scholarly journals Reproductive Seasonality Affects In Vitro Embryo Production Outcomes in Adult Goats

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 873
Author(s):  
Joanna M.G. Souza-Fabjan ◽  
Lucas F.L. Correia ◽  
Ribrio I.T.P. Batista ◽  
Yann Locatelli ◽  
Vicente J.F. Freitas ◽  
...  
Keyword(s):  
Breeding Season ◽  
Assisted Reproductive Technologies ◽  
Formation Rate ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Developmental Competence ◽  
Reproductive Seasonality ◽  
Cleavage Rate

Reproductive seasonality may have a considerable influence on the efficiency of assisted reproductive technologies in seasonal species. This study evaluated the effect of season on cleavage, blastocyst rates and quality of in vitro produced (IVP) goat embryos. In total, 2348 cumulus–oocyte complexes (COCs) were recovered from slaughterhouse ovaries and subjected to the same IVP system throughout 1.5 years (49 replicates). The odds ratio (OR) among seasons was calculated from values of cleavage and blastocyst rates in each season. Cleavage rate was lower (p < 0.05) in spring (anestrus), in comparison with either autumn (peak of breeding season) or summer, while the winter had intermediate values. Furthermore, lower OR of cleavage was observed in spring. Blastocyst formation rate (from initial number of COCs) was higher (p < 0.05) in autumn (52 ± 2.5%) when compared with the other seasons (combined rates: 40 ± 1.9%). Moreover, its OR was higher (p < 0.05) in autumn compared to all other seasons and impaired in the spring compared to winter (OR: 0.54) and summer (OR: 0.48). Embryo hatchability and blastocyst cell number were similar (p > 0.05) among seasons. In conclusion, the breeding season leads to improved oocyte developmental competence, resulting in higher cleavage and blastocyst yield, whereas embryo quality remained similar throughout the years.

Effects of milrinone and butyrolactone-I on porcine oocyte meiotic progression and developmental competence

2006 ◽  
Vol 18 (3) ◽  
pp. 309 ◽  
Author(s):  
Christopher G. Grupen ◽  
Maggie Fung ◽  
David T. Armstrong
Keyword(s):  
Formation Rate ◽  
Inhibitory Effect ◽  
Cell Number ◽  
Developmental Competence ◽  
In Vitro Fertilisation ◽  
Cleavage Rate ◽  
Meiotic Progression ◽  
Porcine Oocyte ◽  
Type 3

Inappropriate coordination of oocyte nuclear and cytoplasmic maturation is thought to contribute to the poor efficiency of embryo production in vitro. With the aim of improving this coordination, the effects of milrinone, an inhibitor of type 3 phosphodiesterases, and butyrolactone-I, a selective inhibitor of cdc2 kinases, on porcine oocyte maturation were investigated. Oocytes recovered from slaughterhouse-derived ovaries of prepubertal animals were treated with the inhibitors for 24 h. At concentrations of 50 and 250 μm, milrinone reversibly inhibited meiotic progression in 57% and 71% of oocytes, respectively. The presence or absence of milrinone in the medium used to wash oocytes for 30 min did not alter the inhibitory effect of the 24 h treatment. At concentrations of 25 and 50 μm, butyrolactone-I inhibited meiotic progression in 61% and 66% of oocytes, respectively, but the effect was not fully reversible in the absence of follicle-stimulating hormone (FSH). The presence of FSH during the butyrolactone-I treatment period increased the ability of oocytes to subsequently complete meiosis at 44 h without changing the inhibitory effect at 24 h. Following in vitro fertilisation at 44 and 50 h, treatment with butyrolactone-I and milrinone, alone or in combination, did not alter embryo cleavage rate, blastocyst formation rate or blastocyst cell number. Despite the different actions of milrinone and butyrolactone-I, the present study demonstrates that these reagents inhibit meiotic progression to a similar extent in the presence of FSH while maintaining developmental competence in porcine oocytes.

65 THE EFFECTS OF CUMULUS CELLS ON DEVELOPMENTAL RATES OF VITRIFIED MOUSE OOCYTES IN C57BL/6J STRAIN

2013 ◽  
Vol 25 (1) ◽  
pp. 180
Author(s):  
N. Kashiwazaki ◽  
N. Kohaya ◽  
K. Fujiwara ◽  
K. Furui ◽  
J. Ito
Keyword(s):  
Ethylene Glycol ◽  
Assisted Reproductive Technologies ◽  
Formation Rate ◽  
Cumulus Cells ◽  
Analysis Data ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Mouse Oocytes ◽  
Free Media

Unfertilized oocytes are one of the most desired germ-cell stages for cryopreservation because these cryopreserved oocytes can be used for assisted reproductive technologies, including IVF and intracytoplasmic sperm injection. However, in general, the fertility and developmental ability of cryopreserved oocytes are still low. We have recently reported that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence (Kohaya et al. 2011 J. Reprod. Dev. 57, 675–680). Since the previous study was carried out using ICR mice (closed colony), we examined whether our protocol can be applied for C57BL/6J mice (inbred strain), which are commonly used for production of transgenic and knockout mice. The effect of cumulus cells on the ability of C57BL/6J mouse oocytes to be fertilized and develop in vitro was examined. Cumulus oocyte complexes (COC) derived from female mice with super ovulation were collected by flushing. Cumulus cells were removed for a portion of the oocytes (DO) using hyarulonidase. Oocytes from both treatment groups (COC and DO) were then vitrified according to the protocol we previously reported (Kohaya et al. 2011). After warming, vitrified COC and DO were used for IVF. All percentage data were subjected to arcsine transformation before statistical analysis. Data were analyzed by one-way ANOVA and Tukey’s test. Significance was considered at P < 0.05. The pronuclear formation rate of vitrified DO after IVF (20/58, 33.3%) was reduced compared with vitrified COC (55/90, 62.1%). Vitrified COC showed significantly (P < 0.05) higher developmental ability to develop into the 2-cell (50/90, 57.0%) and blastocyst stages (42/90, 45.9%) compared with vitrified DO [24.8% (16/58) and 18.4% (11/58), respectively]. The vitrified COC developed to term at a high success rate (51/90, 56.7%) being equivalent to the rate obtained with IVF using fresh COC (52/90, 57.8%). Taken together, the current results clearly demonstrate that, in the presence of surrounding cumulus cells, matured mouse oocytes vitrified using calcium-free media and ethylene glycol retain their developmental competence. These findings will contribute to improve oocytes vitrification in not only experimental animals but also in clinical application in human infertility.

82 Invitro embryo production outcomes in adult goats is affected by season

2021 ◽  
Vol 33 (2) ◽  
pp. 149
Author(s):  
P. V. Pereira ◽  
L. Correia ◽  
R. Batista ◽  
V. Freitas ◽  
Y. Locatelli ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Formation Rate ◽  
Calf Serum ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Seasonal Effects ◽  
Hatching Rate ◽  
Initial Number ◽  
Cleavage Rate ◽  
Blastocyst Rate

In seasonal breeders, reproductive seasonality can have a substantial influence on the efficiency of assisted reproductive technologies. This study assessed seasonal effects on cleavage and blastocyst rates, as well as on quality of invitro-produced (IVP) goat embryos over 18 months. In total, 2348 (autumn: 811, spring: 404, summer: 639, and, winter: 494) cumulus–oocyte complexes (COC) were recovered from slaughterhouse ovaries during 49 replicates (autumn: 17, spring: 7, summer: 15, and, winter: 10) and matured in TCM-199 with 10ng mL−1 epidermal growth factor and 100µM cysteamine for 22h at 38.8°C (5% CO2 in air). Matured oocytes were fertilized with frozen/thawed semen in synthetic oviductal fluid (SOF) with 10% oestrus sheep serum. Sperm and oocytes were co-incubated for 16h at 38.8°C in a humidified atmosphere of 5% CO2 in air. Presumptive zygotes were cultured in SOF medium supplemented with bovine serum albumin (3mg mL−1) for 8 days at 38.8°C in a humidified atmosphere of 5% O2, 5% CO2 and 90% N2. At Day 2, 10% fetal calf serum was added to the culture droplets. The embryos produced were fixed and stained with Hoechst to count their total number of cells, under an epifluorescence microscope. The results of cleavage and blastocyst rates, including hatching rate, from each routine of IVEP were considered as replicates. These data were tested for normality by the Shapiro-Wilk test, before being subjected to ANOVA, followed by Tukey HSD test. The odds ratio (OR) among seasons (autumn: breeding; spring: anoestrus) were calculated. Values of P&lt;0.05 were considered as significant, and the data reported are mean±s.e.m. Cleavage rate was lower (P&lt;0.05) in spring (51±7.1%) than in either autumn (72±2.1%) or summer (71±2.0%) while winter (66±4.1%) had an intermediate value, being similar (P&gt; 0.05) to all others. Indeed, greater possibility of cleavage was observed in autumn (OR: 2.43) and summer (OR: 2.39) compared with spring. The blastocyst rate from cleaved embryos was greater (P &lt;0.05) in autumn (73±2.7%) than in spring (55±2.6%; OR: 2.18). As a result, the blastocyst formation rate from the initial number of COC entering IVM was greater (P&lt;0.05) in autumn (52±2.5%) than in spring: 28±4.7%, summer: 45±2.3%, and winter: 42±2.1%; indeed, the spring season resulted in the lowest rate (P&lt;0.05), compared with other seasons. Moreover, the OR in the blastocyst rate from initial number of COC was greater (P&lt;0.05) in autumn compared with all other seasons and lower in spring compared with winter (OR: 0.54) and summer (OR: 0.48). There were no differences (P&gt; 0.05) in the embryo hatching (mean: 66±2.0%) and blastocyst cell number (mean: 193±2.0 cells). In conclusion, the breeding season (autumn) leads to improved oocyte developmental competence, resulting in greater cleavage and blastocyst yield, whereas embryo quality remained similar throughout the year. Further studies at the molecular level might indicate the mechanisms involved and provide clues to alleviate the negative effect of season.

Characterization of isolated bovine preantral follicles based on morphology, diameter and cell number

Zygote ◽  
2020 ◽  
Vol 28 (2) ◽  
pp. 154-159
Author(s):  
Juliana I. Candelaria ◽  
Anna C. Denicol
Keyword(s):  
Granulosa Cells ◽  
Developmental Stages ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Culture Conditions ◽  
Preantral Follicles ◽  
Secondary Stage

SummaryPreantral follicles are a potential reservoir of oocytes to be used in assisted reproductive technologies. With the increasing interest in developing techniques to grow preantral follicles in vitro, and as the bovine emerges as an appropriate model species to understand human folliculogenesis, the establishment of an accurate classification of developmental stages is needed. Classification of bovine preantral follicles has been mostly based on histological analysis and estimation models, which may not translate well to correctly characterize preantral follicles isolated from the ovary. In this study, we classified bovine preantral follicles by morphology upon isolation, determined diameter and number of granulosa cells by direct counting, and compared our results with previous studies reporting bovine preantral follicle classification. Follicles were isolated via homogenization of ovary tissue and classified into primary, early secondary and secondary stage based on morphology and number of layers of granulosa cells. Diameter was individually measured and Hoechst 33342 was used as a nuclear stain to count granulosa cells. We found that follicles classified by morphology into primary, early secondary, and secondary had different mean diameter and cell number (P < 0.01); cell number and diameter were positively correlated, as were cell density and cell number in each developmental stage (P < 0.01). Results obtained here were mostly in agreement with previous classifications based on histological sections and on isolated follicles, with some discrepancies. The present data add accuracy to classification of bovine preantral follicles that is critical to optimize culture conditions to produce developmentally competent oocytes.

scholarly journals How to Achieve High-Quality Oocytes? The Key Role of Myo-Inositol and Melatonin

2016 ◽  
Vol 2016 ◽  
pp. 1-9 ◽  
Author(s):  
Salvatore Giovanni Vitale ◽  
Paola Rossetti ◽  
Francesco Corrado ◽  
Agnese Maria Chiara Rapisarda ◽  
Sandro La Vignera ◽  
...  
Keyword(s):  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Oocyte Quality ◽  
Fertilization Rate ◽  
In Vitro Models ◽  
The One ◽  
High Level

Assisted reproductive technologies (ART) have experienced growing interest from infertile patients seeking to become pregnant. The quality of oocytes plays a pivotal role in determining ART outcomes. Although many authors have studied how supplementation therapy may affect this important parameter for both in vivo and in vitro models, data are not yet robust enough to support firm conclusions. Regarding this last point, in this review our objective has been to evaluate the state of the art regarding supplementation with melatonin and myo-inositol in order to improve oocyte quality during ART. On the one hand, the antioxidant effect of melatonin is well known as being useful during ovulation and oocyte incubation, two occasions with a high level of oxidative stress. On the other hand, myo-inositol is important in cellular structure and in cellular signaling pathways. Our analysis suggests that the use of these two molecules may significantly improve the quality of oocytes and the quality of embryos: melatonin seems to raise the fertilization rate, and myo-inositol improves the pregnancy rate, although all published studies do not fully agree with these conclusions. However, previous studies have demonstrated that cotreatment improves these results compared with melatonin alone or myo-inositol alone. We recommend that further studies be performed in order to confirm these positive outcomes in routine ART treatment.

P–458 A computational biology approach to improve in-vitro folliculogenesis

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
C D Berardino ◽  
N Bernabò ◽  
G Capacchietti ◽  
A Peserico ◽  
G Buoncuore ◽  
...  
Keyword(s):  
Metabolic Control ◽  
Intracellular Signaling ◽  
Assisted Reproductive Technologies ◽  
Computational Modelling ◽  
Reproductive Technologies ◽  
Developmental Competence ◽  
Paracrine Factors ◽  
Oocyte Growth ◽  
Topological Parameters

Abstract Study question Considering the complexity of mechanisms involved in mammalian ovarian folliculogenesis, how about improving the current in-vitro folliculogenesis (ivF) protocols to prolong individual reproductive chance? Summary answer Computational modelling approach based on network theory was used to manage complexity, improve ivF knowledge and discover new molecules to be targeted for innovating assisted-reproductive-technologies. What is known already: Over the past decades, based on the large ovarian-pool of immature-gametes availability, ivF systems were developed in several mammalian species to support oocyte growth in order to preserve human-fertility and contrast endangered species extinction. Only mouse live-births were obtained when primordial/primary follicles were cultured in-vitro, instead the oocyte differentiation is extremely slow in medium-sized mammals. Moreover, the degree of meiotic-competence is quite incomplete if compared to mice, because oocytes must proceed until late antral-follicle stage to acquire a complete developmental competence. These observations denote the importance to adopt further investigations for establishing a complete ivF protocol in translational mammal model. Study design, size, duration Two researchers expert on reproductive biology generated the Web of Science-Mammals-Made in-vitro folliculogenesis (WoS_MMivF) database including 1111 manuscripts published in peer-reviewed international papers indexed selected in Advanced Search of WoS “Core-collection” by carrying out an independent analysis. Two additional researchers verified the correctness of the records. Participants/materials, setting, methods WoS_MMivF network was built up using Cytoscape 2.6.3 software. The network was analyzed for topological parameters (closeness-centrality, betweenness-centrality and edge count) and to identify key controllers (Hub.BN). Bidimensional-kernel-density-estimation (2D KDE) identifies Hub.BN controllers; Search-Tool-for-the-Retrieval-of-Interacting-Genes/Proteins (STRING) were used to enrich the network with new proteins. Main results and the role of chance The analysis of topological parameters demonstrated that the network is scale-free according to Barabási-Albert-model with a high-degree of robustness-against-random-damage, great controllability and navigability. The network reproduces a coherent framework identifying cross-talking molecules playing a key role in the inter-follicular/intra (somatic and germinal compartment) dialogue. The network allows to organize signalling transduction events/molecules by stratifying them in three layers: input-layer recognizes molecules generating the information flux working as systemic endocrine (pituitary/chorion/enteric-related endocrine hormones) and local paracrine-factors (TGFbeta-superfamily-members and growth-factors) exerting either intrafollicular control or remote feedback on reproductive-cycle. Processing-layer presents molecules able to elaborate/amplify the endocrine/paracrine controllers of ovarian functions, including components of codified intracellular-signaling-pathways like PI3K, KIT and MAPK and second messengers cAMP and Ca2+. These cascades are necessary to promote in-vitro reproducible follicular functions and modulate steroidogenesis, representing molecular events stratified in the output-layer. STRING analysis allowed to extend the regulatory flow of information towards two major biological action contexts: metabolic-control (paracrine-factors and signal-transduction) and angiogenesis. Metabolic-control mediated by mTOR and its interactor cognates FOXO1, FOXO3/SIRT1 plays a key role for ivF, representing the energy sensors of the reproductive cells in hypothalamic-pituitary-ovarian-axis first regulating the status of follicle quiescence/activation and then fate of the structure (specialization or apoptosis). Limitations, reasons for caution - Wider implications of the findings: STRING identified mTOR as key pathway of folliculogenesis, which might act as a molecular-switch to be pharmacologically targeted for potential new in-vitro strategies modulating follicular fate. These results suggest that computational approach in biology might offer perspective in identifying unknown signals, implementing research questions and innovative protocols to face female-fertility. Trial registration number Not applicable

scholarly journals DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Sebastian Canovas ◽  
Elena Ivanova ◽  
Raquel Romar ◽  
Soledad García-Martínez ◽  
Cristina Soriano-Úbeda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Cell Number ◽  
Rna Seq ◽  
Methylation Analysis ◽  
Number Of Children ◽  
Methylation Patterns

The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first whole-genome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.

119 HETEROLOGOUS BOVINE IN VITRO FERTILIZATION USING CRYOPRESERVED BOTTLENOSE DOLPHIN SPERMATOZOA

2012 ◽  
Vol 24 (1) ◽  
pp. 172 ◽  
Author(s):  
M. J. Sanchez-Calabuig ◽  
P. Beltran-Brena ◽  
E. Martinez-Nevado ◽  
D. Rizos ◽  
J. F. Perez-Gutierrez ◽  
...  
Keyword(s):  
Bottlenose Dolphin ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Sperm Chromatin ◽  
Cleavage Rate ◽  
Vitro Fertilization ◽  
Sperm Penetration ◽  
Pronuclear Formation ◽  
Bovine Oocytes

Assisted reproductive technologies are of great importance for increasing genetic diversity in captive animals without displacing them. The development and improvement of these techniques require accurate methods to assess sperm function. The ability of the sperm to bind the zona pellucida and the formation of a male pronucleus have been shown to have a high predictive value for fertilization outcome. The use of zona-intact bovine in vitro–matured oocytes in heterologous fertilization with dolphin spermatozoa could provide valuable information on its fertilizing ability. The aim of the present study was to evaluate male pronuclear formation in zona-intact bovine oocytes after coincubation with frozen-thawed bottlenose dolphin spermatozoa. A total of 1546 immature cumulus oocytes complexes (COC) were obtained from bovine ovaries collected at slaughter. The COC were matured for 24 h in TCM-199 supplemented with 10 ng mL–1 of epidermal growth factor and 10% FCS. Matured COC were inseminated with frozen-thawed Bovi-pure (Nidacon International, Mölndal, Sweden) separated bovine (control) or dolphin spermatozoa. At 18, 20, 22, 24, 26 and 28 h post-insemination (hpi), half of the presumptive zygotes from each group were fixed and stained with Hoechst 33342 to examine sperm penetration, polyspermy and pronuclear formation and the remainder were cultured in synthetic oviduct fluid supplemented with 5% FCS for evaluating fertilization rates by cleavage on Days 2 and 4 (Day 0 = day of IVF). As expected, in the control a higher percentage of 2 pronuclear formation was observed at 18 hpi (74.5%), with a decrease at 20 and 22 hpi (57.4 and 43.2%, respectively) and was significantly lower (P ≤ 0.001) at 24 hpi (13.3%), reaching the lowest values at 26 and 28 hpi. However, in the heterologous group significantly less oocytes with both pronuclear formed (P ≤ 0.001) were observed at 18, 20 and 22 hpi (1.2, 3.4 and 3.0%, respectively) compared with 24, 26 and 28 hpi (22.5, 11.4 and 8.9%, respectively). No polyspermy was detected in oocytes coincubated with dolphin spermatozoa. Moreover, the cleavage rate at Day 2 and 4 in heterologous fertilization was 13.0 and 34.8%, respectively, whereas for the control it was 90.0%. In conclusion, these results indicate that dolphin spermatozoa can penetrate bovine oocytes and induce the block to polyspermy and the differences found regarding pronuclear formation times between the 2 species could be due to distinct sperm chromatin organisation or condensation. In conclusion, our preliminary results show that heterologous fertilization using bovine oocytes is useful for characterising the viability of dolphin thawed spermatozoa, which also could be helpful in performing a more complete sperm evaluation. Further studies are necessary to provide more consistent evidence of the efficiency of this test. The authors thank the staff at Zoo Aquarium Madrid for their dedicated work toward dolphin semen collection.

18 DEVELOPMENTAL COMPETENCE OF CLONED PORCINE EMBRYOS PRODUCED WITH DIFFERENT CLONING PROCEDURES

2014 ◽  
Vol 26 (1) ◽  
pp. 123
Author(s):  
Y. Liu ◽  
A. Lucas-Hahn ◽  
B. Petersen ◽  
R. Li ◽  
P. Hassel ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Nuclear Transfer ◽  
Cell Number ◽  
Culture Conditions ◽  
Developmental Competence ◽  
Cleavage Rate ◽  
Cell Numbers ◽  
Blastocyst Rate ◽  
Handmade Cloning

Two nuclear transfer (NT) techniques are routinely used to produce cloned animals, traditional cloning (TC) and handmade cloning (HMC). The TC embryos keep their zona and can be transferred at early stages, whereas HMC embryos are zona-free and must be cultured to the morula/blastocyst stage before transfer. Some studies have shown that in vitro culture reduces embryo development and quality, but it is not known whether embryos produced by TC or HMC differ because of the NT method or the in vitro culture. Therefore, we investigated the developmental competence and histone acetylation (H3K18ac) of porcine NT embryos produced by TC and HMC with (Day 5 and 6) or without (Day 0) in vitro culture. Nuclear transfer experiments were performed on same day (Day 0), using same batch of porcine oocytes and donor cells and same in vitro culture conditions. Cloning procedures were previously described (TC : Cloning Stem Cells 10 : 355; HMC : Zygote 20 : 61). Parthenogenetically activated embryos (PA) were used as control of activation and culture conditions. Embryos from all groups were collected for immunostaining of H3K18ac on Days 0, 5, and 6. The normalized H3K18ac level was calculated as previously described (Epigenetics 6 : 177). Cell numbers per blastocyst in each group were counted on Days 5 and 6. The cleavage rate (Day 2) and blastocyst rates (Days 5 and 6) between groups were analysed by Chi-squared test, whereas cell number per blastocysts and H3K18ac level between groups and days were analysed by ANOVA (SAS version 9.2; SAS Institute Inc., Cary, NC, USA). Cleavage rate of HMC embryos was lower than that of TC embryos, but blastocyst rate and cell number per blastocyst were higher in the HMC group compared with TC (Table 1). Differences of H3K18ac level between HMC, TC, and PA groups were only observed on Day 6 but not on Day 0 or Day 5. Within HMC and TC groups, there was no difference in H3K18ac level between Day 0 and Day 5, but the level was lower on Day 6 compared with Day 5 in the HMC group, whereas the TC group displayed the opposite pattern. In conclusion, NT embryos produced by HMC show higher blastocyst rate and cell number per blastocyst compared with TC embryos. Both in vitro culture and the NT method result in differences of the normalized H3K18ac levels. Further study is needed to investigate putative differences between NT embryos produced by HMC and TC compared to in vivo embryos also after transfer to recipients. Table 1.Cleavage and blastocyst rate, cell numbers, and normalized H3K18ac level for handmade cloning (HMC), traditional cloning (TC), and parthenogenetically activated (PA) embryos1

246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 239 ◽  
Author(s):  
R. Krisher ◽  
A. Auer ◽  
K. Clark ◽  
K. Emsweller ◽  
S. Rogers ◽  
...  
Keyword(s):  
South Africa ◽  
Oocyte Maturation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Genetic Management ◽  
Blastocyst Development ◽  
Antidorcas Marsupialis

The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P &lt; 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.

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