Bovine embryo technologies

doi:10.1016/S0093-691X(02)01243-8

Theriogenology

Volume 59, Issue 2, 15 January 2003, Pages 599-616

https://doi.org/10.1016/S0093-691X(02)01243-8 Get rights and content

Abstract

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.

Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.

Introduction

The cattle industry is undergoing a major reorganization especially in Europe, primarily as a result of the BSE and Foot and Mouth disease outbreaks that had a devastating effect in 2001, and also as a consequence of increased competition worldwide. Because of these economic difficulties the application of advanced reproductive techniques in cattle breeding has been declining. Research in farm animal reproduction is also shrinking and changing. Increasing animal production is certainly not a priority in Europe today and economic resources are diverted to more “green areas” such as sustainable agriculture and animal welfare. In this situation the main drivers and users of embryo technologies are not farmers but genetic companies or breeder’s co-operatives that earn their revenues from the sale of genetics (for example semen, embryos and animals).

A second problem that Europe is facing is a general negative attitude towards biotechnology products, and embryo technologies are identified in this area. It is not uncommon today to find retailers that will distribute, for example, beef that has not been produced by embryo technologies, even by simple multiple ovulation and embryo transfer (MOET), let alone in vitro embryo production (IVP) or cloning. An important objective today is to prove more convincingly the role and the value of advanced reproductive technologies in cattle breeding. More research is needed towards this goal.

The scope of this paper is to review the state of the art in the different embryo technologies used by the cattle industry for reproduction and selection, relative advantages and disadvantages, bottlenecks of some technologies and the science needed to overcome them. Some indication will be given for possible future research directions.

Section snippets

MOET

MOET is a well-established technology and is used to obtain over 80% of the embryos produced for commercial purposes [1]. The initial use of undefined superovulation products like eCG has been replaced by pituitary extracts (porcine and ovine) and in some cases by human menopausal gonadotropins (HMG). These latter products have a shorter half-life and require multiple injections but have the advantages of lacking side effects such as over-stimulation, failure of ovulation and persistent

In vitro embryo production

An alternative system to produce cattle embryos is to use immature oocytes collected from the ovaries of donors of various age and physiological status [4]. Reliable procedures allow maturation and fertilization of bovine oocytes in vitro and several culture protocols can be used to grow them for about a week up to the stage suitable for transfer or freezing. IVP was developed initially as a research tool and was applied to rescue follicular oocytes of slaughtered donors. In cattle, besides

Conclusions

Embryo technologies applied to animal breeding have the important role of increasing the impact of superior genotypes in the population. However, a more widespread and competent use of the available techniques is required in order to gain the most benefit from their application. Future developments, linked to the newest area of research such as somatic cloning and embryo genotyping, are expected to find a role in advanced animal breeding.

Together with the requirement for continuous scientific

Acknowledgements

The Authors acknowledge the technical support of Mr. Massimo Iazzi, Dr. Fabio Ferrara and Dr. Stefano Allodi. Part of this work was supported by EU CT98-0032, EU FAIR-CT98-4339, EU QLTK3-CT-1999-00104, MIPAF-RAIZ.

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An experiment was conducted to determine whether administration of mycobacterium cell wall fraction (MCWF; Amplimune, NovaVive) could enhance embryo developmental competence following in vitro embryo production (IVP) and pregnancy establishment after embryo transfer (ET). Nulliparous, Holstein heifers (n = 40; age 8–15 months) were submitted to two rounds of ovum pick-up (OPU) and IVP in a crossover design. Thirty-six h after follicle wave synchronization, treatments (saline or MCWF, 5 mL, im) were administered in conjunction with a single dose of follicle stimulating hormone (175 IU) and OPU was performed 48–52 h later. Recovered cumulus-oocyte complexes were used for IVP to assess embryo development. For ET, nulliparous, Holstein heifers (n = 225; age 12–18 months) were used as recipients. At 12–24 h after detection of spontaneous estrus, recipients were randomly treated with either saline or MCWF (5 mL, im). The effect of MCWF on pregnancy per ET (P/ET) was assessed in a 2 × 2 factorial design with recipients treated with or without MCWF receiving a fresh IVP embryo from a donor treated with or without MCWF at day 7 or 8 after detected estrus. Blood samples were collected from a subset of donors (n = 8) and recipients (n = 26 to 33 per treatment) prior to treatment and at 6 and 24 h post-treatment to determine serum concentration of interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and interferon-γ. Blood samples were also collected from a group of recipients (n = 31 to 39 per treatment) to assess serum concentration of progesterone at days 4, 7, and 16 post-treatment. Pregnancy status was determined at days 40 and 100 of gestation. Donor treatment with MCWF tended (P < 0.07) to increase the proportion of oocytes that developed into transferable embryos, but there was no effect of MCWF on other parameters of embryo development. The P/ET at days 40 and 100 of gestation and pregnancy loss were not affected by donor treatment or recipient treatment with MCWF and there was no interaction. Serum concentration of proinflammatory cytokines among donors and recipients and serum concentration of progesterone among recipients were not increased by treatment with MCWF. Results of the present study indicate that treatment of donors with MCWF has minimal impact on subsequent embryo development following IVP. Moreover, regardless of whether donors or recipients were treated with MCWF, there was no effect on P/ET following transfer of IVP embryos.
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Only capacitated spermatozoa have the capacity to fertilize the mature oocyte (Gervasi and Visconti, 2016). The process of capacitation has been mimicked in various species in in vitro conditions, yielding standard procedures for in vitro fertilization and embryo production using human, cattle, pig and mice gametes (Steptoe and Edwards, 1992; Galli et al., 2003; Betteridge, 2006; Perry, 2014). In general, capacitation is induced in vitro by HCO3-, Ca2+ and a cholesterol acceptor such as albumin (review: Leemans et al., 2019a).
Conventional in vitro fertilization is not efficacious when working with equine gametes. Although stallion spermatozoa bind to the zona pellucida in vitro, these gametes fail to initiate the acrosome reaction in the vicinity of the oocyte and cannot, therefore, penetrate into the perivitelline space. Failure of sperm penetration most likely relates to the absence of optimized in vitro fertilization media containing molecules essential to support stallion sperm capacitation. In vivo, the female reproductive tract, especially the oviductal lumen, provides an environmental milieu that appropriately regulates interactions between the gametes and promotes fertilization. Identifying these ‘fertilization supporting factors’ would be a great contribution for development of equine in vitro fertilization media. In this review, a description of the current understanding of the interactions stallion spermatozoa undergo during passage through the female genital tract, and related specific molecular changes that occur at the sperm plasma membrane is provided. Understanding these molecular changes may hold essential clues to achieving successful in vitro fertilization with equine gametes.
Review: Environmental impact on early embryonic development in the bovine species
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Assisted reproduction techniques (ARTs) provide access to early stage embryos whose analysis and assessment deliver valuable information. The handling of embryos, including the in vitro production of bovine embryos, is a rapidly evolving area which nonetheless exposes the embryos to unnatural conditions for a period of time. The Fallopian tube provides innumerable quantitative and qualitative factors, all of which guarantee the successful development of the embryo. It is well known that the Fallopian tube can be bypassed, using embryo transfer, resulting in successful implantation in the target recipient animal and the birth of calves. However, the question arises as to whether such circumvention has a negative impact on the embryo during this sensitive development period. First crosstalk between the embryo and its environment confirms mutual recognition activities and indicate bilateral effects. Nowadays, in vitro production of bovine embryos is a well-established technology. However, it is still evident that in vitro generated embryos are not qualitatively comparable to embryos obtained ex vivo. To counteract these differences, comparative studies between in vitro and ex vivo embryos are advantageous, as embryos grown in their physiological environment can provide a blueprint or gold standard against which to compare embryos produced in vitro. Attempts to harness the bovine oviduct were sometimes very invasive and did not result in wide acceptance and routine use. Long-term development and refinement of transvaginal endoscopy for accessing the bovine oviduct has meanwhile been routinely applied for research as well as in practice. Comparative studies combining in vitro development with development in the cattle oviduct revealed that the environmental conditions to which the embryo is exposed before activation of the embryonic genome can have detrimental and lasting effects on its further development. These effects are manifested as deviations in gene expression profiles and methylation signatures as well as frequency of whole chromosomal or segmental aberrations. Furthermore, it was shown that hormonal superstimulation (multiple ovulation and embryo transfer), varying progesterone concentrations as well as metabolic disorders caused by high milk production, markedly affected embryo development in the postpartum period. Assisted reproductive techniques that allow the production and handling of extra numbers of generated embryos promise to have a very high impact on scientific and practical application. Any influence on the early embryonic life, both in animals and in vitro, is accompanied by a sensitive change in embryonic activity and should be assessed in vivo on the basis of physiological conditions before being used for ART.
Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods
2020, Animal
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Embryos can be derived by IVP from open-cycling heifers and cows as well as females that do not respond properly to superovulatory treatments, have abnormalities in their reproductive tract compromising gamete transport, or are in terminal conditions (age, health, accident). Likewise, pregnant animals during the first trimester of pregnancy, postpartum cows (lactation) and from pre-pubertal calves can be employed for IVP, as well as ovaries collected at abattoirs (Galli et al., 2003). The first calves obtained exclusively by IVP, that is, in vitro maturation (IVM) of oocytes, in vitro fertilization (IVF) as well as in vitro embryo development (IVD), were reported in the late 1980s (Goto et al., 1988).
In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo–produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.
Effect of the interval from follicle aspiration to initiation of lengthened FSH treatment on follicular superstimulatory and superovulatory responses and embryo production in lactating Simmental cows
2019, Theriogenology
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The most important restricting factor in the success of superovulation has been and continues to be the unpredictability in the ovarian response to gonadotropin stimulation, due to high inter-individual variability [41]. It has been stated that one-third of the donors treated do not answer to superovulation, another third yields an average of one to three embryos and only one-third actually superovulates giving a large number of embryos [42,43]. All donor cows in the study responded to superovulation by producing more than 4 CL.
The present study evaluated follicular superstimulatory (FSS) and superovulatory (SOV) responses and in vivo embryo production in lactating Simmental cows treated with FSH starting 1 or 2 days after follicle aspiration (FA). The performance of a lengthened superovulation program, named 6dFSH-P36-hCG60, is described. At random stages of the estrous cycle, cows (n = 52) were subjected to ultrasound-guided transvaginal aspiration of all follicles ≥ 5 mm. After FA, cows were randomly assigned to one of two groups in which FSH treatments started 1 or 2 days after FA (groups FA-1D and FA-2D, respectively). Cows were superstimulated with a total of 500 μg pFSH over 6 days on a decreasing dose schedule and were pre-treated with a single dose of 400 IU of eCG 24 h before the start of FSH treatments. Follicular superstimulatory (the mean numbers of follicles ≥ 8 mm on the day of hCG treatment) and SOV responses (the mean numbers of CL and cows with ≥ 3 CL at the time of collection) were similar in FA-1D and FA-2D groups. However, when compared to FA-1D group, the number of unfertilized ova tended to decrease (0.4 vs 1.7; P = 0.065) and percentage of fertilized ova tended to increase (95.8% vs 84.6%; P = 0.066) in FA-2D group. Moreover, the mean numbers and percentages of both transferable embryos (8.0 and 77.6% vs 6.4 and 57.7%) and freezable embryos (5.3 and 51.5% vs 3.5 and 31.1%) were numerically higher in FA-2D group than FA-1D group.
The results of the study suggest that starting a lengthened superovulation programs in Simmental cows 2 days after FA has potential to increase percentage of fertilized ova and the number of transferable and freezable embryos, although new studies may be needed to confirm this findings.
Treatment with chemical delipidation forskolin prior to cryopreservation improves the survival rates of swamp buffalo (Bubalus bubalis) and bovine (Bos indicus) in vitro produced embryos
2018, Cryobiology
The cryopreservation of embryos is a technology developed for long-term genetic preservation. However, high sensitivity to low temperatures due to a large number of intracellular lipids within ruminant embryos compromises the success of this technique. The aim of this study was to examine the effects of using of lipolytic chemical agent forskolin, during in vitro producing of buffalo and bovine embryos on lipid contents, cryotolerance and subsequent developmental competence of these embryos. Buffalo and bovine oocytes were collected by the aspiration technique from follicles and submitted for in vitro fertilisation; the embryos were later divided into four experiments. Experiment 1, buffalo and bovine embryos were pre-treated in the presence and absence of 10 μM forskolin for 24 h. Lipid contents were determined by Nile red staining and confocal microscopy. We found that 10 μM forskolin was capable to reduce lipid contents within developing embryos in both of species (P < 0.01). Lipid contents within Day 2 embryos exhibited greater fluorescence intensity than did Day 7 embryos in both animal species. The purpose of Experiment 2 was to investigate the adverse effects of 10 μM forskolin on embryo development. In Experiments 3 and 4, Day 2 (4- to 8-cell stage) and Day 7 (blastocyst stage) embryos were pre-treated with 10 μM forskolin for 24 h and further cryopreserved with a controlled-rate freezing technique. The successful cryopreservation was determined by post-thawed embryonic development in vitro. The results showed that the blastocyst rate of the 4–8 cell stage in the forskolin-treated group had increased in both species, while the hatching and hatched blastocyst rates of forskolin-treated day 7 bovine embryos were significantly higher than those of the non-treated group (52.1% vs. 39.4%; P < 0.05). However, delipidation with forskolin did not affect the developmental rate of the day 7 buffalo embryos (P = 0.73). Our studies showed that delipidation by forskolin treatment increased the survival rate of cryopreservation in buffalo and bovine in vitro produced embryos.

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