scholarly journals Use of a colloid to optimize centrifugation in the selection of bovine sperm for IVF

2018 ◽  
Vol 39 (4) ◽  
pp. 1607 ◽  
Author(s):  
Cibele Garcia Moreira Gonçalves ◽  
Fábio Gallas Leivas ◽  
Daniele Missio ◽  
Francielli Weber Santos ◽  
Eduardo Brum Schwengber ◽  
...  
Keyword(s):  
Recovery Rate ◽  
Sperm Quality ◽  
Ros Production ◽  
Sperm Selection ◽  
Percoll Gradient ◽  
Bovine Embryos ◽  
Cleavage Rate ◽  
The Impact ◽  
Normal Fertilization

This study aimed to evaluate the effect of the force and duration of centrifugation and the impact of cushioned centrifugation on sperm selection by Percoll gradient, on sperm quality and development kinetics of in vitro produced bovine embryos. Two experiments were performed. In Experiment I, a pool of semen was selected by Percoll gradients and the pellet was divided into four groups and distributed in a 2 × 2 factorial, with two forces (2200 × g or 9000 × g) and two durations (1 min or 3 min) of centrifugation. In Experiment II, semen was divided into two groups and selected by Percoll gradient with Cushion Fluid (CF) or without CF (Control) in the second centrifugation. The morphofunctionality, biochemical characteristics and fertilizing capacity of the selected sperms were evaluated. In addition, the development of the resulting bovine embryos was monitored for 48 h post-insemination. Duncan and Chi-square tests (P < 0.05) were used to compare the means. In Experiment I, there was a significant increase in sperm vigor (P < 0.05) after sperm selection in all treatments. The force and duration of centrifugation did not have any effect on sperm motility, vigor, and recovery rate among the different treatments (P > 0.05). In Experiment II, the recovery rate and reactive oxygen species (ROS) production in semen were similar among treatments (P > 0.05) although a higher ROS production was observed in the CF fertilization medium. Total fertilization rate was superior in the CF group (65.4 ± 5.3%) compared to that in Control (39.6 ± 4.9%). However, the normal fertilization and cleavage rate did not differ between the Control (94 ± 6.3% and 58.3 ± 8.3%) and CF (89 ± 7.1% and 75.0 ± 7.3%) groups. The reduction in the force and duration of centrifugation did not decrease the sperm recovery during selection by the Percoll gradient and the use of CF in the second centrifugation did not affect the normal fertilization and development of bovine IVF embryos up to 48 h.

68 Comparison of two Percoll gradients for selection of frozen semen for invitro production of ovine embryos

2021 ◽  
Vol 33 (2) ◽  
pp. 141
Author(s):  
A. Velázquez-Roque ◽  
F. Villaseñor-González ◽  
G. Márquez-Márquez ◽  
M. Kjelland ◽  
H. Álvarez-Gallardo ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Physiological Saline ◽  
Bottom Layer ◽  
Penicillin G ◽  
Sperm Selection ◽  
Percoll Gradient ◽  
Cleavage Rate ◽  
Frozen Semen ◽  
Percoll Gradients ◽  
Α Level

Sperm selection methods are routinely applied to prepare semen for IVF in animal species. These procedures are used to improve sperm quality characteristics as well as to remove other background material and debris. Percoll gradient is widely used in animal IVF laboratories. Different Percoll gradient concentrations and volumes can be used to improve the sperm sample motility percentage. The objective of this research was to evaluate the effect of 2 different Percoll concentrations for ovine IVF sperm selection and effects, if any, on invitro embryo production (IVP). Specifically, Percoll gradients consisted of Group 1 (G1) 40–80% and Group 2 (G2) 45–90%, 400µL each. The research was carried out in the reproduction laboratory at Palominos Ranch (Jalisco, México). The IVP was performed with a continuous invitro culture system. Ovaries (n=157) were collected from a slaughterhouse (León, México) and transported to the laboratory within 2h in physiological saline solution (0.9% NaCl) supplemented with penicillin G (100IU mL−1) and streptomycin sulphate (100µg mL−1). For IVP, IVF Bioscience™ media were used for IVM, IVF, and invitro culture (IVC). For IVM, the cumulus–oocyte complexes (COCs) were selected (only grades 1 and 2) and matured for 24h at 38.5°C in 5% CO2 in air and 100% humidity. Matured oocytes (n=800, divided equally into 4 replicates) were divided into 2 groups, G1 and G2. The IVF process was conducted with semen selected through a mini-Percoll technique with gradients at a concentration of 45% (top layer) and 90% (bottom layer) or 40% (top layer) and 80% (bottom layer) for G1 and G2, respectively, using frozen–thawed semen from the same ram, at a concentration of 2×106 sperm mL−1, for 18h in 38.5°C, 5% CO2 in air, and 100% humidity. The presumptive zygotes were denuded by pipetting and set in IVC until Day 7 at 38.5°C, 5% CO2, 5% O2, and 90% N2 at 100% humidity. The percentages of cleavage, embryos with more than 6 cells, and blastocysts on Day 7 of culture were evaluated, based on the initial number of oocytes entering into IVM. The statistical analyses were carried out with the GLM procedure of SAS software (version 9.3; SAS Institute Inc.) to evaluate the results of G1 versus G2 (α level=0.05). Cleavage rate was 47.8%±2.5 and 55.9%±2.5, respectively, in G1 and G2. The percentages of embryos with more than 6 cells were 38.1%±2.2 and 43.5%±2.2, respectively, in G1 and G2. The percentage of blastocysts on Day 7 was 27.4%±1.1 and 37.3%±1.1, respectively, in G1 and G2. There were no significant differences between groups (P&gt;0.05) for percentage of cleavage and embryos with more than 6 cells, although the percentage of cleavage tended to be greater for G2 (P=0.06). Additionally, G2 had a larger percentage of blastocysts on Day 7 compared with G1 (P&lt;0.05). In conclusion, under the conditions of this research, the use of a Percoll gradient at a concentration of 40–80% increased the percentage of blastocysts for ovine IVP.

scholarly journals In vivo culture of bovine embryos and quality assessment of in vivo vs. in vitro produced embryos

2012 ◽  
Vol 50 (No. 4) ◽  
pp. 149-158 ◽  
Author(s):  
V. Havlicek ◽  
M. Lopatarova ◽  
S. Cech ◽  
R. Dolezel ◽  
T. Huber ◽  
...  
Keyword(s):  
Quality Assessment ◽  
Recovery Rate ◽  
Bovine Embryos ◽  
In Vivo Culture ◽  
Blastocyst Rate ◽  
Culture Procedure ◽  
Recovery Methods

Routine access to the bovine oviduct for in vivo culture accomplishes various demands on embryo production for scientific as well as commercial purposes. The experiments conducted in the present study focused on the efficiency of recovery methods after temporary in vivo culture of bovine embryos in oviducts of the homologous species using transvaginal endoscopy (Experiment I) and on the quality assessment of recovered blastocysts (Experiment II). In Experiment I in vitro matured oocytes were fertilized, cultured for 1 to 3 days and transferred unilaterally into the ipsilateral oviducts of 54 heifers by the means of transvaginal endoscopy. After 4 to 6 days of in vivo culture embryos were re-collected either by non-surgical flushing of uterine horns (U-group) or by combined flushing of the oviducts and uterine horns (OU-group). In total the recovery rate was 38.4% (780/2029). After flushing at day seven, 106 blastocysts (blastocyst rate: 13.6% ) were found. The additional 24 h of in vitro culture (day eight) resulted in 153 blastocysts (blastocyst rate: 19.6% ). The recovery rate in the OU-group was twice as efficient as in the U-group (390/1358 vs. 390/671, P &lt; 0.01). The recovery rates among the different stages of transferred embryos did not differ significantly; likewise cross-effects among the stages and the recovery methods were non-significant. The recovery methods (P &lt; 0.001) and the interaction between the recovery methods and the stages of transferred embryos (P &lt; 0.01) had an influence on blastocyst yields on day seven (U-group 37/1358 vs. OU-group 69/671) and day eight (U-group 48/1358 vs. OU-group 105/671). In Experiment II embryo quality was assessed by the survival rate of blastocysts after freezing in ethylene glycol. Day seven embryos were produced in vitro (in vitro group D7) or by IVM/IVF followed by a combined culture procedure (2 to 3 days in vitro prior to 4&nbsp;to 5 days in vivo) (in vivo group D7) or after superovulation and collection at day seven (superovulation group). Embryos from in vitro group D7 re-expanded only for 6 h after thawing, embryos from in vivo group D7 and superovulation group were alive for 24 h and 72 h of culture, respectively. Only embryos derived by superovulation showed hatching activity. Blastocysts from the in vitro group D7 and the in vivo group D7 that were held in culture medium for additional 24 h (day eight) showed an analogous post-thawing culture behaviour. In conclusion, the results of the present study demonstrated that some embryos transferred for in vivo culture remain in the oviduct even at day seven. Hence, combined flushing of oviducts and uterine horns after in vivo culture in the bovine oviduct is necessary for effective embryo re-collection. The quality of recovered embryos after temporary in vivo culture assessed by cryotolerance was in-between those produced in vitro or recovered after superovulation.

256 EFFECT OF SPERM SELECTION ON THE RATE OF IN VITRO FERTILIZATION IN ALPACA (VICUGNA PACOS)

2015 ◽  
Vol 27 (1) ◽  
pp. 217
Author(s):  
E. Mellisho ◽  
V. Rivas ◽  
J. Ruiz ◽  
G. Mamani
Keyword(s):  
Extended Period ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Cleavage Rate ◽  
Slow Cooling ◽  
Vitro Fertilization ◽  
Progressive Motility ◽  
Frozen Semen ◽  
Washing Method

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small size of the testes, extended period of ejaculation, and low quality of semen. This study was designed to determine the effect of 2 sperm preparation treatments before IVF on the cleavage rate. The sperm was obtained by slicing the head of the epididymis of slaughtered male alpacas (n = 8), diluting in Tris-yolk-glycerol, and freezing with the slow-cooling method. Frozen semen straws per each male were thawed in a water bath at 37°C for 15 s and evaluated for percentage of progressive motility (32 ± 8.6%) and concentration (66.5 ± 24 × 106 sperm mL–1) post-thawing. Sperm selection by the swim-up method was performed by centrifugation at 1077 × g for 5 min with washing sperm medium eliminating the supernatant; sperm were settled in inclined tube with fertilization medium (without capacitating agent) for 60 min, after which 100 μL from the surface was recovered for use in IVF. The washing method consisted in repeated washing (twice) of sperm in washing sperm medium and fertilization medium by centrifugation at 1077 × g for 5 and 3 min, respectively, and recovery of 50 μL from the bottom of the tube for use in IVF. Sperm selected by swim-up or washing methods had similar characteristics of progressive motility (18 and 23%); however, the concentration was higher for the washing v. swim-up method (52 v. 14 × 106 sperm mL–1, respectively). Cumulus-oocyte complexes (COC) were recovered from 278 ovaries of alpacas killed at abattoirs and classified (Grade 1 and 2) for in vitro maturation (38.5°C at 5% CO2 in air for 27 h in 50 μL of 10 COC per drop). A total of 839 oocytes cultured for 27 h in maturation medium were partially stripped out of cumulus cells by gentle aspiration with a pipette. Sperm suspensions in Fert TALP medium (5 μL) from each treatment group were added to each fertilization drop with 10 oocytes per drop of 45 μL obtaining a final concentration of 10 × 106 sperm mL–1 and cultivated for 72 h until their evaluation. The data for the 13 repetitions of the rate of cleavage (2 to 8 cells) were converted to angular values (angle = arcsin √%) with the object of normalizing the distribution of the data; the analysis of variance was performed (complete randomised design with sub-sampling, P < 0.05) using SAS® version 8.0 for Windows. The rate of cleavage (cell division) did not show statistical differences (P = 0.67) for the swim-up method (37%; 155/421) v. washing method (35%; 147/418). The methods of sperm selection (swim-up and washing) did not affect the rate of IVF.

134 Effects of Gas Tension During Culture upon Development of Bovine Embryos from Beef (Nellore) and Dairy (Girolando) Breeds

2018 ◽  
Vol 30 (1) ◽  
pp. 207
Author(s):  
J. G. V. Grázia ◽  
L. G. Lacerda ◽  
L. G. B. Siqueira ◽  
C. A. G. Pellegrino ◽  
L. S. Grapiuna ◽  
...  
Keyword(s):  
Oxygen Tension ◽  
Initial Period ◽  
Bos Indicus ◽  
Southeastern Brazil ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Cleavage Rate ◽  
High O2

Culture of bovine embryos is a critical step during in vitro embryo production (IVEP) and, as such, has been the focus of numerous studies on cattle IVEP. Improvements of culture conditions to mimic the in vivo maternal microenvironment involves studying the optimal gas tension for pre-implantation embryonic development. In the commercial conditions, there is great variability in results, in part because of the difference between breeds and donors. The objective of this study was to evaluate the effects of culture in high or low oxygen tension upon the development of embryos from a crossbred dairy breed (Girolando F1; Gir × Holstein) and a beef Bos indicus breed, Nellore. We collected data from an IVEP commercial operation located in a tropical area of southeastern Brazil (Minas Gerais State) from February to May 2017. The study was designed in a 2 × 2 factorial arrangement of treatments: 2 O2 tensions during culture (5%, low O2 v. 20%, high O2) and 2 breeds (Nellore, beef v. Girolando F1, dairy). Thus, the following 4 groups were studied: Nellore-high O2 (n = 86 donors), Nellore-low O2 (n = 107 donors), Girolando F1-high O2 (n = 114 donors), and Girolando F1-low O2 (n = 110 donors). Outcome variables were the number of cleaved embryos 72 h post-insemination (hpi), cleavage rate relative to the total number cumulus–oocyte complexes (COC) put in culture, number and percentage of blastocysts 192 hpi relative to the structures kept in culture. Variables that were not normally distributed were transformed using the formula log(y + 0.05). Data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA) for the main effects of gas tension (low v. high O2) and breed (Girolando F1 v. Nelore). Results are shown as mean ± SEM. Gas tension affected the number of cleaved embryos (10.52 ± 0.92 v. 8.33 ± 0.72 for high and low O2, respectively; P < 0.01) and cleavage rates (40.58 ± 2.49 v. 44.41 ± 2.88 for high and low O2; P < 0.01 in Nellore), but did not affect these variables in Girolando F1 donors (13.23 ± 1.33 v. 10.76 ± 0.76 cleaved embryos, for high and low O2; P = 0.63; 58.01 ± 2.00 v. 60.19 ± 1.97 cleavage rate, for high and low O2; P = 0.80). Nonetheless, the number and percentage of blastocysts were not affected by gas tension in either breed. Results for Nellore were 4.99 ± 0.56 v. 3.51 ± 0.38 blastocysts in high and low O2, respectively (P = 0.051) and 41.92 ± 3.91% v. 39.81 ± 3.77% blastocysts, in high and low O2 (P = 0.11). For Girolando F1, numbers of blastocysts were 5.84 ± 0.66 v. 4.24 ± 0.39 in high and low O2 (P = 0.19) and percentage of blastocysts 49.14 ± 2.97% v. 49.11 ± 3.40% in high and low O2 (P = 0.46). These results suggest that oxygen tension during culture affects IVEP differently depending on breed. The initial period of culture, recognised as critical in IVEP, seemed more sensitive to high O2 tension, particularly in Nellore.

Influence of sperm fertilising concentration, sperm selection method and sperm capacitation procedure on the incidence of numerical chromosomal abnormalities in IVF early bovine embryos

2015 ◽  
Vol 27 (2) ◽  
pp. 351 ◽  
Author(s):  
Sebastián Demyda-Peyrás ◽  
Jesús Dorado ◽  
Manuel Hidalgo ◽  
Miguel Moreno-Millán
Keyword(s):  
Chromosomal Abnormalities ◽  
Selection Method ◽  
Sperm Selection ◽  
Bovine Embryos ◽  
Sperm Capacitation ◽  
Related Factors ◽  
Selection Technique ◽  
Key Factor ◽  
Sequential Experiments

The occurrence of numerical chromosomal aberrations, widely described as a major cause of mortality in in vitro-produced (IVP) embryos, has been linked to several factors. In the present study we investigated the effect of sperm fertilising concentration and semen handling (sperm selection and capacitation) before IVF on the rate of numerical chromosomal abnormalities in bovine embryos. In all, 466 IVP cattle embryos were karyotyped throughout three sequential experiments, analysing the effects of sperm fertilising concentration (0.1, 1.0 or 10 × 106 spermatozoa mL–1), selection method (unselected or Percoll-selected spermatozoa) and capacitation medium (bovine serum albumin (BSA), heparin or their combination). The percentage of normal (diploid) and aberrant (haploid, polyploid or aneuploid) embryos was noted in each experiment. The rate of numerical chromosomal abnormalities was mainly affected by sperm fertilising concentration (P < 0.01) and, to a lesser extent, by the sperm capacitation medium (P < 0.05). Polyploidy and haploidy rates were only affected by sperm fertilising concentration (P < 0.05). Interestingly, the sperm selection technique used in the present study did not reduce the incidence of chromosome abnormalities in IVP cattle embryos (P > 0.05). Finally, aneuploidy rates were not affected during the experiments (P > 0.05), which suggests that they are not related to sperm-related factors. On the basis of these results, we conclude that sperm fertilising concentration is the ‘paternal’ key factor that affects the rate of numerical chromosomal abnormalities in IVP bovine embryos. By making small adjustments to fertilising protocols, the rate of cytogenetically aberrant embryos can be markedly reduced.

scholarly journals EFFECT OF THE USE OF TWO SPERM SELECTION TECHNIQUES FOR IN VITRO PRODUCTION OF ALPACA EMBRYOS

SPERMOVA ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 67-72
Author(s):  
Mijail Contreras Huamani ◽  
◽  
Mary Naveros ◽  
Cesar Olaguivel
Keyword(s):  
In Vitro Fertilization ◽  
Cumulus Cells ◽  
Sperm Selection ◽  
Percoll Gradient ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Medium ◽  
Blastocyst Rate ◽  
Vitro Production

The objective of this research was to evaluate the effect of the use of two sperm selection techniques for in vitro production of alpaca embryos. The ovaries and testis were collected from the local slaughterhouse and transport to 37 ° C in saline solution (0.9%) supplemented with gentamicin. Quality I, II and II oocytes were incubated in a maturation medium for 32 h at 38.5 ° C and 5% O2 and 5% CO2. For in vitro fertilization, sperm from the epididymis were selected using the Percoll gradient and Swim up technique. 18h after the oocytes were incubated with the sperm, these were denuded from the cumulus cells and cultured in SOFaa culture medium for 7 days. Morula and blastocyst rate and their morphological quality are evaluated at day 7 of culture. From a total of 370 ovaries, 1,137 oocytes were recovered, making an average of 3.6 oocytes / ovary. After the maturation and fertilization process and in vitro culture, the blastocyst rate was 8.43 ± 6.04% and 3.89 ± 1.75%, for oocytes fertilized with sperm selected with Percoll gradient and Swim up, respectively, not finding significant statistical differences (p> 0.05), between the groups. In conclusion, the in vitro fertilization of alpaca oocytes with spermatozoa selected with two selection techniques (percoll and swim up) did not significantly influence the quantity and quality of morulae and blastocysts at day 7 of embryo culture.

Production of in vitro bovine embryos supplemented with l-carnitine in different oxygen tensions and the relation to nitric oxide

Zygote ◽  
2020 ◽  
Vol 28 (5) ◽  
pp. 403-408
Author(s):  
Daniela Moraes Pereira ◽  
Christopher Junior Tavares Cardoso ◽  
Wilian Aparecido Leite da Silva ◽  
Mirela Brochado Souza-Cáceres ◽  
Mariana Santos ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Embryo Development ◽  
Calf Serum ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Low Oxygen ◽  
Cleavage Rate ◽  
Treatment Groups ◽  
Oxygen Tensions

SummaryThe aim of this study was to evaluate the production of bovine embryos in vitro when supplemented with l-carnitine for 24 h beginning on day 5 (d 5) under two different oxygen tensions (20% or 5%) and the relationship of nitric oxide (NO) in in vitro culture (IVC) medium to embryo development. Cumulus–oocyte complexes (COC; n = 837) were matured in vitro for 24 h and fertilization was performed for 18 h. Zygotes were cultured in vitro for 9 days after in vitro fertilization in synthetic oviductal fluid (SOF) medium with 5% fetal calf serum. At d 5 the plates were assigned to one of four treatment groups: high (20%) or low (5%) O2 tension either with or without the addition of 3.03 mM l-carnitine (High-Cont, High-Lcar, Low-Cont, Low-Lcar). The concentration of NO in the culture medium was evaluated on d 5, d 6 and d 9. On d 7, parts of the embryos were submitted for evaluation of intracellular lipid droplets. The cleavage rate was similar (P > 0.05) between high and low O2 tension and the blastocyst rate was similar in all conditions evaluated. The hatching rate was higher (P < 0.05) for Low-Cont. The NO concentration was higher at d 9 under low O2 tension (P < 0.1). The addition of 3.03 mM l-carnitine between d 5 and d 6 of IVC was not efficient in reducing cytoplasmic lipid content of bovine embryos. Additionally, IVC at a low oxygen tension without l-carnitine promoted better conditions for embryo development. A higher concentration of NO in medium was observed under low O2 tension.

scholarly journals Vitrification of Human In-Vitro Matured Oocytes: Effects on Mitochondrial Ultrastructure and Oxygen Consumption

2019 ◽  
Vol 01 (03) ◽  
pp. 131-135
Author(s):  
Shubiao Han ◽  
Wei Han ◽  
Xiaodong Zhang ◽  
Junxia Liu ◽  
Guoning Huang
Keyword(s):  
Oxygen Consumption ◽  
Formation Rate ◽  
Control Group ◽  
Mitochondrial Morphology ◽  
Blastocyst Development ◽  
Dynamic Changes ◽  
The Mean ◽  
The Impact ◽  
Normal Fertilization

Background: This study was conducted to evaluate the impact of vitrification on mitochondrial of human IVM oocytes. Methods: A total of 401 immature oocytes were obtained from ovarian stimulated cycles, which were randomly divided into fresh and vitrification groups after IVM. According to the cultured time after thawing, the vitrification groups were divided into 0 hours (0 h), 2 hours (2 h), or 4 hours (4 h) subgroups. Mitochondrial morphology and oxygen consumption were compared among the four groups. After fertilization by ICSI, normal fertilization, cleaved embryos, and blastocyst formation rate were also calculated. Results: The mean gray value of mitochondria structure was significantly decreased in 0 h and 2 h groups when compared to control group (0.48 ± 0.09, 0.50 ± 0.36 vs. 0.61 ± 0.12, respectively; P [Formula: see text] 0.05), and recovered (0.61 ± 0.24 vs. 0.61 ± 0.12, P [Formula: see text] 0.05) in 4 h group. In addition, oxygen consumption was also significantly decreased in 0 h and 2 h groups compared to fresh (2.91 ± 0.77 fmol/s, 3.26 ± 1.34 fmol/s vs. 3.96 ± 1.44 fmol/s, respectively; P [Formula: see text] 0.05), and recovered after 4 h culture (3.96 ± 1.44 fmol/s vs. 4.41 ± 1.38 fmol/s, respectively; P [Formula: see text] 0.05). The percentage of normal fertilization and cleaved embryos were no differences among the four groups, however, blastocyst development rate was significantly lower in 0 h group. Conclusion: These results indicate that during the vitrification process, the oxygen consumption and mitochondrial structure of oocytes may undergo temporary dynamic changes, but appear to recover by 4 hours.

171 EFFECT OF LONG-TERM TRANSPORTATION OF OVARIES ON THE DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 193
Author(s):  
M. Nakatate ◽  
K. Tsuchiya ◽  
I. Adachi ◽  
K. Takahashi ◽  
A. Aisan ◽  
...  
Keyword(s):  
Bovine Embryos ◽  
Cleavage Rate ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Thermos Flask ◽  
Blastocyst Rate ◽  
Functional Peptides ◽  
Number Of Cells

The transportation of bovine ovaries would allow the shipment of oocytes for research purposes after the slaughter of valuable cows. The objective of this study was to investigate the effect of long-term transportation of ovaries on the development of in vitro-produced bovine embryos. After collection of the ovaries from a slaughterhouse, they were placed inside a thermos flask and transported to the laboratory. The thermos flask was covered with a freezer pack in a foam polystyrene box. The transportation time was 17–18 h, and the temperature of the thermos flask changed from 20°C to 28°C (average 23.8°C) during the transportation. Cumulus–oocyte complexes (COCs) were collected by the aspiration of follicles with a diameter of 2–6 mm. The COCs were matured for 20 h in IVMD101 (RIFP: Research Institute for the Functional Peptides, Yamagata, Japan) containing DM199 supplemented with 5.56 mM glucose, 0.91 mM pyruvate, 5 mM taurine, 5 mM selenium, 5 mM HEPES, and 10 µg/mL gentamicin at 38.5°C under an atmosphere of 5% CO2 in air (Hoshi 2003 Theriogenology 59, 675–685). The matured COCs were inseminated with 5 × 106 sperm/mL in IVF100 (RIFP) medium comprising a modified BO medium supplemented with 1.25 mM sodium pyruvate, 0.5 mM cysteine, 5 mg/mL BSA, 7.5 µg/mL sodium heparin, 5 mM caffeine, and 10 µg/mL gentamicin. After 6 h of gamete co-culture, the presumed zygotes were cultured in IVD101 (RIFP) medium comprising DM199, 2.48 mM lactate, 0.27 mM pyruvate, and 2.22 mM of glucose for 9 days at 38.5°C under an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. As controls, bovine ovaries were transported to the laboratory within 1–1.5 h. Embryo development was evaluated based on the cleavage rate, blastocyst rate, and total number of cells on Days 7–9 after in vitro fertilization. The experiment was replicated five times, and data were analyzed by chi-square test and ANOVA. Results are presented in Table 1. There were no differences in the cleavage rate between the treatments. The blastocyst rate and the number of cells in the blastocyst after long-term transportation of ovaries were significantly lower than those in the controls. These results suggest that the long-term transportation of bovine ovaries does not affect on the cleavage; however, the blastocyst rate and the quality of blastocysts may be affected. Therefore, additional experiments are required to determine suitable conditions for long-term transportation of bovine ovaries. Table 1. Effect of long-term transportation of ovaries on the development of bovine IVM/IVF embryos

353 IN VITRO FERTILIZATION WITH FLOW-SORTED BUFFALO SPERM

2006 ◽  
Vol 18 (2) ◽  
pp. 283 ◽  
Author(s):  
M. Zhang ◽  
X. W. Liang ◽  
Y. Q. Lu ◽  
K. H. Lu
Keyword(s):  
Mineral Oil ◽  
Motile Sperm ◽  
Percoll Gradient ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Blastocyst Rate ◽  
Maximum Humidity ◽  
Cattle And Sheep

Flow cytometrically sorted X and Y sperm have been successfully used for IVF and the production of offspring in cattle and sheep (Maxwell et al. 2004 Anim. Reprod. Sci. 82–83, 79–95).The objective of this study was to test the feasibility of flow sorted buffalo sperm used in IVF systems and to establish a suitable IVF system for sorted buffalo sperm. Oocytes were aspirated from 2–6 mm follicles on the buffalo ovaries from a slaughterhouse and matured for 22–24 h in a 1-mL dish containing TCM199 + 10% OCS + 3% BFF (bovine folliciular fluid) + hormones at 38.5°C, 5% CO2 in air with maximum humidity. Semen was sorted by a flow-sorter (Clontech, Mountain View, CA, USA) into X- and Y-chromosome bearing sperm at 90% accuracy and stored at 4°C for later use with IVF. Sorted sperm were prepared for IVF by centrifugation (700g, 20 min) through a Percoll gradient (90%:45%), and washed (centrifugation at 700g, 5 min) in mTALP-BSA. For IVF, groups of 10–15 oocytes were transferred to 40-μL drops of mTALP-BSA and incubated with motile sperm at a concentration of 2 × 106 sperm mL−1 in each fertilization drop for 8–10 h under mineral oil. Presumptive zygotes were cultured until Day 8 in 25-µL drops of TCM–199 supplemented with 0.33 mM pyruvate and 10% NCS with granulosa cells at 38.5°C under 5% CO2 in air. Cleavage and blastocyst rates per oocyte insemination were recorded on Day 2 and Days 6–8 after insemination, respectively. Data were analyzed by ANOVA procedures with replicates and treatments in the model. There were significant differences in cleavage rate (42.23% vs. 52.28%) and blastocyst rate (20.62% vs. 27.67%) between sorted and unsorted sperm, respectively (Table 1). There were no significant differences in the proportions of blastocyst development rates on Days 6, 7, or 8 after insemination with sorted and unsorted sperm. The results indicate that sorted buffalo sperm from two bulls have been successfully used in an IVF system to produce sex-controlled embryos. Table 1. Cleavage and blastocyst rates with different sperm types This research was supported by grants from the National Natural Science Foundation of China (30360073) and the Guangxi Department of Science and Technology (0330004–13). The authors (M. Z. and X.W. L.) contributed equally to this work.

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