Days 6 to 9 post-insemination and rates calculated from 8-cell embryos. In Exp. I, ETOH tended to have a detrimental effect with significantly fewer (P < 0.05) embryos (53%) developing to the blastocyst stage on Day 7 compared with the control (62%) and P4 (71%) groups. At Day 7, significantly more embryos cultured in P4 (71%) developed to the blastocyst stage compared with the control group (62%). P4 treatment significantly increased the number of Grade 1 blastocysts (25%) on Day 7 compared with vehicle (15%) and control (17%) groups. At the end of culture, there were also significantly more Day 9 hatched blastocysts in the P4 group (33%) compared with vehicle (22%) and control (21%) groups. Supplementing P4 in the culture medium increased the rate of development, resulting in significantly more blastocysts (8%) on Day 6 and hatched blastocysts (21%) on Day 8 compared with vehicle (3% and 12%) and control (0% and 8%) groups, respectively. In Exp. II, there were no significant differences between treatment groups for Day 7 blastocysts (control 54%, DMSO 61%, P4 57%) and Day 9 hatched blastocysts (control 46%, DMSO 51%, P4 46%). However, there were significantly more Grade 1 blastocysts in the P4 group (22% and 36%) on Days 6 and 8 compared with vehicle (11% and 23%) and control (13% and 23%) groups, respectively. The lack of improvement in Day 7 blastocysts and Day 9 hatched blastocysts rates leads to further uncertainty in understanding the P4 vehicle interactions. In conclusion, the results of these two experiments indicate that P4 can exert a direct effect on the developing IVF-derived bovine embryo; however, due to P4 vehicle interactions; other inert vehicles need to be explored to further evaluate the direct effects of P4 on the developing bovine embryo.

346 BOVINE EMBRYO DEVELOPMENT AFTER IVM/IVF/IVC OF OOCYTES STORED FOR 22 H IN VARIOUS MEDIA

2007 ◽  
Vol 19 (1) ◽  
pp. 288
Author(s):  
C. Kubota ◽  
T. Kojima ◽  
T. Nagai ◽  
X. Tian ◽  
X. Yang
Keyword(s):  
Subsequent Development ◽  
Industrial Applications ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Control Group ◽  
Bovine Embryo ◽  
Becton Dickinson ◽  
In Vitro Storage ◽  
Bovine Oocytes

The timing of IVM–IVF–IVC is restricted by the onset of oocyte maturation, and sometimes oocytes must be treated at midnight. If we could regulate the timing of IVM of oocytes without decreasing their developmental competence, the IVM–IVF–IVC system could be a more applied technology. The present study was performed to examine the effects of in vitro storage of bovine oocytes in simple media prior to maturation culture to manipulate the start of IVM. Bovine follicular fluid (bFF), Dulbecco&apos;s PBS (PBS), M199 Earle salts (M199), and Earle salts supplemented with 5 mM NaHCO3 (M199A) were used as the fundamental media, after an addition of antibiotics, for in vitro storage of bovine cumulus&ndash;oocyte complexes (COCs) collected from ovaries obtained at the slaughterhouse. The fundamental media except for bFF were supplemented with 10&percnt; fetal bovine serum (FBS) or 1 mg mL&minus;1 polyvinyl alcohol (PVA). COCs were collected from follicles (3&ndash;8 mm in diameter) and washed twice in each medium; then approximately 50 COCs were submerged in 1 mL of each medium in cryotubes (Falcon #2812, 2.5 mL; Becton Dickinson Labware, Lincoln, NJ, USA), which were stored in a container kept at 38.5&deg;C for 22 h under air-closed condition (in vitro storage: IVS). Subsequently, the stored COCs were in vitro-matured (IVM) for 22 h in M199 with 10&percnt; FBS and 20 &micro;g mL&minus;1 estradiol, fertilized (IVF), and cultured in CR1aa (IVC) for examination of their development to the blastocyst stage (Kubota et al. 1998 Mol. Reprod. Dev. 51, 281&ndash;286). Fresh oocytes without IVS were used as controls. The nuclear status of oocytes after IVS&ndash;IVM was compared to that of control oocytes by aceto-orcein stain. Their developmental rates to the blastocyst stage after IVM&ndash;IVF&ndash;IVC were compared between experimental and control groups. The experiment was repeated more than 3 times, and results were statistically analyzed using Student&apos;s t-test. When bFF and PBS supplemented with FBS or PVA were used for IVS, the rates of survived COCs after IVS and the development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC (bFF (n &equals; 87): 0&percnt;, 0&percnt;; PBS/FBS (n &equals; 72): 84&percnt;, 1&percnt;; and PBS/PVA (n &equals; 81): 89&percnt;, 6&percnt;, respectively) were significantly lower than those of the control group (n &equals; 406; 97&percnt; and 29&percnt;, respectively). On the other hand, when M199A supplemented with FBS or PVA was used for IVS, the survival rate after IVS and the developmental rate to the blastocyst stage after IVS&ndash;IVM&ndash;IVF (M199A/FBS (n &equals; 97): 82&percnt;, 28&percnt;; and M199A/PVA (n &equals; 111): 98&percnt;, 31&percnt;, respectively) did not differ from those of the control group. After IVS, cumulus expansion was not seen and most of the oocyte nuclei reached the GVBD stage. These results suggest that the nuclear maturation progress of bovine oocytes can be regulated for at least 22 h in M199A without any deleterious influence on the number of oocytes surviving at an immature state after the storage and their subsequent development to the blastocyst stage after IVM&ndash;IVF&ndash;IVC. The delayed maturation allows a flexible fertilization schedule which is advantageous in research and industrial applications.

83 VITAMIN K2 SUPPLEMENTATION IMPROVES BLASTOCYST RATE BY RECOVERY OF MITOCHONDRIA IN IN VITRO-CULTURED BOVINE EMBRYOS

2014 ◽  
Vol 26 (1) ◽  
pp. 155
Author(s):  
L. Baldoceda ◽  
C. Vigneault ◽  
P. Blondin ◽  
C. Robert
Keyword(s):  
In Vitro Culture ◽  
Fluorescent Microscopy ◽  
Vitamin K2 ◽  
Control Group ◽  
Mitochondrial Activity ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Rate ◽  
Mitotracker Red

Mitochondria play an important role during early mammalian embryo development through their diverse cellular functions, in particular creating balance between production of ATP by electron transport chain and oxidative stress. Embryonic mitochondria are inherited maternally and independently of the nuclear genome. They show limited activity during the early developmental stages before embryonic genome activation. It has been shown that in vitro culture (IVC) has an adverse effect on mitochondrial function in embryos. So far several attempts have been performed to improve and rescue the impaired mitochondria. It has been shown that vitamin K2 (a membrane-bound electron carrier, similar to ubiquinone) was used to rescue mitochondrial dysfunction and resulted in more efficient ATP production in eukaryotic cells (Vos et al. 2012 Science 336, 1306–1310). Therefore, the aim of the present study was to investigate the effects of supplementation of vitamin K2 on mitochondrial activity and blastocyst rate. Cumulus–oocytes complexes (n = 687) recovered from slaughtered animals, were matured and fertilized in vitro according to our standard procedures. After fertilization, zygotes were cultured in SOF media supplemented with 10 mg mL–1 BSA. At 96 h post-fertilization, vitamin K2 was added to the culture media (n = 448 oocytes). On Day 7, treatment embryos were compared with untreated controls (n = 239 oocytes). In vitro culture was carried out at 38.5°C under 5% CO2, 7% O2, and 88% N2. Differences among groups in blastocyst yield were analysed by ANOVA. Mitochondrial activity data was analysed by unpaired 2-tailed t-tests. Results show that the vitamin K2-treated group had a significantly (P < 0.05) higher blastocyst rate (+8.6%), expanded blastocyst rate (+7.8%), as well as better morphological quality compared with the control group. Furthermore, to evaluate mitochondria activity, pools of embryos of each treatment were labelled with a specific dye for active mitochondria (Mitotracker Red). A significantly higher intensity of Mitotracker Red (P < 0.05) was observed in the vitamin K2 treatment versus control group, as measured by fluorescent microscopy. In conclusion, for the first time, our data prove that supplementation of vitamin K2 during IVC of bovine embryos increases blastocyst rates and embryo quality. Future studies will focus on gene expression to identify targets implicated in impaired mitochondrial activity in in vitro bovine embryo production.

7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 104
Author(s):  
J. T. Aaltonen ◽  
K. J. Mattson ◽  
N. M. Loskutoff
Keyword(s):  
Embryo Development ◽  
Disease Transmission ◽  
Bos Taurus ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Human Sequence ◽  
Bovine Sperm

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

Reduced levels of intracellular reactive oxygen species and apoptotic status are not correlated with increases in cryotolerance of bovine embryos produced in vitro in the presence of antioxidants

2014 ◽  
Vol 26 (6) ◽  
pp. 797 ◽  
Author(s):  
Nathália A. S. Rocha-Frigoni ◽  
Beatriz C. S. Leão ◽  
Ériklis Nogueira ◽  
Mônica F. Accorsi ◽  
Gisele Z. Mingoti
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Intracellular Reactive Oxygen Species ◽  
Control Group ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Antioxidant Supplementation ◽  
In Vitro Production ◽  
Intracellular Ros

The effects of intracellular (cysteine and β-mercaptoethanol) and extracellular (catalase) antioxidant supplementation at different times during in vitro production (IVM and/or in vitro culture (IVC)) on bovine embryo development, intracellular reactive oxygen species (ROS) levels, apoptosis and re-expansion rates after a vitrification–thawing process were examined. Blastocyst frequencies were not affected by either antioxidant supplementation (40.5%–56.4%) or the timing of supplementation (41.7%–55.4%) compared with control (48.7%; P > 0.05). Similarly, antioxidants and the moment of supplementation did not affect (P > 0.05) the total number of blastomeres (86.2–90.5 and 84.4–90.5, respectively) compared with control (85.7). However, the percentage of apoptotic cells was reduced (P < 0.05) in groups supplemented during IVM (1.7%), IVC (2.0%) or both (1.8%) compared with control (4.3%). Intracellular ROS levels measured in Day 7 blastocysts were reduced (P < 0.05) in all groups (0.60–0.78), with the exception of the group supplemented with β-mercaptoethanol during IVC (0.88), which did not differ (P > 0.05) from that in the control group (1.00). Re-expansion rates were not affected (P > 0.05) by the treatments (50.0%–93.0%). In conclusion, antioxidant supplementation during IVM and/or IVC reduces intracellular ROS and the rate of apoptosis; however, supplementation does not increase embryonic development and survival after vitrification.

372 X-CHROMOSOME-BEARING SPERM SEPARATION BY Percoll™ DISCONTINUOUS DENSITY GRADIENT AND SEX RATIO DEVIATION IN IN VITRO PRODUCED BOVINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 342
Author(s):  
A. P. Perini ◽  
A. C. Lucio ◽  
A. S. Carmo ◽  
M. C. V. Miguel ◽  
L. Z. Oliveira ◽  
...  
Keyword(s):  
Density Gradient ◽  
X Chromosome ◽  
Cumulus Cells ◽  
Embryonic Cell ◽  
Control Group ◽  
Density Gradients ◽  
Cleavage Rate ◽  
Female Embryo ◽  
Pcr Multiplex

The aims of this study were to separate X-chromosome-bearing bovine sperm by discontinuous Percoll ™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradients, validate the sexing of resultant IVF embryos by PCR, replace the bovine fetal serum (BFS) with BSA in the culture medium, to decrease male development advantage, and verify whether the gradient can be used in an IVF laboratory routine. The gradient was prepared by mixing Dubelcco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) with Percoll™ isotonic solution with 0.3% BSA, for different densities obtained ranging from 1.110 to 1.123 g mL-1, disposed in 3 layers into 15-mL conical tubes. For sexing, 40 million thawed sperm were overlaid on density gradients. The tubes were centrifuged at 500 g, for 15 min, at 22°C. After centrifugation, sperm sediment was used for IVF. For the control group, a Percoll™ 45, 90% gradient was used. The oocytes were selected from ovaries from slaughterhouse and maturated for 24 h in TCM-199 medium. After fertilization, oocytes and sperm were incubated for 20 h in 5% CO2, in humidified air at 38.5°C. Presumptive zygotes were denuded of cumulus cells, and washed in modified SOF medium and then transferred to 500 μL SOF in four well dishes. Embryo culture was carried out under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5°C, and the cleavage assessed at 46 h and development to the blastocyst stage at Day 7. To obtain embryonic cell DNA for sex determination by PCR, 115 embryos of the sexed and 82 of the control group were used. Two pairs of primers of Y-specific sequences were split in two distinct samples. The first pair detected a sequence of 210 bp, and the second one 196 bp of the bovine Y-chromosome. A third one detected an autosomal sequence of 280 bp, indicating the presence of bovine genomic DNA. PCR multiplex was carried out in the same tube with first and third primers and the PCR of the second one was carried out in another tube. The results were analyzed by X2. Of the sexed group, from a total of 373 oocytes, the cleavage rate was 58.2% (n = 217); 35.6% (n = 133) produced embryos; 36.5% (n = 42) were male embryos and the female embryo rate was 63.5% (n = 73). From a total of 268 control oocytes, the cleavage rate was 63.8% (n = 171); produced embryos 37.3% (n = 100); 57.3% (n = 47) were male embryos and the female rate was 42.7% (n = 35). The Percoll™ density gradient for sperm sexing altered the proportion of IVF embryos toward more females. Because of fast and easy preparation, the gradient can be used routinely in an IVF laboratory and also, BSA can replace FBS for the IVF. FAPESP process number 59357-9 and CAPES

258 FACTORS AFFECTING COMMERCIAL SEXING PROGRAM AND MULTIPLE GENETIC ANALYSIS PERSPECTIVES OF IN VITRO-PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 227
Author(s):  
R. V. Alonso ◽  
J. A. A. Hellú ◽  
S. H. V. Perri ◽  
J. A. Visintin ◽  
J. F. Garcia
Keyword(s):  
Mortality Rate ◽  
Sex Ratio ◽  
Genetic Analysis ◽  
Genetic Material ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Chi Square ◽  
Male And Female ◽  
Embryo Stage

The present study aimed to evaluate the interactions among different factors on the viability and sex ratio of in vitro-produced (IVP) bovine embryos, submitted to a large scale sexing program. Additionally, whole genome amplification (WGA) technology was used to amplify genomic DNA from IVP bovine embryo biopsies, in order to perform multiple genetic analyses. The survey was performed in a 4650 IVP bovine sexed embryo database. Embryos were biopsied by a microaspiration technique and sex was determined by PCR of DNA from the biopsy. Only female embryos were transferred to synchronized recipients. Pregnancy diagnosis and fetal sex determination were carried out by ultrasound. The variables were classified in accordance with embryo sex (male, female, and indeterminate), five laboratories (A, B, C, D, and E), six bovine breeds (Nellore, Brahman, Girolando, Simmental, Holstein, and Jersey), embryo stage (MO, EB, BL, XB, and HB), embryo quality (1, 2, and 3) and biopsy quality (“standard” and “nonstandard”). The statistical analysis was carried out by association chi-square test, chi-square for a 1:1 ratio, and logistic regression analysis (PROC LOGISTIC) of SAS. PCR showed 93.3% efficiency, 93.2% accuracy, and male and female rates of 52.9% and 47.1%, respectively. Mortality rate of biopsied embryos was 10.3% and pregnancy rate was 31.7%. Significant differences were not observed between male and female viability, although indeterminate embryos resulted in more death after micromanipulation. For quality 2 and 3 embryos, the mortality rate after biopsy was 3.19 and 11.37 fold higher, respectively, than for quality 1 embryos. For embryos whose biopsies were classified as nonstandard, the embryonic mortality rate was 3.6-fold higher than standard ones. Mortality rate was not affected by embryo stage at biopsy (P > 0.05). Although sex ratio was significantly skewed to male embryos, differences were not observed among laboratories (P > 0.05) and breeds (P > 0.05) on the sex ratio of IVP bovine embryos. To test the feasibility of using WGA method for multiple genetic analysis, biopsies from 28 IVP embryos were submitted to the GenomePlex Single Cell System (Sigma-Aldrich, St. Louis, MO, USA). Aliquots from each DNA sample were purified using column chromatography and submitted to PCR using sexing primers BRY4a, SRY, UMN0920, and S4B. PCR was successful and in agreement among tested DNA aliquots from each single biopsy. The WGA strategy used herein was a useful tool for applications involving restricted amounts of starting genetic material (DNA), such as in preimplantation genetic diagnosis using IVP bovine embryos. To FAPESP and UNESP.

Effect of spermatozoa motility hyperactivation factors and gamete coincubation duration on in vitro bovine embryo development using flow cytometrically sorted spermatozoa

2017 ◽  
Vol 29 (4) ◽  
pp. 805 ◽  
Author(s):  
Luis B. Ferré ◽  
Yanina Bogliotti ◽  
James L. Chitwood ◽  
Cristóbal Fresno ◽  
Hugo H. Ortega ◽  
...  
Keyword(s):  
Embryo Development ◽  
X Chromosome ◽  
Embryo Quality ◽  
Bos Taurus ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
Spermatozoa Motility

The aim of the present study was to evaluate the effects of sperm motility enhancers and different IVF times on cleavage, polyspermy, blastocyst formation, embryo quality and hatching ability. In Experiment 1, sex-sorted X chromosome-bearing Bos taurus spermatozoa were incubated for 30 min before 18 h fertilisation with hyperactivating factors, namely 10 mM caffeine (CA), 5 mM theophylline (TH), 10 mM caffeine and 5 mM theophylline (CA + TH); and untreated spermatozoa (control). In Experiment 2, matured B. taurus oocytes were fertilised using a short (8 h) or standard (18 h) fertilisation length, comparing two different fertilisation media, namely synthetic oviducal fluid (SOF) fertilisation medium (SOF-FERT) and M199 fertilisation medium (M199-FERT). Cleavage and blastocyst formation rates were significantly higher in the CA + TH group (77% and 27%, respectively) compared with the control group (71% and 21%, respectively). Cleavage rates and blastocyst formation were significantly lower for the shortest fertilisation time (8 h) in M199-FERT medium (42% and 12%, respectively). The SOF-FERT medium with an 8 h fertilisation time resulted in the highest cleavage rates and blastocyst formation (74% and 29%, respectively). The SOF-FERT medium produced the highest embryo quality (50% Grade 1) and hatching rate (66%). Motility enhancers did not affect polyspermy rates, whereas polyspermy was affected when fertilisation length was extended from 8 h (3%) to 18 h (9%) and in M199-FERT (14%) compared with SOF-FERT (6%). We conclude that adding the motility enhancers CA and TH to sex sorted spermatozoa and Tyrode’s albumin lactate pyruvate (TALP)-Sperm can improve cleavage and embryo development rates without increasing polyspermy. In addition, shortening the oocyte–sperm coincubation time (8 h) resulted in similar overall embryo performance rates compared with the prolonged (18 h) interval.

scholarly journals Evaluation of in vitro production rates of bovine embryos using melatonin-supplemented culture medium

2021 ◽  
Vol 10 (6) ◽  
pp. e19010615544
Author(s):  
Ricardo Magalhães ◽  
Carlos Renato de Freitas Guaitolini ◽  
Marcio Luiz Denck Tramontin ◽  
Danielle Andressa Oliveira Sestari ◽  
Bruno Argenton de Barros ◽  
...  
Keyword(s):  
Culture Medium ◽  
Statistical Significance ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Blastocyst Formation ◽  
In Vitro Production ◽  
Embryo Production ◽  
Vitro Fertilization

In this study, we aimed to evaluate the rate of bovine embryo production by using 50 ng/mL melatonin supplementation in in vitro culture medium. For this, oocytes from slaughterhouse ovaries were matured in vitro in TCM-199 medium with Earle’s balanced salt solution + 10% SFB, FSH, and LH in an atmosphere of 5% CO2. Twenty-four hours after IVM, the oocytes underwent in vitro fertilization in human tubal fluid under the same conditions as above, for 18 h. Semen was fractionated by Percoll gradient centrifugation and the concentration of sperm was adjusted to 1 × 106/mL. Probable zygotes were then divided into two groups: the control group grown in drops of 90 μL SOFaa medium + 0.6% BSA + 2.5% SFB, in an atmosphere of 5% CO2, 90% N2, and a melatonin group (Mel), similarly cultured in 90 μL drops of SOFaa medium + 0.6% BSA + 2.5% SFB + 50 ng/mL melatonin. Cleavage rates were assessed on day 3 (D3). On D7, blastocyst formation rates were evaluated. Eight routines were performed (320 oocytes per routine). Data were analyzed with ANOVA, followed by Tukey’s range test using a general linear model. The level of statistical significance was set at 5%. There were no differences in the rates of cleavage or blastocyst formation between the control and melatonin groups (P > 0.05). Thus, under the conditions used in this study, supplementation with melatonin did not yield benefits in increasing the rate of in vitro bovine embryo production.

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