171 IN VIVO AND IN VITRO MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS–OOCYTE COMPLEXES DURING THE OVULATORY SEASON

2014 ◽  
Vol 26 (1) ◽  
pp. 199
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion ◽  
Nuclear Maturation ◽  
Wood Bison

Technologies are being developed to conserve the genetic diversity of wood bison. Knowledge of the characteristics of in vivo and in vitro maturation of the cumulus–oocyte complex (COC) are needed in wood bison to design efficient in vitro embryo production protocols. The objectives were to (1) determine the optimal interval after hCG treatment for in vivo maturation of COC in superstimulated wood bison, and (2) compare the characteristics of COC after in vitro and in vivo maturation. Ovarian synchronization was induced in 25 bison during October and November by giving a luteolytic dose of prostaglandin followed 8 days later by follicular ablation (Day –1). Ovarian superstimulation was induced with FSH (Folltropin-V) given i.m. on Day 0 (300 mg) and Day 2 (100 mg). A second luteolytic dose of prostaglandin was given on Day 3. Bison were assigned randomly to 5 groups (n = 5/group). The COC were collected by transvaginal follicle aspiration on Day 4 and were either assessed immediately (0 h, control), or matured in vitro for 24 or 30 h (in vitro maturation), or collected on Day 5 (in vivo maturation), 24 or 30 h after bison were given 2000 IU of hCG i.m. on Day 4. In vitro maturation was done in TCM-199 with 5% calf serum, 5 μg mL–1 LH, 0.5 μg mL–1 FSH, and 0.05 μg mL–1 gentamicin, at 38.5°C and in a 5% CO2 humidified atmosphere. Nuclear maturation was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), or metaphase II (MII) with anti-lamin AC/DAPI staining. Groups were compared by analysis of variance and Fisher's exact test (Table 1). A mean (±s.e.m.) of 7.3 ± 1.7 COC were collected per bison, with no difference among groups. The COC in the control (0 h) group were at the nonexpanded GV stage. Cumulus cells were more expanded after in vivo than in vitro maturation, and the percentage of fully expanded COC was the highest in the 30-h in vivo maturation group (87%; P < 0.05). The greatest number of oocytes reached MII stage after 24 h of in vitro maturation, and 30 h of in vivo maturation. In conclusion, nuclear maturation occurred more quickly in vitro compared with in vivo, but the degree and incidence of cumulus expansion was greater after in vivo maturation. The competence of oocytes to undergo fertilization and develop into embryos remains to be investigated. Table 1.Cumulus expansion and nuclear maturation of wood bison oocytes

271 NUCLEAR MATURATION OF WOOD BISON (BISON BISON ATHABASCAE) CUMULUS - OOCYTE COMPLEXES

2013 ◽  
Vol 25 (1) ◽  
pp. 283
Author(s):  
M. P. Cervantes ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
J. M. Palomino ◽  
G. P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Transvaginal Ultrasound ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Bison Bison ◽  
Nuclear Maturation ◽  
Wood Bison

Methods of producing wood bison embryos in vivo and in vitro are being developed in an effort to preserve the genetic diversity of this threatened species. Previous data from our laboratory suggest that oocytes collected 24 h after LH treatment had not yet achieved nuclear maturation. The objectives of this study were (1) to determine the optimal interval of time after hCG treatment required for in vivo maturation of cumulus–oocyte complexes (COC) in wood bison, and (2) to compare the maturational characteristics of COC after in vitro v. in vivo maturation. Follicular wave emergence was synchronized among bison cows (n = 25) by follicular ablation (Day –1) from May to June. Ovarian superstimulation was induced with FSH IM diluted in 5 mg mL–1 of hyaluronan (MAP-5, Bioniche, Belleville, Ontario, Canada) given on Day 0 (300 mg) and Day 2 (100 mg). Superstimulated cows were assigned randomly to 5 groups (n = 5/group): COC collected on Day 4 with no maturation (control), or matured in vitro for 24 or 30 h, or collected 24 or 30 h after treatment with 2000 IU of hCG IM on Day 4. The COC were collected by transvaginal ultrasound-guided follicle aspiration. In vitro maturation was done in TCM-199 with 5% calf serum, 5 µg mL–1 of LH, 0.5 µg mL–1 of FSH, and 0.05 µg mL–1 of gentamicin, at 38.5°C and in 5% CO2. To assess nuclear maturation, oocytes were stained with anti-lamin AC/DAPI (4′,6-diamidino-2-phenylindole). Nuclear stages were classified as germinal vesicle (GV), GV breakdown (GVBD), metaphase I (MI), or metaphase II (MII). Comparisons among groups were made by ANOVA and Fisher’s exact test (Table 1). A mean (± SEM) of 7.6 ± 0.6 COC was collected per bison; no differences were observed among groups (P = 0.37). Cumulus cell expansion was more extensive after in vivo than in vitro maturation, and the percentage of fully expanded COC was highest in the in vivo 30-h group (97%; P < 0.05). No COC were expanded in the control (0 h) group, and none reached MI. Maximal nuclear maturation was achieved in vitro by 24 h; that is, there was no difference in the proportion of MII-stage COC at 24 versus 30 h. However, between 24 and 30 h of in vivo maturation, the percentage of nuclear stages GV + GVBD decreased from 54 to 24% (P < 0.05), whereas nuclear stages MI + MII increased from 39 to 74% (P < 0.05). In conclusion, nuclear maturation occurred earlier in vitro versus in vivo, but the consequences of this difference are unknown. Although more than one-third of oocytes matured in vivo for 30 h were mature enough to permit immediate IVF, whether additional in vivo maturation time would be beneficial to fertilization rates remains to be tested. Table 1.Nuclear status of wood bison oocytes after in vitro or in vivo maturation Thanks to Bioniche Canada.

326 EVALUATION OF CULTURE DURATION AND PARTIAL CUMULUS CELL REMOVAL DURING CANINE OOCYTE MATURATION

2006 ◽  
Vol 18 (2) ◽  
pp. 270
Author(s):  
C. Hanna ◽  
C. Long ◽  
M. Westhusin ◽  
D. Kraemer
Keyword(s):  
In Vitro Maturation ◽  
Calf Serum ◽  
Cumulus Cell ◽  
Germinal Vesicle ◽  
Cumulus Cells ◽  
Culture Conditions ◽  
Germinal Vesicle Breakdown ◽  
Significant Difference ◽  
Culture Duration

The objectives of this study were to determine whether the percentage of canine oocytes that resume meiosis during in vitro maturation could be increased by either increasing culture duration or by removing approximately one-half of the cumulus cells 24 h after oocytes were placed into culture. Canine female reproductive tracts were collected from a local clinic and ovaries were minced in warm TL-HEPES. Oocytes with a consistently dark ooplasm and at least two layers of cumulus cells were selected, cultured in a basic canine oocyte in vitro maturation medium consisting of TCM-199 with Earl's salts, 2.92 mM Ca-lactate, 20 mM pyruvic acid, 4.43 mM HEPES, 10% fetal calf serum, 1% Penicillin/Streptomycin (GibcoBRL, Grand Island, NY, USA), and 5 μg/mL porcine somatotropin, and incubated at 38.5°C in 5% CO2 in humidified air. Treatment groups were randomly assigned and oocytes were cultured for 60, 84, or 132 h (Basic). From each of these groups, one-half of the oocytes were pipetted through a fine bore pipette to partially remove the cumulus cells 24 h after the start of culture (Basic–1/2). At the end of culture, all oocytes were denuded and the nuclear status was observed with Hoechst 33342 under ultraviolet fluorescence. All data were analyzed by ANOVA with P < 0.05. Since the canine oocyte is ovulated at the germinal vesicle (GV) stage of meiosis and requires up to five days to mature in the oviduct, it was hypothesized that an increased culture time would allow for more oocytes to undergo nuclear maturation to metaphase II (MII). It was also hypothesized that partial removal of cumulus cells would decrease the cumulus cell component in the ooplasm that sustains meiotic arrest, allowing for more oocytes to resume meiosis (RM = germinal vesicle breakdown to MII). Results within each treatment group indicate that there is no significant difference between culture duration and the percent of oocytes that mature to MII. Additionally, there was no significance in the percent of oocytes that resumed meiosis after partial cumulus cell removal. Taken together, these data suggest that neither treatment is effective in canine in vitro maturation systems, given the current maturation culture conditions. Table 1. Nuclear status* of oocytes for three time periods with or without partial cumulus cell removal

337 EFFECTS OF CANINE SYNTHETIC OVIDUCT FLUID MEDIUM SUPPLEMENTED WITH THE VARIOUS ENERGY SUBSTRATES ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 276
Author(s):  
H. J. Oh ◽  
M. K. Kim ◽  
Y. H . Fibrianto ◽  
G. Jang ◽  
H. J. Kim ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Germinal Vesicle ◽  
The Other ◽  
Germinal Vesicle Breakdown ◽  
Energy Substrates ◽  
Fluid Medium ◽  
Nuclear Maturation ◽  
First Polar Body ◽  
Oviduct Fluid

In most mammals, maturation occurs within the ovarian follicle, and preovulatory oocytes are ovulated and ready for fertilization within the oviduct. In contrast, bitch ovulate primary oocytes, over a three day period, undergo both maturation and fertilization within the oviduct. The present study was conducted to evaluate the effects of canine synthetic oviduct fluid (cSOF) supplemented with the various energy substrates on in vitro maturation of canine oocytes. Oocytes were recovered by mincing ovaries collected after ovariohysterectomy in bitches at the follicular stage. Only oocytes with more than two layers of cumulus cells and with homogeneous cytoplasm >100 mm in diameter were selected. Then, oocytes cultured in tissue culture medium (TCM)-199 (control) or cSOF supplemented with various concentrations of glucose (0, 1.11, 3.89, or 5.56 mM, Exp. 1) or fructose (0, 1.11, 3.89, or 5.56 mM, Exp. 1), pyruvate (0, 0.1, 0.25, or 0.5 mM, Exp. 2) or lactate (0, 0.5, 1.0, or 5.0 mM, Exp. 3). In Exp. 4, the combined effects of glucose (1.11 mM), pyruvate (0.5 mM) and lactate (5.0 mM) on nuclear maturation of canine oocytes were investigated. A total of 2990 canine oocytes from 205 ovaries were used for experiments with replication at least three times. The oocytes were cultured for 72 h at 38.5�C in a humidified atmosphere of 5% CO2 in air. After 72 h, the oocytes were stained with 1.9 �g/mL Hoechst 33342 in glycerol and then evaluated under UV light to determine the stage of meiosis as follows: germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII) with first polar body. The results of Exp. 1 showed that maturation of canine oocytes to MII was significantly higher (P < 0.05) in medium supplemented with 1.11 mM glucose (4.8%) than for the control (1.8%) and the other glucose-supplemented groups (0 to 1.8%). In Exp. 2, oocytes cultured in cSOF supplemented with 0.5 mM pyruvate showed a significantly higher (P < 0.05) maturation rate to MII (6.3%) than did the other pyruvate-supplemented (0, 0.8, or 2.5%) groups or the control (2.4%). In Exp. 3, more oocytes were matured to the MII stage in cSOF supplemented with 5.0 mM lactate (7.3%) than were the other lactate-supplemented groups (0 to 2.4%) or the control (2.5%). Results of Exp. 4 showed more oocytes progressed to MII in cSOF supplemented with 0.5 mM pyruvate (8.2%), 1.11 mM glucose + 0.5 mM pyruvate (7.4%), or 1.11 mM glucose + 0.5 mM pyruvate 0.5 + 5.0 mM lactate (7.3%) than did the other combination groups (2.2 to 5.2%). In conclusion, the present study demonstrated that supplementing cSOF with 1.11 mM glucose, 0.5 mM pyruvate, or 5.0 mM lactate significantly increased the maturation of canine oocytes to MII, and the combined supplementation of 1.11 mM glucose, 0.5 mM pyruvate, and 5.0 mM lactate further promoted oocyte nuclear maturation compared to 1.11 mM glucose alone and the control. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

Effects of follicle-stimulating hormone, bovine somototrophin and okadaic acid on cumulus expansion and nuclear maturation of Blue fox (Alopex lagopus) oocytes in vitro

Zygote ◽  
1998 ◽  
Vol 6 (4) ◽  
pp. 299-309 ◽  
Author(s):  
Vlastimil Sršeň ◽  
Jaroslav Kalous ◽  
Eva Nagyova ◽  
Peter šutovský ◽  
W. Allan King ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Okadaic Acid ◽  
Germinal Vesicle ◽  
Alopex Lagopus ◽  
Germinal Vesicle Breakdown ◽  
Cumulus Expansion ◽  
Corona Radiata ◽  
Blue Fox

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte–cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 μm in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 µm in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

250 CYTOPLASMIC DYNEIN INTERMEDIATE CHAIN AND DYNACTIN p150Glued EXHIBIT DISTINCT SPATIAL AND TEMPORAL MICROTUBULE ASSOCIATIONS DURING BOVINE IN VITRO MATURATION AND ARE AFFECTED BY FOLLICLE SIZE

2008 ◽  
Vol 20 (1) ◽  
pp. 204
Author(s):  
S. E. Racedo ◽  
M. C. Branzini ◽  
D. Salamone ◽  
V. Y. Rawe ◽  
H. Niemann
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Chromatin Condensation ◽  
Calf Serum ◽  
Germinal Vesicle ◽  
Cytoplasmic Dynein ◽  
Meiotic Spindle ◽  
Germinal Vesicle Breakdown ◽  
Intermediate Chain

Microtubule molecular motors are critically involved in transporting vesicles during interphase, in building and maintaining spindles during mitosis and meiosis, and also in the localization of various organelles. DYNC1I1 (cytoplasmic dynein 1 intermediate chain) and its cofactor DCTN1 (dynactin p150Glued) are crucial for oocyte maturation but their role during mammalian female meiosis is not yet known. The goal of this study was to analyze the dynamics of these proteins in oocytes collected from different-size follicles at different stages of in vitro maturation (IVM), i.e., germinal vesicle stage (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), and metaphase II (MII), and their association with microtubules. Ovaries were collected at a local abattoir. Cumulus–oocyte complexes (COCs) were aspirated from follicles either <2 mm or 2–8 mm in size and matured in M199, supplemented with 1% fatty acid-free BSA, 10 UI pregnant mare serum gonadotropin (PMSG)/5 UI HCG, and 100 µm cysteamine, at 39�C and 5% CO2. Follicle sizes and time points for fixation were: GV-0 h; GVBD-8 h for oocytes <2 mm and 9 h for oocytes 2–8 mm; MI-15 h; MII-24 h (Racedo et al. 2007, pub. online: 10.1002/mrd.20770). The distribution of the proteins was assessed by immunocytochemistry and laser confocal microscopy. The attached cumulus cells and zona pellucida of oocytes were removed in TALP-HEPES medium containing 1 mg mL–1 hyaluronidase and 2 mg mL–1 pronase, respectively. The oocytes were then incubated in a fixation–permeabilization solution containing 2% formaldehyde and 0.1%Triton X-100 for 1 h. Samples were then blocked for 1 h in 10 mm PBS + 0.3% BSA + 1% fetal calf serum (ICC blocking solution). The primary antibody was applied over night at 4�C, followed by treatment with fluorochrome-conjugated secondary antibodies for 1 h at 37�C in the dark. After RNase treatment, oocytes were incubated with TOTO-3 (Invitrogen, Carlsbad, CA, USA) to visualize the DNA. The material was mounted in an anti-fade medium (Vectashield�, Vector Laboratories, Burlingame, CA, USA) and imaged with a Zeiss laser scanning microscope. Immediately after chromatin condensation (GVBD), dynactin was in close association with the DNA and interacting with the spindles in MI and MII oocytes recovered from large follicles. No clear association with the DNA was observed in GVBD oocytes obtained from small follicles; little dynactin was found in MI and MII spindles. Dynein localization did not differ from dynactin in GVs and was homogeneously distributed in the cytoplasm of both groups of follicles. Dynein was not associated with the DNA in the GVBD stage while at MI and MII it was associated with the meiotic spindle. The association of dynein with microtubules was weak at the MI stage in oocytes from small follicles. Results provide insight into the regulatory mechanisms of oocyte maturation and a possible relationship with oocyte competence.

140 EFFECTIVENESS OF WASHING PROCEDURES FOR REMOVING BRUCELLA ABORTUS FROM IN VIVO-DERIVED WOOD BISON EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 162
Author(s):  
J. M. Palomino ◽  
M. P. Cervantes ◽  
G. Mastromonaco ◽  
R. J. Mapletoft ◽  
B. Allan ◽  
...  
Keyword(s):  
Zona Pellucida ◽  
Brucella Abortus ◽  
Calf Serum ◽  
Petri Dish ◽  
Bison Bison ◽  
Genetic Reserve ◽  
Wood Bison ◽  
Wood Buffalo National Park

Endemic brucellosis threatens wild herds of wood bison (Bison bison athabascae) in and around Wood Buffalo National Park, the largest genetic reserve of wood bison in the world. The overall goal of our project was to produce and preserve disease-free embryos for the purpose of conserving the genetic diversity of this species. The aim of the present experiment was to determine the effectiveness of washing procedures for removing Brucella bacteria from in vivo-derived wood bison embryos exposed in vitro to the pathogen. Wood bison cows were given 300 mg im of Folltropin diluted in 0.5% hyaluronan on the day of follicle wave emergence (Day 0) and 100 mg im of hyaluronan on Day 2, and then given 2500 IU im of hCG on Day 5 and inseminated 12 and 24 h later. Embryos were collected on Day 13. The experiment was done in 6 replicates (n = 4 bison/replicate) and an average of 9 embryos/replicate were collected. Zona pellucida-intact embryos were kept in holding medium (PBS + 2% fetal calf serum) and transported to a Biosafety Level 3 laboratory at the International Vaccine Centre, University of Saskatchewan. Embryos were transferred through 5 aliquots of holding medium to remove any contaminant before exposure to Brucella. Embryos were divided equally into 2 Petri dishes (representing later wash groups with v. without antibiotics) containing 2.7 mL of holding medium (n = 2 to 7 embryos per dish/replicate). In a Class II biosafety cabinet, Brucella abortus biovar 1 (1 × 107 to 1 × 109 CFU mL–1 in 0.3 mL) was added to each Petri dish and incubated for 2 h at 37°C in 8% CO2. A sample of holding medium was taken before exposure and after incubation for culture as negative and positive controls, respectively. After incubation, embryos in each Petri dish were subjected to a 10-step washing procedure (according to the IETS Manual, 2010) using wash medium (PBS + 0.4% BSA) without antibiotics or with antibiotics (100 IU mL–1 of penicillin + 100 μg mL–1 of streptomycin). The embryo wash medium was cultured at wash steps 1, 3, 6, and 9. After the tenth wash, the zona pellucida of each embryo was ruptured mechanically using a glass pipette and embryos were cultured individually. Culturing of samples was done on sheep blood agar and specific identification of Brucella organisms was done by PCR. Brucella abortus was detected in 3 embryos from the group washed in medium without antibiotics (3/27), whereas all embryos washed in medium with antibiotics were culture negative (0/27). Brucella abortus was not detected in wash media after the third wash in either group (with or without antibiotics). In summary, Brucella abortus was removed from 89% of in vitro-exposed wood bison embryos using the washing procedure without antibiotics, and from 100% using the washing procedure with antibiotics. Results validate the embryo washing technique for producing Brucella-free wood bison embryos. Thanks to the Canadian Food Inspection Agency for the field strain of Brucella abortus, Bioniche AH for Folltropin and embryo collection supplies, Merck AH for hCG (Chorulon), and Intervac/VIDO for technical and logistical support.

183 IN VITRO EMBRYO PRODUCTION: A TOOL TO PRESERVE THE THREATENED WOOD BISON (BISON BISON ATHABASCAE)

2016 ◽  
Vol 28 (2) ◽  
pp. 222
Author(s):  
M. P. Cervantes ◽  
J. M. Palomino ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
G. Mastromonaco ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Animal Health ◽  
Developmental Competence ◽  
Bison Bison ◽  
High Humidity ◽  
Embryo Production ◽  
Wood Bison ◽  
In Vitro Embryo Production

In vitro embryo production is being developed as a tool to restore genetic diversity and eliminate endemic disease in wood bison. In a recent study in wood bison, we found that more oocytes reached maturity after 30 h v. 24 h of in vivo maturation following hCG treatment (Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). An additional 4 h of in vitro maturation after an in vivo maturation period of 30 h also had a positive effect on developmental competence. The present study was designed to test the hypothesis that extending the in vivo maturation time (i.e. extending the interval between hCG treatment and cumulus-oocyte complex (COC) collection) from 30 to 34 h will improve in vitro embryo production in wood bison. Follicular wave development was synchronised among female wood bison (n = 28, 6 to 10 years old) by transvaginal follicular ablation. The study was done in 4 replicates (n = 7 bison per replicate). Bison were given FSH 1 day (300 mg) and 3 days (100 mg) after ablation for ovarian superstimulation, and hCG (2500 IU) 5 days after ablation to induce COC maturation in vivo. Bison were divided randomly into 2 groups (n = 14/group) in which COC were collected transvaginally at either 30 h or 34 h after hCG treatment. Expanded COC from the 30 h group were fertilised after 4 h of in vitro maturation, while expanded COC from the 34 h group were fertilised immediately. Oocytes and sperm were co-incubated (Day 0 = day of fertilization) for 18 h at 38.5°C in 5% CO2 in air and high humidity. Presumptive zygotes were cultured in 4-well dishes containing 500 μL well–1 of CR1aa medium at 38.5°C, 5% CO2, 5% O2, 90% N2 and high humidity, and assessed on Days 3, 7, and 8 (Day 0 = day of fertilization). Data were compared between groups by Chi-squared analysis. No effect of replicate was found. Compared to the 30 h group, the 34 h group had a greater cleavage rate [55/74 (74%) v. 49/86 (57%); (P < 0.05)], and a greater blastocyst rate on Day 7 [25/74 (34%) v. 9/86 (10%); (P < 0.05)] and Day 8 [(40/74 (54.1%) v. 32/86 (37.2%); (P < 0.05)]. We concluded that an extended period of in vivo maturation is beneficial for embryo production after in vitro fertilization in wood bison. We thank Vetoquinol Canada for providing FSH (Folltropin-V) and hyaluronan (MAP-5) and thank Merck Animal Health for hCG (Chorulon).

scholarly journals The effect of interaction between macromolecule supplement and oxygen tension on bovine oocytes and embryos cultured in vitro

Zygote ◽  
2009 ◽  
Vol 17 (4) ◽  
pp. 321-328 ◽  
Author(s):  
G.Z. Mingoti ◽  
V.S.D. Caiado Castro ◽  
S.C. Méo ◽  
L.S.S. Barretto ◽  
J.M. Garcia
Keyword(s):  
Oxygen Tension ◽  
Embryo Development ◽  
Calf Serum ◽  
Atmospheric Condition ◽  
Germinal Vesicle ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Germinal Vesicle Breakdown ◽  
Nuclear Maturation

SummaryAiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin – BSA, polyvinyl alcohol – PVA, polyvinyl pyrrolidone – PVP, Ficoll, KnockoutSR, or fetal calf serum – FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

In vivo and in vitro maturation of oocytes collected from superstimulated wood bison ( Bison bison athabascae ) during the anovulatory and ovulatory seasons

2016 ◽  
Vol 173 ◽  
pp. 87-96 ◽  
Author(s):  
Miriam P. Cervantes ◽  
J. Manuel Palomino ◽  
Muhammad Anzar ◽  
Reuben J. Mapletoft ◽  
Gregg P. Adams
Keyword(s):  
In Vitro Maturation ◽  
Bison Bison ◽  
Wood Bison

248 IN VITRO EMBRYO PRODUCTION USING IN VIVO-MATURED OOCYTES COLLECTED TRANSVAGINALLY FROM WOOD BISON (BISON BISON ATHABASCAE)

2015 ◽  
Vol 27 (1) ◽  
pp. 213
Author(s):  
M. P. Cervantes ◽  
J. M. Palomino ◽  
M. Anzar ◽  
R. J. Mapletoft ◽  
G. Mastromonaco ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Animal Health ◽  
Calf Serum ◽  
Developmental Competence ◽  
High Humidity ◽  
Embryo Production ◽  
Wood Bison ◽  
In Vitro Embryo Production

Reproductive technologies are being developed to help conserve the genetic diversity of wood bison, a threatened species. To date, the efficiency of in vitro embryo production in bison is very low and appears to be related to inadequate in vitro conditions for oocyte maturation. Recently, we have attempted to circumvent the problem by inducing oocyte maturation in vivo and found that more than one-third of superstimulated oocytes collected 30 h after administration of hCG were at metaphase II (Cervantes et al. 2013 Reprod. Fertil. Dev. 25, 283; Cervantes et al. 2014 Reprod. Fertil. Dev. 26, 199). We hypothesise that additional maturation time in vitro, after in vivo maturation, will allow the remaining oocytes to reach the MII stage, and thus improve in vitro embryo production in wood bison. The objective of this study was to determine the effect of an additional 4 h of in vitro maturation on the developmental competence of oocytes collected 30 h after hCG treatment. Wood bison cows (n = 24) were superstimulated by the administration of 300 mg of FSH (Folltropin-V) diluted in 0.05% hyaluronan on the day of follicular wave emergence and 100 mg of FSH in hyaluronan 2 days later. Bison were administered 2500 IU of hCG (Chorulon) IM 2 days after the last dose of FSH. Transvaginal ultrasound-guided follicle aspiration was performed 30 h after hCG treatment to collect cumulus-oocyte complexes (COC). Expanded COC (with no evidence of degeneration) were selected and assigned randomly to 2 groups (n = 38 COC/group) in which IVF was done immediately, or after 4 h of in vitro maturation in TCM 199 with 5% calf serum, 5 μg mL–1 pLH, 0.5 μg mL–1 pFSH, and 0.05 μg mL–1 gentamicin, at 38.5°C, 5% CO2 and high humidity. In vitro fertilization (Day 0) was done with frozen-thawed wood bison semen (dose 5 × 106 sperm mL–1) in Brackett-Oliphant medium at 38.5°C, 5% CO2, and high humidity. Presumptive zygotes were cultured in CR1aa plus 5% calf serum, at 38.5°C and in 5% CO2, 5% O2, and 90% N2 and high humidity. Cleavage was recorded on Day 3, and blastocyst formation was recorded on Days 7 and 8. Cleavage and blastocyst rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-square analysis. No difference was detected between groups (immediate fertilization v. after an additional 4 h in vitro) in cleavage rate on Day 3 (55.3 v. 60.5%, respectively, P = 0.82), or blastocyst rate on Day 7 (13.2 v. 23.7%, respectively, P = 0.37). However, the blastocyst rate on Day 8 was higher in the COC group exposed to an additional 4 h of in vitro maturation (18.4 v. 44.7%, respectively, P = 0.03). Results support the hypothesis that an additional short period of in vitro maturation improves the developmental competence of oocytes collected after 30 h of in vivo maturation.We thank Bioniche Animal Health for providing FSH (Folltropin-V) and hyaluronan (MAP-5), and Merck Animal Health for hCG (Chorulon).

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