Intracytoplasmic sperm injection improves in vitro embryo production from poor quality bovine oocytes

An in vitro embryo production system (IVP), in which a single oocyte can be tracked from the moment of retrieval up to the blastocyst stage, would be a valuable tool for studies linking developmental competence and embryo metabolism to oocyte quality and follicular environment. Unfortunately, to date, data on individual IVP are inconsistent, and in most cases show unsatisfactory blastocyst rates. Earlier studies revealed that individual culture on a cumulus cell (CC) monolayer resulted in comparable numbers of good-quality embryos as obtained after regular group culture (Goovaerts et al. 2008 Reprod. Dom. Anim. 43 (Suppl. 3), 190). Because, in the latter study, single culture was performed after group maturation and fertilization, the aim of this study was to develop and test an individual IVP system using bovine oocytes or zygotes obtained after single maturation and single fertilization. Therefore, 532 grade I COC from slaughterhouse ovaries (3 replicates) were randomly assigned to 1 of 2 treatments: a complete individual IVP protocol, or a routine group IVP as a control. Individual maturation (TCM-199 + 20% serum) and fertilization were performed in 20-μL droplets under oil in 24-well plates. Subsequently, each zygote was cultured in 20 μL of medium (SOF + 5% serum, 90% N2, 5% CO2, 5% O2) on a 6-day-old monolayer of matured CC (5% CO2 in air). Group maturation and fertilization were carried out per 100 COC in 500 μL, whereas group culture was performed per 25 zygotes in 50-μL droplets under oil. Cleavage, blastocyst, and hatching rates were assessed 2, 8, and 10 days postfertilization, respectively. Possible effects of individual and group culture were evaluated with binary logistic regression (SPSS 15.0). No interactions between replicate and treatment could be found (P > 0.05). Cleavage and blastocyst rates were significantly lower after individual IVP, compared with group IVP, whereas the blastocyst rates on cleaved zygotes and the hatching rates did not differ significantly (Table 1). In conclusion, acceptable blastocyst rates (25.1%) could be obtained after individual IVP. The lower blastocyst rates were associated with the lower cleavage rates, and no effect of the individual embryo culture system on embryo development could be found. Table 1.Cleavage, blastocyst, and hatching rates after individual and group in vitro embryo production (IVP)

Download Full-text

159 Effect of Commercial Embryo Holding Medium on Development and Quality of Immature Bovine Oocytes

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab159 ◽

2018 ◽

Vol 30 (1) ◽

pp. 219

Author(s):

M. Catteeuw ◽

O. B. Pascottini ◽

G. Opsomer ◽

A. Van Soom

Keyword(s):

Low Cost ◽

Laboratory Work ◽

Blastocyst Development ◽

Embryo Production ◽

Immature Oocytes ◽

Developmental Capacity ◽

In Vitro Embryo Production ◽

Assess Quality ◽

Bovine Oocytes

Bovine in vitro embryo production following ovum pick-up (OPU) in the field is hampered due to large time gaps between first and last OPU sessions. As oocytes will start immediate maturation, scheduling further manipulations makes the laboratory work laborious. There is a need for an easy and low-cost method that conserves the oocytes with full developmental capacity and allows scheduling laboratory work. In this regard, a commercial embryo holding medium (EHM; Syngro®; Bioniche Inc., WA, USA) was evaluated for conserving immature oocytes. Bovine immature oocytes (n = 2160) were collected from slaughterhouse animals by follicle aspiration and grouped per 60 and stored in 1 mL of EHM in 1-mL sterile glass osmometer tubes. Then, different temperatures [4°C, room temperature (RT), and 38.5°C] and different storage times (6, 10, 14 h) were assessed. After storage, oocytes were matured in TCM-199 supplemented with epidermal growth factor and gentamycin for 22 h. Fertilization was performed and zygotes (n = 1786) were cultured per 25 in 50 µL of SOF medium supplemented with 0.4% BSA and insulin, transferrin, and selenium. Control groups were included: immature oocytes (n = 1080) were not stored in EHM but immediately matured; fertilized and zygotes (n = 896) were cultured. Further, differential apoptotic staining was performed on a random subgroup of blastocysts to assess quality. Generalized fixed effect models were computed using R studio. Storage for 6 h showed a decrease in cleavage and blastocyst rate at 38.5°C (50 ± 3.9%; 11 ± 1.8%) compared with the control (78 ± 3.0%; 36 ± 2.8%). When increasing storage time, 38.5°C was not included; here, 4°C had a lower cleavage and blastocyst yield (47 ± 2.9%; 20 ± 3.3%) compared with the control (75 ± 2.5%; 41 ± 4.6%). For both 6 and 10 h, storage at RT resulted in similar cleavage (76 ± 3.4%; 74 ± 2.6%) and blastocyst rates (35 ± 2.7%; 40 ± 4.5%) as the control (P > 0.05). However, increasing storage to 14 h at RT decreased cleavage (61 ± 2.8%) and blastocyst yield (26 ± 2.5%) compared with the control (78 ± 2.4%; 39 ± 2.8%; P < 0.05). Evaluating embryo quality in all groups, no significant differences were found for any holding time or temperature of the EHM. To simulate OPU settings, EHM was also tested in 38 small groups of 10 immature oocytes that were subsequently matured, fertilized, and cultured. Based on the previous results, EHM storage was performed for 6 and 10 h at RT. Blastocyst development was not different between RT (19.8 ± 3.5%; 18.8 ± 3.6%) and the control (20.6 ± 3.6%; 18.3 ± 3.4%; P ≥ 0.05). To conclude, a commercial EHM can be used to conserve immature bovine oocytes without losing developmental capacity. Storage is recommended for no longer than 10 h and at RT in EHM. It opens new perspectives for practitioners, because this method is simple and low-cost; moreover, the start of maturation and subsequent in vitro embryo production process can be scheduled to avoid evening or night work at the laboratory.

Download Full-text

Update on advanced semen-processing technologies and their application for in vitro embryo production in horses

Reproduction Fertility and Development ◽

10.1071/rd19301 ◽

2019 ◽

Vol 31 (12) ◽

pp. 1771

Author(s):

Lee H. Morris ◽

Lisa J. Maclellan

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Reproductive Technologies ◽

Breeding Program ◽

Embryo Production ◽

Processing Technologies ◽

Advanced Technologies ◽

The Status ◽

In Vitro Embryo Production ◽

Semen Processing

The increased commercialisation of intracytoplasmic sperm injection (ICSI) in horses creates more opportunities to incorporate advanced reproductive technologies, such as sex-sorted, refrozen and lyophilised spermatozoa, into a breeding program. This paper reviews the status of these semen-handling technologies in light of their use in equine ICSI programs. Pregnancies have been achieved from each of these advanced technologies when combined with ICSI in horses, but refinements in the semen-handling processes underpinning these technologies are currently being explored to produce more reliable and practical improvements in the results from equine ICSI.

Download Full-text

Male Factors Affecting the Success of Equine In Vitro Embryo Production by Ovum Pickup-Intracytoplasmic Sperm Injection in a Clinical Setting

Journal of Equine Veterinary Science ◽

10.1016/j.jevs.2016.05.014 ◽

2016 ◽

Vol 43 ◽

pp. S6-S10 ◽

Cited By ~ 12

Author(s):

Cesare Galli ◽

Silvia Colleoni ◽

Roberto Duchi ◽

Giovanna Lazzari

Keyword(s):

Intracytoplasmic Sperm Injection ◽

Clinical Setting ◽

Embryo Production ◽

Factors Affecting ◽

In Vitro Embryo Production ◽

Male Factors

Download Full-text

Laparoscopic Ovum Pick-Up Followed by In Vitro Embryo Production and Transfer in Assisted Breeding Programs for Ruminants

Animals ◽

10.3390/ani11010216 ◽

2021 ◽

Vol 11 (1) ◽

pp. 216

Author(s):

Hernan Baldassarre

Keyword(s):

Pregnancy Rate ◽

Commercial Application ◽

Embryo Production ◽

Breeding Programs ◽

Sheep And Goats ◽

Embryo Recovery ◽

Marker Selection ◽

Holstein Calves ◽

In Vitro Embryo Production

The potential of laparoscopic ovum pick-up (LOPU) followed by in vitro embryo production (IVEP) as a tool for accelerated genetic programs in ruminants is reviewed in this article. In sheep and goats, the LOPU-IVEP platform offers the possibility of producing more offspring from elite females, as the procedure is minimally invasive and can be repeated more times and more frequently in the same animals compared with conventional surgical embryo recovery. On average, ~10 and ~14 viable oocytes are recovered by LOPU from sheep and goats, respectively, which results in 3–5 transferable embryos and >50% pregnancy rate after transfer. LOPU-IVEP has also been applied to prepubertal ruminants of 2–6 months of age, including bovine and buffalo calves. In dairy cattle, the technology has gained momentum in the past few years stemming from the development of genetic marker selection that has allowed predicting the production phenotype of dairy females from shortly after birth. In Holstein calves, we obtained an average of ~22 viable oocytes and ~20% transferable blastocyst rate, followed by >50% pregnancy rate after transfer, declaring the platform ready for commercial application. The present and future of this technology are discussed with a focus on improvements and research needed.

Download Full-text