102 IDENTIFICATION OF CONTRASTED PHENOTYPES IN THE BOVINE FROM REPEATED IN VIVO AND IN VITRO EMBRYO PRODUCTION FOLLOWING SUPEROVULATION

2008 ◽  
Vol 20 (1) ◽  
pp. 131 ◽  
Author(s):  
C. Guyader-Joly ◽  
S. Ponchon ◽  
C. Gonzalez ◽  
B. Marquant-Le Guienne ◽  
L. Clément ◽  
...  
Keyword(s):  
Developmental Competence ◽  
Marker Genes ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Cell Monolayers ◽  
In Vitro Embryo Production ◽  
Vitro Development

This study was initiated to evaluate maternal influence on in vivo and in vitro bovine embryo production and identify animals with contrasted phenotypes for reproductive parameters. Nine Montbéliard cows raised on the same farm and with various genetic origins were included in the study. In vivo-derived embryos were collected nonsurgically from superovulated cows on day 7 after AI (34 collections). Immature oocytes were collected by ovum pickup from the same (superovulated) cows (36 sessions) then matured, fertilized (day 0) with the same bull, and cultured in vitro until day 7 on Vero cell monolayers in B2 medium. Grade 1 to 3 in vivo and grade 1 and 2 in vitro produced embryos deemed viable according to IETS criteria. The mean numbers of blastocysts and viable blastocysts per session per cow were, respectively, 8.3 ± 5.5 and 4.8 ± 3.6 in the in vivo system and 2.5 ± 2.6 and 1.8 ± 2.2 in the in vitro system. Individual cow data of in vivo and in vitro embryo production were analyzed by ANOVA (GLM program in SAS; SAS Institute Inc., Cary, NC, USA). Results are presented in Table 1: mean ± SD. Quantity and quality of produced embryos varied significantly among females, and production in vivo and in vitro was not systematically related. Contrasted phenotypes were identified according to their viable blastocyst rates in both systems (in vivo: no viable/recovered; in vitro: no viable/inseminated). Two females presented a relatively high percentage of viable blastocysts in both systems (over 30% in vitro and over 70% in vivo, Table 1). On the contrary, 2 females showed low percentages of blastocysts in the 2 systems (<10% in vitro and <50% in vivo). For most other females, the percentage of in vivo-produced blastocysts was relatively high (>50%), but in vitro development rates were low. Only one female (C3) presented the inverse situation. Oocytes collected from animals with contrasted phenotypes will be analysed for gene expression to identify marker genes associated with oocyte developmental competence. Table 1. This study was conducted with financial support of ‘Genanimal’ – French Ministry of Research (#03P409) and Apis-Gene.

159 IN VITRO EMBRYO PRODUCTION FROM HOLSTEIN CALF OOCYTES RECOVERED BY LAPAROSCOPIC OVUM PICKUP

2016 ◽  
Vol 28 (2) ◽  
pp. 209
Author(s):  
H. Baldassarre ◽  
L. Currin ◽  
L. Michalovic ◽  
W. Glanzner ◽  
K. Gutierrez ◽  
...  
Keyword(s):  
Reproductive Organs ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Oocyte Competence ◽  
Veterinary Practitioner ◽  
Collection Tube ◽  
In Vitro Embryo Production

Oocyte competence and reproductive biology in prepubertal heifer calves are not fully understood. Multiple publications have reported high oocyte yields recovered from calves aged 2–6 months old but low embryo development rates following in vitro embryo production. The objective of this study was to characterise the developmental competence of oocytes from young calves. We report herein the oocyte/embryo yields obtained from 6 Holstein heifer calves that were subjected to gonadotropin stimulation and laparoscopic ovum pick-up (LOPU) every 2 weeks, starting at 2 months of age and ending at 5 months of age. The LOPU was conducted under general anaesthesia with the animal lying in dorsal recumbency on a table with a 45-degree angle to facilitate the visualisation of reproductive organs. Briefly, looking through the laparoscope, the ovarian surface was exposed by pulling from the fimbria with an atraumatic grasping forceps. The follicle contents were aspirated using a 20 G needle mounted on a pipette, which was connected to a collection tube and a vacuum pump. Media and procedures for aspiration, in vitro maturation (IVM), IVF, and in vitro culture (IVC) were standard in use for commercial adult bovine embryo production. Because of the small number of animals and the multifactorial variables in play (age, number of previous treatments and aspirations, etc.), in this preliminary study we focused on the overall oocyte/embryo yield and the potential effects of LOPU on ovarian integrity. A total of 766 follicles were aspirated (avg. 17/calf per session) resulting in 625 cumulus-oocyte complexes (COC) recovered (avg. 14/calf per session; 82% recovery rate). A total of 457 (73%) COC were graded eligible for IVM, of which 353 cleaved (77%) and 109 (24%) reached a viable blastocyst stage at the end of IVC, of which 42 (38.5%) were graded as freezable. In balance, ~2 viable blastocysts/calf per session were produced. No adhesions or sequels were observed in the animals up to the last LOPU session, as well as 2 weeks after the last LOPU when the animals were evaluated by rectal palpation by an experienced OPU veterinary practitioner. Further studies will look into other aspects of oocyte developmental competence to better understand this biological process.

249 AN EFFICIENT AND REPEATABLE IN VITRO FERTILIZATION TECHNIQUE IN THE EQUINE FOR PRESERVATION OF ENDANGERED SPECIES

2015 ◽  
Vol 27 (1) ◽  
pp. 214
Author(s):  
C. Douet ◽  
O. Parodi ◽  
F. Reigner ◽  
P. Barrière ◽  
G. Goudet
Keyword(s):  
Embryo Development ◽  
Intracytoplasmic Sperm Injection ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
In Vitro Development ◽  
Vitro Development

Most wild equids are currently endangered or threatened, as mentioned in the International Union for the Conservation of Nature Red List, and several domestic horse breeds are at risk of extinction. Genome resource banking requires cryoconservation of semen, oocytes, and/or embryos. Embryo production in equids is limited in vivo because routine induction of multiple ovulation is still ineffective. Embryo production in vitro allows the production of several embryos per cycle that could easily be frozen because of their small size. Intracytoplasmic sperm injection has been widely adopted to generate horse embryos in vitro; however, intracytoplasmic sperm injection is time-consuming and requires expensive equipment and expertise in micromanipulation. Several attempts to establish an efficient IVF technique in the equine were performed, but reported IVF rates remain quite low and no repeatable equine IVF technique was available. Our objective was to develop an efficient and repeatable IVF technique in the equine. Immature cumulus-oocyte complexes (COC) were collected either from slaughtered mares in a local slaughterhouse or from our experimental mares by ovum pick up (OPU). The COC were cultured for 26 h in an in vitro maturation (IVM) medium or in preovulatory follicular fluid (FF) collected by OPU, pre-incubated for 30 min in oviducal fluid collected from slaughtered females, co-incubated for 18 h with fresh spermatozoa treated with procain, and cultured in SOF for 30 h. They were fixed and analysed either after 18 h IVF (experiment 1) or after 30 h in vitro development (experiment 2). In experiment 1, COC were collected from slaughtered mares and analysed after 18 h IVF. Zygotes with 2 pronuclei were observed. The IVF rate was similar for oocytes matured in IVM medium (22/33, 67%) or FF (24/42, 57%; chi-square test, P > 0.05). In experiment 2, COC were collected from slaughtered mares and from experimental mares and analysed after 30 h of in vitro development. We observed zygotes with 2 highly decondensed pronuclei, pronuclei decondensation being the first step of embryo development. For oocytes collected from slaughtered mares, the percentage of zygotes was similar for oocytes matured in IVM medium (8/11, 73%) or FF (10/15, 67%). For oocytes collected by ovum pickup, the percentage was similar for IVM medium (3/5, 60%) or FF (6/8, 75%). We also observed some embryonic structures with several nuclei, but the quality of these embryos was poor. In conclusion, we have established an efficient IVM-IVF technique that allows the first step of embryo development. Because we obtained similar results for 4 years, we consider that this efficient technique is repeatable. Further experiments are in progress to improve the quality of the embryos.

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).

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).

293 EFFECT OF GUAIAZULENE ON IN VITRO BOVINE EMBRYO PRODUCTION

2007 ◽  
Vol 19 (1) ◽  
pp. 262 ◽  
Author(s):  
I. Dimitriadis ◽  
E. A. Rekka ◽  
E. Vainas ◽  
G. S. Amiridis ◽  
C. A. Rekkas
Keyword(s):  
Lipid Peroxidation ◽  
Embryo Development ◽  
Antioxidant Properties ◽  
Thiobarbituric Acid ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Embryo Production ◽  
Reactive Material

The substrates used in in vitro embryo production (IVP) mimic the in vivo fluids in which oocytes mature, oocytes are fertilized, and the early embryos develop (follicular and oviductal fluid). It is well established that oxidative stress negatively affects in vitro culture (IVC) outcomes. Guaiazulene (G) is a component of chamomile species oil with known antioxidant properties. In the present study, all IVP media were modified by the addition of G solutions so that the former exhibited a total protection against induced lipid peroxidation (TPaLP) similar to that of the respective in vivo environment. The IVP outcomes were then compared between G-processed and control oocytes. Bovine preovulatory follicular (BF) and oviductal (BO) fluid samples were collected from 10 Holstein 4- to 5-year-old cows in estrus. TPaLP was assessed according to the samples&apos; ability to inhibit rat hepatic microsomal lipid peroxidation, by determination of the 2-thiobarbituric acid reactive material. TPaLP (mean % � SEM) of the BF and BO were 70.63 � 10.03 and 16.33 � 4.33, respectively, whereas those of the IVP [in vitro-matured (IVM), in vitro-fertilized (IVF), and IVC] media were lower (17.94 � 1.66, -1.82 � 0.78, and 14.57 � 1.26, respectively). TPaLP of the 0.1 mM G-modified IVP medium increased to 67.2 � 5.85, 19.98 � 2.49, and 69.19 � 6.22, respectively. A total of 2041 class A oocytes were used. The proportion of cleavage, early embryo development (embryos with more than 4 cells), or both after IVP (18 h IVM–5% CO2 in air, and 18 h IVF, 48 h IVC–5% CO2, 10% O2, 85% N) in the presence of G (n = 1237) during each of the IVP phases or any possible combination of IVP phases was compared with the respective control (C, n = 804). Statistical analysis was performed by a chi-squared test; P &lt; 0.05 was considered significant. G improved cleavage and embryo development rates when present during IVM (79.4 and 57.8% vs. 64.5 and 38.2% for C) or both IVM and IVC (78.0 and 60.7% vs. 57.8 and 36.5%, respectively). When present only during 18 h of IVF, G had no effect on embryo production. However, an increased embryo development rate resulted from the combined exposure to G during IVF and IVM (56.4 vs. 29.6%), during IVF and IVC (55.3 vs. 35.5%), or at all IVP phases (56.6 vs. 34.9%). The latter effect resembled the one obtained after G addition only to the IVC medium (62.5 vs. 39.7%, respectively). We concluded that the addition of G to IVP substrates, at concentrations that mimic the in vivo TPaLP conditions, could promote bovine IVP efficiency.

238 KNOCKING DOWN OF THYROID HORMONE RECEPTORS INHIBITS DEVELOPMENT OF EARLY BOVINE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 267
Author(s):  
N. Y. Rho ◽  
F. A. Ashkar ◽  
T. Revay ◽  
P. Madan ◽  
W. A. King
Keyword(s):  
Embryo Development ◽  
Reference Genes ◽  
Messenger Rna ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Blastocyst Formation ◽  
Embryo Production ◽  
Mrna Transcripts ◽  
In Vitro Embryo Production

Thyroid hormones (TH) play an important role in the physiology of vertebrates, ranging from the regulation of metabolic processes to cell proliferation, differentiation, and embryo development. We have previously shown a beneficial effect of supplementing TH in in vitro embryo production media. Recently, detection of TH receptors (TR) in oocytes and early stages of pre-implantation embryos indicated a possible regulatory role for TH in these stages (unpublished data). The objective of this study was to investigate the importance of TR expression in the pre-attachment bovine embryo in vitro. Bovine embryos, produced by standard in vitro embryo production procedures, were microinjected at the zygote stage with small interfering RNA (siRNA) specifically designed for knocking down either TR-α or TR-β. In addition, groups of zygotes were microinjected with scrambled siRNA (SI) or were not injected (NI), and these groups served as controls. Embryo developmental rates were assessed using light microscopy for blastocyst formation rates and expression of TR messenger RNA (mRNA) transcripts at the blastocyst stage was assessed by quantitative PCR across all groups. Expression of TR mRNA was normalized against glyceraldehyde 3-phosphate dehydrogenase, H2a, and 18S as reference genes. There was a significant decrease in blastocyst formation rates in both embryo groups injected with either TR-α (P < 0.002) and TR-β (P < 0.001) siRNA compared with the NI and SI groups. Moreover, the TR-β knockdown group exhibited a lower developmental rate than the TR-α knockdown group, which indicates a stronger inhibitory role for TR-β. Quantification of the level of TR mRNA expression in four groups normalized with three different reference genes shows a consistent significant reduction in the levels of TR-α (P < 0.05) and TR-β (P < 0.02) mRNA transcripts compared with the NI and SI groups. However, TR-β expression was inhibited more than was TR-α expression. In conclusion, the results indicate that knocking down either TR-α or TR-β restrains embryo development. This suggests that TH play a vital role in the regulation of embryo development through their receptors during bovine early embryogenesis. The specific role of each of these receptors and their mechanism of action in mediating development needs to be further elucidated. Funding was provided by CRC, NSERC, and the EmbryoGENE network.

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

1997 ◽  
Vol 47 (1) ◽  
pp. 259 ◽  
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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