Bovine oviductal and uterine fluid support in vitro embryo development

2018 ◽  
Vol 30 (7) ◽  
pp. 935 ◽  
Author(s):  
Meriem Hamdi ◽  
Ricaurte Lopera-Vasquez ◽  
Veronica Maillo ◽  
Maria Jesus Sanchez-Calabuig ◽  
Carolina Núnez ◽  
...  
Keyword(s):  
Embryo Development ◽  
Dna Methyltransferase ◽  
Calf Serum ◽  
Stress Genes ◽  
Low Concentrations ◽  
Uterine Fluid ◽  
Oviduct Fluid ◽  
Oviductal Fluid ◽  
Intracellular Channel

In order to mimic the maternal oviductal environment, we evaluated the effect of oviductal fluid (OF) and/or uterine fluid (UF) supplementation on in vitro embryo development and quality. In vitro-produced zygotes were cultured with 1.25% OF from Day 1 to Day 4 after insemination (OF group), 1.25% OF from Day 1 to Day 4 followed by 1.25% UF from Day 4 to Day 9 (OF+UF group) or 1.25% UF only from Day 4 to Day 9 (UF group). Control groups were cultured in the presence of synthetic oviduct fluid (SOF) supplemented with 3 mg mL−1 bovine serum albumin (BSA) or 5% fetal calf serum (FCS). Supplementation of the culture medium with OF and/or UF (both at 1.25%) supported embryo development (Day 9 blastocyst rate 28.2–30.6%). At 72 h after vitrification–warming, the survival of blastocysts from the OF and OF+UF groups was similar to that of blastocysts in the SOF+BSA group (61.0 ± 5.7% and 62.8 ± 6.4% vs 64.8 ± 6.4% respectively), but significantly higher than that of blastocysts from the SOF+FCS group (31.6 ± 4.9%; P < 0.001). Blastocysts from the OF group exhibited upregulation of epigenetic genes (i.e. DNA methyltransferase 3α (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R)), compared with expression in the SOF+FCS group (P < 0.05). Whereas those from OF+UF and UF groups exhibited downregulation of oxidative stress genes compared to SOF+BSA and OF groups for glutathione peroxidase (GPX1) and to SOF+FCS, SOF+BSA and OF groups for chloride intracellular channel 1 (CLIC1) (P < 0.05). In addition, accumulation of reactive oxygen species was lower in blastocysts from the OF, OF+UF and UF groups. In conclusion, the use of low concentrations of OF and UF in in vitro serum-free culture supports embryo development, with OF providing a better control of embryo methylation, whereas UF may have antioxidant activity.

Effect of bovine oviductal fluid on development and quality of bovine embryos produced in vitro

2017 ◽  
Vol 29 (3) ◽  
pp. 621 ◽  
Author(s):  
Ricaurte Lopera-Vasquez ◽  
Meriem Hamdi ◽  
Veronica Maillo ◽  
Valeriano Lloreda ◽  
Pilar Coy ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Embryo Development ◽  
Cell Mass ◽  
Water Channel ◽  
Calf Serum ◽  
Cell Counting ◽  
Bovine Embryos ◽  
Oviductal Fluid

To evaluate the effect of bovine oviductal fluid (OF) supplementation during in vitro culture of bovine embryos on their development and quality, in vitro-produced zygotes were cultured in synthetic oviductal fluid (SOF; negative control; C–) supplemented with OF or 5% fetal calf serum (positive control; C+). Embryo development was recorded on Days 7–9 after insemination and blastocyst quality was assessed through cryotolerance, differential cell counting of the inner cell mass and trophectoderm, and gene expression. OF was added to the culture medium at concentrations ranging from 0.625% to 25%. The higher OF concentrations (5%, 10% and 25%) had a detrimental effect on embryo development. Lower OF concentrations (1.25% and 0.625%) supported embryo development until Day 9 (27.5%) and produced higher-quality blastocysts, as reflected by their cryotolerance (53.6% and 57.7% survival at 72 h, respectively, vs 25.9% in C+) and total cell number (mean (± s.e.m.) 165.1 ± 4.7 and 156.2 ± 4.2, respectively, vs 127.7 ± 4.9 in C– and 143.1 ± 4.9 in C+). Consistent with these data, upregulation of the water channel aquaporin 3 (AQP3) mRNA was observed in blastocysts supplemented with 1.25% OF compared with C– and C+. Serum supplementation resulted in a reduction in the expression of glucose and lipid metabolism-related genes and downregulation of the epigenetic-related genes DNA methyltransferase 3A (DNMT3A) and insulin-like growth factor 2 receptor (IGF2R). In conclusion, in vitro culture with low concentrations of OF has a positive effect on the development and quality of bovine embryos.

scholarly journals Effect of bovine oviductal extracellular vesicles on embryo development and quality in vitro

Reproduction ◽  
2017 ◽  
Vol 153 (4) ◽  
pp. 461-470 ◽  
Author(s):  
Ricaurte Lopera-Vasquez ◽  
Meriem Hamdi ◽  
Veronica Maillo ◽  
Alfonso Gutierrez-Adan ◽  
Pablo Bermejo-Alvarez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Extracellular Vesicles ◽  
Water Channel ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Analysis System ◽  
Oviductal Fluid

The aim of this study was to evaluate the effect of extracellular vesicles (EV) from oviductal fluid (OF), either from the ampulla or isthmus, on the development and quality of in vitro-cultured bovine embryos. Zygotes were cultured in synthetic oviduct fluid (SOF + 3 mg/mL BSA) without calf serum (C− group), in the presence of 3 × 105 EV/mL from ampullary or isthmic OF at either 1 × 104 g (10 K) or 1 × 105 g (100 K), and compared with SOF + 5% FCS (C+ group). OF-EV size and concentration were assessed by electron microscopy and nanotracking analysis system. Embryo development was recorded on Days 7–9, and blastocyst quality was assessed through cryotolerance and gene expression analysis. Lower blastocyst yield was observed on Day 7 in the C− and OF-EV groups (12.0–14.3%) compared with C+ (20.6%); however, these differences were compensated at Days 8 and 9 (Day 9: 28.5–30.8%). Importantly, the survival rate of blastocysts produced with isthmic 100 K OF-EV was higher than that of C+ and C− group at 72 h after vitrification and warming (80.1 vs 34.5 and 50.5% respectively, P < 0.05). In terms of gene expression, blastocysts produced in the presence of 100 K isthmic OF-EV upregulated the water channel AQP3 and DNMT3A and SNRPN transcripts compared with the C+, with the expression in C− being intermediate. The lipid receptor LDLR was downregulated in C+ compared with all other groups. In conclusion, the addition of oviductal fluid extracellular vesicles from isthmus, to in vitro culture of bovine embryos in the absence of serum improves the development and quality of the embryos produced.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

161 EFFECT OF OVIDUCTAL FLUID ON BOVINE OOCYTE ZONA PELLUCIDA HARDENING AND SPERM BINDING, AND ON EARLY EMBRYO DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 210
Author(s):  
P. Hugon ◽  
J. Lamy ◽  
E. Corbin ◽  
P. Mermillod ◽  
M. Saint-Dizier
Keyword(s):  
Corpus Luteum ◽  
Embryo Development ◽  
Zona Pellucida ◽  
Developmental Stages ◽  
Early Embryo ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
Oviductal Fluid

This study was designed to evaluate the effects of oviductal fluid at different periovulatory times on oocyte maturation, modification of the zona pellucida (ZP), fertilization and embryo development. Bovine oviducts were collected at a slaughterhouse and classified as preovulatory (pre-ov: 1 pre-ov follicle and a regressing corpus luteum) or post-ovulatory (post-ov: a corpus haemorrhagicum or recent corpus luteum; n = 10 cows/stage). Both oviducts were flushed with 1 mL of sterile TCM-199, and oviductal flushes (OF) were aliquoted and stored at –80°C. Abattoir-derived bovine ovaries were aspirated and cumulus‐oocyte complexes (COC) with at least 3 cumulus layers and homogeneous oocyte cytoplasm were in vitro matured for 22 h in standard maturation medium (control group, n = 319) or in standard medium with 2× concentrated additives supplemented (50% v/v) with pre-ov OF (n = 255) or post-ov OF (n = 248). After in vitro maturation (IVM), subgroups of COC were denuded, and the time of digestion of the ZP by pronase 0.1% (v/v in TCM-199) was determined to evaluate ZP hardening. After IVM, COC were fertilised in vitro for 18–20 h at a final concentration of 1.106 million spermatozoa (spz)/mL. After in vitro fertilization (IVF), COC were denuded, washed twice and cultured for 8 days more under standard conditions. After IVM, IVF, and embryo culture, oocytes/embryos were fixed with ethanol, stained with Hoescht, and examined under fluorescence microscopy for determination of (1) maturation and developmental stages, (2) numbers of fertilised and polyspermic oocytes, and (3) spz bound to the ZP. Percentages were compared between groups by chi-square. Times of ZP digestion were compared by Kruskal‐Wallis test. Numbers of spz bound to the ZP were compared by ANOVA on normalised data followed by Newman-Keuls tests. Data are presented as mean ± SEM. A P < 0.05 was considered significant. Addition of OF during IVM had no effect on maturation rates compared with the control. However, the digestion time of the ZP by pronase was reduced after IVM with pre-ov OF (313 ± 21 s; n = 26) compared with post-ov OF (459 ± 23 s; n = 23) but not with the control (416 ± 30 s; n = 25). After IVF, the number of spermatozoa bound to the ZP was increased after IVM with pre-ov OF (57 ± 5 spz/oocyte; n = 67) and decreased after IVM with post-ov OF (34 ± 3 spz/oocyte; n = 76) compared with the control (42 ± 5 spz/oocyte; n = 60). Addition of OF during IVM had no effect on rates of IVF and polyspermia. However, the rate of development to the blastocyst stage was less after IVM with post-ov OF (10%, n = 97 cleaved oocytes) compared with control (24%, n = 130) and pre-ov OF (29%, n = 101). In conclusion, the OF collected before ovulation decreased the resistance of the ZP to protease digestion and increased its ability to bind spz, whereas it was the opposite for the post-ov OF. Furthermore, the post-ov OF decreased the developmental competence of fertilised oocytes.

150 DEVELOPMENT OF ZONA-FREE AND WITH ZONA PARTHENOGENETIC GOAT EMBRYOS IN DIFFERENT MEDIA

2010 ◽  
Vol 22 (1) ◽  
pp. 233
Author(s):  
M. K. Jena ◽  
D. Malakar ◽  
A. K. De ◽  
S. Garg ◽  
Y. S. Akshey
Keyword(s):  
Embryo Development ◽  
Chemical Activation ◽  
Formation Rate ◽  
Electrical Activation ◽  
Cleavage Rate ◽  
Maturation Medium ◽  
Parthenogenetic Embryo ◽  
Oviductal Fluid ◽  
Phosphate Buffered Saline

The present study was carried out to see the developmental efficiency of zona-free and with zona parthenogenetic goat embryos cultured in Research Vitro Cleave from Cook Australia (RVCL), Embryo Development Media (EDM), modified synthetic oviductal fluid (mSOF), and modified Charles Rosenkrans media (mCR2a). Zona-free embryos were cultured in 4 media, whereas with zona embryos were cultured in 3 media except mCR2a. Ovaries were collected from slaughterhouse and oocytes were isolated by puncturing the follicles in medium containing Dulbecco’s phosphate-buffered saline, 3% BSA, and 50 μg mL-1 gentamicin. Oocytes were matured in maturation medium containing TCM-199 (HEPES modified), 0.05 mg mL-1 Na pyruvate, 0.003 mg mL-1 L-glutamine, 5.5 mg mL-1 glucose, 3 mg mL-1 BSA, 5 μg mL-1 FSH, 10 μg mL-1 LH, 1 μg mL-1 estradiol-17β, 50 μg mL-1 gentamicin, and 10% FBS in 5% CO2 in air at 38.5°C. The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 27 h. Matured oocytes were made cumulus free by treatment with hyaluronidase (0.5 mg mL-1) and zona-free by pronase (2 mg mL-1) in zona-free parthenogenesis. Then the oocytes were activated by 5 μM Ca ionophore for 5 min in a CO2 incubator and then treated with 2 mM 6-DMAP for 4 h. Activation was also done by electrical activation with DC 1.78 kV cm-1, 20 μs, and 2 pulses. Then the zona-free oocytes were kept for in vitro culture in 4 types of media such as RVCL, EDM, mSOF, andm CR2a for 7 days in 5% CO2 in air at 38.5°C. The cleavage rate andmorulae formation were observed in RVCL 40.95%, 13.95%, in EDM 46.92%, 14.75%, in mCR2a 56.66%, 5.88%, and in mSOF 48.23%, 14.63%, respectively. The cleavage rate and morulae formation were also found 55.9%, 14.63% during chemical activation and 32%, 12.5% in electrical activation. Hence, better result was found in chemical activation than electrical activation. For with zona parthenogenesis, the matured oocytes were chemically activated by 5 μM Ca ionophore for 5 min and 2 mM 6-DMAP for 4 h. Then the oocytes were cultured in RVCL, EDM, and mSOF in 100-μL micro-drops media for 7 days. The cleavage, morulae, and early blastocyst production rate were as follows: cleavage rate 75.68%, 72.03%, and 57.11%; morulae 44.61%, 30.29%, and 40.22%; and early blastocyst 17.49%, 11.88%, and 25.01% in RVCL, EDM, and mSOF, respectively. Hatched blastocyst formation rate was 6.75%, 5.48%, and 1.15% in RVCL, EDM, and mSOF, respectively. It could be concluded that zona-free parthenogenetic embryos were produced better in EDM medium and with chemical activation. With zona parthenogenetic embryo development was significantly (P < 0.05) higher in RVCL and EDM media.

197 DOES DURATION OF BOVINE OOCYTE MATURATION IN VITRO AFFECT THE SPEED OF EMBRYO DEVELOPMENT IN VITRO AND SEX RATIO AT THE TWO-CELL OR BLASTOCYST STAGE?

2008 ◽  
Vol 20 (1) ◽  
pp. 177
Author(s):  
P. Bermejo-Álvarez ◽  
A. Gutiérrez-Adán ◽  
P. Lonergan ◽  
D. Rizos
Keyword(s):  
Sex Ratio ◽  
Embryo Development ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Blastocyst Development ◽  
Maturation In Vitro ◽  
Maturational Stage ◽  
Development In Vitro ◽  
Oviduct Fluid

The faster-developing blastocysts in IVC systems are generally considered more viable and better able to survive following cryopreservation or embryo transfer than those that develop more slowly. However, evidence from several species indicates that embryos that reach the blastocyst stage earliest are more likely to be males than females. The aim of this study was to determine whether the duration of maturation could affect early embryo development and, furthermore, the sex ratio of early- or late-cleaved embryos and blastocysts. Cumulus–oocyte complexes were matured in vitro for 16 h (n = 2198) or 24 h (n = 2204). Following IVF, presumptive zygotes from each group were examined every 4 h between 24 and 48 h postinsemination (hpi) for cleavage, and all embryos were cultured to Day 8 in synthetic oviduct fluid to assess blastocyst development. Two-cell embryos at each time point and blastocysts on Days 6, 7, and 8 from both groups were snap-frozen individually for sexing. Sexing was performed with a single PCR using a specific primer BRY. There was a significantly lower number of cleaved embryos from the 16-h compared with the 24-h maturation group at 28 (10.0 � 1.51 v. 28.8 � 3.57%), 32 (35.3 � 1.48 v. 57.6 � 3.33%), 36 (54.8 � 1.76 v. 67.4 � 2.81%), 40 (63.3 � 1.82 v. 72.0 � 2.54%), and 48 (70.6 � 1.78 v. 77.1 � 2.18%) hpi, respectively (mean � SEM; P d 0.05). However, the blastocyst yields on Day 6 (17.1 � 3.11 v. 16.4 � 2.11%), 7 (30.6 � 4.10 v. 34.6 � 3.51%), or 8 (34.1 � 3.90 v. 39.4 � 4.26%) were similar for both groups (mean � SEM; 16 v. 24 h, respectively). Significantly more 2-cell early cleaved embryos (up to 32 hpi) were male compared with the expected 1:1 ratio from both groups (16 h: 1.24:0.76 v. 24 h: 1.17:0.83, P ≤ 0.05); however, the overall sex ratio among 2-cell embryos was significantly different from the expected 1:1 in favor of males only for the 16-h group (1.18:0.82, P ≤ 0.05). The sex ratio of blastocysts on Day 6, 7, or 8 from both groups was not different from the expected 1:1. However, the total number of male blastocysts obtained after 8 days of culture from the 24-h group was significantly different from the expected 1:1 (1.19:0.81, P ≤ 0.05) and approached significance in the 16-h group. These results show that the maturational stage of the oocyte at the time of fertilization has an effect on the kinetics of early cleavage divisions but not on blastocyst yield. Furthermore, irrespective of the duration of maturation, the sex ratio of early-cleaving 2-cell embryos was weighted in favor of males, and this observation was maintained at the blastocyst stage.

112 EFFECTS OF GUAIAZULENE ON IN VITRO CULTURE OF BOVINE ZYGOTES, AND ON mRNA TRANSCRIPTS RELATED TO EMBRYO QUALITY

2009 ◽  
Vol 21 (1) ◽  
pp. 156
Author(s):  
E. Dovolou ◽  
M. Clemente ◽  
G. S. Amiridis ◽  
I. Messinis ◽  
A. Kalitsaris ◽  
...  
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Early Embryo ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Early Embryo Development ◽  
Mrna Transcripts ◽  
Oviductal Fluid ◽  
Maximum Humidity

We have previously shown that follicular and oviductal fluid provide greater total protection against lipid peroxidation than the respective media used for the in vitro embryo production. Reactive oxygen species (ROS) production has been implicated as a major cause for the reduced in vitro bovine embryo production; it is believed that they participate in meiotic arrest of oocytes, embryonic block and cell death. The aim of this study was to determine whether guaiazulene (G), an exogenous antioxidant, added in the post fertilization culture medium would affect the early embryo development and the quality of the produced blastocysts in terms of mRNA expression of several important genes. In a previous study we had shown that media modified with 0.01 mm of G provided the same antioxidant protection as the respective in vivo environments (i.e. the follicular and the oviductal fluid). Bovine cumulus–oocyte complexes (COC) were aspirated from ovaries derived from slaughtered cows and matured in groups of 50 in 500 μL in TCM199 with 10% fetal calf serum and 10 ng mL–1 Epidermal Growth factor at 39°C in an atmosphere of 5% CO2 in air and maximum humidity. Twenty-four hours later matured oocytes were inseminated with frozen/thawed bull semen and co-incubated in the same conditions as maturation. Presumptive zygotes were divided into 4 groups and cultured in groups of 25 in 25 μL of SOF with 5% FCS (Control–, n = 355), supplemented with 0.01 mm of G (n = 344) or 0.1 mm of G (n = 345) or 0.05% DMSO – the G diluent–(Control+, n = 347) at 39°C in an atmosphere of 5% CO2, 5% O2 and maximum humidity. Blastocyst yield was recorded on Days 6, 7, 8 and 9; Day 7 blastocysts from each group were snap frozen and stored at –80°C for mRNA extraction. Quantification of transcripts for aldose reductase mRNA (AKRIBI), prostaglandin G/H synthase-2 (PGHS-2, COX-2), glyceraldehyde 3-phosphate dehydrogenase (GADPH), facilitated glucose/fructose transporter, member 5 (GLUT-5) genes related to metabolism, glutathione peroxidase 1 (GPX1), glucose-6-phosphate dehydrogenase (G6PD) antioxidant enzymes and placenta-specific 8 (PLAC8) related to implantation was carried out by real-time quantitative RT-PCR. Data for embryo development and on transcript abundance were analyzed by chi square and ANOVA, respectively. Cleavage rate tended to be higher in 0.01 mm group than in Control– (77.87% v. 71.41%, P = 0.07). Barring that, no other differences were detected in cleavage rate (Control+: 71.32%; 0.1 mm: 72.75%) or in the overall blastocyst yield on Day 9 (Control–: 25.50%; Control+: 26.71%; 0.1 mm: 25.75%; 0.01 mm: 29.58%). The relative abundance of genes studied varied among groups, but these differences were not significant. We infer that under the current culture conditions, G as an antioxidant has no serious direct effect on early embryo development or on embryo quality at least on the mRNA transcripts studied. Further studies using the same antioxidant in different atmospheric conditions are planed. ED and GSA were sponsored by COST (FAO702) and OECD fellowships, respectively.

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Monolayers ◽  
High Concentrations ◽  
Oviduct Fluid ◽  
Bovine Oocytes

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

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

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

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

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