Reproductive Technologies in Animals

Reproductive Technologies in Animals provides the most updated and comprehensive knowledge on the various aspects and applications of reproductive technologies in production animals as well as companion, wild, exotic, and laboratory animals and birds. The text synthesizes historical information and recent discoveries, while dealing with economical and geographical issues related to the implementation of the same technologies. It also presents the effects of reproductive technology implementation on animal welfare and the possible threat of pathogen transmission.

Reproductive Technologies in Animals is an important resource for academics, researchers, professionals in public and private animal business, and students at the undergraduate and graduate levels, as it gives a full and detailed first-hand analysis of all species subjected to the use of reproductive technologies.

• Provides research from a team of scientists and researchers whose expertise spans all aspects of animal reproductive technologies
• Addresses the use of reproductive technologies in a wide range of animal species
• Offers a complete description and historical background for each species described
• Discusses successes and failure as well as future challenges in reproductive technologies

• Language: English
• Publisher: Academic Press
• Release date: May 28, 2020
• ISBN: 9780128171080

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Chapter 1

Reproductive technologies in cattle

J. Richard Pursley and Jose Cibelli,    Michigan State University, East Lansing, MI, United States

Abstract

In this chapter, a particular focus of attention has been given to the use of reproductive technologies for the manipulation of ovarian function and physiology. The implementation of fertility programs based on Ovsynch have been shown to be instrumental for enhancing reproductive performance and controlling calving interval. The adoption of such newly developed reproductive technologies has undoubtedly given proof to be valuable tools for increasing productivity in dairy cattle. In the second part of this chapter, more complex technologies such as cloning through somatic cell nuclear transfer will be discussed. They give the possibility to replicate the genome of highly valuable animals of superior genetics while underlining the limits, costs, and hurdles to be found in the various steps of the entire process. Finally, a vision will be given to the reader on the need to obtain more information on cellular reprogramming and cell plasticity, and on the new technical challenge regarding emerging assisted reproductive techniques, from the study and development of DNA markers for shortening generation interval and selection to the production of functional gametes from embryonic and primordial germ cells into sperm and eggs.

Keywords

Cattle; fertility programs; Ovsynch; cloning; emerging ARTs

1.1 Introduction

Reproductive technologies are critical for genetic modification and management efficiencies that directly impact sustainability of dairy and beef herds. These technologies provide ways to improve individual production and health traits as well as manipulate production cycles to maximize herd performance parameters.

This chapter describes current reproductive technologies from whole animal to cloning in cattle. Section 1 focuses on recent technologies that have transformed reproduction in cattle industries. Technologies that control ovarian function and timing of ovulation for the purpose of timed-artificial insemination (AI) are discussed in depth. These technologies succinctly control the time of ovulation and can improve fertility. The increases in reproductive efficiency from these technologies have had major positive impacts on profit of herds that utilize them. In addition, new electronic technologies that enhance detection of estrus and new ways to diagnose early pregnancies in cattle are discussed. Lastly, this section will introduce new ways to control ovarian development during ovarian stimulation with follicle-stimulating hormone (FSH).

Section 2 discusses bovine cloning and emerging reproductive technologies to improve cattle genomic parameters. It includes a brief description on the origins of cloning cattle, and the current techniques implemented commercially. We also discuss new technologies that, coupled with somatic cell nuclear transfer (SCNT), have the potential to exponentially increase genetic selection in a very short period of time.

Section 1: Increasing the efficiency of pregnancy production utilizing technologies that control ovarian function in cattle

1.1.1 Controlling ovarian development to enhance pregnancy production following artificial insemination

1.1.1.1 Physiological basis for ovarian manipulation of the estrous cycle

Technologies that manipulate ovarian structures in systematic ways are important for a number of reasons. The primary purpose of manipulating ovarian function is to synchronize timing of estrus and/or ovulation for preparation to AI. Additionally, synchronizing estrus and/or ovulation is important for recipients of embryos in addition to donor cows being prepared for superovulation or ovum pickup (OPU).

Follicles and corpora lutea (CL) growth and function during an estrous cycle are very well understood. Follicles grow in waves following estrus [1]. A cohort of antral follicles begins growing due to an FSH surge that occurs simultaneously with the luteinizing hormone (LH) surge at the onset of estrus. The largest of these follicles continues to grow and develop after the remainder of the follicles in the wave become atretic. This follicle has been termed the dominant follicle (DF) because the inhibin and estradiol that it produces inhibits the release of FSH, thus dominating the ovary as the only growing follicle until it either becomes atretic or, in the case of most second- or third-wave DFs, ovulates. FSH will surge once lack of inhibitory substances, inhibin and estradiol, are no longer being produced from the DF [2–4]. This natural FSH surge will induce a new follicular wave. Once the DF becomes atretic or ovulates a new wave of follicles starts the process again.

Acquisition of LH receptors in granulosa cells of the DF occurs when the DF deviates in growth from the other follicles in the wave [5]. This critical change in functionality to a follicle with LH receptors makes it possible to manipulate this follicle with LH, human chorionic gonadotropin (hCG), or an exogenously induced LH surge using gonadotropin-releasing hormone (GnRH). Understanding this mechanism opened doors to novel ways to manipulate ovarian function and control time to estrus and ovulation. For example, gaining a greater understanding of these concepts led to the discovery of Ovsynch (Fig. 1.1 [6]).

Figure 1.1 Description of how Ovsynch controls follicle and corpora lutea development in cattle to synchronize the timing of ovulation in an 8-hour period. Adapted from Pursley JR, Mee MO, Wiltbank MC. Synchronization of ovulation in dairy cows using PGF2alpha and GnRH. Theriogenology 1995;44:915–23. doi:10.1016/0093-691x(95)00279-h.

Historically, four types of hormones have been utilized to manipulate ovarian function: GnRH type products including the direct impacts of LH or hCG, prostaglandin F2-alpha (PG), progesterone (P4), and pharmaceutical estrogens. The earliest technology derived from one of these hormones was the systematic use of PG to induce and schedule estrus in cattle [3,7]. This technology enhanced the percentage of cows detected in estrus and provided a management tool to organize labor more efficiently.

1.1.1.2 Importance of inducing a new follicular wave

The most important step in assuring success of timed-AI technologies is the induction of a new follicular wave. It is a critical step in manipulating ovarian development to control time of ovulation to allow for timed-AI. A new follicular wave can be pharmacologically induced following ovulation with an endogenous or exogenous surge of LH, estrogen-induced atresia of functional follicles, or ablation of functional follicles with ultrasound-guided aspiration [8].

Asynchrony of follicle development relative to luteolysis and induction of ovulation is likely to occur if a new follicular wave is not induced in timed-AI technologies. DFs can become atretic prior to PG induced luteolysis. This would result in an untimely new wave of follicles near the time of the final induction of ovulation. Thus follicles from this untimely new wave may lack sufficient maturity to respond to the final LH surge and ovulate. Delayed time to estrus and ovulation would occur 3–4 days later.

GnRH products induce an LH surge from the anterior pituitary. Functional DFs generally ovulate approximately 28 hours following an LH surge even in the presence of progesterone. An FSH surge occurs at the same time as the LH surge, and stimulates a new wave of follicular development. Numbers of follicles in this new wave are positively associated with circulating levels of anti-Müllerian hormone [9].

1.1.2 Ovsynch technologies

Ovsynch technologies utilize GnRH and PG in timely fashions to synchronize ovulation during a period of low progesterone imitating as closely as possible the physiological environment of a natural estrus [6]. Ovsynch technologies control follicle and CL development in order to synchronize ovulation amongst groups of cattle for fixed timed-AI [10,11]. Most countries have approved GnRH and PG products available for use. GnRH products are decapeptides that may or may not have a hydrophobic peptide for a longer half-life [12]. Most PG products utilized in Ovsynch technologies contain either cloprostenol sodium or dinoprost tromethamine. Ovsynch-based technologies generally include Ovsynch, Double Ovsynch, G6G, and Presynch/Ovsynch protocols. Ovsynch may include the use of a progesterone-releasing device during the period between the GnRH and PG.

1.1.2.1 The basis for development of Ovsynch-based technologies

Embryonic survival continues to be a critical problem in lactating dairy cows, limiting thus profitability and sustainability of dairy farms [13]. The inability of the lactating dairy cow to foster complete embryonic and fetal development is escalating due to greater genetic pressure on milk production. Pregnancies per AI at 32 days following a detected estrus are approximately 30% in Holstein cows compared to 60% in virgin dairy heifers with similar genetic makeup [14].

Remarkable changes in follicular dynamics occur in dairy cattle during transition from nulliparous to primiparous and multiparous. Size of the ovulatory follicle, length of follicular dominance, number of double ovulations (twinning rates), and ovarian cysts [15] are greater in cows versus nulliparous heifers. Nulliparous heifers have approximately double the progesterone concentrations during mid-luteal phase of the estrous cycle compared to cows. The considerable decrease in fertility and increase in twinning rates transitioning from nulliparous heifer to cow negatively affects farm profit. Fertility programs (Fig. 1.2) were developed in the past 15 years to increase pregnancies per AI and reduce the chance for twinning compared to Ovsynch alone or natural estrus [16–18]. Fertility programs utilize pre-Ovsynch hormonal manipulations to increase levels of progesterone and manipulate the age and size of ovulatory follicles in cows treated with Ovsynch [19].

Figure 1.2 The fertility program G6G presynchronizes most cows to start Ovsynch on day 6 of the estrous cycle. Cows on day 6 of the cycle have a 95% chance of ovulating a first-wave DF, inducing an accessory CL, and starting a new follicular wave (Bello et al., 2006).

1.1.2.2 First gonadotropin-releasing hormone of Ovsynch

Fertility program technologies in dairy cows are designed to presynchronize, so Ovsynch can be implemented on day 6 or 7 of the estrous cycle. Ovsynch is based on three treatments. The first treatment is GnRH. The intent of the GnRH-induced LH surge is to cause ovulation of the day 6 or 7 DF(s). Cows on day 6 or 7 of the estrous cycle have >95% chance of ovulating the DF. Ovulation of the DF induces the subsequent emergence of a new follicular wave ~1.5 days later [6] followed by the growth and development of both a new DF and an accessory CL during the next 7 days. The new DF has approximately a 95% chance of remaining functional during the 7 days leading up to the PG and then continuing on to ovulation following the final GnRH-induced LH surge [17].

Cows that have not been presynchronized with a fertility program and receive Ovsynch at a random stage of the estrous cycle have approximately a 30% chance to be in early stages of follicular development (first or second wave) when granulosa cells have not yet acquired sufficient LH receptors to respond to the GnRH-induced LH surge [5]. In this case, the first GnRH does not induce ovulation and the potential DF continues to grow, deviate from subordinates, and develop as a DF. This DF has approximately an 80% chance of remaining functional prior to the PG-induced luteolysis and subsequent increase in LH pulsatility that allows further development. If this DF remains functional until the time of PG, it will continue to develop into a pre-ovulatory follicle and has a 97% chance of ovulation following the final injection of GnRH [17]. However, this follicle could be as much as 12 days from emergence. Thus fertility of this follicle could be compromised due to the antral age of the follicle with an oocyte developing under insufficient levels of progesterone. Conversely, approximately 20% of these follicles become atretic prior to the PG. If this happens, a new wave will develop generally just prior to the PG and the new potential DF will emerge, deviate from subordinates, and become a DF. But this follicle will likely not have deviated from subordinate follicles and will not have acquired LH receptors prior to the final GnRH-induced LH surge. In this scenario, cows will likely have natural estrus that may or may not be detected 3–4 days after the timed-AI as the new DF develops under basal concentrations of progesterone into an ovulatory follicle. Conception rates from the timed-AI in this case would be near 0%. Thus it is critical to control ovulation of a DF in response to the first GnRH of Ovsynch to not only induce an accessory CL to increase P4 but to control the age of the DF to control ovulation to the final GnRH-induced LH surge and avoid the asynchrony of ovulation just described. To ensure that cows respond to the first GnRH-induced LH surge of Ovsynch, cows must be on day 6 or 7 of the estrous cycle [10]. This is the value of fertility programs that presynchronize cows to day 6 or 7 of the estrous cycle at the time of the first GnRH of Ovsynch.

1.1.2.3 Prostaglandin F2-alpha of Ovsynch

PG is administered to induce luteolysis, thus enabling the DF of the new follicular wave to develop into a preovulatory follicle. Unfortunately, lactating dairy cows have approximately an 80% chance of complete luteolysis prior to the final GnRH following a single dose of PG [20]. Even though cows have a high likelihood of responding to the final GnRH and ovulating, data indicate that chances of a pregnancy were only 5% in cows that do not decrease to <0.5 ng/mL P4 at the time of the final GnRH [21]. Studies indicate that a second PG 24 hours later resolves this problem [22].

When Ovsynch is initiated late in the estrous cycle, there is a high likelihood that CL may undergo natural luteolysis prior to the PG. If this happens, cows may have a natural estrus and ovulate early. In this case, if cows are not detected in estrus at this time, usually around the time of PG, conception rates are significantly less due to the asynchrony of AI and ovulation, that is, timed-AI may occur well after ovulation.

1.1.2.4 Final gonadotropin-releasing hormone of Ovsynch

This additional GnRH treatment is administered 48–60 hours after the PG to induce a preovulatory LH surge, trigger ovulation of the functional DF 24–32 hours later [6], and release the oocyte to be fertilized following AI. The chance of ovulation to this treatment is >95% even if luteolysis is not complete prior to this injection [20]. As mentioned above, cows can have a synchronized ovulation but still not have a chance to become pregnant due to incomplete or prolonged time to luteolysis.

1.1.3 Synchronization technologies for cows diagnosed not-pregnant, anovular, or cystic

Reducing the time between inseminations in cows diagnosed not-pregnant is essential to control herd calving interval. Cows that become pregnant later in lactation (>150 days in milk) are at greater risk for poorer body condition at the next calving, and subsequently more health issues and reduced fertility during the next lactation [23]. Resynchronization technologies can be initiated before or after pregnancy diagnosis [24,25]. Generally, these protocols are shorter and simpler to implement and maintain compliance compared with first-service programs. Pregnancies per AI achieved are often less than first-service programs. These technologies take into consideration the importance of limiting time between inseminations to ensure pregnancy occurs earlier in lactation.

The most common program for re-synchronization is Ovsynch (Fig. 1.1). But, programs can be implemented based on veterinarian diagnoses of ovarian status at time of a negative pregnancy diagnosis. Cows that have a CL at time of a negative pregnancy diagnosis should be treated with Ovsynch or Ovsynch plus an intra-vaginal progesterone-releasing insert. Cows that do not have CL, including cows with large cysts, should be administered the GnRH 7 days ahead of the start of the Ovsynch program. This would create a new CL and start a new follicular wave similar to fertility programs so that cows can start Ovsynch on day 6 or 7 of the new cycle. This program can be used as a treatment for anovular and cystic ovaries with acceptable pregnancy rates per AI. Pregnancy rates can be increased using fertility and resynchronization technologies if management can be highly accurate utilizing estrus detection prior to pregnancy diagnoses.

1.1.4 Detection of estrus technologies

Electronic technologies that assist management in detection of estrus are key tools that can improve reproductive management of herds. Many of these technologies have additional features that monitor other cow health parameters. These technologies are highly efficient at determining estrus. Most data report these technologies are effective at finding 70% of eligible cows in estrus during a 21-day period [26]. These technologies are very helpful in determining the percentage of cows that are anovular (not cycling). This type of information can lead to making herd management changes that increase the percentage of cows that have ovulated and are cycling during the breeding period. The downsides to these technologies include the inability to induce cyclicity in anovular cows, and in addition they do not enhance fertility of cows when compared to fertility programs.

1.1.5 Technologies to evaluate pregnancy status

Accurate determination of pregnancy at the earliest possible time following AI or embryo transfer followed with re-examinations to detect losses are critical management interventions that are necessary to control calving intervals. Ultrasound technologies have been used for decades to determine pregnancy, but newer ultrasound technologies with color Doppler may become essential for predictions of pregnancy loss. Advantages of ultrasound technologies are the ability to determine a heartbeat, evaluate embryonic or fetal age and health, and evaluate ovarian function and uterine diseases in nonpregnant cattle. Other advantages occur when the veterinarian is present at time of pregnancy diagnosis and can evaluate the health status of the whole animal.

A newer technology that is highly accurate is the blood test for pregnancy-associated glycoproteins and pregnancy-specific protein-B [27]. These proteins are produced from binucleate cells of the trophectoderm of the conceptus. Detection of these proteins in blood is direct evidence of a recent viable pregnancy. These large proteins may remain elevated in circulation following embryonic or fetal death for 1–2 weeks allowing for false positives to occur in these animals. New ways to utilize this technology are being reported in the literature and hold promise for detection of pregnancy just following uterine attachment of the conceptus [28].

1.1.6 Embryo transfer technologies

Producers can make faster genetic progress and at the same time enhance reproductive measures utilizing embryo transfer technologies. Currently, embryos for transfer are produced through in vivo and in vitro means. In vivo embryos are collected following superovulation. In vitro produced embryos are made via oocytes collected from either oocyte pickup (OPU) or ovaries collected from abattoirs. Yet, follicle development during FSH stimulation of ovaries clearly influences outcomes from these technologies.

1.1.6.1 In vivo embryo production through follicle stimulating hormone-stimulated ovulations

In in vivo production of embryos, numbers of embryos collected following superovulation are variable and highly correlated, as expected, with the number of ovulations as measured from number of corpora lutea at time of embryo retrieval [29,30]. Thus embryo production is dependent upon the ovulation of a large cohort of mature FSH-stimulated follicles. FSH-stimulated follicles must grow to ovulatory size (approximately ≥10 mm in diameter) and be capable of responding to an LH surge to ensure ovulation of these follicles [31]. Initiating FSH treatment at the onset of a new follicular wave increases consistency of ovulation rates. This can be accomplished in a laboratory setting with ultrasound-guided follicle ablation of dominant and mid-sized follicles. Because the use of ultrasound-guided aspiration can be limited due to lack of equipment or logistics (time), superovulation protocols are generally initiated between day 8 and 12 of the estrous cycle to coincide with emergence of the second follicular wave. Unfortunately, the onset of the second follicular wave is quite variable in cows and can range from day 8 to 14 of the cycle. Thus the FSH treatment period could begin several days before or after the second follicular wave. In these cases, it is not clear how the DF or the largest follicles in a new follicular wave may affect the FSH-stimulated new wave of follicles or superovulation outcome. Data suggest that if FSH is administered at the time of a DF, a new wave of follicles will be induced in response to the FSH. This DF may stay functional after induced luteolysis, and if so may induce an LH surge before the FSH-induced follicles have sufficient time mature enough to acquire LH receptors and ovulate. Once an LH surge occurs, the growing but not yet mature follicles will become atretic (cell death). In this scenario, the cow or heifer would likely show signs of estrus following FSH stimulation but would have just one or two ovulations.

1.1.6.2 New ideas to increase ovulations in follicle stimulating hormone–treated cattle

Progesterone-releasing devices have been utilized in superovulation studies [29], but in most cases the removal of progesterone coincided with timing of induced luteolysis on the third day of FSH stimulation. Yet, maintaining a very low level of progesterone device in the vagina until the final treatment with FSH appears to prolong the time of the LH surge long enough to allow a greater number of superstimulated follicles to reach the ovulatory pool of follicles. In this case, the low-level progesterone device would allow more follicles to continue to grow, mature, and eventually ovulate due to the attenuation of an LH surge from subluteal circulating levels of progesterone. So, as in the previous example, if a DF is still functional after luteolysis, this follicle could not cause the positive feedback mechanism that induces an LH surge due to the presence of the low level of progesterone and would allow more follicles to continue to grow and reach the ovulatory pool of follicles prior to the removal of the progesterone device. It is important to note that circulating concentrations of progesterone must be low to have this effect. Only progesterone devices used for approximately 14 days that are cleaned and sanitized should be considered for use in this case [32].

1.1.7 Section 1—Summary

The reproductive technologies outlined in the section can completely control physiological processes in cattle to allow for fixed-time AI, greater fertility, timely pregnancy diagnoses, precise estrus information, and a greater chance for embryo production in FSH-stimulated donor cows. Control of reproductive processes allows for precise management of calving intervals and length of lactations of dairy cows. These management tools increase meat and milk production of cattle herds without increasing cattle numbers through greater control of reproduction.

Section 2: Bovine cloning and emerging reproductive technologies to improve cattle genomic parameters

1.1.8 Brief history of cattle cloning

Making genetically identical cattle using embryonic cells as donors has been possible since the 1970s [33]. This was done using embryo splitting, in which a fertilized embryo between the eight-cell and the late morula stage is split into two under a microscope [34]. The halved embryos are then placed in two emptied zona pellucida before transferring them to recipient cows. In 1987, the birth of cloned calves using nuclear transfer, in which a cell from a preimplantation embryo is introduced into an enucleated oocyte, was reported [33]. The potential to generate revenue from this new bovine assisted reproductive technique (ART) enticed investors to create companies based on such promise [35]. The idea was to produce multiple individuals from a single embryo, also called multigenerational embryo cloning [36,37]. In theory, one could take a 4- 8- or 16-cell fertilized embryo and generate 4, 8, or 16 cloned embryos that, in turn, could be re-cloned multiple times to generate an unlimited number of embryos [38]. In practice, though, this did not work as planned. The two main unaccounted roadblocks were (1) wide variation in the in vivo development of the clones depending on the generation, that is, second, third, or fourth round, and (2) the absence of a reliable cryopreservation protocol that would allow for proper storage and distribution of embryos [39,40]. Another roadblock not appreciated at the time that compromised the developmental potential of cloned embryos was our poor understanding of the need to coordinate the cell cycle of the donor nucleus with that of the oocyte [41].

Fast forward 10 years and Dolly, the cloned sheep, was born from a differentiated epithelial cell [42]. Soon after, multiple laboratories started working on translating the breakthrough into various species. In 1998, the birth of the first transgenic calves generated from fetal fibroblasts was published, and soon after that, Tsunoda’s laboratory showed that it was feasible to clone cows from adult somatic cells [43,44].

1.1.9 Reprogramming somatic cells into embryos

In the field of SCNT, reprogramming is defined as the process of transforming the nucleus of a somatic cell into the nucleus of a preimplantation embryo. The factors that influence the efficiency of this man-made process can be classified as technical and biological ones.

The technical factors include the expertise of the person performing the procedure, the experimental protocol, and the tools and reagents used. We should also add the knowledge and skills of the farm crew managing the recipient heifers and performing embryo transfers.

Details of the most common protocols used to clone cows have been described as elsewhere [45–47]. For the purposes of this chapter, we will focus on the SCNT procedure that our laboratory has successfully implemented to generate cloned calves repeatedly [44,47–49].

We isolate dermal fibroblasts from a small, full-thickness skin biopsy of 3 mm in diameter taken from the donor animal. We culture the epidermis and dermis using fibroblast culture medium at 38.5°C in 5% CO2 in air. Provided that the tissue is attached to the culture plate, somatic cells start to proliferate within a week. We usually first observe a mix of epithelial and fibroblast cells. However, after two to three passages, only dermal fibroblasts remain. At passage 4, we perform karyotypic analysis that confirms that they have normal ploidy and freeze and store the rest of the cells until needed. Approximately a week before the SCNT procedure is scheduled, we thaw the cells and culture them under the same culture conditions described above. The day that SCNT is done, fibroblasts are enzymatically dissociated and resuspended as single cells in culture media until used. This section of the protocol—the preparation of the cell before fusion/injection with the oocyte—varies between laboratories depending on the specific cell cycle stage the somatic cell is synchronized [50].

We isolate the recipient oocytes from ovaries recovered from the slaughterhouse. Immediately after we remove the oocytes from the reproductive tract, we rinse them with phosphate-buffered saline and maintain them at room temperature during transportation to the laboratory. Upon arrival, we aspirate the immature oocytes at the germinal vesicle stage with their granulosa cells from follicles that have a diameter between 2 and 8 mm [51]. We perform a brief selection of aspirated oocytes to eliminate those that do not have granulosa cells attached to them, otherwise called denuded [52]. Alternatively, others have reported using oocytes isolated in vivo from the ovaries using ovum pickup and matured in the laboratory [53]. Regardless of the source of oocytes, before we can use them for SCNT, they have to resume meiosis and reach metaphase II, the point at which an oocyte is ready to be fertilized. To accomplish this, we place the immature oocytes in media containing FSH and LH and aseptically incubate them at 38.5°C with 5% CO2 in air for a period of 17–20 hours. We then take the oocytes with their expanded cumulus cells and place them in a solution of hyaluronidase, we vigorously pipette or vortex them until they are devoid of any somatic cell attached to its zone pellucida. If, after this process, we see some oocytes that still have granulosa cells attached to them, we assume they are not properly matured, and we discard them. Denuded—or naked—oocytes are placed in embryo culture medium at 38.5°C and stained with a fluorescent, nontoxic, DNA intercalator such as Hoechst or a similar compound [47]. For the process of oocyte enucleation and cell transfer, we use an inverted microscope with 20X objective. Brief exposures of ultraviolet light allow us to confirm that the eggs have extruded the second polar body and remove the maternal chromosomes arranged in metaphase using a glass needle. The same needle is subsequently used to deliver the somatic cells underneath the zona pellucida, in close contact with the oocyte’s cell membrane [47].

We fuse the somatic cell with the oocyte by incubating the eggs with inactivated Sendai virus; alternatively, we use electrical current [54,55].

From this point forward, the eggs have a diploid nucleus (in G1 or G0 stage of the cell cycle) and a cytosol arrested at metaphase stage.

After a period of incubation of 2–3 hours, the somatic nucleus starts to remodel, and while the typical mitotic spindle of a cell at metaphase is not observed, the nuclear envelope disappears, and its chromatin condenses [56].

For the eggs to divide, though, we must induce the resumption of the first cell cycle like the sperm would do, but without letting the oocyte extrude the second polar body. We accomplish this by incubating the eggs in a HEPES-buffered solution containing ionomycin, a cell-permeable calcium ionophore, that triggers intracellular calcium release. Subsequently, we place the eggs for four hours into a solution containing compounds that can lower maturation-promoting factors. From this point onward, embryos are treated as conventional-fertilized embryos until they are transferred into recipient cows. Phenotypically, these embryos are indistinguishable from fertilized ones [47].

1.1.10 Biological factors of bovine cloning with somatic cells

One-cell embryos, produced by fertilization, are generated using sperm and eggs, two highly specialized cells that start their developmental process months in advance, during postimplantation embryonic development. Briefly, primordial germ cells (PGCs), originated in the embryonic epiblast, reach the genital ridge—developing gonads—when the fetus is 35 days old [57]. PGCs continue to divide, and start differentiation into oogonia in the female and spermatogonia in male. Final maturation into fertile eggs and sperm occurs, on average, at 9 months after birth, depending on factors such as breed and nutrition [58–61]. Throughout that time, the genomic DNA of the eggs and sperm undergo extensive epigenetic reprogramming, including the erasure and reestablishment of gene imprinting. Basically, it takes months to prepare the genome of a gamete to form a zygote that will develop into a healthy calf [62,63]. One-cell embryos, created by SCNT, have no oocyte’s or sperm’s genomic DNA. Instead, they have a nucleus carrying the epigenetic signature of a somatic cell. When taking into account the molecular processes required for gametogenesis, it is surprising that SCNT works at all.

Skin fibroblasts are easy to isolate and culture in vitro, making them the cell of choice for SCNT. Notwithstanding, a fibroblast’s function is to provide structure to tissues, extracellular matrix, and participate in wound healing. It stands to reason that the epigenetic state of a fibroblast’s DNA is not prepared to turn, in one cell division, into a fully functional zygote. However, this does happen. When data from multiple laboratories is compiled, the chances of obtaining a healthy clone calf hover between 10% and 15%. That is, for every 7%–10% recipient cows that received a cloned embryo, one calf will be born healthy. This efficiency pales in comparison to pregnancy rates by embryo transfer using in vitro fertilized embryos that can reach up to 60%, with a rate of healthy calves born healthy in the 40%–50% range, over the total number of cows transferred [64].

There are reasons to be optimistic about the prospects of having a bovine cloning efficiency that is the same as in vitro fertilization embryos. Recent experimental results in other mammalian species have provided a better understanding of the epigenetic changes that take place in the cloned embryos. Molecules responsible for a somatic nucleus resistance to reprogramming have been identified. These are histones and histone-residues accountable for conveying a somatic cell its phenotypic identity and provide an opportunity to manipulate these targets to increase cell plasticity.

Wakayama’s laboratory conducted one of the first studies in 2006, where they demonstrated that the addition of Trichostatin A in the culture medium of mouse-cloned embryos for only 10 hours after oocyte activation increased the efficiency of production of cloned pups up to five times [65,66].

Trichostatin A (TSA) is a potent histone deacetylase inhibitor, targeting a large number of proteins, including H3K4, H3K5, and H3K9 histones [67–69]. Subsequent studies in bovine indicate that TSA, when used after cell fusion, and before the first embryo cleavage, improves embryo quality and survival of cloned calves [69,70].

Another relevant histone repressive mark, H3K9me3, was found to be responsible for the main failures of reprogramming in mouse-cloned embryos. Matoba and colleagues elegantly demonstrated this in 2014 [71]. They showed that the efficiency of mouse SCNT could be increased when mRNA for Kdm4d, the enzyme responsible for removing methyl residues from lysine 9, was injected in cloned embryos at the one-cell stage. These results have been replicated in vitro with human- and bovine-cloned embryos [72].

These are just a handful of studies that still require further validation by other laboratories. Nonetheless, encouraging data continue to pour in on new histone targets that when modified, SCNT efficiency is improved.

1.1.11 Need for more basic research to improve the efficiency of bovine somatic cell nuclear transfer

Synchronized recipient cows that received a cloned bovine embryo have a slightly lower pregnancy rate and a higher incidence of abortion [73]. Pregnancies are lost either in the first trimester or near parturition [73]. Incidences of perinatal deaths are also above normal [74–76]. The signs and symptoms present in abnormal calves are collectively called large calf syndrome (LCS), that was initially reported in embryos that were in vitro fertilized and cultured in the presence of serum or with coculture of cells [77].

A pregnant cow carrying a fetus that is developing LCS displays a progressively large abdominal cavity to the point that, in the most severe cases, compromises the life of the dam [73,78].

We still do not fully understand the pathogenesis of LCS. The working hypothesis is that it starts with a defective trophoblast differentiation in the early embryo, leading to a dysfunctional placenta, which in turn compromises placenta’s blood circulation triggering edema, fluid retention, hydroallantois, and placental necrosis. Parts of the placenta that are still functional develop larger cotyledons to compensate for the ones dying. A dysfunctional placenta negatively impacts the fetal circulatory system, that is, the fetal heart must work harder to maintain normal blood circulation. There is an increase in fetal pulmonary blood pressure, which contributes to pulmonary edema, heart hypertrophia, and dilated cardiomyopathy [75,79]. Liver steatosis and enlarged kidney are reported as well [80].

Newborn cloned calves that had a faulty placenta during pregnancy are born with an abnormally sizeable umbilical cord that can reach up to three times its average diameter. Such overgrowth interferes with the physiological occlusion of umbilical blood vessels after parturition [73,80,81]. The recommendation is to surgically cut the cord and tie the vein and artery permanently.

Independent from placentation failures, some cloned fetuses are much larger than average. They show excessive bone development, forcing the leg joints to bend inside the constrained space of the uterus. Upon birth, cloned must be aided to stand on their own, and in the worst cases, euthanized.

Despite these associated pathologies observed in some cloned fetuses and calves, the vast majority of cloned calves born alive are healthy, requiring minimal support during the first days of their life [82]. Once they reach 3 months of age, they have a health- and life-span equivalent to that of animals produced by fertilization, a phenomenon also reported in sheep [80,83].

Producing clone cattle at a higher efficiency will: (1) lower the cost of making cloned calves, (2) mitigate some animal welfare concerns, (3) accelerate genetic progress, and (4) increase food security.

The economics of producing cloned calves at efficiencies of 10%–15% restricts the use of cloning to selected groups of breeders that sell genetically superior cows and bulls that are then used as founder animals. At the moment of this writing, the cost of cloning cattle is 20,000 American dollars per animal. For commercial farmers that would want to make genetic copies of animals for food production purposes, cloning is untenable.

There are valid concerns regarding the welfare of cloned calves and the recipient dam. The most recent evidence indicates that, while the efficiency of cloning remains low, there is a fewer incidence of hydrops in recipient cows and neonatal death of cloned calves [84]. Nonetheless, the origin of these problems remains poorly understood and may still occur, something that could be construed as an iatrogenic phenomenon to take into account before broadly deploying this technology [85,86].

Genetically modified (GM) organisms, such as plants and prokaryotic species, are an essential component of our food supply, especially in developing countries where food sources are scarce. There is still a need, however, to generate enough animal protein to feed the estimated 2 billion undernourished people by 2050, as stated in the United Nations Zero Hunger Challenge (www.zerohungerchallenge.org). In November 2019, AquAdvantage became the first GM animal—Atlantic salmon—to be approved by the Food and Drug Administration for human consumption. This is interpreted as a sign that regulatory agencies, and the public at large, are becoming more aware of the need for more aggressive strategies to cope with the expected food shortages. GM cattle as a source of food should be considered a valid option. Transgenic calves produced using SCNT were first reported in 1998. More recently, SCNT, coupled with precise gene-editing tools, was used to generate prion-free and polled calves [87,88]. It is reasonable to envision genetically engineered cattle that have increased production traits without compromising the welfare of the animal and that can be raised in an environmentally sustainable manner.

1.1.12 Emerging assisted reproductive techniques in cattle

The broad implementation of reliable genomic markers for the selection of genetically superior animals is the main factor that contributed to the improvement of production traits over the last decade. In the past, the genetic merit of an animal—mostly males—was determined when the production traits of its progeny were measured, a process that would take 4–5 years. With the implementation of genetic selection based on DNA markers, the genomic estimated breeding value (GEBV) of an animal can be determined at birth, shortening the generation interval by 3 years [89,90]. The critical issue is to rapidly generate new genotypes from founders with the desired GEBV. In practice, the selection of an individual that has high GEBV can be made as soon as diploid cells are isolated from a calf, a fetus, or an embryo. Once these cells are determined to have a superior genotype, it is possible to produce a new founder animal by SCNT, shortening the generation interval even further. Nonetheless, at present, we still need a whole animal to produce the gametes for a calf, fetus, or embryo, to start the process, for now that is.

Experiments in mice have demonstrated that functional gametes can be obtained by differentiating embryonic and PGCs into sperm and eggs, embryos such produced, when transferred to a recipient female, have generated viable offspring [91–95]. In vitro produced bovine gametes, coupled with SCNT, could have an extraordinary impact on bovine genetic selection. Producers would have, at their disposal, a method to create unique genomes over multiple generations without the need to make an animal.

1.1.13 Section 2—Summary

Bovine cloning is an ART currently used to replicate the genome of genetically superior animals. At present, cloned cattle are used as gamete donors for the generation of fertilized embryos. The entry barrier for commercial producers is its cost, a consequence of the low efficiency of the procedure as measured by the number of healthy clones born over the number of recipient cows transferred. Since first reported two decades ago, the efficiency of cloning cattle using somatic cells have been in the low teens. Recent reports describing the molecular mechanisms of cellular reprogramming have unveiled new genes and gene products that can increase the plasticity of the donor cells. These new findings give credence to the notion that shortly, embryos produced using SCNT could reach the same developmental competence as those generated using in vitro fertilization, allowing for the use of bovine cloning in commercial herds.

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