Achievements and future perspectives of embryo transfer technology in pigs

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Achievements and future perspectives of embryo

transfer technology in pigs

Emilio A. Martinez, Cristina Martinez-Serrano, Josep M. Cambra, Carolina Maside, Xiomara Lucas, Jose L. Vazquez, Juan Maria Vazquez, Jordi Roca, Heriberto

Rodriguez-Martinez, Maria Antonia Gil, Inmaculada Parrilla and Cristina Cuello

The self-archived postprint version of this journal article is available at Linköping University Institutional Repository (DiVA):

http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-161617

N.B.: When citing this work, cite the original publication.

Martinez, E. A., Martinez-Serrano, C., Cambra, J. M., Maside, C., Lucas, X., Vazquez, J. L., Maria Vazquez, J., Roca, J., Rodriguez-Martinez, H., Antonia Gil, M., Parrilla, I., Cuello, C., (2019), Achievements and future perspectives of embryo transfer technology in pigs, Reproduction in

domestic animals, 54, . https://doi.org/10.1111/rda.13465

Original publication available at:

https://doi.org/10.1111/rda.13465

Copyright: Wiley (12 months)

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Achievements and future perspectives of embryo transfer technology in pigs E.A. Martinez1,2_{, C.A. Martinez}1,3_{, J.M. Cambra}1,2_{, C. Maside}1,2_{, X. Lucas} 1,2_{, J.L.} Vazquez1,2_{, J.M. Vazquez}1,2_{, J. Roca}1,2_{, H. Rodriguez-Martinez}3_{, M.A. Gil}1,2_{, I.} Parrilla1,2_{*, C. Cuello}1,2

1 _{Department of} _{Medicine and Animal Surgery, Faculty of Veterinary Medicine,} International Excellence Campus for Higher Education and Research “Campus Mare Nostrum”, University of Murcia, 30100, Murcia, Spain; 2_{Institute for Biomedical} Research of Murcia (IMIB-Arrixaca), Campus de Ciencias de la Salud, Carretera Buenavista s/n, 30120 El Palmar, Murcia, Spain; 3_{Department of Clinical & Experimental} Medicine (IKE), Linköping University, Campus US, 58183, Linköping, Sweden. Corresponding author: Inmaculada Parrilla

E-mail address: parrilla@um.es Running title: Embryo transfer in pigs Contents

Commercial embryo transfer (ET) has unprecedented productive and economic implications for the pig sector. However, pig ET has been considered utopian for decades mainly because of the requirements of surgical techniques for embryo collection and embryo deposition into recipients, alongside challenges to preserve embryos. This situation has drastically changed in the last decade since the current technology allows nonsurgical ET and short- and long-term embryo preservation. Here, we provide a brief review of the improvements in porcine ET achieved by our laboratory in the past 20 years. This review includes several aspects of nonsurgical ET technology and different issues affecting ET programs and embryo preservation systems. The future perspectives of ET technology are also considered. We will refer only to embryos produced in vivo since they are the only type of embryos with possible short-term use in pig production. KEYWORDS: porcine, embryo transfer, embryo storage, vitrification.

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More than 65 years have passed since the first litters produced by embryo transfer (ET) were born (Kvasnickii, 1950). Today, porcine ET technology is mandatory for highly relevant biotechnologies, such as cloning, gene editing and interspecies blastocyst complementation technologies.

On the other hand, although ET technology presents fundamental applications in pig production through the transfer of high-genetic-value embryos with minimal risk of disease transmission, reduced transportation cost and lack of an effect on animal welfare during transport, its commercial use in pigs has been and still is insignificant compared to ET in other species, as cattle.

The main factor required to expand ET technology is the use of nonsurgical methods for embryo collection and transfer under field conditions, an absolute pre-requirement to attend welfare issues. In cattle, for instance, the development of nonsurgical ET procedures has led to a dramatic expansion of the ET industry, which is today a blooming worldwide business (Rodriguez-Martinez, 2012). Unfortunately, the special anatomy of the swine genital tract has made it difficult to develop easy nonsurgical procedures for transfer the embryos into recipients, precluding their application in the field. Moreover, difficulties encountered in the storage of porcine embryos and the human and material requirements to work with in vivo-derived embryos have discouraged many researchers. Despite these constrains, new procedures have been developed in the past decade that enable successful nonsurgical ET and efficient embryo preservation. Their description is the core of the present review.

2. THE NONSURGICAL DEEP UTERINE ET TECHNIQUE

In the early 2000s, we designed procedures for the nonsurgical transfer of embryos in the depth of a uterine horn (NsDU-ET) of gilts and sows (Martinez, Vazquez, Roca, Lucas, Gil, & Vazquez, 2001). The various aspects related to the design, development, safety and effectiveness of this procedure have been recently reviewed (Martinez, Cuello, et al., 2016; Martinez, Cuello, et al., 2013; Martinez, Gil, et al., 2013). Therefore, herein, we shall only briefly summarize the most important findings achieved from these studies. The NsDU-ET catheter can be rapidly and correctly inserted through the cervix into a uterine horn in more than 90% of non-sedated recipients (gilts and sows). The procedure does not disturb the welfare of the recipient female and, with increasing practice, it can be quickly accomplished without any procedural problems. In addition, the use of adequate aseptic protocols during the procedure avoids uterine infections after ET. In the

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first attempts at NsDU-ET, acceptable fertility and prolificacy was achieved using fresh (71.4% farrowing rate and 6.9 piglets born) and vitrified-warmed (42.9% farrowing rate and 5.4 piglets born) embryos (Cuello et al., 2005; Martinez et al., 2004). With the development and improvement of the procedure, the reproductive performance of recipients was significantly increased with both fresh embryos stored for 0-24 h (80-90% farrowing rate and 9.0-9.5 piglets born) and vitrified-warmed embryos (50-75% farrowing rate and 9.0-10 piglets born) (Martinez et al., 2014, 2015). Because of the potential of NsDU-ET technology, other experiments were and are being conducted to develop a safe, practical and efficient ET procedure to be widely used by the pig industry. 3. FACTORS AFFECTING THE ET TECHNOLOGY

There are many factors related to embryo-producers (“donors”) and embryo-recipients that can influence the reproductive outcomes after NsDU-ET.

3.1. Selection of embryo donors and recipients

A key factor affecting the efficiency and final cost of an ET program is the selection of the donors of embryos, which is commonly based on their health condition, breeding history and, essentially, their genetic value. However, other factors, including parity, the weaning-to-estrus interval (WEI) or the season of the year, can decisively influence donor embryo production outcomes and affect the entire ET success. Because there was hardly any information regarding the impact of these factors on embryo production and quality, we investigated whether parity (2 to 7), WEI (estrus within 3 to 4 days) and season (fall, winter and spring) had any influence on the number of corpora lutea and ovarian cysts, number and quality of collected embryos, embryo developmental stages and recovery and fertilization rates among embryo-producing donors (Nohalez et al., 2017). The results of this study showed a lack of signiﬁcant effects of all variables evaluated or their interactions on any of the reproductive and embryonic parameters examined. These results opened the door to use most sows included in the genetic breeding stock of farms as donors for as long their genetic value and health allows, thus contributing to the widespread use of ET technology.

The strict selection of embryo recipients, a fundamental element in ET programs, has unfortunately received very little attention. Gilts and sows without health problems and with good body-condition scoring are usually selected as recipients; despite other factors could jeopardize the success of ETs. Age and parity inﬂuence reproductive performance

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after artificial insemination (AI), with gilts and primiparous sows being less efficient than multiparous sows (Clark, Schinckel, Singleton, Einstein & Teclaw, 1989). Multiparous sows are ideal as recipients because the insertion of the NsDU-ET catheter is easier, their conduct during the transfers is better (Martinez, Cuello, et al., 2013) and their estruses are naturally and effectively synchronized by managing weaning. All of the abovementioned observations led us to evaluate the influence of recipient parity (parity 1 to 5) on NsDU-ET outcomes (Martinez, Nohalez, et al., 2016). The results indicated that neither the efficiency of the NsDU-ET procedure nor the reproductive performance of the recipients were affected by parity. The use of recipients with a large parity range simplifies commercial ET programs because of the high accessibility of this kind of sow on farms.

3.2. Superovulation of embryo donors

The normal numbers of oocytes released at ovulation in combination with other aspects (Martinez, Gil, et al., 2013) determine that the number of embryo donors usually doubles the number of recipients in the ET programs. A strategy to reduce the donor:recipient ratio is to use superovulated donors. As such, our laboratory investigated the efficacy of several superovulation regimes in post-weaning Duroc sows (Angel et al., 2014a). These experiments showed that 1000 IU eCG administered 24 h post-weaning followed by 750 IU hCG at the onset of estrus increased the number of transferable embryos and, contrary to results previously reported (Holtz & Schlieper, 1991), the incidence of ovarian cysts, the quality of embryos or the number of unfertilized oocytes were unaffected by the treatment. In addition, the farrowing rates and litter sizes of recipients of superovulated and non-superovulated embryos were similar. In another set of experiments, it was found that the ovarian superovulatory response varied among breeds; the response in Pietrain sows was comparable to that of Duroc sows, and in Danish DanBred sows, the response was much more pronounced. Taken together, these results demonstrated that the donor:recipient ratio could be decreased from 2:1 to 1.5-1:1, which is of great benefit for genetic companies, through the use of superovulated donors.

3.3. Synchrony between the stage of embryo development and the recipients

Little (and contradictory) information has been available regarding the optimal synchrony between the developmental stage of embryos to be transferred and the recipients. Thus, several studies involving surgical ETs showed that the highest pregnancy rates were

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achieved when the recipients were in estrus after the donors (Blum-reckow & Holtz, 1991; Polge, 1982; Wilde, Xie, Day, & Pope, 1988). In contrast, a study using nonsurgical ET into the uterine body suggested the contrary (Hazeleger, Noordhuizen, & Kemp, 2000). To clarify this apparent discrepancy, we performed a large trial involving more than 130 NsDU-ETs (Angel et al., 2014b). In this study, the lowest farrowing rates (<5%) were obtained in recipients that started estrus 24 h before the donors, and the highest farrowing rates (82%) were found in recipients in estrus 24 h after the donors, irrespective of the stage of the embryos transferred (morulae or blastocysts). These results confirmed those achieved in the surgical experiments mentioned above. Although still mechanistically unclear, the causes for the discrepancies between these studies might involve dissimilarities in the uterine location where the embryos were deposited, which is different for each ET procedure (tip, middle and bottom of the uterine horn for surgical, NsDU-ETs and nonsurgical uterine body ETs, respectively).

3.4. Future reproductive performance of donors and recipients of embryos

At present, it is only possible to obtain pig embryos from donors after slaughter or by surgical intervention. In contrast, today it is possible to transfer the embryos deep into a uterine horn with nonsurgical techniques (Martinez, Cuello, et al., 2013; Martinez, Gil, et al., 2013). A usual concern of genetic and production companies is the impact that surgical embryo collection and NsDU-ET procedures have on the future reproductive performance of the donors and recipients, respectively, which previous studies never examined. Recently, we evaluated the reproductive performance of donors and recipients after AI subsequent to surgical embryo collection and NsDU-ET procedures, respectively (Martinez et al., 2017). We found that while the farrowing rate of donors was not affected by surgery, prolificacy was deeply reduced (two piglets less). It is clear that more efforts and surgical refinement are needed to decrease the negative effect of the surgical approach on the donor sows. We also found that the NsDU-ET procedure did not affect subsequent reproductive performance, indicating the safety of this technology in the ET programs.

4. RESEARCHES ON EMBRYO VITRIFICATION

Although several investigations in the 1990s showed that frozen/thawed porcine embryos were able to develop to term following transfer to recipients (Berthelot, Martinat-Botté,

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Vajta, & Terqui, 2003), the traditional slow-freezing cryopreservation method was largely ineffective when applied to pig embryos (Berthelot, Martinat-Botté, Vajta, & Terqui, 2003; Martinat-Botté, Berthelot, Plat, & Madec, 2006). Pig embryos show high sensitivity to chilling injury (Wilmut, 1972), which limits their ability to be cryopreserved by conventional slow-freezing methods. By the end of the 1990s and at the beginning of the 2000s, vitrification was applied as an alternative to the traditional freezing method. The development of vitrification systems resulted in piglets born after the transfer of vitrified/warmed embryos in several laboratories (Martinez, Gil, et al., 2013). During the 2000s, numerous studies aimed to determine which factors were pivotal to improve the survival of vitrified/warmed embryos. The improvements achieved in the vitrification protocols, especially the Open Pulled Straw (OPS) method (Vatja, Holm, Greeve, & Callesen, 1997), resulted in high in vitro embryonic survival rates and acceptable pregnancy and farrowing rates after surgical transfers (Martinez, Vazquez, et al., 2016; Martinez, Gil, et al., 2013). Next, we describe those factors studied by our research group since then.

4.1. Embryo donor variability for embryo vitrification

Fujino, Yonemura, Suzuki, & Misumi (2003) reported the individual effect of the donor on the post-warming survival of embryos. These results indicated that the donor might be important in the vitrification ability of embryos, similar to the boar effect on sperm freezability (Roca, Hernández, Carvajal, Vázquez, & Martínez, 2006). To confirm this statement, we demonstrated the individual effect of the donor on the post-warming viability of embryos at different stages of development, including zygotes (Gomis et al., 2013a), morulae and blastocysts (Cuello et al., 2004b; Sanchez-Osorio et al., 2008). Because morulae showed more post-warming variability among donors than blastocysts, we suggested that donor differences in resilience to vitriﬁcation might be more marked at earlier stages of embryo development (Sanchez-Osorio et al., 2008). Although donor variability in embryo vitrification ability may be influenced by many factors, such as individual differences in the response to sucrose (Weber & Youngs, 1994) or genotype (Berthelot, Martinat-Botté, Locatelli, Perreau, & Terqui, 2000), the main causes remain unknown. To diminish this donor effect, it would be advisable to mix vitrified-warmed embryos from two o more donors for ET. According to this suggestion, our group, in collaboration with the Martinat-Botte group (INRA, Tours, France), demonstrated the

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beneficial effects of pooling warmed embryos from two donors on the in vivo embryonic efficiency after ET (Cuello, Berthelot, et al., 2004).

4.2. Cooling rates and cryoprotectant concentrations

Initial studies on embryo vitrification were performed using 0.25 mL straws, which limited the cooling rate to 2,500 °C/min. The success of vitrification was considerably augmented by increasing the cooling rate through the development of smaller straws, such as the OPS (20,000 ºC/min; Vatja, Holm, Greeve, & Callesen, 1997) and superfine OPS straws (SOPS) (Isachenko et al., 2003) or by using other vitrification procedures, including the Vit-Master technique, microscopic grids (Martino, Songsasen, & Leibo, 1996), cryotops (Du et al., 2007) or cryoloops (Lane, Schoolcraft, Gardner, & Phil, 1999). When we compared three vitrification systems (OPS, SOPS, and Vit-Master-SOPS) to investigate whether the cooling rate influenced the post-warming embryo viability, we found that cooling rates above 20,000 ºC/min did not improve post-warming outcomes (Cuello et al., 2004b). However, the use of higher cooling rates made it possible to reduce the concentration of cryoprotectants in the vitrification media. Thus, we successfully decreased the concentration of ethylene glycol and dimethyl sulfoxide in the vitrification media from the typical 18% to 16% using the SOPS-vitrification procedure (Cuello et al., 2008). Since cryoprotectants are toxic to embryos at high concentration, their reduction might be favorable for embryo survival. Unfortunately, there are no studies comparing the development to term of embryos vitrified under different concentrations of cryoprotectants.

4.3. One-step embryo warming procedure

The warming procedure for vitrified embryos traditionally involved three steps to eliminate the cryoprotectants added to vitrification media. However, this procedure was successfully replaced by a simple (one-step) protocol for bovine and ovine embryos (Otoi, Takagi, Boediono, Sumantri, & Suzuki, 1996; Isachenko et al., 2003). To explore the possibility of developing a one-step warming method for vitrified porcine embryos, we designed a procedure involving direct (one-step dilution) warming in a 1-mL syringe procedure, which could be directly connected to the NsDU-ET catheter without the need for any special equipment. This procedure may be particularly useful for developing the NsDU-ET technology under field conditions. We found that the one-step warming procedure did not show any differences in the in vitro embryo viability and hatching of

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OPS-vitrified embryos compared to the traditional three-step warming method (Cuello et al., 2004a). Moreover, we obtained the first piglets produced by NsDU transfers of OPS-vitriﬁed, direct warmed blastocysts (Cuello et al., 2005), although fertility was below expectations. Surprisingly, when we used the SOPS procedure instead of the OPS procedure, we found that the syringe warming method was detrimental for embryo development post-warming, either in vitro or in vivo (Gomis et al., 2012). In the direct warming procedure, the tip of the straw is inserted through the small tip of the syringe, which implies managing difficulties and, therefore, an increased time for the contact of the cryopreserved straws (embryos) with the warming medium. This situation is especially important when the vitrification volume is very low (~1μL), as in the SOPS method, because it can cause devitrification and, consequently, embryonic death. This might explain the detrimental effects of direct warming on SOPS-vitrified embryos. Later modifications of the one-step warming method by using a dish prior to inserting the embryos into the syringe resulted in promising farrowing rates and litter sizes after NsDU-ET of SOPS-vitrified morulae and blastocysts (Gomis et al., 2012; Martinez et al., 2015).

4.4. Postwarming morphological evaluation of embryos

In vitro embryo survival rates post-warming are generally very high, mainly for embryos at the blastocyst stage; in many cases there are no differences between vitrified blastocysts and fresh blastocysts (Martinez, Gil, et al., 2013). Typically, embryonic post-warming survival is assessed by stereomicroscopic morphological evaluation. Nevertheless, studies in ovines showed that vitrified embryos with good morphology after warming failed to develop in vivo after ET (Cocero, Díaz de la Espina, & Aguilar, 2002). Moreover, other studies in bovine and porcine species demonstrated ultrastructural alterations that were unobserved by stereomicroscopy (Fabian, Gjørret, Berthelot, Martinat-Botté, & Maddox-Hyttel, 2005; Vatja, Holm, Greeve, & Callesen, 1997). To validate the morphological evaluation, we performed a study comparing morphology of vitrified embryos and fresh embryos with parallel observations of ultrastructure and cell death (Cuello, Berthelot, et al., 2007). We observed that while a high percentage of post-warmed, expanded blastocysts morphologically viable by stereomicroscopy exhibited important ultrastructural abnormalities and increased levels of cell death compared to their fresh control counterpart hatching blastocysts did not differ from fresh hatching blastocysts. These results clearly indicated that hatching rates are a more valid parameter

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than development progression or embryo re-expansion for the morphological quality evaluation of vitrified-warmed embryos.

4.5. Vitrification of embryos at different developmental stages

Because NsDU-ETs are performed in a uterine horn, the morulae and unhatched blastocysts are the most suitable embryonic developmental stages to be transferred. It is widely recognized that the content of intracytoplasmic lipids, which decreases as the embryo development stage increases, plays a key role in the cooling tolerance of porcine embryos (Nagashima, Kashiwazaki, Ashman, Grupen, Seamark, & Nottle, 1994). Accordingly, our studies indicated that while expanded blastocysts showed the highest postwarming survival and hatching rates (similar to those of fresh blastocysts), the vitrification ability of early blastocysts was superior to that of the morulae (Cuello, et al., 2004b), and two-to-four cell embryos displayed the lowest survival and hatching rates (Sanchez-Osorio et al., 2008).

The lack of a successful methodology to vitrify porcine embryos in the very early stages of development was one of our main concerns in the mid-2000s. In certain sanitary crises involving the sacrifice of all animals within a security perimeter and the prohibition to move animals outside that area, embryo vitrification may allow genetic rescue of affected herds. Nevertheless, under these circumstances, the selection of the day to obtain embryos at the morula and blastocyst stages, the preferred stages for vitrification and transfers, is unlikely to be an option. The use of ultra-rapid vitrification of untreated two-to-four cell embryos was ineffective (Cuello, Gil, et al., 2007). However, when two-to-four cell embryos were cultured for 3 to 4 days, the resulting blastocysts displayed successful post-warming survival and hatching rates (Cuello, Gil, et al., 2007). This was not surprising, as it had been previously reported in ovine embryos (Garcia-Garcia, Gonzalez-Bulnes, Dominguez, Veiga-Lopez, & Cocero, 2005). We also worked on intracytoplasmic lipids at several levels. In vivo-derived zygotes treated with forskolin, a lipolytic agent that increased the survival of in vitro-derived blastocysts (Cuello et al., 2013), prior to vitriﬁcation enhanced their vitrification ability, resulting in a post-warming blastocyst formation rate of 75% (Gomis et al., 2013b). In addition, lipid polarization pretreatments before vitrification also increased the post-warming capacity of in vivo-derived zygotes to reach the blastocyst stage (Gomis et al., 2013a). Unfortunately, other pretreatments, including centrifugation or the use of hyperosmotic media, failed to improve the resistance to vitrification of very early porcine embryos (Gomis et al., 2013a). More

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research is needed to develop adequate protocols for vitrification and warming of zygotes and two-to-four cell embryos.

4.6. Pretreatments of embryos prior to vitrification

In the early stages of embryo vitrification, it was reported that the traditional vitrification procedures (0.25 mL straws) produced damage in the cytoskeleton that caused alterations in embryonic development (Dobrinsky, Pursel, Long, & Johnson, 2000). To solve this problem, several strategies were proposed, including pretreatments with cytoskeleton stabilizers such as cytochalasin B, alone or in combination with centrifugation to polarize cytoplasmic lipids (Beebe, Cameron, Blackshaw, & Keates, 2005; Dobrinsky, Pursel, Long, & Johnson, 2000). However, with the development of OPS and other ultra-rapid vitrification procedures, it was possible to achieve very high post-warming survival rates, similar to those of fresh blastocysts, using non-pretreated in vivo-derived blastocysts (Cuello, et al., 2004b; Sanchez-Osorio et al., 2008). In these studies, the embryo survival rates were morphologically assessed by stereomicroscopy; however, as mentioned above, this evaluation procedure is not able to detect structural abnormalities and cytoskeleton alterations in the embryos. Therefore, we investigated cytoskeletal alterations in SOPS-vitrified blastocysts and evaluated the effects of pretreatments of embryos with cytochalasin B and centrifugation on post-warming embryo survival and integrity of the actin cytoskeleton (Cuello et al., 2010). In this study, high numbers of untreated blastocysts survived to warming and displayed a low cell death index and high cytoskeletal integrity, indicating that the pretreatments utilized were not needed for the SOPS vitrification of blastocysts. These results were of unquestionable value at a practical level since the absence of treatments prior to vitrification reduces time and increases the practicality of the procedure.

4.7. Length of embryo storage

In 2010, we became interested in the effects of storage length in liquid nitrogen (LN2) on the viability of vitrified-warmed porcine embryos. Surprisingly, to that date, only a limited number of studies had been conducted on the long-term storage of embryos in human and mice, with inconsistent results (Mozdarani & Moradi, 2007; Riggs, Mayer, Dowling-Lacey, Chi, Jones, & Oehninger, 2010), and -to the best of our knowledge- no data was available for domestic species. We conducted a retrospective analysis of the data collected in our laboratory on post-warming survival and hatching rates of OPS- or

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SOPS-vitriﬁed embryos at the morula and blastocyst stages (Sanchez-Osorio, Cuello, Gil, Parrilla, Almiñana, et al., 2010). The main conclusion achieved was that these embryos could be stored for at least three years without affecting their post-warming development ability. Moreover, we recently obtained piglets born alive from vitrified embryos that had been cryopreserved for 5 years. These results agree with those of subsequent studies in bovine, ovine and leporine species (Fang et al., 2014; Lavara, Baselga, & Vicente, 2011; Marco-Jiménez, Baselga, & Vicente, 2018; Yao, Qi, Lu, Wang, Li, & Han, 2012) and indicate the possibility of building cryobanks with embryos from valuable genetic lines for future use in ET programs, which may be important for the re-establishment of specific pig populations.

4.8. Vitrification and warming with chemically defined media

Vitrification and warming media usually contain serum or serum derivatives, which reduce post-warming results reliability and increase the risk of infectious disease transmission (Guerin, Nibart, Marquant Le Guienne, & Humblot, 1997; Wrathall, 1995). Accordingly, we demonstrated that polyvinyl alcohol (PVA), a macromolecule previously used for the vitrification and freezing of embryos of several species with variable results, could replace serum in vitriﬁcation and warming media without affecting the post-warming developmental capacity of in vivo-derived porcine morulae and blastocysts (Sanchez-Osorio, Cuello, Gil, Parrilla, Maside, et al., 2010). From a practical point of view, we also considered replacing the TCM-199 medium, which has a carbonate buffer and is traditionally used as basal medium for vitrification and warming, with a pH-stable medium that does not require CO2. Tyrode’s lactate (TL)-HEPES- PVA medium (TL-PVA), which is used routinely for the collection and transfer of embryos, was tested. There was no difference in either post-warming in vitro or in vivo development between the TL-PVA medium and the traditional TCM-199 medium (Cuello et al., 2016). Therefore, at present, a defined, pH-stable (without CO2 gassing) medium for the collection, handling, vitrification, warming and transfer of porcine embryos is available, which is ideal for ET programs under field conditions.

4.9. Effectiveness of non-surgical transfer with vitrified embryos

In 2015, to determine the total effectiveness of the NsDU-ET of vitrified-warmed embryos, our group performed a large trial involving more than 100 ETs (Martinez et al.,

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2015). That study compared the efficiency of NsDU-ETs using vitrified, in vivo-derived morulae and blastocysts on the reproductive outcomes of the recipients. Nonsurgical transfers were performed with either 30 or 40 embryos. Recipients of 30 surgically transferred embryos were used as controls. Regardless of embryonic stage, the number of embryos transferred was relevant, since NsDU-ETs with 30 embryos resulted in decreased farrowing rates and litter sizes, while NsDU-ETs with 40 embryos achieved comparable fertility and prolificacy to the traditional surgical procedure. These results represent significant progress for the worldwide trade of porcine embryos and for the extensive use of ET in the pig industry.

4.10. Embryo re-vitrification

Although embryo re-vitrification had successfully been attempted in several species, including the human, mice and sheep (Chang, Shapiro, Bernal, Wright, Kort, & Nagy, 2008; Isachenko et al., 2003; Kumasako, Otsu, Utsunomiya, & Araki, 2009; Leoni, Bogliolo, Pintus, Ledda, & Naitana, 2003), until last year nothing was known about it in pigs. We showed that although re-vitrification adversely affected embryo viability, more than 60% of vitrified morulae and blastocysts were effectively re-vitrified and rewarmed (Nohalez et al., 2018). This fact may be of particular importance in the case of having more warmed embryos than needed, for instance when a recipient cannot receive a transfer because of health or ET methodological issues.

4.11. Commercial flight embryo transport in the vapor phase of LN2

An important concern for the commercial flight transport of vitrified embryos is the prohibition of LN2 tanks. Currently, a unique method for the air transport of cryopreserved embryos is to use vapor phase dewars (dry shippers; DSs), which maintain the vapor phase of LN2 at -150 ºC (Bielanski, 2005). The DSs have been used for cryopreserved spermatozoa, oocytes and embryos of several species (AbdelHafez, Xu, Goldberg, & Desai, 2011; Cobo, Romero, Pérez, De Los Santos, Meseguer, & Remohí, 2010; Lim, Shin, Song, Bak, Yoon, & Lee, 2010; Punyatanasakchai, Sophonsritsuk, Weerakiet, Wansumrit, & Chompurat, 2008). However, we could not find any report on the efficacy of DSs for the transport of vitriﬁed porcine embryos. In addition, the particular sensitivity of porcine embryos to temperature variation (Dobrinski, 2002) and certain known problems, such as failure of the DS (Tomlinson & Morroll, 2008) and decreased viability of the transported human embryos (Desmet et al., 2009), encouraged

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us to evaluate the efficacy of the DS dewars to store vitrified porcine embryos for 3 days (Nohalez et al., 2018). Embryo survival and hatching rates and total cell numbers of vitrified-warmed embryos (morulae and blastocysts) did not differ between the DS-stored embryos and the control embryos (storage in a LN2 tank). From these results, it is clear that the use of DS dewars could allow the secure air transport of porcine embryos; however, further studies are necessary to determine the in vivo results after ET of the DS-transported embryos.

5. RESEARCH ON EMBRYO STORAGE IN THE LIQUID STATE

As an alternative to the use of cryopreserved embryos, the preservation of porcine embryos in liquid state has been proposed. We evaluated two storage media and two storage temperatures and showed that more than 95% of the porcine morulae and early blastocysts progressed to the unhatched blastocyst stage, a sanitary prerequisite for ET, after 24 h of liquid storage in a deﬁned medium (TL-PVA) that did not require CO2. Furthermore, despite the embryonic developmental delay observed at the end of storage, these embryos generated high fertility rates and proliﬁcacy after NsDU-ET into recipients (Martinez et al., 2014). Later, we described CO2-free storage conditions to support the survival of morulae (Martinez et al., 2018) and blastocysts (unpublished data) for 72 h and 48 h, respectively, and to allow their development to the unhatched blastocyst stage. These conditions included NCSU-23 medium with 0.4% BSA at 37 ºC (morulae) or 25 ºC (blastocysts). Although these storage conditions maintained the in vitro functionality (hatching capacity after exposure to conventional culture conditions) and the developmental competence after ET (normal fetuses by day 38 of pregnancy) of the embryos, many of the stored blastocysts hatched after 72 h in storage. Thus, we evaluated another strategy for the preservation of embryos in the liquid state for up to 72 h. We investigated whether NCSU-23 medium containing high concentrations of either fetal calf serum (FCS; 50%) or bovine serum albumin (BSA; 4%) at temperatures of 17 ºC or 20 ºC preserved morulae and blastocysts alive and unhatched during for up to 72 h. The results indicated that FCS and BSA were unable to reduce the adverse effects of low temperature (17 ºC) on embryo viability after storage. In contrast, medium containing 4% BSA at 20 ºC arrested the development of both morulae and unhatched blastocysts for 72 h. Furthermore, 80% of the arrested embryos resumed development in conventional culture and progressed to further embryonic stages, including hatching (REF!!! Martinez et al 2019???). Although the results of these studies indicate that the liquid preservation

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of porcine embryos may be an alternative method to cryopreservation, further studies are needed to determine the potential of these embryos to develop to term after NsDU-ET. 6. FROM THE RESEARCH BENCH TO ON-FARM COMMERCIAL APPLICATION

The first commercial non-surgical embryo transfer programs have been recently performed using embryos preserved for 24 h in liquid state or vitrified embryos. Although in both cases the number of transfers has been very limited, some conclusions can already be drawn about the effectiveness of the procedure. In all these programs, the embryos showed an excellent or very good morphological quality immediately before the transfers. Despite this, there was a wide variability among farms in the reproductive performance of the recipients following NsDU-ETs, with farrowing rates and litter sizes ranging from 50% to 85% and from 6.5 to 10.5 piglets, respectively. Likewise, while in some recipient farms there were no problems of pregnancy losses post-ETs in others a percentage of recipients lost pregnancy beyond day 25. Although the variability among farms may be due to numerous factors, including differences in the genotype of the recipients and their capacity to maintain the pregnancy to term, sanitary status, detection of estrus procedures or post-transfer management, the differences in the hygienic condition during and after ETs is likely the most important. While in these ET programs, the recipients received the embryos while kept in their normal usual stalls in the pregnancy room without any special care paid to hygiene, in all our studies the ETs were performed, although field conditions, in specifically dedicated and clean rooms where the recipients were kept al least until day 35 of pregnancy. Because the challenge now is to translate the ET achievements into reliable on-farm practices, further research is required to determine those factors that can play a key role when ET is applied under commercial conditions.

7. FUTURE PERSPECTIVES

Although significant progress in porcine ET technology has been made, many issues remain unresolved. The currently main limiting factor for the worldwide dissemination of this technology is, undoubtedly, the procedure of embryo collection, as in vivo-derived embryos can only be obtained from either slaughtered donors or through surgery. Both alternatives significantly reduce the efficiency of the embryo donor since both procedures limit the number of embryos obtained from each donor. The development of nonsurgical methods would enable the cyclic collection of embryos from the same donor, which

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would substantially increase the donor efficiency in the ET programs. However, due to the anatomical difficulties of the sow's genital tract, it has not been possible to develop such technology until now. Currently, some researchers are attempting to develop nonsurgical embryo collection methods using donors with surgically shunted uterine horns, a technique that was used 30 years ago with little success (Hazeleger, van der Meulen, & van der Lende, 1989; Kobayashi, Hayashi, Ohtubo, Honda, Mizuno, & Hirano, 1989). Further developments in nonsurgical embryo collection procedures are imperatively necessary for the wide and efficient use of ET in the pig industry.

Among the numerous factors affecting fertility and prolificacy, embryonic and fetal survival play an unprecedented role and, therefore, are the main concerns in pig production. Almost 40% of the embryos produced by natural or artificial breeding will not develop to term because of the marked embryonic mortality that occurs mainly during the peri-implantation period in this species (Pope, 1994). The situation is even more prevalent in recipient sows after ET, as approximately 70% of transferred embryos die in the reproductive tract of recipients after ET (Martinez et al., 2014). Physiologically, the embryo(s) is hemi-allogeneic to the mother since it contains 50% of paternal genetic material and, therefore, produces proteins that are unknown to the maternal immune system. Although immune maternal recognition of the hemi-allogeneic embryo occurs, the maternal immune system is locally downregulated, at least in humans, which favors embryo implantation (Tilburgs, Roelen, van der Mast, de Groot-Swings, Kleijburg, Scherjon, & Claas, 2008). The regulatory mechanisms of the maternal immune response to the embryo presence may be more complex and less efficient in the case of ET pregnancies, where the transferred embryos are allogeneic (they contain both paternal and maternal material different from the recipient female), jeopardizing the ability of many of these embryos to develop to term. Therefore, it is necessary to evaluate the impact of the transferred allogeneic embryos on the stimulation of the recipient uterus, which may be an essential key to understanding and eradicating different factors involved in female infertility and/or embryonic death. The study of molecular interactions between the mother and the allogeneic embryo during the peri-implantation period will facilitate the implementation of new strategies to improve embryo-maternal communication, increasing the gestation rate and decreasing embryonic losses after ET.

The improvement of the combined protocols for the synchronization of estrus and superovulation aiming to obtain high percentages of transferable embryos and, therefore, attain maximum efficiency of the gilts and sows used as donors in ET programs, or the

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development of an aseptic and safe vitrification system that allows the simultaneous vitrification of a sufficient number of embryos in a single device to carry out a single on-farm transfer are important factors that have to be solved for the application of this technology under field conditions.

8. CONCLUSIONS

Non-surgical ET is a potent technology widely demanded by the pig industry. Many questions affecting the NsDU-ET technique and vitrification and liquid state embryo preservation procedures have been answered in recent years. Although still in its infancy, the recent advances achieved have enabled the expansion of ET technology to levels unimaginable a short time ago. Today, the combined use of NsDU-ET and preserved embryos allows the application of ET by the pig industry, as evidenced by the growing number of ET programs established by several genetics companies.

ACKNOWLEDGEMENTS

The authors are grateful to Prof. Billy N. Day for his constant help and invaluable advice over the years. We also thank the staff of Agropor SA (Murcia, Spain), Porcisan (Murcia, Spain) and Selección Batalle SA (Gerona, Spain), for the excellent management of the donors and recipients in the studies reported here. Some of the studies presented in this review were supported by MICINN-FEDER (Madrid, Spain; AGL2004–07546 and AGL2009–12091), MINECO-FEDER (Madrid, Spain; AGL2012-38621 and AGL2015-69735-R), CDTI (Madrid, Spain; IDI-20140140 and IDI-20140142), the Fundación Séneca (Murcia, Spain; GERM 04543/07 and 19892/GERM/15) and the Research Council FORMAS, Stockholm, Sweden (Project 2017-00946). CAM is supported by a postdoctoral grant from the Fundación Séneca (Murcia, Spain) and JMC is supported by a predoctoral grant from the MINECO (Madrid, Spain; BES-2016-077869).

CONFLICTS OF INTEREST

The authors have no conflict of interest to declare. AUTHOR CONTRIBUTIONS

All authors contributed to writing the paper and approved the final version of the manuscript.

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