Advances in production of embryos in vitro from juvenile and prepubertal oocytes from the calf and lamb

1997 ◽  
Vol 9 (3) ◽  
pp. 333 ◽  
Author(s):  
D. T. Armstrong ◽  
P. J. Kotaras ◽  
C. R. Earl
Keyword(s):  
Embryo Production ◽  
Vitro Fertilization ◽  
Oocyte Collection ◽  
Domestic Livestock ◽  
Normal Offspring ◽  
Calf Oocytes ◽  
Collection Methods ◽  
Fresh Transfer

The use of juvenile donors in embryo-transfer (ET) programmes offers considerable potential for accelerated genetic gain in domestic livestock through reduced generation interval. The present paper reviews recent research aimed at optimizing embryo production from oocytes collected from young calves and lambs using in vitro methods of embryo production. Emphasis is placed on criteria for donor selection, oocyte-collection methods, and hormone-stimulation methods designed to produce maximum yields of viable oocytes. In vitro fertilization (IVF) rates of calf and lamb oocytes did not differ significantly whether matured in vivo or in vitro, and rates of development to blastocyst stages in culture were similar to those observed for embryos derived from adult donors. Blastocysts produced by IVF of lamb and calf oocytes established ET pregnancies at rates of 30–45%. Pregnant recipients have reached full term and delivered normal offspring at rates similar to those expected following ET of embryos produced in vivo from superovulated donors. On the basis of current follicle-stimulation protocols, on rates of blastocyst production in vitro under optimal conditions, and on observed pregnancy rates from fresh transfer of IVF embryos, 8–10 pregnancies may be expected per oocyte collection from 10–12-week-old calves and from 6–8-week-old lambs.

17 SPERM FERTILITY RATE ASSESSED BY EMBRYO PRODUCTION IN VIVO AND IN VITRO IN SOUTH AFRICAN BULLS

2017 ◽  
Vol 29 (1) ◽  
pp. 116
Author(s):  
M. H. Mapeka ◽  
F. V. Ramukhithi ◽  
C. M. Pilane ◽  
D. Norris ◽  
C. Banga ◽  
...  
Keyword(s):  
South African ◽  
Sperm Motility ◽  
Developmental Stages ◽  
Fertility Rate ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Recovery Methods

The aim of this study was to determine the sperm fertility rate by embryo production in vivo and in vitro in South African bulls and further compare the embryo quality developed from different oocyte recovery methods. A total of 15 frozen semen straws (5 Bonsmara; 5 Nguni; 5 Boran) were thawed and evaluated for sperm motility characteristics using sperm class analyzer. The fertilizing ability of frozen–thawed semen was assessed by performing AI and in vitro fertilization. For AI, 6 cows were superovulated and inseminated with frozen–thawed semen followed by flushing on Day 7 post-insemination and then evaluated for embryo developmental stages. For IVF, oocytes were retrieved using two recovery methods namely ovum pick-up (OPU) and ovary aspiration. A total of 383 (106, OPU; 277, ovary aspiration) oocytes were matured in M199 + 10% fetal bovine serum (FBS) maturation medium at 38.5°C for 24h. Oocytes were washed in Bracket and Oliphant’s fertilization medium, co-incubated with frozen–thawed (Boran) semen at 38.5°C for 6 h, and then cultured in SOF-BSA medium, incubated at 38.5°C, 5% CO2 for 7 days, and further evaluated for embryo development. Data were analysed by ANOVA. Total sperm motility was >70% in all breeds. Boran had a significantly (P < 0.05) higher total post-thaw sperm motility (93.2 ± 3.6) compared with Nguni (75.1 ± 4.2) and Bonsmara (80.7 ± 6.9). Furthermore, Boran had higher (P < 0.05) progressive motility (39.7 ± 3.4) and rapid motility (36.1 ± 5.9) compared with other breeds. Interestingly, Boran produced significantly (P < 0.05) higher blastocyst rate (56.34%) compared with Bonsmara (38.03%) Nguni (31.08%). Superovulation and OPU resulted in a significantly higher (P < 0.05) number of blastocysts (10.5 ± 3.3 and 10.5 ± 3.3) respectively, compared with aspiration (1.3 ± 3.3). Moreover, the OPU method yielded a significantly higher (P < 0.05) number of grade 2 blastocyst (3.0 ± 0.1) compared with aspiration (0.50 ± 0.1). However, there was no significant (P > 0.05) difference in the number of grade 1 and grade 3 blastocysts obtained when the 3 recovery methods were used. In conclusion, the Boran breed showed better a sperm fertility rate following in vivo and in vitro embryo production. The superovulation and OPU methods resulted in higher numbers and better quality blastocysts compared with aspiration.

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.

122 A New Maturation Medium Improves Porcine Embryo Production In Vitro

2018 ◽  
Vol 30 (1) ◽  
pp. 200 ◽  
Author(s):  
A. Lucas-Hahn ◽  
B. Petersen ◽  
M. Nowak-Imialek ◽  
U. Baulain ◽  
R. Becker ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Number ◽  
Developmental Competence ◽  
Gestation Length ◽  
Cleavage Rate ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Maturation Medium

Recently (Spate et al. 2017 Reprod. Fertil. Dev. 29, 150), a new medium [TCM-199 supplemented with hCG 10 IU, pregnant mare serum gonadotropin (PMSG) 10 IU mL−1, fibroblast growth factor (FGF) 40 ng mL−1, leukemia inhibitory factor (LIF) 2000 U mL−1, IGF-1 20 ng mL−1, epidermal growth factor (EGF) 10 ng mL−1], termed FLI medium, was demonstrated to improve porcine oocyte maturation in vitro. The effects on embryo development and quality have not yet been investigated. The purpose of the present study was to compare the FLI medium in porcine in vitro embryo production (IVP) with our standard maturation medium (DMEM supplemented with 10 IU mL−1 PMSG and hCG, 50 ng mL−1 EGF, 100 ng mL−1 IGF1, and 5 ng mL−1 FGF). Briefly, gilt oocytes were collected via aspiration of follicles from abattoir ovaries and matured for 44 h in either FLI or standard DMEM medium at 39°C, 5% CO2 in humidified air. In vitro fertilization was performed with freshly ejaculated sperm (250,000 mL−1) of a multi-transgenic boar (GGTA1-KO/hCD46/hCD55/hCD59/hHO-1/hA20) by co-incubation with the matured oocytes in PGMTac4 medium for 4 h. Zygotes were washed twice and then cultured for 6 days in PZM3 medium. Development to the blastocyst stage was recorded at Day 6 of culture. Blastocysts were fixed and Hoechst33342 stained for counting the nuclei. Each of the experiments was repeated 3 times. In a second step, Day 5 blastocysts derived from the FLI medium were transferred to synchronized pubertal gilts to test the in vivo developmental competence of the IVF embryos. Maturation of oocytes in FLI medium resulted in a significantly higher blastocyst rate (49.3 vs. 13.5; P ≤ 0.001, Chi-squared test) and nuclei number (41.3 ± 12.2 vs. 35.3 ± 10.8; P ≤ 0.001, one-way ANOVA) compared with the standard medium, whereas the cleavage rate was not affected. Transfer of Day 5 blastocysts (average 35 embryos/recipient) derived from the FLI system using 8 recipients resulted in 7 pregnancies (87.5%) as determined by ultrasound scanning on Day 25 of gestation. At the time of writing, one recipient had delivered 5 healthy piglets after a gestation length of 114 days. Results indicate that the FLI medium significantly improves blastocyst rates and the cell number of the resulting blastocysts (Table 1) and yields pig IVF embryos with a high developmental capacity in vivo. By producing high-quality porcine embryos, this FLI-based IVF system provides an efficient method to modify the porcine genome by cytoplasmic microinjection of CRISPR/Cas molecules into IVF-derived zygotes. Table 1.Results of maturation of oocytes in FLI medium compared with DMEM

scholarly journals Evidence of oocyte donor cow effect over oocyte production and embryo development in vitro

Reproduction ◽  
2003 ◽  
pp. 629-637 ◽  
Author(s):  
M Tamassia ◽  
Y Heyman ◽  
Y Lavergne ◽  
C Richard ◽  
V Gelin ◽  
...  
Keyword(s):  
Formation Rate ◽  
Maternal Influence ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Oocyte Donor ◽  
Oocyte Collection ◽  
Study Support ◽  
The Mean ◽  
Development In Vitro

There have been few studies on a possible maternal influence on in vitro embryo production in cows. The objective of this study was to evaluate the maternal influence on oocyte production and in vitro blastocyst formation rate using repeated ovum pick-up and in vitro fertilization. Six contemporary cows raised on the same farm and with varied genetic origins were submitted to 42 weeks of ovum pick-up organized into four series. Collected oocytes were fertilized in vitro with spermatozoa from a different bull for each series. In total, 1933 oocytes were recovered from 3936 follicles with a recovery rate of 57.2% and a mean oocyte collection of 4.6+/-0.2 (mean+/-SEM) per animal per session. Animals were ranked according to their oocyte production. The best oocyte donor was the same female in all four series. No relationship was identified between oocyte production and blastocyst production rate (r=-0.08). The mean blastocyst rate was 28.8% with significant variation among animals. The best and the worst blastocyst producers were always the same animals independent of the semen used. The results of the present study support the hypothesis that in cattle, the oocyte donor influences the production of blastocysts. Furthermore, they demonstrate that oocyte and embryo production are independent factors. Further studies are necessary to identify the maternal or oocyte factors responsible for such differences.

scholarly journals Seminal Plasma: Relevant for Fertility?

2021 ◽  
Vol 22 (9) ◽  
pp. 4368
Author(s):  
Heriberto Rodriguez-Martinez ◽  
Emilio A. Martinez ◽  
Juan J. Calvete ◽  
Fernando J. Peña Vega ◽  
Jordi Roca
Keyword(s):  
Seminal Plasma ◽  
Current Knowledge ◽  
Inorganic Ions ◽  
Vitro Fertilization ◽  
Dna And Rna ◽  
Model Animal ◽  
Sexual Glands ◽  
Species Specific

Seminal plasma (SP), the non-cellular component of semen, is a heterogeneous composite fluid built by secretions of the testis, the epididymis and the accessory sexual glands. Its composition, despite species-specific anatomical peculiarities, consistently contains inorganic ions, specific hormones, proteins and peptides, including cytokines and enzymes, cholesterol, DNA and RNA—the latter often protected within epididymis- or prostate-derived extracellular vesicles. It is beyond question that the SP participates in diverse aspects of sperm function pre-fertilization events. The SP also interacts with the various compartments of the tubular genital tract, triggering changes in gene function that prepares for an eventual successful pregnancy; thus, it ultimately modulates fertility. Despite these concepts, it is imperative to remember that SP-free spermatozoa (epididymal or washed ejaculated) are still fertile, so this review shall focus on the differences between the in vivo roles of the SP following semen deposition in the female and those regarding additions of SP on spermatozoa handled for artificial reproduction, including cryopreservation, from artificial insemination to in vitro fertilization. This review attempts, including our own results on model animal species, to critically summarize the current knowledge of the reproductive roles played by SP components, particularly in our own species, which is increasingly affected by infertility. The ultimate goal is to reconcile the delicate balance between the SP molecular concentration and their concerted effects after temporal exposure in vivo. We aim to appraise the functions of the SP components, their relevance as diagnostic biomarkers and their value as eventual additives to refine reproductive strategies, including biotechnologies, in livestock models and humans.

scholarly journals Graphene and Reproduction: A Love-Hate Relationship

Nanomaterials ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 547
Author(s):  
Marina Ramal-Sanchez ◽  
Antonella Fontana ◽  
Luca Valbonetti ◽  
Alessandra Ordinelli ◽  
Nicola Bernabò ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Reproductive Function ◽  
Scientometric Analysis ◽  
Potential Toxicity ◽  
Good Tool ◽  
Vitro Fertilization ◽  
Number Of Publications ◽  
The Individual

Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species (from Archea to mammals). Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way.

scholarly journals Novel Insights on the Role of Nitric Oxide in the Ovary: A Review of the Literature

2021 ◽  
Vol 18 (3) ◽  
pp. 980
Author(s):  
Maria Cristina Budani ◽  
Gian Mario Tiboni
Keyword(s):  
Nitric Oxide ◽  
In Vivo Studies ◽  
Published Data ◽  
Vitro Fertilization ◽  
Peripheral Neurons ◽  
Relevant Role ◽  
Oocyte Meiotic Maturation

Nitric oxide (NO) is formed during the oxidation of L-arginine to L-citrulline by the action of multiple isoenzymes of NO synthase (NOS): neuronal NOS (nNOS), endotelial NOS (eNOS), and inducible NOS (iNOS). NO plays a relevant role in the vascular endothelium, in central and peripheral neurons, and in immunity and inflammatory systems. In addition, several authors showed a consistent contribution of NO to different aspects of the reproductive physiology. The aim of the present review is to analyse the published data on the role of NO within the ovary. It has been demonstrated that the multiple isoenzymes of NOS are expressed and localized in the ovary of different species. More to the point, a consistent role was ascribed to NO in the processes of steroidogenesis, folliculogenesis, and oocyte meiotic maturation in in vitro and in vivo studies using animal models. Unfortunately, there are few nitric oxide data for humans; there are preliminary data on the implication of nitric oxide for oocyte/embryo quality and in-vitro fertilization/embryo transfer (IVF/ET) parameters. NO plays a remarkable role in the ovary, but more investigation is needed, in particular in the context of human ovarian physiology.

scholarly journals 252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF

2005 ◽  
Vol 17 (2) ◽  
pp. 276 ◽  
Author(s):  
J. Pryor ◽  
S. Romo ◽  
D.D. Varner ◽  
K. Hinrichs ◽  
C.R. Looney
Keyword(s):  
Sperm Concentration ◽  
Embryo Production ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Before And After ◽  
Chi Square Test ◽  
Group 2 ◽  
Live Sperm ◽  
Sperm Separation

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.

scholarly journals FOS, a Critical Downstream Mediator of PGR and EGF Signaling Necessary for Ovulatory Prostaglandins in the Human Ovary

2018 ◽  
Vol 103 (11) ◽  
pp. 4241-4252 ◽  
Author(s):  
Yohan Choi ◽  
Katherine L Rosewell ◽  
Mats Brännström ◽  
James W Akin ◽  
Thomas E Curry ◽  
...  
Keyword(s):  
Target Genes ◽  
Activator Protein 1 ◽  
Egf Receptors ◽  
Human Ovary ◽  
Promoter Regions ◽  
Vitro Fertilization ◽  
Egf Signaling ◽  
Periovulatory Follicles

Abstract Context Fos null mice failed to ovulate and form a corpus luteum (CL) even when given exogenous gonadotropins, suggesting that ovarian Fos expression is critical for successful ovulation and CL formation. However, little is known about FOS in the human ovary. Objectives To determine the expression, regulation, and function of FOS in human periovulatory follicles. Design/Participants Timed periovulatory follicles were obtained from normally cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures The in vivo expression after human chorionic gonadotropin (hCG) administration and in vitro regulation of FOS, JUN, JUNB, and JUND was evaluated at the mRNA and protein level. Binding of progesterone receptor (PGR) and FOS to their target genes was assessed by chromatin immunoprecipitation analyses. Prostaglandin E2 (PGE2) and progesterone were measured. Results The expression of FOS, JUNB, and JUND drastically increased in ovulatory follicles after hCG administration. In human granulosa/lutein cell cultures, hCG increased the expression of FOS and JUN proteins. Inhibitors of PGR and epidermal growth factor (EGF) receptors reduced hCG-induced increases in the expression and phosphorylation of FOS. PGR bound to the FOS gene. A selective FOS inhibitor blocked hCG-induced increases in PGE2 and the expression of prostaglandin (PG) synthases and transporters (PTGES, SLCO2A1, and ABCC1). FOS bound to the promoter regions of these genes. Conclusions The increase of FOS/activator protein 1 in human periovulatory follicles after hCG administration is mediated by collaborative actions of PGR and EGF signaling and critical for the upregulated expression of key ovulatory genes required for the rise in ovulatory PG in human granulosa cells.

scholarly journals Efficient Correction of a Hypertrophic Cardiomyopathy Mutation by ABEmax-NG

2021 ◽  
Author(s):  
Shuhong Ma ◽  
Wenjian Jiang ◽  
Xujie Liu ◽  
Wen-Jing Lu ◽  
Tao Qi ◽  
...  
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Mouse Model ◽  
Pathogenic Mutation ◽  
Adeno Associated Virus ◽  
Vitro Fertilization ◽  
Base Editing ◽  
Dna And Rna ◽  
Hereditary Disorders

Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. Methods and Results: Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation (Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5-70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the post-natal mice as well as their offspring. In addition, the same sgRNA was also used to target an intronic locus (TGG PAM) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different PAMs (NG) and genomic distribution in vivo. Compared with CRISPR/ssODN-mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 (AAV9) encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off target lesions, thus safely eliminating HCM in derived mice and their progeny.

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