Current status of nonsurgical embryo transfer in swine

Introduction

Embryo transfer (ET) is a valuable tool in pig production. It facilitates the movement of desirable genetics with a marginal possibility of disease spread. Compared to transporting live animals, ET offers significant advantages, including reduced transportation costs and the avoidance of negative impacts on animal welfare. ET also plays a crucial role in the use of new genetic tools for genome editing (GE) in pigs. This technology allows targeted genetic modifications to alter the phenotype of pigs, which serves two key purposes. First, the creation of pigs with improved production traits relevant to agriculture, such as enhanced meat quality (Zou et al. 2018) and increased disease resistance (Aigner et al. 2010; Whitworth et al. 2014). Second, it facilitates the development of valuable biomedical pig models to advance disease research, regenerative medicine, and organ transplantation in humans (Hryhorowicz et al. 2020).

Despite these important applications and although the first litters from ET in pigs were obtained almost 75 years ago (Kvasnickii 2001), the commercial use of ET has been limited because of the need for surgical procedures for embryo collection and transfer. Additionally, the challenges associated with cryopreservation further restricted its commercial development. However, the situation has changed in the last two decades, primarily due to the development of a nonsurgical deep uterine (NsDU) ET method and vitrification procedures as an excellent alternative to the conventional slow freezing cryopreservation method. These advancements have opened the door for a more practical and efficient use of ET in the pig industry.

This review focuses on the practical applications of porcine NsDU-ET technology. We explore the fundamentals of this technology and its current achievements. We also discuss the key factors impacting the success rates of NsDU-ET and the experimental results achieved under field conditions. Additionally, we present the first findings from the implementation of this technology in commercial pig production programs and highlight the observed differences from the experimental results. Finally, we address several future indications for the commercial application of this technology. For the purposes of this review, we focus mainly on in vivo-derived embryos because they are currently the only type of embryo with potential for use in pig production.

A historical perspective on nonsurgical embryo transfer in pigs

Nonsurgical embryo transfer deep into the uterine horn

A key limitation of these nonsurgical ET procedures is that they only deposit embryos within the UB, which is not the natural location for transferred embryos. Surgical ET studies have shown significantly lower pregnancy rates (12%) when expanded blastocysts are placed in the UB compared to placement in the middle (88%) or the tip (81%) of the uterine horn (Wallenhorst and Holtz 1999). These findings suggest that the UB is not the optimal location for embryo deposition. To address this and other limitations associated with UB nonsurgical ET methods, we developed a research program in 1998. Our goal was to develop a novel nonsurgical method for transferring porcine morulae and blastocysts deeper into the uterine horns of non-sedated gilts and sows.

The greatest challenge in placing a catheter deep into a pig’s uterus without surgery is the cervix. Its firmness changes throughout the estrous cycle, and it is easier to pass the catheter through during metestrus. Additionally, the uterus itself is long and curved, which adds another obstacle. In 1999, we developed a special endoscope technique for NsDU insemination in non-sedated sows (Martinez et al. 2001) that overcomes these challenges. The endoscope was inserted via an artificial insemination (AI) spirette, crossed the cervical canal, and reached deep into the uterine horn. This procedure offered a safe and efficient approach and enabled rapid access to a uterine horn in a high proportion of sows. Despite its effectiveness, the cost of the endoscope and its delicate nature encouraged the development of a more practical solution: a flexible catheter inspired by the endoscope’s design (Fig. 1). This catheter was less expensive, easier to use, and worked in gilts and sows during a wider estrous cycle window, including days 4–6 of the estrous cycle (Martinez et al. 2004). The design was further optimized for ET. The tip was enlarged to prevent accidental perforation of the cervical and uterine walls during embryo placement. Additionally, high-quality material was used for the inner tubing to safeguard the embryos (Cuello et al. 2005). In 2008, Japanese researchers introduced a new design for an NsDU-ET catheter. Despite its innovative approach, the prototype has seen scarce use (Nakazawa et al. 2008; Yoshioka et al. 2012).

Fig. 1.

(a) Catheter designed for nonsurgical deep uterine embryo transfer (NsDU-ET) in gilts and sows during days 4–6 of the estrous cycle. The ET catheter is equipped with a soft, enlarged tip (ETCT) to minimize the risk of cervical or uterine wall injury. (b, c) Reproductive tract of a sow obtained from a slaughterhouse, longitudinally opened to reveal the uterine body (UB), cervical folds (CF), cervix entry (EC), and vagina. (d) Position of the artificial insemination catheter tip (AICT) at the cervix entry. (e) Detailed view of the ET catheter passing through the cervical folds and its correct placement within a uterine horn (arrowhead).

Accuracy of NsDU catheter placement

Successful NsDU-ET relies on the efficient insertion of the catheter and ensuring proper placement within the pig’s uterus. This catheter effectively passes through the cervical canal and reaches deep into a uterine horn in a high proportion of gilts and sows. Notably, it achieves this feat without causing any cervical or uterine perforations (Martinez et al. 2004).

One important factor in nonsurgical ET programs is the type of recipient. The number of previous estrous cycles in gilts is crucial for successful insertion, with higher success rates in gilts with more than two previous estrous cycles (Cuello et al. 2005). Weaning sows, especially sows with a greater number of previous parities, typically exhibit easier passage of the catheter through the cervix and into the uterus. Catheter insertion presented minimal difficulties in a high proportion of both gilts and sows. Notably, the procedure typically took less than 3 min, with no observable difference in time between gilts and sows (Martinez et al. 2013; Martinez et al. 2016a).

Correct catheter placement inside the uterine horn is essential for successful NsDU-ET in pigs. Previous studies demonstrated that it was possible to correctly insert a flexible catheter and reach the second and third quarters of the uterine horn in 90% of recipients (Martinez et al. 2004; Cuello et al. 2005). Furthermore, the ability to accurately identify potential catheter misplacement minimizes the number of wasted embryos and improves pregnancy rates.

Several other key factors influence the effectiveness and potential of the NsDU procedure, including operator training requirements and animal welfare considerations (Martinez et al. 2013). While the NsDU-ET procedure appears simple, operator training is needed. This training promotes the development of essential skills and ultimately leads to improved farrowing rates and faster insertion times. On the other hand, more than 90% of females exhibited good or moderate behavior throughout the procedure, with gilts showing slightly less tolerance than sows. This result suggests minimal discomfort and good overall acceptance of the NsDU-ET process. Furthermore, the absence of bleeding or trauma during and after insertion indicates a low risk of injury to the reproductive tract.

Factors influencing the efficiency of ET

Experimental results obtained under field conditions using nonsurgical deep uterine embryo transfer

In addition to the previously discussed factors, several other factors can influence NsDU-ET outcomes. Among these factors, the state of the embryos for transfer is notable, whether fresh (recently collected), stored in a liquid state for up to 24 h, or vitrified.

While information on the surgical transfer of fresh embryos is scarce, existing studies report that farrowing rates and litter sizes are ~60–80% with 7–8 piglets born, respectively (Cameron et al. 1989; Galvin et al. 1994). Compared to surgical procedures, the nonsurgical transfer of embryos directly into the UB results in lower success rates. Farrowing rates using this method typically range from 33–40%, with an average litter size of 6.7–7.4 piglets (Hazeleger and Kemp 1994; Ducro-Steverink et al. 2004). Similarly, early attempts using the NsDU-ET technique achieved a 70% farrowing rate and an average litter size of 7 piglets (Martinez et al. 2004). Further improvements in the NsDU-ET procedure, including aseptic protocols, synchronized estrous cycles between donors and recipients, and proper operator training (as detailed in Martinez et al. 2013), significantly improved its effectiveness. Currently, NsDU-ET achieves farrowing rates of ~80% and a litter size of ~9.5 piglets per litter, which makes it the most successful nonsurgical ET method for pigs (Angel et al. 2014a, 2014b; Martinez et al. 2014).

The use of embryos stored for short durations (24–30 h) can impact the success of ET procedures. Surgical ET with embryos transported internationally (in vitro culture for 24–30 h) resulted in acceptable farrowing rates (50–60%) and litter sizes (5–8 piglets) (James et al. 1980; Niemann et al. 1989). However, more recently, NsDU-ET using fresh morulae cultured for 24 h achieved significantly better outcomes (90% farrowing rates with 9.0 piglets per litter) (Martinez et al. 2014). Several factors may contribute to the observed variations in success rates, including the number of embryos transferred, differences in culture media used, the developmental stage of the embryos at transfer, recipient characteristics, and the specific facilities and practices used on individual farms, particularly embryo handling and transfer procedures.

Recent advancements in vitrification protocols have significantly improved the post-warming survival rates of untreated morulae and blastocysts. Promising results have been reported in farrowing rates and litter sizes following the surgical transfer of vitrified embryos, but the number of studies is limited due to the need for large numbers of embryos at specific times (Martinez et al. 2013). The use of NsDU-ET and embryo vitrification is important for the broader application of porcine ET. Preliminary experiments in our laboratory combining these technologies yielded acceptable farrowing rates (40–50%) and litter sizes (5–10 piglets) (Cuello et al. 2005; Gomis et al. 2012), albeit with a low number of recipients. Later, we performed a separate study comparing surgical and NsDU-ET procedures for vitrified embryos under farm conditions in 109 sows. Our focus was to evaluate how the number of nonsurgically transferred vitrified embryos impacted the reproductive performance of recipients (Martinez et al. 2015). NsDU-ET with 30 vitrified–warmed embryos resulted in significantly lower farrowing rates and litter sizes compared to surgical and NsDU-ET with 30 and 40 vitrified embryos, respectively. We concluded that NsDU-ET achieved similar success rates to surgical ET when the appropriate number of embryos per transfer was used. The greater number of vitrified embryos needed for NsDU-ET than for surgical ET is likely due to differences in embryo deposition sites within the uterus. In surgical ET, embryos are deposited into the tip of a uterine horn (Polge 1982), whereas they are placed deeper in the uterine horn in NsDU-ET. Physiologically, pig embryos remain near the horn tip until days 6 or 7. Therefore, transferring morulae and blastocysts to the horn tip may be more beneficial. It is likely that the uterine environment in more caudal locations is less favorable for embryo development, which reduces survival rates.

From research to commercial farming applications

Several commercial programs for NsDU-ET have been implemented using embryos preserved for 24 h in a liquid state or using vitrified embryos. Despite the limited number of transfers, some observations can be made about the effectiveness of the procedure. In these programs, the embryos exhibited excellent or very good morphological quality just before transfer. However, compared to the results achieved under experimental conditions, reproductive performance varied significantly between farms. The farrowing rates ranged from 30.0 to 83.3%, and the litter sizes varied from 4.2 to 11.2 piglets. Notably, some farms experienced no pregnancy losses after transfers, whereas others reported losses beyond day 25 (Table 1). Additionally, piglet production efficiency exhibited significant variability. When the ratio of live-born piglets to the total number of embryos transferred was calculated, the efficiency ranged from 5.6 to 31.7%. When calculated using only farrowed recipients, the efficiency ranged from 13.5 to 38.5%. These results show much greater variation than the data obtained under experimental conditions (18.7 to 28.8% and 26.5 to 32.0%, respectively) (Fig. 2).

Fig. 2.

Box-and-whisker plots with individual data points overlaid illustrate the efficiency of piglet production following nonsurgical deep uterine embryo transfer under experimental conditions (published literature: Martinez et al. 2016a, 2019) and commercial situations (EA Martinez, J Sanchez-Osorio, I Parrilla, C Cuello, MA Gil, unpubl. data). Piglet production efficiency is defined as the ratio of live-born piglets to the total number of transferred embryos, considering only farrowed or transferred recipients. The boxes represent the interquartile range (25th to 75th percentiles), the solid line indicates the median, the dashed line marks the mean, and the whiskers show the maximum and minimum values.

This variability can arise from a complex interplay of factors, such as the recipient’s genetics, sanitary standards, methods of detecting estrus, and management after transfer. However, the recipient’s uterine environment is likely one of the most influential factors. Studies have established a clear link between hygiene conditions, the estrous cycle stage, and the ability of the uterus to resist infections because gilts inoculated with bacteria during estrus exhibited much higher resistance to endometritis than gilts inoculated during diestrus (de Winter et al. 1994). These findings suggest that elevated estrogen levels during estrus offer a protective benefit. In contrast, the period following estrus, which is dominated by progesterone, increases the likelihood of infection (de Winter et al. 1992; Wulster-Radcliffe et al. 2003). Therefore, recipients are particularly vulnerable to uterine infections during and shortly after NsDU-ET (approximately days 5–6 of the cycle), which makes rigorous aseptic measures essential to prevent infections. The absence of these precautions may account for the pregnancy losses observed in some commercial farms following NsDU-ET. In fact, many sows that were initially confirmed to be pregnant via ultrasound but subsequently lost their pregnancies experienced vaginal discharge or symptoms of endometritis. In contrast to commercial NsDU-ET programs that housed recipients in their standard stalls without specific hygiene protocols, our experimental studies, although performed under field conditions, involved performing ETs in separate, meticulously maintained, and clean rooms where the recipients were kept until day 28 of pregnancy. This approach effectively prevented uterine infections and pregnancy losses (Fig. 3). Therefore, ensuring good health through biosecurity measures, proper facilities, effective management practices, accurate estrus detection, and stringent hygiene protocols is crucial for achieving optimal fertility and prolificacy after NsDU-ET. However, further research is necessary to identify and address the key factors that affect the commercial viability of this technology.

Fig. 3.

Box-and-whisker plots with individual data points overlaid illustrate the pregnancy and farrow rates, as well as litter sizes, following nonsurgical deep uterine embryo transfer under experimental (published literature: Martinez et al. 2016a, 2019) and commercial (EA Martinez, J Sanchez-Osorio, I Parrilla, C Cuello, MA Gil, unpubl. data) conditions. The boxes represent the interquartile range (25th to 75th percentiles), the solid line indicates the median, the dashed line marks the mean, and the whiskers show the maximum and minimum values.

Upcoming directions

Despite significant advancements in porcine ET technology, several challenges remain. The primary obstacle to widespread adoption is the embryo collection method because in vivo-derived embryos require slaughtering the donors or performing surgery, which limit donor efficiency. The development of nonsurgical methods for embryo collection would allow repeated use of the same donor, which would greatly enhance efficiency. However, because of the complex anatomy of the sow’s reproductive tract, nonsurgical collection has not been successfully implemented. Researchers are currently revisiting methods used 30 years ago with limited success, including techniques involving surgically shunted uterine horns, but progress is still needed.

Another alternative to surgical embryo collection in NsDU-ET programs is the use of in vitro-produced (IVP) embryos. Many immature oocytes can be retrieved from slaughtered animals then matured and fertilized under controlled conditions. While cost-effective and time-saving, current IVP systems in pigs face challenges because of suboptimal culture conditions that lead to a variety of issues, such as polyspermy and poor embryo development (Gil et al. 2010). A successful and efficient IVP program, such as programs in cattle (Dyck et al. 2014), could enable the global exchange of valuable genetic material and accelerate breeding programs. However, a limitation of current IVP methods is the use of immature oocytes collected from slaughtered prepubertal gilts. These oocytes have low genetic value, and their use results in minimal impact on genetic progress. A promising approach to overcome that limitation is the use of Ovum Pick-Up (OPU), which is a well-established technology for retrieving oocytes from live animals with high genetic value that is commonly used in cows and mares. Although OPU in pigs is less documented due to anatomical challenges (Okuyama et al. 2017), advancements, such as sedation and epidural anesthesia during transvaginal OPU (Yoshioka et al. 2020; Oltedal et al. 2024a), address animal welfare concerns and make the procedure easier to perform. These refinements yield an average of over nine oocytes per session, with no reported difference in subsequent fertility or prolificacy compared to control sows (Oltedal et al. 2024b). These findings suggest the viability of OPU for oocyte retrieval in live sows and pave the way for the use of genetically superior oocytes in IVP protocols. This use could significantly accelerate genetic improvement programs in swine breeding through NsDU-ET technology.

Another major challenge facing current ET programs is post-transfer embryonic loss. The reproductive efficiency of sows is an economic factor that directly impacts the productivity of the swine industry. Numerous physiological and environmental factors affect fertility and prolificacy. Among these factors, embryonic and fetal survival play unprecedented roles (Spötter and Distl 2006) because prenatal mortality is the main determinant of litter size (Pope 1994; Spötter and Distl 2006), and therefore, it is one of the main concerns of pig companies. Under physiological conditions, ~20–30% of pig embryos produced after fertilization die before, during, or shortly after implantation, and another 10–15% die at mid-pregnancy (Vallet et al. 2011). Therefore, almost half of the embryos derived from natural breeding or AI do not progress to full term (Pope 1994). These data are even more pronounced after ET. The fertility and prolificacy results available in the literature after surgical or nonsurgical ET range between 50% and 80% with 6 to 10 piglets born per litter, respectively (Martinez et al. 2016a, Martinez et al. 2019). That is, fertility values are 10 to 30 points lower and litter sizes of 4 to 6 piglets lower than the values obtained by AI (Roca et al. 2016). Furthermore, the efficiency of piglet production after ET is ~30% (Angel et al. 2014b; Martinez et al. 2014), which means that 70% of the embryos used in each ET die in the uterus of the recipient. Several studies attempted to understand some of the causes of embryonic losses, including nutritional, genetic, environmental, and placental factors (Ford 1997; Wilson et al. 1998; Miles et al. 2008; Foxcroft et al. 2009). Other factors, such as immunity (Croy et al. 2009) and inadequate maternal–embryo communication (Wolf et al. 2003), also play fundamental roles in embryonic loss and pregnancy failure. Embryos produced under natural conditions (including AI) are hemiallogeneic to the mother because they contain genetic material from the father that generates unfamiliar proteins for the mother’s immune system. However, embryos are usually not rejected by the mother during pregnancy without affecting the preservation of effective immunity against pathogens because the immune system of the mother is controlled (or regulated locally) to favor embryo implantation (Parrilla et al. 2022). The regulation of the maternal immune response to the embryo may be more complicated and inefficient in pregnancies produced by ET because the embryos in these cases are allogeneic (they contain paternal and maternal genetic material unknown to the recipient female), which would reduce the developmental possibilities of many embryos. This supposition is supported by previous results obtained in our laboratory on cytokine levels and gene expression. We showed that after the transfer of allogeneic embryos, pig pregnancies show clear dysregulation of pro- and anti-inflammatory cytokine levels (Martinez et al. 2020), a lack of sufficient endometrial levels of leukemia inhibitory factor (Cambra et al. 2020), and severe downregulation of interferon-stimulated genes in the peri-implantation endometrium (Martinez et al. 2022), all of which could be associated with impaired maternal immune tolerance and could partially explain the high embryonic mortality associated with ET programs. Understanding the molecular omics interactions between mothers and embryos during the peri-implantation period could lead to strategies that improve embryo–maternal communication, increase gestation rates and reduce embryonic loss.

Another challenge is the cryopreservation procedure. Vitrification is currently the only efficient method for cryopreserving pig embryos (Martinez et al. 2019; Xingzhu et al. 2021). The most common technique is superfine open pulled straw (SOPS), which yields good embryo survival rates in vitro (Cuello et al. 2007, 2008; Sanchez-Osorio et al. 2008) and successful pregnancies after surgical and nonsurgical ET (Cuello et al. 2005, 2016; Martinez et al. 2015). However, SOPS has a significant practical limitation: only four to six embryos can be vitrified at a time per device without compromising the necessary volume for optimal preservation. This limitation is impractical for ET, which typically requires 20–40 embryos per recipient (Cuello et al. 2005, 2016; Martinez et al. 2015). To address this issue, the open Cryotop system has been introduced, which allows for the simultaneous vitrification of up to 20 embryos (Gonzalez-Plaza et al. 2022). This technique provides a significant procedural advantage by simplifying the vitrification, warming, and transfer process in species such as pigs. This method yields similar embryo survival rates and quality markers to fresh embryos and even outperforms the SOPS method (Gonzalez-Plaza et al. 2022). Additionally, it minimally affects embryo gene expression (Gonzalez-Plaza et al. 2023). However, further research is needed to understand the impact of Cryotop vitrification on embryo development after ET.