scholarly journals LÍQUIDO FOLICULAR FRESCO OU CONGELADO NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS

2003 ◽  
Vol 8 (1) ◽  
Author(s):  
L.P. RAUBER ◽  
D.F. ALVES ◽  
F.D. MOZZAQUATRO ◽  
J.V. TESSMANN ◽  
M.L. BERNARDI ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Freezing Process ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cumulus Oocyte Complex ◽  
Follicle Size ◽  
Blastocyst Rate ◽  
Bovine Oocytes

A manutenção dos complexos cumulus-oócitos (CCO) em líquido folicular (LF) antes da sua maturação, além de visar a capacitação, viabiliza o transporte até o laboratório por ser de baixo custo, de fácil aquisição e o congelamento do LF permite seu armazenamento para futura utilização. Neste experimento avaliou-se o efeito do congelamento do LF obtido de folículos de 2-8mm e de folículos >8mm, sobre a taxa de produção embrionária. Oócitos foram aspirados de folículos de 2 a 8mm de ovários provenientes de abatedouro. No grupo controle (n=295) os CCO foram maturados por 24h. Nos tratamentos GF (n=297) e GC (n=282), os CCO foram mantidos por 6h a 30ºC em LF fresco ou congelado, respectivamente, de folículos >8mm. Já no tratamento PF(n=278) e PC (n=281), os CCO foram mantidos em LF fresco ou congelado, respectivamente, de folículos de 2-8mm. Posteriormente, os CCO dos tratamentos GF, GC, PF e PC foram maturados por 18h. Não houve efeito negativo do congelamento do líquido folicular e nem do tamanho dos folículos sobre as taxas de clivagem e produção embrionária em D7 e D9 (P>0,05). No entanto, o congelamento do LF de folículos de 2 a 8mm resultou em redução da taxa de eclosão e do número de células dos blastocistos. A manutenção de oócitos bovinos por 6h a 30ºC, antes da maturação, pode ser efetuada em líquido folicular de folículos >8mm, fresco ou congelado. Fresh or frozen follicular fluid in vitro bovine embryo production Abstract In addition to the capacitation, the maintenance of cumulus-oocyte complex (COC) in follicular fluid (FF) before maturation, allows the transport to the laboratory, being a practical and less expensive media. The FF can be stored after freezing to future use. Oocytes aspirated from bovine slaughterhouse ovaries, were used to evaluate the effect of maintaining the oocytes in fresh or frozen bovine FF (from 2-8mm and >8mm follicles) on the blastocyst rate. In the control group (n=259) the COC were matured for 24h. On treatments GF (n=297) and GC (n=282) the COC were held for 6h at 30°C in fresh or frozen FF from >8mm follicles, respectively. In treatments PF (n=278) and PC (n=281) the COC were held in fresh or frozen FF from 2-8mm follicles, respectively. Later, the COC from GF, GC, PF and PC were matured for 18h. The freezing process as well as the follicle size had no effect on the cleavage, D7 or D9 blastocyst rates (P>0,05). Nevertheless, the frozen FF from 2-8mm follicles resulted in a reduced hatching rate and lower ICM cells. Fresh or frozen follicular fluid of >8mm follicles could be used for a 6h transport of bovine oocytes before maturation for 18h.

78 SUPPLEMENTATION WITH CARNOSINE DURING IN VITRO CULTURE IMPROVES THE QUALITY OF IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 146
Author(s):  
D. Le Bourhis ◽  
M. Verachten ◽  
P. Salvetti ◽  
M. Hochet ◽  
L. Schibler
Keyword(s):  
Reactive Oxygen Species ◽  
Reactive Oxygen ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Oxygen Species ◽  
Cleavage Rate ◽  
Cell Stage ◽  
Blastocyst Rate

The objective of the present study was to determine the effect of supplementation of culture medium with carnosine (β-alanyl-l-histidine; Sigma, St-Quentin Fallavier, France), a reactive oxygen species scavenger, on in vitro bovine embryo development and survival following cryopreservation. Abattoir-derived bovine oocytes (4 replicates) were in vitro matured and fertilized with frozen-thawed semen of one bull, according to our standard procedures. In Experiment 1, 20 h after IVF, groups of presumptive zygotes were cultured in 30 μL of SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 205) or 5 μg mL−1 of carnosine (n = 209) under humidified air with 5% CO2, 5% O2, and 88% N2. Cleavage rates were determined on Day 2, and the blastocyst rates and grade were assessed on Day 7 according to IETS classification. Day 7 grade 1 expanded blastocysts (n = 25 control and n = 27 carnosine) were frozen in 1.5 M ethylene glycol + 0.1 M sucrose. Embryos were thawed and then cultured for 72 h in SOF-BSAaa + 1% oestrus cow serum for re-expansion and hatching rate assessments at +24 h, +48 h, and +72 h post-thawing. In Experiment 2, presumed zygotes were cultured in SOF BSAaa + 1% oestrus cow serum with 0 (control; n = 48) or 5 μg mL−1 of carnosine (n = 48) in a WOW dish and observed with Time Laps Cinematography (Primo Vision®, VitroLife, Göteborg, Sweden). Images were recorded every 15 min for up to 168 h post-insemination. For embryos that reached the blastocyst stage, mean timing of the first cleavage (C1; 2-cell stage), second cleavage (C2; 4-cell stage), second cleavage to compaction (C3), and blastocoel cavity appearance (B4) were recorded. Chi-square test for Experiment 1 and Student’s t-test for Experiment 2 were used, and differences were considered significant at P < 0.05. In Experiment 1, no differences were observed in cleavage rate, blastocyst rate on Day 7, and grade 1 blastocyst rate between both control and carnosine groups (84.0 ± 4.2 v.85.2 ± 3.8, P = 0.7; 46.9 ± 7.1 v. 45.0 ± 7.5, P = 0.7; 24.1 ± 2.0 v. 24.0 ± 6.5, P = 0.6; respectively). After thawing, the re-expansion at +24 h was not different between groups (74.1 v. 48.0% for carnosine and control groups, respectively; P = 0.06). However, at +48 h and +72 h, the survival rate of carnosine treated blastocysts was significantly higher than that of blastocysts in the control group: 70.4 ± 4.5% v. 40.0 ± 3.8% and 59.3 ± 3.8% v. 24.0 ± 3.6%, respectively. Results from Experiment 2 indicated no difference between control and carnosine groups for C1 (32.1 ± 3.9 v. 33.8 ± 6.1; P = 0.3), C2 (8.2 ± 8.9 v. 8.9 ± 0.9; P = 0.07), and B4 (147.0 ± 9.5 v. 145.4 ± 11.6; P = 0.6), whereas C3 was significantly different within groups: 59.9 ± 9.6 v. 51.8 ± 6.7 (P = 0.008). In conclusion, bovine blastocysts derived from zygotes cultured in the presence of 5 μg mL−1 carnosine possess a significantly faster kinetic from 4-cell stage to compaction and show a higher post-thawing viability. However, further analyses are still needed to clarify the relationship between the reactive oxygen species intracellular levels after carnosine treatment and in vitro bovine embryo quality. This work was supported by FECUND European project (grant agreement number 312097).

scholarly journals Follicular Fluid from Infertile Women with Mild Endometriosis Impairs In Vitro Bovine Embryo Development: Potential Role of Oxidative Stress

2021 ◽  
Author(s):  
Vanessa Silvestre Innocenti Giorgi ◽  
Rui Alberto Ferriani ◽  
Paula Andrea Navarro
Keyword(s):  
Oxidative Stress ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Formation Rate ◽  
Fertilization In Vitro ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Infertile Women ◽  
Bovine Oocytes

Abstract Objective To investigate whether follicular fluid (FF) from infertile women with mild endometriosis (ME) alters in vitro bovine embryo development, and whether the antioxidants N-acetyl-cysteine (NAC) and/or L-carnitine (LC) could prevent such damages. Methods Follicular fluid was obtained from infertile women (11 with ME and 11 control). Bovine oocytes were matured in vitro divided in: No-FF, with 1% of FF from control women (CFF) or ME women (MEFF); with 1.5 mM NAC (CFF + NAC, MEFF + NAC), with 0.6 mg/mL LC (CFF + LC, MEFF + LC), or both antioxidants (CFF + NAC + LC, MEFF + NAC + LC). After in vitro fertilization, in vitro embryo culture was performed for 9 days. Results A total of 883 presumptive zygotes were cultured in vitro. No differences were observed in cleavage rate (p = 0.5376) and blastocyst formation rate (p = 0.4249). However, the MEFF group (12.5%) had lower hatching rate than the No-FF (42.1%, p = 0.029) and CFF (42.9%, p = 0.036) groups. Addition of antioxidants in the group with CFF did not alter hatching rate (p ≥ 0.56), and in groups with MEFF, just NAC increased the hatching rate [(MEFF: 12.5% versus MEFF + NAC: 44.4% (p = 0.02); vs MEFF + LC: 18.8% (p = 0.79); versus MEFF + NAC + LC: 30.8% (p = 0.22)]. Conclusion Therefore, FF from infertile women with ME added to medium of in vitro maturation of bovine oocytes impairs hatching rate, and NAC prevented these damages, suggesting involvement of oxidative stress in worst of oocyte and embryo quality of women with ME.

scholarly journals Three-step in vitro maturation culture of bovine oocytes imitating temporal changes of estradiol-17<i>β</i> and progesterone concentrations in preovulatory follicular fluid

2017 ◽  
Vol 60 (4) ◽  
pp. 385-390 ◽  
Author(s):  
Minami Matsuo ◽  
Kazuma Sumitomo ◽  
Chihiro Ogino ◽  
Yosuke Gunji ◽  
Ryo Nishimura ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Culture System ◽  
In Vitro Maturation ◽  
Formation Rate ◽  
Temporal Changes ◽  
Control Group ◽  
Bovine Embryo ◽  
Developmental Potential ◽  
Bovine Oocytes

Abstract. The objective of the article is to evaluate the effect of three-step in vitro maturation (IVM) culture system imitating estradiol-17β (E2) and progesterone (P4) concentrations in preovulatory follicles on in vitro bovine embryo production. The cumulus–oocyte complexes (COCs) were collected from follicles (2 to 8 mm in diameter) of bovine ovaries obtained from a local slaughterhouse. For IVM, the COCs were cultured for 22 h in a three-step system: (1) culture in medium 199, containing 700 ng mL−1 E2 and 50 ng mL−1 P4, for 5 h, followed by the medium containing 150 ng mL−1 E2 and 150 ng mL−1 P4 for 11 h, and then the medium containing 20 ng mL−1 E2 and 300 ng mL−1 P4 for 6 h (EP group); (2) culture in the medium containing 700 ng mL−1 E2 for 5 h, followed by the medium containing 150 ng mL−1 E2 for 11 h, and then the medium containing 20 ng mL−1 E2 for 6 h (E group); or (3) culture in the medium containing 50 ng mL−1 P4 for 5 h, followed by the medium containing 150 ng mL−1 P4 for 11 h, and then the medium containing 300 ng mL−1 P4 for 6 h (P group). The COCs were cultured in the medium containing 1000 ng mL−1 E2 for 22 h (control group). After IVM, the COCs were co-incubated with sperm and further cultured. At 48 h after insemination, the cleavage rate of embryos was not different among the groups. At 192 h after insemination, the blastocyst formation rate of EP group was significantly higher than that of the other groups. The total cell number of blastocysts did not differ among the groups. In conclusion, these results demonstrate that the three-step IVM culture system of bovine oocytes imitating temporal changes of E2 and P4 concentrations in preovulatory follicular fluid improves the developmental potential of embryos in vitro.

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 LÍQUIDO FOLICULAR EQÜINO NA MATURAÇÃO IN VITRO DE OÓCITOS BOVINOS

2004 ◽  
Vol 9 (1) ◽  
Author(s):  
M.G.L. PINTO ◽  
M.I.B. RUBIN ◽  
C.A.M. SILVA ◽  
T.F. HILGERT ◽  
M.F. SÁ FILHO ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
In Vitro Maturation ◽  
Mineral Oil ◽  
Control Group ◽  
Sodium Pyruvate ◽  
Vitro Fertilization ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Bovine Oocytes

O desenvolvimento embrionário de oócitos bovinos maturados in vitro (MIV) foi avaliado em meio suplementado com líquido folicular eqüino (Lfe). Foram distribuídos 1045 oócitos em 11 repetições formando três grupos tratamentos (T1, T2, T3) e um controle (C). O meio de maturação utilizado foi o TCM-199 acrescido de piruvato de sódio, hormônio folículo estimulante recombinante (rFSHh) e hormônio luteinizante equino (LHe). Suplementou-se esse meio com 10% de soro de égua em estro para o grupo controle e para T1, T2 e T3, o meio foi suplementado com 5, 10, e 20% de LFe, respectivamente. Os oócitos foram maturados in vitro (MIV) por 24h. A fecundação in vitro (FIV) foi realizada em meio Talp-Fert. A MIV e a FIV foram realizadas em estufa a 39ºC com 5% de CO2 em ar e umidade saturada. Os zigotos foram cultivados em meio SOFaaci, sob óleo mineral no interior de bolsas plásticas gaseificadas. As taxas de clivagem e de blastocistos foram observadas diariamente (D), e em D7, foram superiores (P0,05) às do grupo controle. Em D9, a taxa de blastocistos do T2 foi superior (P0,05). O LFe, na concentração de 10% pode ser utilizado, em substituição ao soro de égua em estro para suplementar o meio de MIV de oócitos bovinos. Equine follicular fluid on in vitro maturation of bovine oocytes Abstract Embryo development of bovine oocytes was evaluated using maturation medium supplemented with equine follicular fluid (eFF). One thousand and forty five (1045) oocytes were distributed in 11 replications forming three treatment groups (T1, T2 e T3) and one Control (C). TCM-199 added with sodium pyruvate, rFSHh and LHe was used as maturation medium. This medium was supplemented with 10% estrous mare serum for Control group, and 5, 10, and 20% eFF, respectively, for T1, T2 e T3 groups. In vitro maturation (IVM) of all groups was performed during 24h. In vitro fertilization (IVF) was performed in TALP-FERT medium. IVM and IVF were carried out in an incubator at 39ºC with 5% CO2 in air and saturated humidity. Zygotes were cultured in SOFaaci medium, under mineral oil in gasified bags. Cleavage and blastocyst rates were daily observed (D), and at D7, were higher (P0.05) for those from control group. At D9, blastocyst rate of T2 was higher (P0.05). The eFF, at a 10% concentration, can replace the use of estrous mare serum to supplement the IVM medium of bovine oocytes.

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.

294 EFFECTS OF FOLLICULAR FLUID OBTAINED FROM REPEAT BREEDER DAIRY HEIFER ON MATURATION OF BOVINE OOCYTES IN VITRO

2015 ◽  
Vol 27 (1) ◽  
pp. 236
Author(s):  
M. Kafi ◽  
M. R. Divar ◽  
S. Gharib-Zadeh
Keyword(s):  
Follicular Fluid ◽  
Present Experiment ◽  
Calf Serum ◽  
Low Fertility ◽  
Control Group ◽  
Normal Fertility ◽  
Maturation Rate ◽  
Bovine Oocytes ◽  
Repeat Breeder

The cause of repeat breeding syndrome is often difficult to explain in dairy heifers with no clinical abnormalities. The aim of the present experiment was to determine the effect of follicular fluid obtained from the preovulatory follicle of repeat breeder heifers on maturation of bovine oocytes in vitro. Holstein virgin heifers either with normal fertility (VH, n = 5) or repeat breeder syndrome (RB, n = 5) were used in the present experiment. The RB heifers had a history of at least 5 unsuccessful consequent artificial breeding. The reason for using such RB heifers was to exclude the possibility of the presence of usual causes of infertility in heifers. Oestrus cycles of all heifers were synchronized using 2 injections of PGF2a 11 days apart. Six to 12 h after oestrus detection, clear follicular fluid samples from the ovulatory follicles were collected transrectally using a long fine-needle covered by a hard plastic tube. Follicular fluid samples were pooled, centrifuged, and frozen until used in the maturation medium. A total of 483 good or excellent quality bovine cumulus-oocytes complexes (COC) were obtained from 2 to 6 mm follicles in diameter from slaughterhouse ovaries and randomly allocated in 3 groups; in group 1 (control, n = 180), oocytes were cultured in TCM-199 supplemented with 10% heat-treated fetal calf serum and hormones (5 IU mL–1 of hCG plus 0.1 IU mL–1 of rFSH); in group 2 (n = 126), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of VH without hormones; in group 3 (n = 177), oocytes were cultured in TCM-199 supplemented with 10% filtered follicular fluid of RB heifers without hormones. All oocytes were cultured for 24 h at 39°C in an atmosphere of 5% CO2 under 90% humidity. At the end of maturation, the degree of cumulus expansion was evaluated and scored under a stereomicroscope. Then, oocytes were mechanically denuded using 3% sodium citrate and repeated pipetting and were fixed in ethanol/acetic acid (3 : 1) for 24 h. The oocytes were subsequently stained with 1% aceto-orcein and evaluated for meiotic resumption. Proportions were statistically analysed using a Chi-squared test (significant at P < 0.05; SPSS program, 11.5). The percentages of fully expanded COC differed among groups (P < 0.001). The maturation rate (MII stage) was 83% (150/180) in oocytes that were cultured in the presence of FCS as the control group. However, a reduction in the maturation rate was observed when oocytes were cultured either in VH follicular fluid (71.4%, 90/126; P < 0.01) or RB follicular fluid (59.3%, 105/177; P < 0.001) compared to the control group. The percentages of matured oocytes were also different between VH and RB follicular fluid (71.4 v. 59.3%; P < 0.01, respectively). In conclusion, the quality of follicular fluid of the preovulatory follicles of repeat breeder heifers is lower than that of the virgin heifers with normal fertility. This may explain the cause of the low fertility in some repeat breeder Holstein heifers.

32 Vitrification of in vitro-matured bovine oocytes in triacetate cellulose hollow fibres

2019 ◽  
Vol 31 (1) ◽  
pp. 142
Author(s):  
E. V. Kornienko ◽  
A. B. Romanova ◽  
M. A. Ikonopistseva ◽  
G. P. Malenko
Keyword(s):  
Ethylene Glycol ◽  
Fertilization Rate ◽  
Control Group ◽  
Hatching Rate ◽  
High Survival ◽  
Hollow Fibres ◽  
Group 2 ◽  
Bovine Oocytes ◽  
Group 1

A prospective method of vitrification in triacetate cellulose hollow fibres (HF) introduced by Matsunari et al. (2012 J. Reprod. Dev. 58, 599-608) allowed significant simplification and standardization of vitrification/warming procedures and was successfully used for group cryopreservation of various pre-implantation mammalian embryos. The goal of the current work was to evaluate the effectiveness of the HF vitrification method for cryopreservation of in vitro-matured bovine oocytes. The base medium for all the vitrification and rewarming solutions was calcium-free TBP-like protein-HEPES supplied with 20% of fetal bovine serum. Groups of 15 morphologically normal in vitro-matured bovine oocytes were equilibrated with 3% (vol/vol) ethylene glycol for 15min, loaded into HF, and transferred into vitrification solution containing 30% ethylene glycol and either 0.5M (Group 1) or 1.0M (Group 2) sucrose. Hollow fibres were incubated for either 60s (Group 1) or 30s (Group 2) and immediately plunged into LN. Rewarming was conducted at 39°C. Oocytes within HF were placed in decreasing concentrations of sucrose solutions to remove cryoprotectants. Then, oocytes were subjected to IVF. Non-vitrified denuded oocytes were used as a control. Survival rates were evaluated at 21h post-rewarming. Part of the presumptive zygotes were fixed and stained with acetolacmoid for fertilization rate. The remaining zygotes were cultured for 10 days. Developmental rates were evaluated at 44h and 7 and 10 days post-IVF. All results are presented as mean percentage±standard deviation. Data were analysed using Mann-Whitney U test. Significance was set at P&lt;0.05. Survival rate was significantly lower in Group 1 (79.0±8.0%) and Group 2 (75.0±5.0%) compared with the control group (97.0±4.0%). Fertilization rate in Group 1 differed significantly from the control (80.5±18.3% v. 95.5±9.1%). Cleavage rates in Groups 1 and 2 did not differ significantly from the control (42.5±15.7% v. 60.7±11.1% v. 63.0±15.8%, respectively). Blastocyst yields at 7 days post-IVF were 0.9±2.3 (1/116) and 9.6±5.4% (6/65) in Groups 1 and 2, respectively. The former was significantly lower than in the control group (17.0±10.3%, 23/154). It should be noted that hatching in the control group started at 8 days post-IVF and was delayed in Groups 1 and 2 for at least 24h. Day 10 blastocyst yields were 3.0±3.3 (P&lt;0.05), 20.9±13.8, and 30.4±9.6% in Groups 1 and 2 and the control group, respectively. All obtained Day 10 blastocysts (3/116) in Group 1 hatched. Hatching rate in Group 2 was significantly lower than in the control group. Both Groups 1 and 2 showed relatively high survival and fertilization rates, but embryo development rates in both groups had a tendency to be lower than in the control. However, the obtained results indicate that the modifications of the protocol may increase the effectiveness of HF vitrification. The HF vitrification method remains a prospective option for simultaneous cryopreservation of a group of bovine oocytes.

scholarly journals Developmental competence and expression of the MATER and ZAR1 genes in immature bovine oocytes selected by brilliant cresyl blue

Zygote ◽  
2009 ◽  
Vol 18 (3) ◽  
pp. 209-216 ◽  
Author(s):  
Gustavo Bruno Mota ◽  
Ribrio Ivan Tavares Pereira Batista ◽  
Raquel Varella Serapião ◽  
Mariana Cortes Boité ◽  
João Henrique Moreira Viana ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Control Group ◽  
Blue Dye ◽  
Brilliant Cresyl Blue ◽  
Immature Oocytes ◽  
Relative Expression ◽  
Cresyl Blue ◽  
Blastocyst Rate ◽  
Bovine Oocytes

SummaryThe objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus–oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 °C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB− (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB− group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB− (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB− (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB− groups. Despite the relative expression of MATER in holding control, BCB+ and BCB− were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.

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