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Recent Advances in In Vitro Fertilization Recent Advances in In Vitro Fertilization

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IntroductionFor the past few decades, reproductive biotechnolo-

gies such as artificial insemination (AI), embryo trans-fer (ET), and embryo production via in vitro fertilisation (IVF), have undergone incredible advances.

The first IVF of a mammalian oocyte, confirmed cy-tologically, was performed in a rabbit using spermatozoa that had capacitated in the uterus and recently ovulated oocytes [1]. Five years later, the birth of baby rabbits con-firmed the biological normality of in vitro fertilisation. For practical reasons, but also to understand the mecha-nisms of capacitation, fertilisations were then attempted using spermatozoa that had capacitated in vitro. The first IVF under these conditions were achieved in rodents (hamsters, 1963; mice, 1968), then in man in 1978 [2], and much later in large domestic mammals: the first IVF calf was born in 1982 [3], then the first goats in 1985, lambs and piglets in 1986, and the first foal in 1990 [4]. Some time later, IVF was achieved using oocytes that had been matured in vitro in various species, with the first calf born under these conditions in 1986, the first piglet in 1988, and the first lamb in 1990 [4].

In reality, IVF is already an integral part of the em-bryo transfer (ET) process used for the industrial produc-tion of bovine embryos; this process involves in vitro oo-cyte maturation, IVF, in vitro embryo development, and finally the in utero transplantation of the embryo in the recipient dam. IVF is also a valuable means of learning more about the mechanisms involved in vivo fertilisation.

Chapter 2

In Vitro Fertilization (IVF)

Diego MORENO GARCIA1, Alberto NERA1, Lamia BRIAND1, Emmanuel TOPIE1, Djemil BENCHARIF1 and Daniel TAINTURIER1

1Laboratory of Biotechnology and Pathology of Repro-duction, Nantes-Atlantic National College of Veterinary Medicine, France

*Corresponding Author: Prof. Daniel TAINTURIER, Laboratory of Biotechnology and Pathology of Repro-duction, Nantes-Atlantic National College of Veterinary Medicine, Food Science and Engineering - Oniris. CS 40706, 44307 NANTES Cedex, France, Email: [email protected]

First Published February 26, 2016

Copyright: © 2016 Daniel TAINTURIER et al.

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source.



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With IVF, one can also study the associated problems: oo-cyte maturation, spermatozoa capacitation, activation of the egg, control of the embryo’s genome, mechanisms of cell division, influence of maternal and paternal genomes, interaction between the nucleus and cytoplasm, etc. IVF also offers the possibility of analysing the mechanism of nuclear differentiation by using nuclear grafts, an essen-tial part of the process needed to develop embryo cloning techniques.

In farm animals, IVF presents several important ad-vantages, such as the production of a large number of em-bryos in the same time period, the ability to use embryos from dead cows or those with reproductive problems, the possibility of producing embryos from a cow during the first third of gestation as well as the production of em-bryos in pre-pubertal heifers.

This technique is currently used intensively in cattle, on pregnant and non-pregnant cows, with or without re-productive abnormalities. Thus IVF is a low-cost means of producing embryos on a massive scale from ovaries obtained at the abattoir. In recent years, this biotechnol-ogy has been widely used in cattle on a commercial scale; according to data collected by the IETS [international em-bryo transfer society), in 2012 a total of 1,143,119 embry-os produced in vivo and in vitro were transferred in cattle. Of this total, 699,586 embryos were produced in vivo, and

443, 533 were fertilised in vitro [5].

In practice, the procedure for producing embryos in vitro (IVP) involves four distinct phases:

1. Harvesting the oocytes (via OPU, Ovum Pick Up, ultrasound guided follicular puncture; or via nee-dle aspiration from ovaries obtained post-mor-tem).

2. In vitro maturation (IVM)

3. In vitro fertilisation (IVF)

4. In vitro development (IVD)

The last three phases (IVM, IVF, and IVD) are per-formed in a specific medium in an incubator at 38.5°C to mimic in utero conditions. Despite numerous studies performed on the three main phases of in vitro embryo production (IVM, IVF, IVD), their efficacy is still low, with only 30 to 40% of oocytes actually developing into blastocysts [6].

Obtaining Immature Oocytes via Ovum Pick Up (OPU)

Oocytes can be obtained post-mortem via follicular aspiration; alternatively, OPU [7], which is directly de-rived from the method of oocyte collection that is used routinely in humans, has enabled considerable advances in the collection of bovine oocytes. This technique enables



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the multiplication of high-performance female descend-ants using oocytes harvested from animals with a known genetic potential. The oocytes are harvested via a needle introduced into the dorsal sac of the vagina under ultra-sound guidance. The ultrasound probe can be used to visualise ovarian follicles of 6 to 8 mm in diameter, which are then punctured. In cows, these probes are usually 5 or 7.5 MHz sectorial probes. The animal is sedated, often with additional epidural anaesthesia, the probe is inserted intravaginally and the ovary grasped via the rectum and held against the head of the probe. The follicles are thus visible on the screen of the ultrasound allowing a needle to be guided through the vaginal wall and into the follicle. An aspiration system into a specific medium (containing PBS and heparin) allows collection of the follicular flu-id and the oocyte surrounded by its cumulus oophorus, composed of follicular cells. This minimally invasive tech-nique enables a high rate of repeated harvest, up to twice a week for several weeks, with no negative effects on the donors [8-9]. As such up to 5 oocytes can be collected at a time and around 35% of the subsequent blastocysts are suitable for in vitro transfer [10]. The OPU technique is also useful for other species such as the bison and horses. In small ruminants, oocytes are usually recovered via lap-arotomy or laparoscopy. Currently, the use of high resolu-tion ultrasound with high frequency transducers (25-70 MHz) enables dynamic examination of the development

of small antral follicles and to observe the Cumulus Oo-cyte Complexes (COC) in vivo [11].

In Vitro Oocyte Maturation (IVM)An oocyte blocked at the germinal vesicle stage re-

quires a maturation phase to enable fertilisation. This in-volves a certain number of modifications: nuclear matura-tion culminates in the emission of the first polar globule and the formation of the second maturation phase (be-tween 18-21h in cows; 24 in the ewe, and 27 hours in goats). Membrane maturation, which enables the oocyte to specifically recognise the spermatozoa of its species, and cytoplasmic maturation, which prevents polyspermia via the cortical reaction, and which ensures the synthesis of the proteins needed for fertilisation [12].

Under natural conditions, the follicle undergoes sig-nificant modification during maturation. Expansion of the cumulus is reliant on the presence of FSH in the folli-cular fluid, whose action is modulated by LH. FSH stimu-lates the synthesis of hyaluronic acid by the cells of the cumulus, which results in the formation of an abundant viscoelastic matrix, which in turn expands the intercellu-lar spaces and thus the cumulus as a whole [13]. In addi-tion, once the inhibitory action of OMI (Oocyte Meiotic Factor) has been lifted by the LH peak, the permeable junctions that unite the oocyte to the cells of cumulus rup-



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ture. In vitro, this inhibition is not an issue as there are no OMI-producing cells [14].

During in vitro oocyte maturation, the nuclear phase normally occurs spontaneously, whereas cytoplasmic as-pects are dependent on the maturation medium [14]. The most widely used medium for maturing oocytes is TCM-199; this medium is buffered with bicarbonate and contains mineral salts, sources of carbon and energy (glu-cose), and amino acids such as cystine and cysteine, im-portant in the metabolism of glutathione and glutamine, which with glucose and pyruvate provide the principal energy sources of the oocyte [15-16]. This medium has similar characteristics to the follicular fluid; its osmotic pressure varies between 280 and 310 mOsm/kg and the pH between 7.0 and 7.6. Normally, the osmotic pressure of follicular fluid lies between 280 and 320 mOsm/kg and the pH between 7.3 and 7.4 [17].

High molecular weight molecules are often added to this medium, such as foetal calf serum [18], or serum from a cow that is in oestrus [19], whose surfactant effect prevents the adhesion of the oocyte cumulus complexes. However, the use of these animal-derived additives pos-es a sanitary risk and hinders the reproducibility of ex-periments. Good results have been achieved by replac-ing these animal proteins with synthetic polymers with the same surfactant properties, such as polyvinyl alcohol

(PVA) [13].

In addition, the gonadotrophic hormones, notably FSH, have been used in oocyte maturation media in cattle by promoting the expansion of the cells of the cumulus in vitro [20-21]. FSH potentiates the action of growth fac-tors such as EGF or PDGF, and stimulates the expression of the LH receptor by the cells of the cumulus; LH has a direct action on the metabolism of the oocyte, increasing glycolyis and oxidative phosphorylation. GH accelerates nuclear maturation, stimulates the expansion of the cu-mulus, and improves the rate of development to the blas-tocyst stage in the absence of serum and gonadotrophic hormones [22]. In addition to the effect on nuclear matu-ration, GH also has an effect on cytoplasmic maturation expressed on the rate of development; This effect results from an acceleration of the migration of cortical granules [13].

Other factors can also intervene during IVM. Epider-mal growth factor (EGF) whose beneficial effect on nu-clear and cytoplasmic maturation has been demonstrated in cattle. This effect was reported on denuded oocytes, suggesting that EGF may act, at least in part, directly on the oocyte [23]. Factors from the insulin family, largely involved in the control of folliculogenesis [24]; notably IGF-1, whose beneficial effect during IVM has been dem-onstrated in horses [25], cats [26], and cattle, where the maximal rates of expansion of the cumulus and nuclear



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maturation were obtained in the presence of IGF-1 and EGF [27]. Another molecule is activin, present in the folli-cular fluid, and whose receptor is expressed by the cumu-lus and by the oocyte; their beneficial effect during IVM has been demonstrated on bovine oocytes [28].

The physical conditions used for in vitro maturation are generally those of the core temperature of the species (37°C in primates and in small rodents, 39°C in domes-tic mammals), in an atmosphere enriched with 5% CO2 saturated in humidity, for a duration of 24 hours (mice, ruminants) to 44 hours (humans, pigs) [13].

Classification of Oocyte Quality for In Vitro Maturation (IVM)

Immature bovine oocytes can be classified into 4 cate-gories depending on the degree of compaction of the cells of the cumulus and the transparency of the cytoplasm [29-30].

Category 1The cumulus oocyte complex (COC) is transparent.

The cumulus (cells of the granulosa) is compact and com-pletely surrounds the oocyte. The cytoplasm has a homo-geneous appearance.

Category 2The COC has the same appearance as in category 1,

but the cytoplasm is more irregular. It has a darker zone visible in the periphery.

Category 3The entire COC is dark, the cumulus is less com-

pact, the cytoplasm is more irregular and presents darker clumps.

Category 4The cumulus is completely disorganised or even ab-

sent (naked oocytes). De Loos et al., 1989 [29], studied the behaviour of 4 categories of immature oocytes during IVM and observed that only category 4 oocytes showed a reduced capacity for growth. Oocytes from categories 1 to 3 demonstrated a similar capacity for development in an IVM system.

In Vitro Fertilisation (IVF)Several studies have tested in vitro fertilisation me-

dia that mimic the composition of the media in the fal-lopian tubes, achieving cleavage rates of between 80 and 85% in ruminants. Various factors are important during IVF: firstly, the elimination of the seminal plasma, which would normally occur during the passage through the cervix or by resorption in the uterus; in vitro, it should be



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performed by centrifugation using a density gradient, or via spontaneous ascending migration in a diluent (swim up). Next is spermatic capacitation, this is achieved in vit-ro through the presence of specific substances in the me-dium in which the spermatozoa are suspended [31].

The most widely used medium for the capacitation of spermatozoa and IVF is FERT-TALP (Fertilization Ty-rode’s Albumin Lactate Pyruvate medium) [32]. This me-dium is often supplemented with heparin in cows or in the serum of ewes that are in oestrus in small ruminants, to capacitate the spermatozoa in vitro.

Heparin leads the efflux of cholesterol and phospho-lipids from the plasma membrane of the spermatozoa, which is an important step in the capacitation process [33-35]. In association with heparin, other acceptors of cholesterol, such as BSA, are often used in the capacita-tion / fertilisation medium. BSA can act by absorbing the cholesterol from the membrane, leading to changes in the plasma membrane [36]. Although capacitation induced by heparin does not require the presence of BSA, the capacitated spermatozoa need the latter to undergo the acrosome reaction, the exact role of BSA during the acro-some reaction is not fully understood [35].

In order for heparin to capacitate the spermatozoa, there needs to be a minimum amount of bicarbonate in the capacitation / fertilisation medium [35]. BSA only in-tervenes on the sub-population of spermatozoa that have

undergone modifications of the membrane induced by bi-carbonate [37]. Bicarbonate alters the plasma membrane inducing the rapid collapse of the transversal asymmetry of the phospholipids, facilitating the elimination of cho-lesterol from the membrane during capacitation [38]. Af-ter 1 to 10 hours, depending on the species, a significant percentage of spermatozoa have capacitated. The times required are similar in vivo and in vitro [31].

Successful in vitro fertilisation is dependent on a high concentration of spermatozoa in contact with the oocyte, yet in vivo only a few spermatozoa make it to the oviduct. Although the spermatozoal density is high, in vitro ca-pacitation is still less effective than in vivo. If capacitation is incomplete when the gametes come into contact, ferti-lisation may not occur until 6 to 8 hours later. This leads to ageing of the oocytes, which prejudices the success of fertilisation and subsequent embryo development [31].

The conditions used for IVF are the same as those used for IVM. Although 37°C is the optimal tempera-ture to fertilise the oocytes of mice and women, it does not enable fertilisation in domestic animals with a core body temperature of 38-39°C. In cattle, IVF is performed in an atmosphere enriched with 5% CO2 for a duration of around 20 hours [4].



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In Vitro Embryo Development (IVD)The secretions of the fallopian tubes and uterus play

an essential role during in vivo embryo development. The liquid in the fallopian tubes has an osmotic pressure of around 290 mOsm/kg and its pH varies between 7.2 and 7.6. It is rich in potassium and bicarbonate ions and has lower concentrations of Na+, Ca2+, Mg2+, and Cl-; it is also rich in energy sources such as glucose, lactate, and pyruvate, amino acids, vitamins A and E, which protect against free radicals, lipids, proteins such as albumin, transferrin, and immunoglobulins G, and growth factors [17]. Under these conditions, the embryo is therefore ca-pable of balancing its endogenous metabolism pools by incorporating certain external components and via the metabolic “turn over” from its own reserves.

In vitro, the pre-implantation development of the em-bryo occurs in close relation with the culture medium. This demands specific balanced conditions in the medium to allow embryonic metabolism. With the exception of man and rabbits, there is an in vitro blockade of the devel-opment of the segmented egg in numerous mammals: at the 2-cell stage in mice, 4-cell stage in pigs, 8-16-cell stage in cows. These blockades correspond to poor synchroni-sation between the fall of maternal mRNA and the start of mRNA transcription from the embryonic genome. This stoppage is linked to inadequate culture conditions for the development of the embryo [39].

Numerous studies have been conducted into culture systems to improve the conditions of in vitro embryo de-velopment. Tervit el al., 1972 [40] proposed SOF (Syn-thetic Oviductal Fluid), a culture system that enables the production of viable blastocysts from oocytes fertilised in ruminants. However, this system does not make it pos-sible to remove the blockade of development observed in ruminants at the 8-16 cell stage. This lead to the creation of co-culture systems. The first experiments with embryo co-culture with tubular or uterine cells were conducted in ewes [41]. This system made it possible to eliminate the blockade in ruminant eggs. The tubular cells act on em-bryo development by reducing the oxygen concentration, by eliminating certain toxic components present in the culture medium such as metal ions and bivalent cations [41]. They also supply growth factors that are required for the in vitro development of the embryo [2].

Subsequently, co-culture was performed in the pres-ence of epithelial cell lines of non-genital origin such as BRL (Buffalo Rat Liver) or Vero cells (renal cells of green monkeys) in an atmosphere enriched with 5% CO2. These cell lines were used successfully for the co-culture of mu-rine [43], bovine [44-45], and human embryos [46].

Later on, these positive effects obtained in co-culture, notably the low oxygen concentration, were conceptual-ised using systems defined under low-oxygen culture con-ditions to limit oxidation reactions [47]. Defined media were created from SOF, such as SOFaa supplemented with



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amino acids [48], KSOM [49], or CR1aa [50]. Currently, the most widely used culture medium is SOF as modified by Holm et al., 1997 [51]. It is supplemented with sodium citrate, myo-inositol, and amino acids. In this system the eggs are cultured at 38.5°C in a humid atmosphere in the presence of serum or bovine serum albumin (BSA) in mi-crodrops covered with mineral oil under 5% CO2, 5% O2, and 90% N2 [12].

Synthetic Media for Embryo CultureOn a practical level, one problems remains, which

has yet to be fully resolved, concerning the use of animal-based products as a source of protein for in vitro culture media (foetal calf serum (FCS), serum from cows in oes-trus, bovine serum albumin, etc.). Despite the beneficial effect of FCS and BSA on embryo development [52], these biological products are associated with certain sanitary disadvantages [53]; they can be a source of viral or other pathogenic agents and can provoke alterations during in vitro embryo development.

Despite the rich composition of FCS, with growth fac-tors, proteins, vitamins, trace elements, hormones, etc. all of which are essential for cell growth, their use is contro-versial for several reasons. Various studies have demon-strated that the presence of FCS in the culture medium has a noxious effect on the embryo during development. These embryos have high concentrations of lipids in the

cells of the embryonic bud and the cells of the trophecto-derm [54, 55]. This accumulation of lipids can affect the tolerance of the embryo to cryopreservation [56-58]. In certain cases, a phenomenon characterised by high birth-weight calves, prolonged gestation, frequent dystocias, high rates of abortion, and perinatal mortality were ob-served [59,60]. From a sanitary point of view, FCS can be contaminated by viruses such as bovine viral diarrhoea [61]. This contamination can be limited by the irradiation of commercial serums, but this procedure is not always applied. Seasonal and continental variation in the compo-sition of FCS results in variations from one batch to an-other provoking differences in the cultures, which leads to variable results [62].

The beneficial effect of BSA on embryo development has been proven [63], which is not surprising given that albumin is an extracellular protein, the most widespread in the mammalian reproductive system. BSA may have a nutritional role by supplying amino acids after hydroly-sis. It can also bind to several low molecular weight com-pounds such as heavy metal ions, free radicals, citrate, steroids, etc. It can protect cell components against the ef-fects of toxins, and regulate the oxydoreductive potential, pH, and osmolality [57, 64]. Yet, despite these advantages, there are variations between batches of BSA, and it also presents a health risk.

Furthermore, it is difficult to elucidate the specific functions of each growth factor or other stimulants, given its undefined composition [52].



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For in vitro embryo production, it has been clearly established that these embryos are morphologically and physiologically different from those fertilised in vivo. For example, there may be differences in the structure of the zona pellucida [65]. The zona pellucida of embryos pro-duced by IVF can be considered as immature and certain pathogenic microbes seem to adhere more easily to the zona pellucida of embryos that are produced in vitro [66-68]. Using electron microscopy, Vanroose et al., 2000 [65], concluded that the intact zona pellucida of an embryo produced in vitro is made in such a way that the bovine vi-ral diarrhoea virus (BVDV) or bovine herpes virus (BHV-1) should not be able to cross it and infect the embryonic cells. However, small viruses such as BVDV can bind to the outer layers of the zona pellucida and contaminate the embryo as it hatches.

For all of these reasons, in addition to the hygiene conditions associated with handling embryos during in vitro production, it is essential to have an appropriate cul-ture medium for the development of the embryo after in vitro fertilisation. Several recent studies have therefore at-tempted to establish defined culture media to replace all molecules of animal origin (FCS, BSA) using synthetic molecules to standardise the embryo production chain and reduce health risks. Several different molecules have been tested for use in culture media.

Molecules Used to Replace Molecules of Animal Origin in In Vitro Embryo Culture Media

Polyvinyl alcohol (PVA) has been proposed as a pre-ferred additive, especially given that this synthetic poly-mer has a similar surfactant activity to albumin. Despite this similarity, the majority of studies have reported gen-erally lower development rates with PVA in comparison with BSA and with BSA and FCS [52].

Polyvinylpyrrolidone (PVP) is another synthetic polymer that has been used as a substitute for BSA in em-bryo culture media. In cattle, blastocyst rates are compa-rable to those obtained with BSA [70, 71]. This molecule has been used more recently as a replacement for FCS in bovine oocyte maturation media, indicating that PVP has a similar effect to FCS as a function of the maturation of oocytes and the development of the embryo [72, 73].

Hyaluronic Acid is the most abundant glycosami-noglycan in the follicular fluid, oviduct, and uterus of the cow [74], mouse [75], and woman [76]. Its receptor (CD44) is expressed on the surface of oocytes, cells of the cumulus, and of the bovine [77] and human [78] embryo, at different stages of development. It is involved in several processes, the proliferation and differentiation of various cell types, dynamic processes carried by the in-teraction with the extracellular matrix, the regulation of



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protein secretion, gene expression [19], and the fixation of several molecules such as lipases or growth factors that affect cell function and morphology [79]. There appears to be a close relationship between hyaluronic acid and GF. Klominek et al., 1989 [80], demonstrated that the pres-ence of PDGF-BB, EGF, bFGF, and TGF-f31, during the culture of human fibroblasts, produce a marked stimula-tion of hyaluronic acid synthesis, an effect that is not ob-served when using these GF and CYK separately. The ef-fect of hyaluronic acid in culture media was first studied several years ago; Hamashima 1982 [81], demonstrated that this molecule promotes the differentiation of the ex-tra-embryonic tissues of mice embryos. More recently, its beneficial effect on embryo development has been proven in pigs where it produced improved blastocyst rates [82]. In cattle, through higher blastocyst rates [83], hyaluronic acid improves the survival rate of embryos after vitrifica-tion, resulting in increased gestation rates after embryo transfer [84]. A reduction in the expression of the genes coding for apoptosis (Bax - SOX) has also been observed in embryos frozen with hyaluronic acid [85].

Preparations of recombinant albumin have recently become available. They are free from all potential biologi-cal contamination such as viruses and prions. In addition, their composition is purer than that of traditional albu-mins, which are purified from blood, and which often con-tain unknown molecules. This is the case of citrate, which

was isolated in batches of BSA that showed embryotrophic activity in the rabbit [86]. In cattle, Lane et al., 2003 [87], used recombinant albumin in association with hyaluronic acid in the culture medium, obtaining blastocyst rates that were comparable to those obtained with BSA.

Tween-80 is a synthetic surfactant with the ability to reduce the surface tension of the culture medium. This polymer can replace the surfactant properties of FCS and BSA in culture media, but does not have their embryo-trophic properties. Palasz et al., 2000 [88], improved the rate of bovine blastocysts produced in vitro thanks to the addition of tween-80.

The effect of ITS was studied by George et al., 2008 [57]. This association of molecules contains an anti-apop-totic agent (insulin), a protein that detoxifies the medium by binding metals (transferrin), and an antioxidant (sele-nium). In addition to SOF, ITS gives a blastocyst rate at 8 days after fertilisation that is comparable to that obtained with SOF supplemented with FCS. The addition of BSA to this SOF-ITS medium gives similar hatching rates to SOF-FCS. This SOF-BSA-ITS combination therefore increases the cryotolerance of embryos comprised between 160 and 180 µm and reduces the quantity of lipids in the embryo in comparison with the medium containing FCS.

Plant peptones have been used as a substitute for pro-teins of animal origin. These peptones have anti-oxidant properties. George et al., 2009 [89], obtained post-thaw



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development, hatching, and survival rates similar to those obtained with BSA. However, they reported a significant reduction in the concentration of glutathione when the embryos were produced with plant peptones. After sup-plementation of the medium with glutathione precursors (cystine and P-mercaptoethanol), the glutathione concen-tration increased significantly.

Growth factors and cytokines used in in vitro embryo culture media

The beneficial effect of growth factors and cytokines for the culture of embryos produced in vitro is linked to the natural production of these molecules in the repro-ductive tract during the peri-implantation period of the embryo.

EGF (Epidermal Growth factor) has been used very little in culture media. Their efficacy during in vitro oo-cyte maturation has been widely demonstrated in the rat [90], man [91], pigs [92], and in cattle [23, 93].

The IGF family (Insulin-like growth factors) has been widely used in culture media with satisfactory results. Several studies have demonstrated a positive effect of IGF-1 in embryo development in the cow [94-97] and in man [98]. An anti-apoptotic effect of IGF-1 in the culture me-dium has been observed in murine [99], porcine [100], bovine [101], and human [98] embryos. An increase in

gestation rates and parturitions following transfers of bo-vine embryos produced in vitro has been demonstrated [102-104]. A reduction in the apoptotic index was also observed in murine embryos cultured in the presence of IGF-11 [105].

FGF (Fibroblast growth Factor) promotes in vitro embryo development in cows [106, 107], the migratory activity of the cells of the trophoblast in sheep [108], and increases the production of interferon-tau by the tropho-blast in cows [109-111].

PDGF-BB (Platelet Derived Growth Factor-SB), stim-ulates in vitro embryo development in cows [112-113]. TG-F131 (Transforming Growth Factor-131), improves the embryo development rate and the rates of embryo im-plantation in mice produced in vitro [114] and promotes in vitro embryo development in cows [107].

Several studies have also demonstrated the beneficial effect of certain cytokines during embryo development. This is the case of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), which improves the in vitro development potential of bovine [115] and porcine em-bryos [116], increases the number of cells in the inner cell mass and gestation rates in cows [117], and stimulates the production of interferon-tau by the trophectoderm in cat-tle [110] and sheep [118].

LIF (Leukocyte Inhibitory Factor) is another cy-



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tokine. it does not improve the blastocyst rate, but increas-es the number of cells in the inner cell mass and the total number of cells of bovine [119] and ovine [120] embryos produced in vitro.

Larson et al., 1992 [107] and Neira et al., 2010 [60], demonstrated a beneficial effect by combining certain growth factors and cytokines with a synergistic action during early in vitro embryo development.

More recently, Moreno et al., 2015 [121], demonstrat-ed the beneficial effect of an association of growth factors and cytokines (IGF-1, IGF-11, bFGF, GM-CSF, PDGF-BB, LIF, and TGF-1), supplemented with hyaluronic acid and recombinant albumin, on in vitro embryo development in cattle. This combination has a synergistic action, with higher blastocyst rates 7 days post-fertilisation in compar-ison with the commonly used culture medium containing FCS, BSA, and ITS. The number of cells in the embryonic bud and the post-thaw survival obtained with this syn-thetic medium made with growth factors and cytokines were compared to those of the medium containing BSA. Finally, the viability of embryos produced in this synthet-ic medium has been demonstrated by the production of pregnancies and neonates born after the transfer of fresh and frozen embryos into recipient heifers.

References1. Dauzier L, Thibault C, Wintenberger S. La fecun-

dation in vitro de l’oeuf de lapine. C.R. Acad. Sci. 1954; 38:1063-1064.

2. Martal J. Les biotechnologies de la reproduction animale. In: Scriban R, editor. Dans Biotechnolo-gie, 5th edn. Paris: Tec. & Doc. 1999; 627-759.

3. Brackett BG, Bousquet D, Boice MI, Donawick WG, Evans JF, et al. Normal development follow-ing in vitro fertilization in the cow. Biol Reprod. 1982; 27:147-158.

4. Crozet N. La fécondation in vivo et in vitro. Dans La reproduction chez les mammifères et l’homme, Thibault C, Levasseur MC, INRA. Edit. Ellipses, Paris:315-338.

5. Perry G. 22nd Annual report of the International Embryo Transfer Society (IETS). 2013.

6. Camargo LSA, Viana JHM, Sá WF, Ferreira AM, Ramos AA, et al. Factors influencing in vitro em-bryo production. Anim. Reprod. 2006; 3: 19-28.

7. Pieterse MC, Kappen KA, Kruip Th. AM, Taverne MAM. Aspiration of bovine oocytes during trans-vaginal ultrasound scanning of the ovaries. The-riogenology. 1988; 30: 751-762.

8. Galli C, Crotti G, Notari C, Turini P, Duchi R, et



www.avidscience.com

al. Embryo production by ovum pick up from live donors. Theriogenology. 2001; 55: 1341-1357.

9. Gibbons JR, Beal WE, Krisher RL, Faber EG, Pear-son RE, et al. Effects of one versus twice weekly transvaginal aspiration on bovine oocyte recovery and embryo development. Theriogenology. 1994; 42: 405-419.

10. De Roover R, Genicot G, Leonard S, Bols P, Dessy F. Ovum pick up and in vitro embryo produc-tion in cows superstimulated with an individually adapted superstimulation protocol. Anim Reprod Sci. 2005; 86: 13-25.

11. Pfeifer LFM, Adams GP, Pierson RA, Singh J. Ul-trasound biomicroscopy: a non-invasive approach for in vivo evaluation of oocytes and small antral follicles in mammals. Reprod Fert Dev. 2014; 26: 48-54.

12. Chemineau P, Cognié Y, Thimonier J. La maîtrise de la reproduction des mammifères domestiques. Dans La reproduction chez les mammifères et l’homme, Thibault C, Levasseur MC, INRA. Edit. Ellipses, Paris. 2001; 792-817.

13. Mermillod P. Croissance et maturation de l’ovocyte in vivo et in vitro. Dans La reproduction chez les mammifères et l’homme, Thibault C, Levasseur

MC, INRA. Edit. Ellipses, Paris. 2001; 348-366.

14. Mermillod P, Marchal R. La maturation de l’ovocyte de mammifères. médecine/sciences. 1999; 15 : 148-156.

15. Mermillod P, Wils C, Massip A, Dessy F. Collec-tion of oocytes and production of blastocysts in vitro from individual slaughtered cows. J Reprod Fertil. 1992; 96: 717-723.

16. Steeves TE, Gardner DK. Metabolism of glucose, pyruvate, and glutamine during the maturation of oocytes derived from pre-pubertal and adult cows. Mol Reprod Dev. 1999; 54: 92-101.

17. Menezo Y, Guérin P. Biochimie de l’environnement in vivo et in vitro de l’ovocyte et du jeune embry-on. Dans La reproduction chez les mammifères et l’homme, Thibault C, Levasseur MC, INRA. Edit. Ellipses, Paris. 2001; 410-424.

18. Nedambale TL, Dinnyés A, Groen W, Dobrinsky JR, Tian XC, et al. Comparison on in vitro ferti-lized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrifica-tion. Theriogenology. 2004; 62: 437-449.

19. Stojkovic M, Kölle S, Peinl S, Stojkovic P, Zakhartchenko V, et al. Effects of high concen-trations of hyaluronan in culture medium on development and survival rates of fresh and fro-



www.avidscience.com

zen–thawed bovine embryos produced in vitro. Reproduction. 2002; 124: 141–153.

20. Calder MD, Caveney AN, Smith LC, Watson AJ. Responsiveness of bovine cumulus-oocyte-com-plexes (COC) to porcine and recombinant human FSH, and the effect of COC quality on gonadotro-pin receptor and Cx43 marker gene mRNAs dur-ing maturation in vitro. Reprod Biol Endocrinol. 2003; 1: 14.

21. Tol HM, Eijk MG, Mummery CL, van den Hurk R, Bevers MM. Influence of FSH and hCG on the resumption of meiosis of bovine oocytes sur-rounded by cumulus cells connected to membra-na granulose. Mol Reprod Dev. 1996; 45: 218–224.

22. Izadyar F, Hage WJ, Colenbrander B, Bevers MM. The promotory effect of growth hormone on the developmental competence of in vitro matured bovine oocytes is due to improved cytoplasmic maturation. Mol Reprod Dev. 1998; 49: 444-453.

23. Lonergan P, Carolan C, Van Langendonckt A, Donnay I, Khatir H, et al. Role of epidermal growth factor in bovine oocyte maturation and preimplantation embryo development. Biol Re-prod. 1996; 54: 1412-1421.

24. Monget P, Monniaux D. Growth factors and the

control of folliculogenesis. J Reprod Fertil. 1995; 49: 321-333.

25. Carneiro G, Lorenzo PL, Pimentel C, Pegoraro L, Bertolini M, et al. Influence of insulin-like growth factor-I and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro matura-tion and parthenogenetic development in equine oocytes. Biol Reprod. 2001; 65: 899-905.

26. Kitiyanant Y, Saikhun J, Pavasuthipaisit K. So-matic cell nuclear transfer in domestic cat oocytes treated with IGF-I for in vitro maturation. Theri-ogenology. 2003; 59: 1775-1786.

27. Lorenzo PL, Illera MJ, Illera JC, Illera M. En-hancement of cumulus expansion and nuclear maturation during bovine oocyte IVM with the addition of epidermal growth factor and insulin-like growth factor-I. J Reprod Fertil. 1994; 101: 697-701.

28. Silva CC, Knight PG. Modulatory actions of ac-tivin-A and follistatin on the developmental com-petence of in vitro-matured bovine oocytes. Biol Reprod. 1998; 58: 558-565.

29. De Loos F, Van Vliet C, Maurik P, Kruip AM. Morphology of immature bovine oocytes. Gamete Research. 1989; 24: 197-204.

30. Hanzen Ch. Biotechnologie : La production



www.avidscience.com

d’embryons in vitro. Université de Liège. Méde-cine Vétérinaire. 2013.

31. Thibault C. La fécondation. Dans La reproduc-tion chez les mammifères et l’homme, Thibault C, Levasseur MC, INRA. Edit. Ellipses, Paris. 2001; 366-389.

32. Parrish JJ, Suski-Parrish JL, Leibried-Rutledge ML, Critser ES, Eyestone WH, et al. Bovine in vit-ro fertilization with frozen-thawed semen. Theri-ogenology. 1986; 35: 547-555.

33. Manjunath P, Therien I. Role of seminal plasma phospholipid binding proteins in sperm mem-brane lipid modification that occurs during ca-pacitation. J Reprod Immunol. 2002; 53: 109–119.

34. Medeiros CMO, Parrish JJ. Changes in lectin binding to bovine sperm during heparin-induced capacitation. Molec Reprod Develop. 1996; 44: 525–532.0

35. Parrish JJ. Bovine in vitro fertilization: In vitro oocyte maturation and sperm capacitation with heparin. Theriogenology. 2014; 81: 67-73.

36. Gadella BM, Luna C. Cell biology and functional dynamics of the mammalian sperm surface. The-riogenology. 2014; 81: 74-84.

37. Flesch FM, Brouwers JF, Nievelstein PF, Verkleij

AJ, van Golde LM, et al. Bicarbonate stimulated phospholipid scrambling induces cholesterol re-distribution and enables cholesterol depletion in the sperm plasma membrane. J Cell Sci. 2001; 114: 3543–3555.

38. Harrison RA, Gadella BM. Bicarbonate-induced membrane processing in sperm capacitation. The-riogenology. 2005; 63: 342-351.

39. Martal J, Chene N, Huynh L, Zourbas S, Rogez C, et al. Les interactions immuno-endocrines en-tre la mère et l’embryon. Dans L’embryon Chez L’homme et l’animal. INSERM – INRA, Paris. 2002; 159-201.

40. Tervit HR, Whittingham DG, Rowson LEA. Suc-cessful culture in-vitro of sheep and cattle ova. J Reprod Fertil. 1972; 30: 493–497.

41. Gandolfi F, Moor R. Stimulation of early embry-onic development in sheep by co-culture with oviduct epthelial cells. J Reprod Fertil. 1987; 81: 23–28.

42. Mclaughlin KJ, Mclean DM, Stevens G, Ashman RA, Lewis PA, et al. Viability of one-cell bovine embryos cultured in synthetic ovidut fluid medi-um. Theriogenology. 1990; 33: 1191-1199.

43. Ouhibi N, Hamadi J, Guillaud J, Ménézo Y. Co-culture of 1-cell mouse embryos on different cell



www.avidscience.com

supports. Human Reprod. 1990; 5: 737-743.

44. Myers MW, Broussard JR, Ménézo Y, Prough SG, Blackwell J, et al. Established cell lines and their conditioned media support bovine embryo devel-opment during in vitro culture. Human Reprod. 1994; 9: 1927-1931.

45. Reed WA, Suh TK, Bunch TD, White KL. Culture of in vitro fertilized bovine embryos with bovine oviductal epithelial cells, buffalo rat liver (BRL) cells, or BRL-cell-conditionned medium. Theri-ogenology. 1996; 45: 439-449.

46. Ménézo Y, Hazout A, Herbaut N, Nicollet B. Co-culture of embryos on Vero cells and transient transfer of blastocysts in humans. Human Reprod. 1992; 7: 101-106.

47. Watson AJ, Watson PH, Warnes D, Walker SK, Armstrong DT, et al. Preimplantation develop-ment of in vitro-matured and in vitro-fertilized ovine zygotes: comparison between coculture on oviduct epithelial cell monolayers and culture un-der low oxygen atmospheres. Biol Reprod. 1994; 50: 715–724.

48. Gardner DK, Lane M, Spitzer A. Enhanced rates of cleavage and development for sheep zygotes cultured to the blastocyst stage in vitro in the ab-

sence of serum and somatic cells: amino acids, vi-tamins and culturing embryos in groups stimulate development. Biol Reprod. 1994; 50: 390-400.

49. Lawitts JA, Biggers JD. Culture of preimplantation embryos. Methods Enzymol. 1993; 225: 153-164.

50. Rosenkrans CF, Zeng GQ, McNamara GT, Schoff P, First NL. Development of embryos in vitro as affected by energy substrates. Biol Reprod. 1993; 49: 459.462.

51. Holm P, Booth P, Vajta G, Callesen H. A protein-free SOF system supplemented with amino ac-ids, sodium citrate and myo-inosito for bovine culture. Report of the 13th scientific Meeting of the European Embro Transfer Association, Lyon, France (abstract 158). 1997.

52. Lim K, Jang G, Ko K, Lee W, Park H, et al. Im-proved in vitro bovine embryo development and increased efficiency in producing viable calves us-ing defined media. Theriogenology. 2007; 67: 293-302.

53. Krisher RL, Lane M, Bavister B. Developmental competence and metabolism of bovine embryos cultured in semi-defined and defined culture me-dia. Biol Reprod. 1999; 60: 1345–1352.

54. Abe H, Yamashita S, Itoh T, Satoh T, Hoshi H. Ul-trastructure of bovine embryos developed from in



www.avidscience.com

vitro–matured and–fertilized oocytes: Compara-tive morphological evaluation of embryos cul-tured either in serum-free medium or in serum-supplemented medium. Mol Reprod Dev. 1999; 53: 325-335.

55. Abe H, Hoshi H. Evaluation of bovine embryos produced in high performance serum-free media. J Reprod Dev. 2003; 49: 193-202.

56. Abe H, Yamashita S, Satoh T, Hoshi H. Accumula-tion of cytoplasmic lipid droplets in bovine em-bryos and cryotolerance of embryos developed in different culture systems using serum-free or serum-containing media. Mol Rep Dev. 2002; 61: 57-66.

57. George F, Daniaux C, Genicot G, Verhaeghe B, Lambert P, Donnay I. Set up of a serum-free cul-ture system for bovine embryos: Embryo develop-ment and quality before and after transient trans-fer. Theriogenology. 2008; 69: 612-623.

58. Rizos D, Gutiérrez-Adán A, Pérez-Garnelo S, De la Fuente J, Boland MP, et al. Bovine embryo cul-ture in the presence or absence of serum: Implica-tions for blastocyst development, cryotolerance, and messenger RNA expression. Biol Reprod. 2003; 68: 236-243.

59. Van Wagtendonk-de Leeuw AM, Aerts BJG, Den Daas JHG. Abnormal offspring following in vitro production of bovine preimplantation embryos: a field study. Theriogenology. 1998; 49: 883-894.

60. Neira JA, Tainturier D, Peña MA, Martal J. Effect of the association of IGF-I, IGF-II, bFGF, TGF-β1, GM-CSF, and LIF on the development of bovine embryos produced in vitro. Theriogenology. 2010; 73: 595-604.

61. Guérin B, Nibart M, Marquant-Leguienne B, Humblot P. Sanitary risks related to embryo trans-fert in domestic species. Theriogenology. 1997; 47: 33-42.

62. Van der Valk J, Brunner D, De Smet K, Fex Sven-ningsen A, Honegger P, et al. Optimization of chemically defined cell culture media – Replacing fetal bovine serum in mammalian in vitro meth-ods. Toxicology in Vitro. 2010; 24: 1053-1063.

63. Thompson JG. In vitro culture and embryo me-tabolism of cattle and sheep embryos—a decade of achievement. Anim Reprod Sci. 2000; 60: 263–275.

64. Bavister BD. Culture of preimplantation embryos: facts and artifacts Hum Reprod Update. 1995; 1: 91–148.

65. Vanroose G, Nauwynck H, Soom AV, Ysebaert



www.avidscience.com

MT, Charlier G, et al. Structural aspects of the zona pellucid of in vitro-produced bovine embry-os: a scanning electron and confocal laser scan-ning microscopic study. Biol Reprod. 2000; 62: 463–469.

66. Le Tallec B, Ponsart C, Marquant-Le Guienne B, Guérin B. Risks of transmissible diseases in rela-tion to embryo transfer. Reprod Nutr Dev. 2001; 41: 439-450.

67. Stringfellow DA, Givens MD, Waldrop JG. Bios-ecurity issues associated with current and emerg-ing technologies. Reprod Fert Dev. 2004; 16: 1-10.

68. Van Soom A, Nauwynck H. Risk associated with bovine embryo transfer and their containment. CAB reviews: Perspectives in Agriculture and Veterinary Science. 2008.

69. Eckert J, Pugh PA, Thompson JG, Niemann H, Tervit HR. Exogenous protein affects develop-mental competence and metabolic activity of bo-vine pre-implantation embryos in vitro. Reprod Fertil Dev. 1998; 10: 327-332.

70. Bredbacka K, Bredbacka P. Effect of polyvinylpyr-rolidone, serum albumin and fetal calf serum on early cleavage of bovine embryos. Theriogenol-ogy. 1995; 43: 174-174.

71. Iwasaki T, Kimura E, Totsukawa K. Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes. Theriogenology. 1999; 51: 709-720.

72. Chung JT, Tosca L, Huang TH, Xu L, Niwa K, et al. Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development. Reprod Biomed Online. 2007; 15: 198-207.

73. Vireque AA, Camargo LS, Serepiao RV, Rosa E Silva AA, Watanabe YF, et al. Preimplantation development and expression of Hsp-70 and Bax genes in bovine blastocyst derived from oocytes matured in alpha-MEM supplemented with growth factors and synthetic macromolecules. Theriogenology. 2009; 71: 620-627.

74. Lee CN, Ax RL. Concentration and composition of glycoaminoglycans in the female bovine repro-ductive tract. J Dairy Sci. 1984; 67: 2006-2009.

75. Sato E, Ishibashi T, Koide SS. Prevention of spon-taneous degeneration of mouse oocytes in cul-ture by ovarian glycosaminoglycans. Biol Reprod. 1987; 37: 371-376.

76. Suchanek E, Simunic V, Juretic D, Grizelj V. Fol-licular fluid contents of hyaluronic acid, follicle-stimulating hormone and steroids relative to the



www.avidscience.com

success of in vitro fertilization of human oocytes. Fertil Steril. 1994; 62: 347-352.

77. Furnus C, Valcarcel A, Dulout F, Errecalde A. The hyaluronic acid receptor (CD44) is expressed in bovine oocytes and early stage embryos. Theriog-enology. 2003; 60: 1633-1644.

78. Campbell S, Swann HR, Aplin JD, Seif MW, Kim-ber SJ, et al. CD44 is expressed throughout preim-plantation human embryo development Human Reproduction. 1995; 10: 425-430.

79. Tzanakakis GN, Karamanos NK, Hjerpe A. Ef-fects on glycosaminoglycan synthesis in cultured human mesothelioma cells of transforming, epi-dermal, and fibroblast growth factors and their combinations with platelet-derived growth factor. Exp Cell Res. 1995; 220: 130-137.

80. Klominek J, Robert KH, Hjerpe A, Wikstrom B, Gahrton G. Serum-dependent Growth Patterns of Two, Newly Established Human Mesothelioma Cell Lines. Cancer Res. 1989; 49: 6118-6122.

81. Hamashima N. Effect of hyaluronic acid on the preimplantational development of mouse embryo in vitro. Dev Growth Differ. 1982; 24: 353-357.

82. Miyoshi K, Umezu M, Sato E. Effect of hyaluronic acid on the development of porcine 1-cell em-

bryos produced by a conventional or new in vitro maturation/fertilization system. Theriogenology. 1999; 51: 777–784.

83. Furnus CC, de Matos DG, Martinez AG. Effect of hyaluronic acid on development of in vitro pro-duced bovine embryos. Theriogenology. 1998; 49: 1489-1499.

84. Block J, Bonilla L, Hansen PJ. Effect of addition of hyaluronan to embryo culture medium on sur-vival of bovine embryos in vitro following vit-rification and establishment of pregnancy after transfer to recipients. Theriogenology. 2009; 71: 1063-1071.

85. Palasz AT, Breña PB, Martinez MF, Perez-Garnelo SS, Ramirez MA, et al. Development, molecular composition and freeze tolerance of bovine em-bryos cultured in TCM-199 supplemented with hyaluronan. Zygote. 2008; 16: 39-47.

86. Gray CW, Morgan P, Kane MT. Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate. J Reprod Fert. 1992; 94: 471-480.

87. Lane M, Maybach JM, Hooper K, Hasler JF, Gard-ner DK. Cryo-survival and development of bovine blastocysts are enhanced by culture with recombi-nant albumin and hyaluronan. Mol Reprod Dev.



www.avidscience.com

2003; 64: 70-78.

88. Palasz AT, Thundathil J, Verrall RE, Mapletoft RJ. The effect of macromolecular supplementation on the surface tension of TCM-199 and the uti-lization of growth factors by bovine oocytes and embryos in culture. Anim Reprod Sci. 2000; 58: 229–240.

89. George F, Kerschen D, Van Nuffel A, Rees JF, Don-nay I. Plant protein hydrolysates (plant peptones) as substitutes for animal proteins in embryo cul-ture medium. Reprod Fert Dev. 2009; 21: 587–598.

90. Ben-Yosef D, Galiani D, Dekel N, Shalgi R. Rat oocytes induced to mature by epidermal growth factor are successfully fertilized. Mol Cell Endo-crinol. 1992; 88: 135-141.

91. Das K, Stout LE, Hensleigh HC, Tagatz GE, Phi-pps WR, et al. Direct positive effect of epidermal growth factor on the cytoplasmic maturation of mouse and human oocytes. Fertil Steril. 1991; 55: 1000-1004.

92. Reed ML, Estrada JL, Illera MJ, Petters RM. Ef-fects of epidermal growth factor, insulin-like growth factor-I, and dialyzed porcine follicular fluid on porcine oocyte maturation in vitro. J Exp Zool. 1993; 266: 74-78.

93. Mermillod P, Lonergan P, Carolan C, Khatir H, Poulin N, et al. Maturation ovocytaire in vitro chez les ruminants domestiques. Contraception Fert Sexualité. 1996; 24: 552-558.

94. Bonilla AQ, Ozawa M, Hansen PJ. Timing and de-pendence upon mitogen-activated protein kinase signaling for pro-developmental actions of insu-lin-like growth factor 1 on the preimplantation bovine embryo. Growth Horm IGF Res. 2011; 21: 107-111.

95. Moreira F, Paula-Lopes FF, Hansen PJ, Badinga L, Thatcher WW. Effects of growth hormone and insulin-like growth factor-I on development of in vitro derived bovine embryos. Theriogenology. 2002; 57: 895–907.

96. Palma GA, Muller M, Brem G. Effect of insulin-like growth factor I (IGF-I) at high concentrations on blastocyst development of bovine embryos produced in vitro. J Reprod Fertil. 1997; 110: 347–353.

97. Prelle K, Stojkovic M, Boxhammer K, Motlik J, Ewald D, et al. Insulin-like growth factor I (IGF-I) and long R(3) IGF-I differently affect develop-ment and messenger ribonucleic acid abundance for IGF-binding proteins and type I IGF receptors in in vitro produced bovine embryos. Endocrinol-ogy. 2001; 142: 1309-1316.



www.avidscience.com

98. Spanos S, Becker DL, Winston RM, Hardy K. An-ti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo develop-ment. Biol. Reprod. 2000; 63: 1413-1420.

99. Fabian D, Il’kova G, Rehak P, Czikkova S, Baran V, et al. Inhibitory effect of IGF-I on induced apop-tosis in mouse preimplantation embryos cultured in vitro. Theriogenology. 2004; 61: 745-755.

100. Cui XS, Jeong YJ, Jun JH, Kim NH. Insu-lin-like growth factor-I alters apoptosis related genes and reduces apoptosis in porcine parthe-notes developing in vitro. Theriogenology. 2005; 63: 1070-1080.

101. Byrne AT, Southgate J, Brison DR, Leese HJ. Analysis of apoptosis in the preimplantation bovine embryo using TUNEL. J Reprod Fertil. 1999; 117: 97-105.

102. Block J, Drost M, Monson RL, Rutledge JJ, Rivera RM, et al. Use of insulin-like growth fac-tor-I during embryo culture and treatment of re-cipients with gonadotropin-releasing hormone to increase pregnancy rates following the transfer of in vitro-produced embryos to heat-stressed, lac-tating cows. J Anim Sci. 2003; 81: 1590-1602.

103. Block J, Hansen PJ. Interaction between

season and culture with insulin-like growth fac-tor-1 on survival of in vitro produced embryos following transfer to lactating dairy cows. Theri-ogenology. 2007; 67: 1518-1529.

104. Block J, Hansen P, Loureiro B, Bonilla L. Improving post-transfer survival of bovine em-bryos produced in vitro: Actions of insulin-like growth factor-1, colony stimulating factor-2 and hyaluronan. Theriogenology. 2011; 76: 1602-1609.

105. Desai N, Kattal N, AbdelHafez F, Szep-tycki-Lawson J, Goldfarb J. Granulocyte-mac-rophage colony stimulating factor (GM-CSF) and co-culture can affect post-thaw development and apoptosis in cryopreserved embryos. J Assist Re-prod Genet. 2007; 24: 215-222.

106. Fields SD, Hansen PJ, Ealy AD. Fibroblast growth factor requirements for in vitro develop-ment of bovine embryos. Theriogenology. 2011; 75: 1466-1475.

107. Larson RC, Ignotz GG, Currie WB. Trans-forming growth factor beta and basic fibroblast growth factor synergistically promote early bo-vine embryo development during the fourth cell cycle. Mol Reprod Dev. 1992; 33: 432-435.

108. Yang QE, Giassetti MI, Ealy AD. Fibroblast growth factors activate mitogen activated protein



www.avidscience.com

kinase pathways to promote migration in ovine trophoblast cells. Reproduction. 2011; 141: 707-714.

109. Cooke FN, Pennington KA, Yang Q, Ealy A. Several fibroblast growth factors are expressed during pre-attachment bovine conceptus develop-ment and regulate interferon-tau expression from trophectoderm. Reproduction. 2009; 137: 259-269.

110. Michael D, Alvarez I, Ocon O, Powell A, Talbot N, et al. Fibroblast Growth Factor-2 is ex-pressed by the bovine uterus and stimulates inter-feron-production in bovine trophectoderm. En-docrinology. 2006; 147: 3571-3579.

111. Yang QE, Johnson SE, Ealy AD. Protein kinase C delta mediates fibroblast growth factor-2-induced interferon-tau expression in bovine trophoblast. Biol Reprod. 2011; 84: 933-943.

112. Eckert J, Niemann H. Effects of Plateled Derived Growth Factor (PDGF) on the in vitro production of bovine embryos in protein-free me-dia. Theriogenology. 1996; 46: 307-320.

113. Larson RC, Ignotz GG, Currie WB. Platelet derived growth factor (PDGF) stimulates devel-opment of bovine embryos during the fourth cell

cycle. Development. 1992; 115: 821-826.

114. Luo S, Yin HN, Li SW. Effect of TGF-be-ta1 on embryo implantation and development in mice in vitro. J Sichuan Univ. 2010; 41: 265-268.

115. Loureiro B, Bonilla L, Block J, Fear J, Bonil-la A, et al. Colony-Stimulating Factor 2 (CSF-2) improves development and post-transfer survival of bovine embryos produced in vitro. Endocrinol-ogy. 2009; 150: 5046-5054.

116. Kwak SS, Jeung SH, Biswas D, Jeon YB, Hyun S. Effects of porcine granulocyte-mac-rophage colony-stimulating factor on porcine in vitro-fertilized embryos. Theriogenology. 2012; 77: 1186-1197.

117. Loureiro B, Block J, Favoreto M, Caram-bula S, Pennington KA, et al. Consequences of conceptus exposure to colony-stimulating factor 2 on survival, elongation, interferon-τ secretion, and gene expression. Reproduction. 2011; 141: 617-624.

118. Rooke J, Ewen M, McEvoy T, Entrican G, Ashworth C. Effect of inclusion of serum and granulocyte-macrophage colony stimulating fac-tor on secretion of interferon-tau during the in vitro culture of ovine embryos. Reprod Fert Dev. 2005; 17: 513-521.

46 www.avidscience.com

Recent Advances in In Vitro Fertilization

119. Sirisathien S, Hernandez-Fonseca HJ, Bosch P, Hollet BR, Lott JD, et al. Effect of leu-kemia inhibitory factor on bovine embryos pro-duced in vitro under chemically defined condi-tions. Theriogenology. 2003; 59: 1751-1763.

120. Ptak G, Lopes F, Matsukawa K, Tischner M, Loi P. Leukaemia inhibitory factor enhances sheep fertilization in vitro via an influence on the oocyte. Theriogenology. 2006; 65: 1891-1899.

121. Moreno D, Neira A, Dubreil L, Liegeois L, Destrumelle S, et al. In vitro bovine embryo pro-duction in a synthetic medium: Embryo develop-ment, cryosurvival, and establishment of preg-nancy. Theriogenology. 2015; 84: 1053-1060.

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