Effect of season and superstimulatory treatment on in vivo and in vitro embryo production in wood bison (
            Bison bison athabascae
            )

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).

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133 In Vitro Embryo Production from Oocytes Collected from Non-Supertimulated Wood Bison (Bison bison athabascae) Following Maturation In Vitro Using Portable Incubators

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab133 ◽

2018 ◽

Vol 30 (1) ◽

pp. 206

Author(s):

M. P. Cervantes ◽

G. P. Adams ◽

M. Anzar ◽

J. M. Palomino ◽

G. F. Mastromonaco

Keyword(s):

Embryo Development ◽

Transvaginal Ultrasound ◽

Calf Serum ◽

Cumulus Cells ◽

Bison Bison ◽

Embryo Production ◽

Commercial Medium ◽

Wood Bison ◽

In Vitro Embryo Production

This study was done to determine the feasibility of in vitro embryo production in wood bison during the anovulatory season, without ovarian superstimulation or follicle wave synchronization, to simulate collection conditions in a wild or field setting. The experiment provided the opportunity to compare embryo development using 2 different maturation media and incubator systems. The cumulus-oocyte complexes (COC) were collected by transvaginal ultrasound-guided follicle aspiration during May from non-superstimulated bison. Compact COC were allocated to 2 groups and matured in standard maturation medium using a portable gassed incubator, or in commercial medium using a portable non-gassed incubator. In the former (Standard), the COC were placed in a round-bottomed tube containing TCM-199 medium with 5% calf serum, 5 μg mL−1 LH, 0.5 μg mL−1 FSH, and 0.05 μg mL−1 gentamicin, and the tube was placed in a portable incubator with 5% CO2. In the latter (Commercial), COC were placed in a round-bottom tube containing the commercial medium (Boviteq, Saint-Hyacinthe, QC, Canada), and placed in a portable incubator without CO2. After 24 h of maturation, oocytes were fertilized in vitro (Day 0) in Brackett-Oliphant medium at 38.5°C in a conventional incubator with 5% CO2 humidified atmosphere. 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 embryo development was recorded on Day 7. Cleavage and transferable embryo rates (calculated from the total number of oocytes submitted to IVF) were compared between groups by chi-squared test. No difference in cleavage rates was observed between Standard and Commercial treatment groups [68.1 (32/47) v. 79.2% (57/72), respectively; P = 0.25], nor in morula plus blastocyst rates on Day 7 (36.2 v. 45.8%, respectively; P = 0.39). However, the rate of transferable embryos (grade 1 and grade 2) on Day 7 was higher in the Commercial group (38.9 v. 12.8%; P < 0.01). Of the COC in the Commercial group, a higher number of morula plus blastocyst were observed to be compact good COC (>3 layers of cumulus cells) than compact regular COC (1-3 layers of cumulus cells) (66.7 v. 31.0% respectively; P < 0.05), along with a higher number of transferable embryos on Day 7 (60.0 v. 23.8%, respectively; P < 0.05). In conclusion, wood bison oocytes collected during the anovulatory season from non-superstimulated, non-synchronized bison and matured in vitro using portable incubators were competent to develop to the morula and blastocyst stages following IVF and culture. These results are important for future plans that require transporting oocytes from remote collection sites to the IVF laboratory, particularly with respect to the effectiveness of commercial maturation media which does not require CO2 supplementation. Research was supported by the Natural Sciences and Engineering Research Council of Canada.

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

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab248 ◽

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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In vivo and in vitro embryo production in goats

Small Ruminant Research ◽

10.1016/j.smallrumres.2009.12.037 ◽

2010 ◽

Vol 89 (2-3) ◽

pp. 144-148 ◽

Cited By ~ 19

Author(s):

M.T. Paramio

Keyword(s):

Embryo Production ◽

In Vitro Embryo Production

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Predictive value of in vitro embryo production characteristics for in vivo fertility of bulls

Theriogenology ◽

10.1016/s0093-691x(97)82386-2 ◽

1997 ◽

Vol 47 (1) ◽

pp. 259 ◽

Cited By ~ 1

Author(s):

L.M.T.E. Lansbergen ◽

E.H.A.T. Hanenberg ◽

A.M. van Wagtendonk-de Leeuw

Keyword(s):

Predictive Value ◽

Embryo Production ◽

In Vitro Embryo Production ◽

Production Characteristics

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In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH

Animal Reproduction Science ◽

10.1016/s0378-4320(00)00186-x ◽

2000 ◽

Vol 63 (3-4) ◽

pp. 145-158 ◽

Cited By ~ 30

Author(s):

K.L Goodhand ◽

M.E Staines ◽

J.S.M Hutchinson ◽

P.J Broadbent

Keyword(s):

Bovine Oocyte ◽

Embryo Production ◽

Oocyte Recovery ◽

In Vitro Embryo Production ◽

Oocyte Donors

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279VASCULAR MORPHOMETRY OF BOVINE PLACENTOMES IN LATE GESTATION FROM EMBRYOS PRODUCED IN VIVO OR IN VITRO

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab279 ◽

2004 ◽

Vol 16 (2) ◽

pp. 259

Author(s):

J.R. Miles ◽

C.E. Farin ◽

K.F. Rodriguez ◽

J.E. Alexander ◽

P.W. Farin

Keyword(s):

Blood Vessels ◽

Maternal Blood ◽

Volume Density ◽

Late Gestation ◽

Embryo Production ◽

Vascular Volume ◽

Treatment Groups ◽

In Vitro Embryo Production

The role of the vascular supply in the development of placentas from embryos produced in vitro is poorly understood. The objective of this study was to determine the effects of in vitro embryo production on morphometry of blood vessels within fetal (cotyledonary) and maternal (caruncular) components of the placentome during late gestation. In vivo-produced embryos were recovered from superovulated Holstein cows on Day 7 after estrus. For in vitro embryo production, oocytes were aspirated from the ovaries of Holstein cows, matured in vitro, and then fertilized. Presumptive zygotes with their cumulus cells were transferred into M-199 with 10% estrus cow serum and cultured for 168h post-insemination. Semen from the same Holstein sire was used for the production of in vivo and in vitro embryos. Single blastocysts from each production system were transferred into the uteri of heifers. On Day 222 of gestation, fetuses and placentas were recovered in utero (in vivo, n=12; in vitro, n=12). Placentomes were collected, fixed and sectioned. Fetal and maternal blood vessels were identified within placentome sections using immunocytochemistry for vascular endothelial growth factor (VEGF) protein. A total of 4.8×105μm2 of tissue were examined from each placentome. Stereological methods were used to determine the volume densities of fetal and maternal blood vessels. Data were analyzed by GLM procedures. Fetuses were heavier (P=0.03) in the in vitro group (20.7±1.0kg, LS mean±SEM) compared to the in vivo group (17.3±1.0kg). Placentas were also heavier (P=0.06) for the in vitro group (2.5±0.2kg) compared to the in vivo group (2.0±0.2kg). Placental efficiency, calculated as fetal weight/placental weight, was similar between the two treatment groups (9.0±0.5 and 8.9±0.5 for in vivo and in vitro, respectively). Fetal vascular volume density in placentomes was not different between the two treatment groups (5.4±0.3% and 5.4±0.3% for in vivo and in vitro, respectively). In contrast, maternal vascular volume density was greater (P=0.02) for placentomes in the in vitro group (5.9±0.3%) compared to in vivo controls (4.9±0.3%). In summary, compared to placentomes from embryos produced in vivo, placentomes from embryos produced in vitro had similar volume density of fetal vessels, but had significantly increased volume density of maternal vessels. Supported by the State of North Carolina.

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97 IN VIVO AND IN VITRO EMBRYO PRODUCTION WITH Y-SEXED SORTED OR CONVENTIONAL SEMEN IN BEEF CATTLE

Reproduction Fertility and Development ◽

10.1071/rdv26n1ab97 ◽

2014 ◽

Vol 26 (1) ◽

pp. 162

Author(s):

H. Tribulo ◽

J. Carcedo ◽

R. Tribulo ◽

J. Menajovsky ◽

B. Bernal ◽

...

Keyword(s):

Animal Health ◽

T Test ◽

High Fertility ◽

In Vitro Production ◽

Embryo Production ◽

Sexed Semen ◽

Chi Squared ◽

In Vitro Embryo Production

An experiment was designed to evaluate in vivo and in vitro embryo production following the use of frozen–thawed conventional or Y-sexed semen from a Brangus bull with known high fertility. For in vivo embryo production, Brangus heifers (n = 12) were superovulated twice in a crossover design and inseminated with sexed or conventional semen. On Day 0, all heifers received an intravaginal progesterone device (DIB 1 g, Syntex S.A., Buenos Aires, Argentina) and 2.5 mg oestradiol benzoate and 50 mg progesterone (Progestar, Syntex S.A.) by intramuscular injection (IM). On Day 4, heifers were superstimulated with 200 mg of NIH-FSH-P1 Folltropin-V (Bioniche Animal Health, Belleville, Ontario, Canada) in twice-daily decreasing doses over 4 days. In the a.m. and p.m. of Day 6, all heifers received PGF2a (Ciclase, Syntex) and DIBs were removed in the p.m.. In the a.m. of Day 8, heifers received 100 μg de Gonadolerin (Gonasyn, Syntex S.A.) and were randomly allocated to receive either one straw of conventional semen (24 × 106 sperm per dose) 12 and 24 h later or two straws of sexed semen (2.4 × 106 sperm per dose) 18 and 24 h after GnRH. Ova/embryos were collected nonsurgically on Day 15 and evaluated following IETS recommendations. Means were compared by t-test. Mean ( ± s.e.m.) number of ova/embryos, fertilized ova, and transferable embryos were 14.8 ± 2.7, 9.4 ± 1.8, and 7.1 ± 1.7 v. 16.8 ± 3.1, 9.9 ± 2.5, and 8.1 ± 2.0 for donors inseminated with conventional or sexed semen, respectively (P > 0.6). For in vitro production, oocytes were obtained from 50 ultrasound-guided follicle aspiration (OPU) sessions that was performed at random stages of the oestrous cycle and without superstimulation in 22 Brangus cows and heifers. Oocytes were classified and matured in TCM-199 medium with NaHCO3 and supplemented with 1% fetal bovine serum. Semen samples from the same bull used for in vivo embryo production were selected using Percoll and capacitated in Fert medium and used at a final concentration of sperm/mL for nonsexed semen and 2 × 106 sperm mL–1 for sexed semen. After 16 h (sexed) or 18 h (conventional) in Fert medium, zygotes were denuded and cultured in SOF supplemented with 0.4% BSA under oil at 37°C, 5% CO2 and saturated humidity for 7 days. The total number of oocytes matured and fertilized was 528 and 318 for conventional and sexed semen, respectively. Means were compared by t-test and proportions by chi-squared test. Mean (± s.e.m.) number of cleaved zygotes and blastocysts produced per OPU session did not differ between conventional (11.0 ± 1.4 and 7.1 ± 1.0) and sexed (8.7 ± 0.8 and 4.9 ± 0.7; P > 0.2) semen. However, the proportion of cleaved zygotes and blastocysts produced were significantly higher (P < 0.05) with conventional semen (61.2%; 329/538 and 39.4%; 212/538) than with sexed semen (54.4%; 173/318 and 30.8%; 98/318), respectively. In conclusion, comparable number of embryos can be obtained in vivo with sexed or conventional semen from a bull with proven high fertility. However, the proportion of blastocysts produced in vitro is likely to be reduced following the use of sexed as compared with conventional semen from the same bull.

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