Some challenges and unrealized opportunities toward widespread use of the in vitro-produced embryo in cattle production

Abstract

The embryo produced by in vitro oocyte maturation, fertilization, and embryonic development is an important resource for genetic improvement and has the potential to improve female fertility and to be programmed to produce offspring with superior ability for health and production. The cultured embryo is also an important component of several realized and potential technologies such as gene editing, somatic cell nuclear cloning, stem cell technologies and gamete generation in vitro. Full realization of the opportunities afforded by the in vitro-produced embryo will require overcoming some technical obstacles to cost-effective implementation of an embryo transfer program. Among the research goals for improving the penetration of embryo transfer in the cattle industry are development of methods to increase the supply of oocytes from genetically elite females, enhance the proportion of oocytes that become transferrable embryos, improve the fraction of embryos that establish pregnancy after transfer, reduce pregnancy wastage after pregnancy diagnosis, and identify culture conditions to optimize postnatal phenotype.

Implications

Embryo transfer using embryos produced in vitro can result in improved genetic selection, fertility, and postnatal phenotypes of cattle. Overcoming inefficiencies in the system of producing and transferring embryos, including those described here, will make the practice more widespread.

Toward the goal of an in vitro-produced embryo in every cow

Henri IV of France (1553–1610) was said to have pledged that every ploughman in his kingdom would have a chicken in his pot. Speaking to Charles-Emmanuel I of Savoy, he said “Si Dieu me prête encore vie, je ferai qu’il n’y aura point de laboureur en mon royaume qui n’ait moyen d’avoir une poule dans son pot” (Hardouin de Péréfixe de Beaumont, 1661). A similar pledge should be made by bovine reproductive biologists. Developing the capacity to put an in vitro-produced (IVP) embryo in every cow rather than relying only on natural or artificial insemination to achieve pregnancy would allow full realization of the promise of embryo technologies for improving the efficiency and sustainability of cattle production.

Embryo transfer is already an important tool for genetic improvement. It can be used to improve the accuracy of genetic selection, increase the intensity of dam-side genetic selection, allow genomic selection as early as the embryo stage, and facilitate the production of beef animals from dairy cattle (VanRaden, 2020; Crowe et al., 2021). The cultured embryo is also an important component of several existing or emerging technologies such as gene editing, somatic cell nuclear cloning, stem cell technologies and gamete generation in vitro. These transformative procedures are discussed in other papers in this volume and elsewhere (Goszczynski et al., 2019; Su et al., 2020). Transfer of the IVP embryo can also improve fertility in certain situations such as during heat stress and for the repeat-breeder cow and, as discussed by Hansen (2020a), improvements in the technologies associated with embryo production could result in fertility enhancement more broadly. Furthermore, the preimplantation embryo is amenable to developmental programming; culture media can be modified to produce offspring with phenotypes desirable for production (Estrada-Cortés et al., 2021a; 2021b).

Refinements in existing technologies and development of new ones are imperative for meeting the contemporary challenges for animal production to increase the economic and biological efficiency of production of meat and milk while also reducing the emission of associated greenhouse gasses. A combination of a growing world population and more economic affluency is driving growth in the numbers of livestock globally (Gordon, 2018; Food and Agriculture Organization of the United Nations and Global Dairy Platform, 2019). Livestock contribute to global climate change through the direct and indirect production of gasses such as methane, carbon dioxide and nitrous oxide. Increasing yield per animal reduces the production of greenhouse gasses per unit of food produced (Balmford et al., 2018; Food and Agriculture Organization of the United Nations and Global Dairy Platform, 2019). Improved fertility also reduces greenhouse gas emissions (Garnsworthy, 2004).

There are constraints to achieving the goal of an IVP embryo in every cow. Technical limitations make it expensive to produce a calf from an IVP embryo. Moreover, the practice of embryo transfer in many countries is constrained by laws that limit cow-side procedures to veterinarians. At the current state of technology, the costs of embryo transfer often exceed gains due to genetic improvement or improved fertility (Kaniyamattam et al., 2018; Ferreira et al., 2021). Despite these circumstances, the use of the IVP embryo in cattle production systems has been rising. The global number of IVP embryos reported as being transferred in cattle increased 2.6-fold between 2012 and 2020 (Viana, 2021). Over 1.4 million embryos globally were reported to be transferred in 2021, with the majority being embryos produced in vitro (1 166 034) as compared to those produced by superovulation (313 780; hereafter referred to as multiple ovulation embryo transfer; MOET) (Viana, 2022). Transfer of IVP embryos exceeded those of MOET embryos for the first time only in 2017 (Viana, 2021). The main drivers of these historical changes are the introduction of single nucleotide polymorphism chips for genotyping cattle in 2009 to allow for accurate estimates of genomic breeding values of females and the advantages of IVP over MOET in maximizing embryo yields. Embryos can be produced by IVP more frequently than by superovulation (as often as twice weekly vs every month or two) and from females that would not be eligible for enrollment in a superovulation program (prepubertal and pregnant animals).

The growth of the embryo transfer industry globally in the 21st Century has occurred despite only incremental changes in the underlying technologies for production and transfer of embryos. Sustained and extensive research to improve embryo technologies is urgently needed to increase the net value of embryo transfer in cattle production systems. The goal of this paper is to highlight some of the constraints to effectiveness of embryo transfer programs based on the IVP embryo and suggest some research avenues that might lead to their elimination or reduction. Space limitations mean that some issues are not included or are discussed in a limited fashion. The reader is directed elsewhere (Sirard, 2016; Ealy et al., 2019; Hansen, 2020a and 2020b; Baruselli et al., 2021; Valente et al., 2022) for treatment of some relevant topics not discussed here, such as recipient management, cryopreservation, and the impact of sire and genetics, as well as for a more detailed analysis of specific topics discussed here.

Overview of the process for production of calves from embryos produced in vitro

Production of a calf from an IVP embryo requires several specific procedures (Fig. 1). The starting point is the collection of semen from bulls and the harvest of oocytes from donor females. Oocytes are typically obtained by ultrasound-guided transvaginal follicular aspiration [i.e., oocyte pickup (OPU)] although oocytes recovered from an abattoir are an alternative source. Following maturation of oocytes in vitro, fertilization takes place in a medium that facilitates sperm capacitation. The resultant embryos are cultured for 7–8 days in one of a variety of media that have been developed for bovine embryos. Embryos for transfer (most frequently, blastocysts) are selected based on developmental and morphological criteria and transferred to recipient females at a comparable stage after ovulation (typically day 7) via transcervical passage into the uterine horn ipsilateral to the side of the corpus luteum. Usually, up to half of the transferred embryos establish pregnancy (Hansen, 2020a). A large fraction of embryos that establish pregnancy continue to term although pregnancy loss has been reported to be higher than following artificial insemination (Stewart et al., 2011; Marques et al., 2020). Indeed, none of the procedures outlined in Fig. 1 have been optimized and major bottlenecks to production of calves exist. The remainder of the paper will be focused on key aspects of the IVP embryo transfer system for which there are potential routes for improvement.

Fig.1.

Overview of the procedures for embryo transfer in cattle using in vitro-produced embryos. None of the steps shown here have been optimized. Among the major bottlenecks to optimization of the system are insufficient supplies of oocytes, low rates of production of transferrable embryos from matured oocytes, lower than expected rates of pregnancy establishment, and high loss of pregnancies before calving. The figure was produced using biorender.com.

Increasing availability of oocytes

Developments in stem cell biology may make it possible to generate oocytes or sperm from stem cells as has been reported for mice (Saitou and Hayashi, 2021). Until these advances are recapitulated in cattle, the production of IVP embryos is dependent on oocytes obtained from females. This is usually performed by OPU whereby follicles visualized by ultrasonic examination of the ovaries are aspirated transvaginally using specialized aspiration needles and pumps. The procedure can be performed as often as twice weekly (Lopes et al., 2006; Saleem et al., 2022). Follicular aspiration can be performed on pregnant cows in the first 100 days of gestation (Aller et al., 2012) and, using laparoscopy, in prepubertal animals as early as 2–4 months of age (Baruselli et al., 2021).

The number of oocytes recovered by OPU varies between animals and breeds. Females of various Bos indicus breeds have more follicles that can be aspirated than those of at least some B. taurus breeds (Baruselli et al., 2021). Accordingly, Bos indicus donors are usually used without hormonal stimulation while B. taurus females are often treated with FSH to increase the number of follicles. Understanding the physiological and molecular basis for genetic differences between breeds in control of folliculogenesis could lead to new procedures for increasing the harvest of oocytes by OPU; this type of research is now beginning (Baldrighi et al., 2022).

Collection of oocytes from abattoir material is also possible. Culled females are sometimes genetically elite although, by definition, such animals are a minority of the national herd. Advances in the electronic identification of animals should make it possible to identify genetically desirable females at a slaughter facility. The number of oocytes in the ovary greatly exceeds the number harvested by follicular aspiration. Research currently underway to understand the control of follicular activation and growth and to culture primordial follicles to a stage suitable for oocyte harvesting (Telfer, 2019) could greatly expand the number of oocytes collected from abattoir-derived ovaries.

Increasing the yield of transferrable embryos

In vivo, the percent of oocytes that become blastocysts varies between ~50 and ~90% depending on the breed and physiological status of the female (Hansen, 2020a). The percent of oocytes that become blastocysts in vitro, in contrast, typically varies between ~20 and 40%. The low yield of blastocysts occurs despite most oocytes subjected to in vitro maturation completing nuclear maturation (>80%) and becoming fertilized (>70%). Thus, the competence of a zygote produced in vitro to develop to the blastocyst stage is compromised.

Experiments by Rizos et al. (2002) illustrated in Fig. 2 can be interpreted as indicating that the greatest limitation to optimal development to the blastocyst stage for IVP are the periods of oocyte maturation and, to a lesser extent, fertilization. When these two events occurred in vivo and embryos were cultured thereafter, 74% of oocytes became blastocysts. Even when fertilization occurred in vitro, the percent of oocytes becoming blastocysts when maturation proceeded in vivo ranged from 58 to 78%. In contrast, placing embryos produced by in vitro maturation and fertilization into the sheep oviduct for development did not increase the percent of oocytes becoming blastocysts (35%) as compared to embryos grown in vitro (34%). Similar results have been obtained by Gad et al. (2012) whereby a greater proportion of embryos became blastocysts if residing in the reproductive tract until the 4-cell stage than if oocyte maturation, fertilization, and early development took place in vitro. The importance of the period of oocyte maturation and fertilization for establishing embryonic competence for further development has also been shown for the mouse, pig and rhesus monkey (Hansen, 2020b).

Fig. 2.

Importance of the period of oocyte maturation and fertilization for the establishment of competence of an embryo to develop to the blastocyst stage. Effects of the in vitro environment on oocyte maturation, fertilization and embryonic development were assessed for the percent of embryos developing to the blastocyst stage and the survival of the blastocysts after vitrification and thawing. Events occurring in vitro are indicated by the petri dish while events in vivo, either in heifers or sheep, are shown by the animal icons. The figure was produced using data from Rizos et al. (2002) and is reproduced from Hansen (2020b) with permission of Annual Reviews.

Little work has been done to understand better the inadequacies of fertilization protocols in vitro but much more effort has been placed on improving oocyte maturation. In part, poor outcomes reflect the fact that most oocytes used for IVP are not harvested from dominant follicles. Thus, they have not undergone all the physiological changes associated with growth of the dominant follicle and may have experienced changes in oocyte-cumulus communication associated with atresia (Sirard, 2016). One approach to create a more functional oocyte for IVP is to manipulate circulating FSH concentrations via the administration of exogenous FSH to donor cows. It has been proposed that creating a period of low circulating concentrations of FSH in the 44–68 hours before OPU mimics the low FSH concentrations in the blood before ovulation and simulates an environment associated with dominant follicle selection (Sirard, 2016). A blastocyst rate at day 7 after insemination as high as 63% has been reported (Blondin et al., 2002) although, in practice, this rate is not usually achieved. There are also indications that regulation of cyclic adenosine monophosphate (cAMP) in the oocyte (a second messenger for FSH) can improve developmental competence. Treatment of abattoir-derived cumulus-oocyte complexes with N6,2′-O-dibutyryladeno sine 3′,5′-cyclicmonophosphate and the cAMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine increased the percent of cleaved embryos that became blastocysts to as high as 70% (Sugimura et al., 2018).

Efforts to enhance blastocyst yield by modifying embryo culture media have been less successful. Addition of serum to culture medium can have a large positive effect on development to the blastocyst stage but may also reduce competence of the resultant embryos to establish pregnancy (Amaral et al., 2022). The percent of embryos becoming blastocysts can also be increased by addition to embryo culture medium of oviductal fluid (Lopera-Vasquez et al., 2017) and specific embryokines produced by the reproductive tract (Wooldridge et al., 2022) but the magnitude of effects is small.

Modifying embryo culture conditions to increase the competence of the embryo to establish pregnancy

One would expect that the pregnancy rate after transfer of an IVP embryo would be greater than after artificial or natural insemination because embryo transfer bypasses causes of pregnancy failure such as misdiagnosis of estrus, anovulation, fertilization failure, and embryonic mortality before day 7 of pregnancy (Hansen, 2020a). In fact, except for situations in which fertility to artificial insemination is low, such as for heat stress or in repeat-breeder cows, the proportion of cows pregnant after transfer of an IVP embryo is not higher than the proportion of cows pregnant after artificial insemination (Hansen, 2020a). Results of an experiment illustrating this situation are illustrated in Fig. 3A.

Fig. 3.

Selected experiments that illustrate (A) pregnancy rate at day 30 of gestation following embryo transfer vs following artificial insemination (total n = 2 874), (B) pregnancy rate at day 30 to day 35 of gestation following transfer of a multiple ovulation embryo transfer (MOET) or in vitro-produced (IVP) embryo (total n = 1 189), and (C) pregnancy rate at day 41 of gestation following the transfer of a MOET or IVP embryo (total n = 8 295). Recipients were lactating dairy cows (A and C) or crossbred beef cows (B). Abbreviations: TAI = timed artificial insemination; TET = timed embryo transfer. Results for A, B, and C are from Oliveira et al. (2019), Pontes et al. (2009), and Ferraz et al. (2016), respectively.

One of the possible causes for the equivalency of pregnancy rates for IVP embryo transfer and artificial insemination is that the IVP embryo has aberrant characteristics unfavorable for pregnancy establishment. Compared to the MOET embryo, the IVP embryo has an increased incidence of chromosomal abnormalities, changes in oxygen consumption, increased accumulation of intracellular lipid, altered gene expression and DNA methylation, and delays in trophoblast elongation (Ealy et al., 2019; Hansen, 2020a and 2020b). Pregnancy rates after the transfer of an IVP embryo are lower than after the transfer of an embryo produced by superovulation (Ealy et al., 2019). Representative examples of this phenomenon are shown in Fig. 3.

Reduced competence of the IVP embryo to establish pregnancy could be caused in part to use of oocytes that are not sufficiently developed or errors in the process of oocyte maturation or fertilization. There is some evidence that the period of embryo culture may also be important for the formation of a blastocyst fully competent for subsequent development. The experiment by Rizos et al. (2002) outlined in Fig. 2, in which various aspects of the embryo production system were carried out in vitro or in vivo, were indicative that the maternal environment enhanced functional characteristics of the blastocyst. Specifically, blastocysts that developed in the sheep oviduct and uterus had superior ability to survive cryopreservation as compared to those that developed in vitro. Further evidence that the molecular and functional characteristics of the blastocyst are shaped by molecules in the reproductive tract comes from studies showing that exposure of the embryo to in vitro conditions for a short period altered gene expression in the blastocyst (Gad et al., 2012).

The results summarized above lead to the hypothesis that altering the culture medium of the embryo to produce an environment more akin to that of the reproductive tract could increase competence of blastocysts to establish pregnancy. One approach tested has been to add embryokines to culture medium. Embryokines are regulatory molecules produced by the cells of the oviduct and endometrium that can regulate specific aspects of development of the embryo. As such, they are not typically present in embryo culture medium unless serum is present or embryos are co-cultured with somatic cells producing specific embryokines. Initial experiments with two embryokines, colony-stimulating factor 2 (CSF2) and dickkopf WNT signaling inhibitor 1, indicated that the addition of either molecule to embryo culture medium increased pregnancy rate after embryo transfer (Loureiro et al., 2009; Denicol et al., 2014). More recently, however, the beneficial effects of these molecules were not observed (Estrada-Cortés et al., 2021b; Amaral et al., 2022). Actions of these or other embryokines may depend on the specific characteristics of the embryo or recipient, as some results indicate (Dobbs et al., 2014; Amaral et al., 2022). Alternatively, the small sample size employed in the experiments resulted in inconsistent outcomes. Indeed, difficulties in performing embryo transfer experiments with sufficiently large numbers to produce robust results have limited progress in improving pregnancy outcomes after embryo transfer.

A total of 175 receptor genes have been identified as expressed by the bovine morula (Sang et al., 2021) and optimal development may require exposure of the embryo to multiple embryokines. Recent developments in organoid and microfluidic culture systems (Ferraz et al., 2018; Bourdon et al., 2021) will provide new opportunities to improve the competence of the IVP embryo for embryo transfer. Co-culture of embryos with organoids of oviductal epithelial cells or endometrial epithelial cells, singly or in serial combination, could represent one approach to provide the embryo with regulatory signals important for optimal development.

Another avenue for improving pregnancy outcomes is to develop new tools for identifying embryos most likely to establish pregnancy. There are promising results for embryo selection based on morphokinetic features of development recorded with time-lapse imaging (Sugimura et al., 2017), screening of embryos for chromosomal abnormalities (Turner et al., 2019) and identification of molecular signatures of the embryo predictive of pregnancy success (Zolini et al., 2020). One drawback of any embryo selection scheme is that the cost of producing a transferrable embryo will increase because of expenses associated with the screening procedure and the amortization of costs of production over fewer embryos (since more embryos will be discarded before transfer).

Reducing pregnancy losses after pregnancy diagnosis

Pregnancies sometimes fail and the incidence can be high in the first trimester of pregnancy (Wiltbank et al., 2016). Inadequate maternal support for pregnancy is one cause of pregnancy loss. Pregnancy failure was greater for cows subjected to timed artificial insemination or embryo transfer in which estrus was not observed than for cows in which estrus was observed (Pereira et al., 2016). Aberrant embryonic development is another cause of pregnancy loss. Pregnancies resulting from the transfer of an IVP embryo have been associated with a range of placental and fetal perturbations (Ealy et al., 2019) so it is reasonable to hypothesize that pregnancy loss after initial pregnancy diagnosis would be greater for the IVP embryo than for the embryo produced by artificial insemination (AI). As shown in Fig. 4, some studies indicate pregnancy loss after day 28 of gestation is greater for pregnancies established by transfer of an IVP embryo (Stewart et al., 2011; Marques et al., 2020). In another study, however, there was no statistically significant difference between IVP and AI (Pereira et al., 2016). Moreover, there was no difference in pregnancy loss between IVP and MOET embryos (Pontes et al., 2009). Given the variation in methods used to produce embryos in vitro, it is possible that embryos from some systems are more prone to pregnancy failure than embryos from other systems. It is possible that changes to culture systems to result in an IVP embryo with greater competence to establish pregnancy will also produce an embryo more likely to develop successfully to term after initial pregnancy diagnosis.

Fig. 4.

Pregnancy loss after initial pregnancy diagnosis in cows as affected by the type of pregnancy. Pregnancy loss was calculated for the period indicated above each set of bars. Recipients were lactating dairy cows (A–C) or crossbred beef cows (D). The total number of cows that were initially pregnant was 143 (A), ~427 (B), 2 845 (C), and 814 (D). Abbreviations: AED = automatic estrous detection device; ET = embryo transfer; IVP = in vitro produced; MOET = multiple ovulation embryo transfer; TAI = timed artificial insemination; TET = timed ET. Results for A, B, C, and D are from Stewart et al. (2011), Marques et al. (2020), Pereira et al. (2016), and Pontes et al. (2009), respectively. Note that the study indicated in B used a mixture of IVP and MOET embryos.

Optimizing postnatal phenotype

It is well established that changes in the environment of the developing embryo or fetus can alter the developmental program to change postnatal phenotype. This phenomenon is probably multicausal but among the mechanisms implicated are changes in epigenome of the embryo, fetus, and placenta (Christoforou and Sferruzzi-Perri, 2020; Lapehn and Paquette, 2022). Developmental programming occurs during the preimplantation period (Hansen et al., 2016) and IVP embryos of various species have been associated with changes in phenotypic characteristics of the resultant offspring (Hansen et al., 2016; Duranthon and Chavatte-Palmer, 2018; Hansen, 2020b). In cattle, in vitro production has been reported to alter the DNA methylome at the blastocyst stage (Canovas et al., 2021), in placenta and fetal muscle and liver at day 86 of gestation (Li et al., 2020), and in hypothalamus, pituitary, testis, adrenal and blood of the newborn calf (Rabaglino et al., 2021).

More research is needed to determine whether these changes in the epigenome or other alterations in development caused by IVP are sufficient to modify the phenotype of the calf. One study indicates alterations in calf survival due to IVP or MOET and reduced milk yield in first-lactation females derived from embryos produced by IVP using reverse-sorted semen (Siqueira et al., 2017). Other research failed to identify any negative consequences of IVP or MOET on age at puberty or first-lactation milk production (Baruselli et al., 2021). More recently, offspring derived from IVP were reported to be slightly more fertile than offspring produced by MOET and to have lower somatic cell count (Mullaart et al., 2022).

Occasionally, IVP is associated with a phenomenon called abnormal offspring syndrome in which there is gross dysregulation of the epigenome and transcriptome, excessive somatic growth, and other developmental abnormalities including enlarged tongues, umbilical hernias, muscle and skeleton malformations, abnormal organ growth and placental development (Li et al., 2019; 2022). Although associated with IVP, the phenomenon of abnormal offspring syndrome can also occur following insemination. The incidence of this syndrome is not often reported and may depend upon the culture system used to produce embryos. A rough estimate of the frequency of the syndrome in IVP calves is <3%. Presence of serum in the embryo culture medium is often claimed to be a causative factor for abnormal offspring syndrome but experimental evidence in cattle is lacking (Hansen, 2020b).

The fact that the developmental program of the preimplantation embryo can be modified by culture conditions means that it is possible to improve postnatal phenotypes of cattle derived from an IVP embryo by manipulation of the culture environment. Thus, an additional benefit of IVP could be to produce animals with superior epigenetic potential for growth, reproduction or milk yield. There is some evidence for this idea. Holstein heifers derived from embryos treated with CSF2 from day 5 to day 7 of development had similar birth weights as heifers derived from control embryos but experienced accelerated body growth after 3 months of age (Kannampuzha-Francis et al., 2015). In another experiment (Estrada-Cortés et al., 2021a), addition of the methyl donor choline to culture medium of IVP embryos resulted in an increase in birth and weaning weight in Brahman cattle (Fig. 5).

Fig. 5.

Programming of 205-day adjusted weaning weight in Brahman calves by addition of choline chloride to culture medium for in vitro-produced embryos. Abbreviations: Cho = choline; Veh = vehicle. The increase in weaning weight due to choline, which was significant, averaged 20 kg (a 9% increase). Data are from Estrada-Cortés et al. (2021a).

Concluding comments – limits to the penetration of the technology

It might seem unrealistic to expect that embryo transfer will become a common practice on commercial farms. Even artificial insemination, which was developed in the early part of the 20th Century, is still not widely utilized in beef cattle production systems in much of the world. Embryo transfer technologies are more complex than AI and more expensive. Undoubtedly, the degree to which embryo transfer penetrates the dairy and beef cattle industries will depend on the economic rates of return that accrue from the procedure. There are two reasons for optimism. The first is that the cattle industry, particularly the dairy industry, has always been willing to adopt new, economically beneficial technologies. Just in the last 40 years, timed artificial insemination protocols and use of ultrasound technologies for reproductive diagnoses have become routine on many dairy farms despite the expenses in equipment and labor involved. Each of these techniques was met with skepticism. Sasser and Ruder (1987) stated that “Ultrasonic devices might provide very early detection in cattle but the expense of a test will limit application”. They also recognized that less expensive devices would become available with time. Nebel and Jobst (1998) used survey data from Virginia veterinarians to calculate that the cost per pregnancy of the Ovsynch timed artificial insemination protocol was sometimes over twice as expensive as that of estrous synchronization programs based on injections of prostaglandin F_2a at 14-day intervals. In many countries, the costs of drugs used for timed artificial insemination have declined since that paper was written. In a more distant time, artificial insemination was met with great skepticism by many including the Shorthorn Society of the United Kingdom of Great Britain and Ireland who put in their minutes in 1923 that the procedure was cumbersome and “would never be widely adopted” (Wilmot, 2007).

This author has sometimes expressed skepticism about new technologies that subsequently have become part of the routine procedures implemented on farms. Lesson learned! Accordingly, this paper is closed with the prediction that many of the technical problems constraining use of the IVP embryo in cattle production systems will be overcome through additional research and that embryo transfer will become a widely used reproductive technique that provides benefits to producers and society at large.

Abbreviations:

IVP

in vitro-produced

MOET

multiple ovulation embryo transfer

OPU

oocyte pickup