89 Influence of sperm preincubation on development and sex ratio of in vitro-produced bovine embryos

2022 ◽  
Vol 34 (2) ◽  
pp. 281
Author(s):  
A. Fries ◽  
B. Zimmer ◽  
B. Rabenau ◽  
F. Kotarski ◽  
C. Wrenzycki
Keyword(s):  
Sex Ratio ◽  
Bovine Embryos

264 TESTOSTERONE IN THE OOCYTE CULTURE DOES NOT ALTER SEX-RATIO OF IN VITRO PRODUCED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 229
Author(s):  
C. Díez ◽  
P. Bermejo-Alvarez ◽  
A. Gutiérrez-Adan ◽  
J. N. Caamaño ◽  
M. Muñoz ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Cumulus Cells ◽  
Basic Medium ◽  
Bovine Embryos ◽  
Experimental Conditions ◽  
Vitro Fertilization ◽  
Grant Support ◽  
High Concentrations

The production of sex-known offspring is a main objective in reproductive biotechnology. It has been reported that bovine ova developed in follicles with high concentrations of testosterone in vivo yielded significantly more male embryos in vitro (Grant V et al. 2008 Biol. Reprod. 78, 812–815). In this work we aimed to test the effects of testosterone on sex ratio of bovine embryos produced in fully in vitro conditions. Immature bovine cumulus–oocyte complexes (COCs; n = 750) from slaughterhouse ovaries were cultured in 199 HNaCO3 with polyvinyl alcohol (PVA) 0.1 mg mL–1 as a basic medium. Culture was made in two steps, a 24 h meiotic arrest (roscovitine 25 μm), and a subsequent in vitro maturation period with FSH-LH for 24 h. Testosterone (T-86500, Sigma-Aldrich, St. Louis, MO, USA) was added throughout the entire oocyte culture at 0, 30, 300, and 1500 nm. After in vitro fertilization (Day 0), zygotes were freed of cumulus cells by pipetting, and subsequently cultured in SOF + 6 g L–1 BSA up to Day 3. At this time, embryo development was recorded, and all embryos having 3 or more cells were treated with pronase to remove the zona pellucida. Zona-free embryos were washed in PBS containing PVA 0.1 mg mL–1 and individually frozen at –80°C until sex analysis by PCR (Bermejo-Alvarez P et al. 2008 Biol. Reprod. doi:10.1095/biolreprod.108.070169). A total of 252 embryos from 5 replicates were sexed. Data for development and sex-ratio are presented as % LSM ± SD. There were no interactions between testosterone treatment, embryonic sex, and embryonic stage analyzed. Testosterone did not affect development rates (P > 0.05) at any stage: cleavage (47.8 ± 6.8, 56.5 ± 6.8; 50.9 ± 6.8; 62.2 ± 6.8), 3 to 4 cells (40.6 ± 5.2, 45.8 ± 5.2; 37.8 ± 5.2; 47.7 ± 5.2) and >5 cells rates (24.5 ± 4; 27.3 ± 4; 21.3 ± 4; 25.3 ± 4) for 0, 30, 300, and 1500 nm testosterone, respectively. Cumulative percentages of male embryos were as follows: 53 ± 8 (n = 56), 42.6 ± 8 (n = 52), 53.6 ± 6 (n = 81) and 57.6 ± 8 (n = 63) for 0, 30, 300, and 1500 nm groups respectively (P > 0.05). These results show that the testosterone effects on oocyte ability to select Y-chromosome bearing spermatozoa are not reproducible in vitro under the present experimental conditions. Grant support: MEC, project AGL2008-01530; RTA2008-0082; M. Muoz is supported by FICYT.

Factors affecting the sex ratio of in-vitro and in-vivo produced bovine embryos

1999 ◽  
Vol 51 (1) ◽  
pp. 396 ◽  
Author(s):  
G Carbonneau ◽  
H Twagiramungu ◽  
N Morin ◽  
C Brisson ◽  
J Durocher ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Bovine Embryos ◽  
Factors Affecting

109 INTRAFOLLICULAR GLUCOSE CONCENTRATION HAS AN INFLUENCE ON THE SEX OF BOVINE BLASTOCYSTS PRODUCED IN VITRO

2011 ◽  
Vol 23 (1) ◽  
pp. 160
Author(s):  
E. Abele ◽  
H. Stinshoff ◽  
A. Hanstedt ◽  
S. Wilkening ◽  
S. Meinecke-Tillmann ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Follicular Fluid ◽  
Glucose Concentration ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Bovine Embryos ◽  
Developmental Rates ◽  
Group 2 ◽  
Group 1

Several factors have been shown to alter the sex ratio of bovine embryos generated in vitro, i.e. the maturity of the oocyte at the time of insemination, the duration of sperm-oocyte co-incubation and the culture conditions after in vitro fertilization. It has been shown that the presence of glucose during in vitro culture reduced the development of female embryos to the blastocyst stage compared with controls cultured in the absence of glucose. The sex ratio of bovine embryos has also been linked with changes in the composition of the follicular fluid in which the oocyte undergoes growth and maturation, i.e. the intrafollicular testosterone concentration. However, no information is available regarding the effect of intrafollicular glucose concentration on the sex ratio of embryos after in vitro production (IVP). The purpose of this study was to determine whether different glucose concentrations in the follicular fluid at the time of cumulus–oocyte complex (COC) collection have an effect on the sex ratio of the resulting blastocysts after IVP. Ovaries from a local abattoir were transported to the laboratory within 2 h of slaughter. Follicles (3–8 mm) were individually dissected and the glucose concentration of each follicle was measured using a blood glucose monitoring system (Freestyle Freedom Lite, Abbott, Germany). Based on a glucose concentration, COC [low glucose: <1.1 mM (group 1) and high glucose: >1.1 mM (group 2)] were pooled in groups and used for blastocyst production employing standard protocols for IVP. Developmental rates were recorded at Day 3 (cleavage) and Day 7/8 (blastocyst stage). Total cell number of blastocysts was determined after Hoechst staining. Sex of the embryos was analysed via PCR using bovine X- and Y-chromosome specific primers. Developmental rates for COC stemming from follicles with different glucose concentrations did not show significant differences (P > 0.05) compared to each other [Cleavage rate: group 1: 81.8 ± 4.7% (93/117); group 2: 79.3 ± 4.9% (94/123); blastocyst rate: group 1: 35.6 ± 5.2% (38/117); group 2: 31.6 ± 5.2% (38/123)]. Total cell numbers were similar in embryos of both groups [Group 1: 117.7 ± 8.1 (n = 18); group 2: 117.2 ± 6.4 (n = 18)]. The overall sex ratio significantly differed (P < 0.05) from 1:1 in favour of females in both groups [Group 1: 85 v. 15% (n = 20); group 2: 63.6 v. 36.4% (n = 22)]. No significant difference (P > 0.05) in the overall sex ratio was detected in blastocysts produced under standard IVP conditions employed in the laboratory [without measurement of follicular glucose concentration, 55.0 v. 45.0%, (n = 20)]. In conclusion, under the conditions used in the present study, the intrafollicular glucose concentration from which the immature COC was collected affects the sex of the resulting embryo after IVP, favouring females. Further studies are needed to confirm these findings in living cows using the ovum pickup technique.

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.

scholarly journals Effect of Maturation Culture Period of Oocytes on the Sex Ratio of In Vitro Fertilized Bovine Embryos

2006 ◽  
Vol 52 (1) ◽  
pp. 123-127 ◽  
Author(s):  
Budiyanto AGUNG ◽  
Takeshige OTOI ◽  
Pimprapar WONGSRIKEAO ◽  
Masayasu TANIGUCHI ◽  
Ryohei SHIMIZU ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Culture Period ◽  
Bovine Embryos

Influence of physiological concentrations of androgens on the developmental competence and sex ratio of in vitro produced bovine embryos

2013 ◽  
Vol 13 (1) ◽  
pp. 41-50 ◽  
Author(s):  
Angus D. Macaulay ◽  
Christine K. Hamilton ◽  
W. Allan King ◽  
Pawel M. Bartlewski
Keyword(s):  
Sex Ratio ◽  
Developmental Competence ◽  
Bovine Embryos

scholarly journals 331INSEMINATION OF OOCYTES WITH AGED SEMEN ALTERS SEX RATIO OF BOVINE EMBRYOS PRODUCED IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 285
Author(s):  
J.L. Edwards ◽  
P. Bredbacka ◽  
A.M. Saxton ◽  
F.N. Schrick
Keyword(s):  
Sex Ratio ◽  
Sperm Motility ◽  
Block Design ◽  
Water Bath ◽  
Bovine Embryos ◽  
Cleavage Stage ◽  
Cell Stage ◽  
Frozen Semen ◽  
Effects Of Aging

Preincubation of semen before insemination may alter the sex ratio of resulting embryos (Lechniak, D. et al., 2003 Reprod. Dom. Anim. 38, 224–227). The overall objective of this study was to evaluate effects of aging sperm before insemination for altering the sex ratio of bovine embryos. In the first experiment oocytes presumed mature were inseminated with frozen semen within minutes post-thaw and percoll-preparation (control) or after aging for 14h post-thaw in a 34.4°C water bath, or after aging for 23h post-thaw at 4°C. Sperm from 4 different bulls, representing 3 different breeds, were utilized (1 bull per experimental replicate). Sperm motility was assessed after aging and percoll preparation. Zona pellucidae were removed from cleaved embryos (43–68h post-insemination; hpi) using 0.5% pronase. Blastomeres were dissociated and counted before transfer to PCR tubes. Sex of cleavage-stage embryos was determined using Ampli-Y™(Bredbacka, P. 1998 Reprod. Nutr. Dev. 38, 605–613). Data were analyzed using a randomized block design with mixed models of SAS (2000) after testing for normality. Aging semen for 14h post-thaw reduced the proportion of motile sperm and compromised the ability of presumptive zygotes to cleave after insemination (Table 1). However, ability of cleaved embryos to develop to the 8–16-cell stage was not affected. Number of blastomeres comprising cleavage-stage embryos that resulted from insemination of oocytes with aged semen was similar to that for controls. Insemination of oocytes with semen aged 14h in a water bath increased the proportion of female embryos (Table 1).To determine if effects of aging semen on altering the sex ratio of bovine embryos was time dependent, oocytes were inseminated with frozen semen within minutes post-thaw (control) or after aging for 8 or 14h post-thaw in a 34.4°C water bath. Sperm from 3 different bulls, representing 2 different breeds were utilized (one bull per experimental replicate). The experiment was replicated 3 times with 195–233 oocytes inseminated within each treatment. Sperm motility averaged 72.7, 65.7 and 45.0% for control and semen aged for 8 and 14h, respectively (SEM=10). Cleavage of inseminated oocytes (67hpi) was similar regardless of sperm treatment (68.5, 70.2 and 70.1%; SEM 14.6, for control semen or after aging for 8 or 14h post-thaw, respectively). Insemination of oocytes with semen aged for 14h tended to increase the proportion of female embryos (51.6 v. 38.3 and 39.1%; sperm aged for 14h v. 8h or control, respectively;; P=0.08; SEM=9.7). Within the seven replicates across both experiments, the differences in percent female for control v. aged were 5.2, 6.3, 14.9, 18.5, 20.0, 29.7 and 60%, with the highest three being significantly different (P&lt;0.05) by Fisher’s Exact test. Bull or replicate variation was noted but the direction of aging for increasing proportion of females was consistent. Preliminary observations suggest that biological differences between X- and Y-bearing sperm may exist such that alternative strategies for altering sex ratio in livestock species may be possible.

288 KINETICS OF FERTILIZATION, DEVELOPMENT, AND SEX RATIO OF IN VITRO-PRODUCED BOVINE EMBRYOS OBTAINED WITH FOUR DIFFERENT BULLS

2007 ◽  
Vol 19 (1) ◽  
pp. 259 ◽  
Author(s):  
M. Alomar ◽  
H. Tasiaux ◽  
S. Remacle ◽  
F. George ◽  
D. Paul ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
In Vitro Production ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test ◽  
Kinetics Of

The between-bulls variation in in vitro fertility and the shift of sex ratio toward male embryos are two problems affecting the in vitro production (IVP) of bovine embryos. Our objective was to evaluate the possible correlation between the kinetics of fertilization, embryo development, and the sex ratio of the resulting embryos. In a first experiment, and using frozen-thawed semen of 4 different AI bulls, the kinetics of pronucleus (PN) formation was evaluated at 8, 12, and 18 h post-in vitro insemination (hpi) after fixation and staining with Hoechst 33342. Fertilized oocytes were classified in 3 PN stages: PN1: showing the first signs of sperm head decondensation; PN2: with two pronuclei of different sizes, the two being far from each other; and PN3: showing two symmetric pronuclei of equal size, close to each other. Differences between bulls were observed at each time point, but were greater at 12 hpi than at 8 or 18 hpi. At 8 hpi and 12 hpi, bull C showed a significantly faster PN formation by comparison with the 3 other bulls (chi-square test: P &lt; 0.05), whereas at 18 hpi, the proportion at each of the PN stages was similar to that of bulls A and D, with bull B showing delayed PN development. In a second experiment, a standard IVP procedure was conducted with the 4 bulls to determine cleavage and blastocyst rates. The timing of first cleavage was measured using time-lapse cinematography. Compared with those of bull B, the embryos generated with bull C led to significantly higher Day 7 blastocyst yields (31.3 � 9.5% vs. 21.9 � 6.7%; ANOVA: P &lt; 0.05). Moreover, the embryos from bull C reaching the blastocyst stage cleaved faster (first cleavage at 23.1 � 2.1 hpi vs. 25.4 � 2.7 hpi for bull B; ANOVA: P &lt; 0.05). In a third experiment, 65 to 76 Day 8 blastocysts were sexed per bull. Embryo sexing was performed by PCR using the co-amplification of a Y-specific bovine SRY sequence and an autosomal btRep-137 sequence. Only blastocysts obtained with bull C showed a shift in sex ratio toward male embryos (76.0% male embryos vs. 53.8% for bull B; chi-square test: P &lt; 0.05), whatever the size of the blastocyst. The shift in sex ratio was already present at the 2-cell stage (64.2% male embryos; n = 53; chi-square test: P &lt; 0.05). In conclusion, for 2 out of 4 bulls, a correlation was observed between the kinetics of PN formation, the timing of first cleavage, and the sex ratio of the resulting embryos.

Impact of in vitro fertilization of bovine oocytes with sex-sorted frozen–thawed spermatozoa on developmental kinetics, quality and sex ratio of developing embryos

Zygote ◽  
2010 ◽  
Vol 18 (3) ◽  
pp. 185-194 ◽  
Author(s):  
J. Peippo ◽  
M. Räty ◽  
K. Korhonen ◽  
M. Eronen ◽  
K. Kananen ◽  
...  
Keyword(s):  
In Vitro Fertilization ◽  
Sex Ratio ◽  
Embryo Development ◽  
Bovine Embryos ◽  
Embryo Production ◽  
Male Embryo ◽  
Vitro Fertilization ◽  
Bovine Oocytes

SummaryWe studied whether bovine embryos developing after in vitro fertilization (IVF) with sex-sorted spermatozoa differed in developmental kinetics, quality and sex ratio from embryos produced with unsorted spermatozoa. Abattoir-derived oocytes were fertilized with X-sorted, Y-sorted or unsorted spermatozoa from a single bull. To evaluate economical use of the sex-sorted spermatozoa, washed spermatozoa from a single straw (2 million spermatozoa) were used to fertilize each batch of collected oocytes without any further isolation steps. Concentration of the unsorted spermatozoa was adjusted accordingly. Fertilizations were assessed by staining sperm asters at 10 hpi and pronuclei at 20 hpi. Embryo development and morphological quality were monitored on days 2, 7, 8 and 9 of the development (IVF = day 0). All embryos were sexed using PCR. Following fertilization, penetration and subsequent cleavage rates were compromised in the X-sorted group compared with the Y-sorted and unsorted groups (penetration: 58.0% vs. 89.8% and 90.0%, cleavage: 65.3% vs. 81.5% and 75.0%). The use of the sex-sorted spermatozoa did not, however, reduce the proportion of transferable embryos (sex-sorted 29.6% vs. unsorted 27.7%) or their quality (quality 1: sex-sorted 36.0% vs. unsorted 19.9%). The Y-sorted spermatozoa produced more transferable embryos of better quality than the X-sorted spermatozoa (days 7–8: 31.9% vs. 26.4%, quality 1: 38.9% vs. 30.6%). On average, out of 10 transferable embryos, nine were of the predicted sex in the X- and Y-sorted spermatozoa groups. These results indicate that low numbers of X- and Y-sorted spermatozoa can be used successfully for female and male embryo production in vitro.

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