EMBRYO TRANSFER IN SWINE – AN INDISPENSABLE KEY FOR THE APPLICATION OF REPRODUCTIVE TECHNIQUES
DOI:10.30825/5.EJPAU.78.2018.21.3

Klaus-Peter Brüssow¹, Jozsef Rátky², Paweł Antosik¹, Bartosz Kempisty³, Jędrzej Maria Jaśkowski^1
1 Centre of Veterinary Sciences, Nicolaus Copernicus University, Toruń, Poland
² Department and Clinic of Reproduction, University of Veterinary Medicine, Budapest, Hungary
³ Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland

ABSTRACT

This paper gives an overview on present stage and application of embryo transfer in swine (ETS). The work steps of selection and stimulation of donor pigs, embryo collection techniques, embryo handling, selection and stimulation of recipient pigs as well as embryo transfer methods are shortly described. Further, factors affecting the application of ETS today and fields of current and future use in pig production and research are named. Hopeful benefits of ETS will be the international exchange of porcine embryos of value breeds instead of live animals, the production of piglets for xenotransplantation and probably the conservation of endangered pig breeds. However, the prospective application of ETS will not become the same significance compared to embryo transfer in other livestock species.

Key words: embryo transfer, pig, practical application.

INTRODUCTION

The historical start point of embryo transfer in swine (ETS) was nearly 70 years ago in 1949. Only some years after the Second World War Kvasnicki performed the first successful recovery and transfer of porcine embryos at the Research Institute in Poltava, Ukraine. The embryo transfer lead to the birth of four piglets after the application of nine embryos into the oviduct of a recipient sow and the results were reported in 1950 and 1951 [23, 24]. Interestingly, this method of interbreed embryo transfer should be used to support the ideological concept of Lysenko that heredity is influenced by environment, but not of genes. In any case, the development of such sophisticated technique was pioneering since his general method of surgical recovery and transfer of embryos is actually used today.

One decade later, in 1960, Pomeroy [38] continued experiments to recover and transfer porcine embryos. At the early and late 1960-ies the collection and transfer methods were improved further [15, 18, 43] and the first attempt of nonsurgical ETS was done by Polge & Day [37]. During the period of 1970–1980 in vitro techniques were worked out and international exchange of porcine embryos were practiced first [34, 51].

Generally, ETS can be defined as a complex of measures which includes the following work steps: (I) selection and stimulation of donor sows, (II) recovery of embryos, (III) embryo manipulation, i.e.  morphological assessment, intermediate storage, cultivation and transport, and (IV) the transfer of recovered embryos into recipients.

WORK STEPS OF PORCINE EMBRYO TRANSFER (ETS)

Selection and stimulation of donor sows
Prepuberal gilts, puberal cycling gilts and adult sows can be successfully used as donors for ET. Often prepuberal gilts are preferable selected because of higher (super-)ovulatory response and easy handling. Cycling gilts and especially sows at the end of their reproductive life are preferred for the propagation of value breeds. 

Normally, about 15 to 20 oocytes are ovulated in the pig. However, for embryo transfer it is desirable to recover a higher number of embryos. To achieve a higher as normally number of ovulated oocytes, a so called "superovulation" (SO) should be stimulated with exogenous gonadotropins. In prepuberal gilts it is induced by application of 1,000–1,500 IU of equine chorionic gonadotropin (eCG) followed by an injection of 500 IU human chorionic gonadotropin (hCG) 72 h after eCG to synchronize ovulation. SO in cycling gilts is commonly induced after estrus synchronization feeding altrenogest (Regumate®) for 15–18 days and by subsequent administration of 1,500 IU eCG and of 500 IU hCG 78 h later. Multiparous sows are injected with 1,000–1,250 IU eCG 24 h after weaning and 500 IU hCG 58 h after eCG. Gilts and sows are fixed-time inseminated 24 h and 38 h after hCG injection. However, there can be a considerable and non-predictable variation in the ovarian response to SO stimulation in prepuberal and cycling gilts, and in sows (Tab. 1). The superovulatory response depends also on breeds. If 20–25 transferable embryos can be flushed from white breeds, their number is significant lower e.g. in fatty pig breeds as Mangalica [41] or Iberico.

Embryo collection techniques
Embryos can be flushed from the genital tract of sows after slaughter or they can be recovered surgically or by means of minimal invasive endoscopy from the oviduct and/or the uterine horns, respectively. Embryo collection after slaughter has the disadvantage of using donor sows only once. Usually, embryo recovery is accomplished surgically under general anesthesia, with the genital tract presented through a mid-ventral incision involving retrograde flushing of the oviducts and/or uterine horns. The disadvantage of invasive surgical procedures to recover embryos can be diminished by minimal-invasive endoscopic flushing technique which was developed by Brüssow & Rátky [10] and Besenfelder et al. [3] and allows to some extent repeated embryo collection. So far, due to the anatomy of the porcine genital tract, no successful nonsurgical embryo collection has been reported in pigs.

Embryos can be recovered at different stages of development. Embryos at the one-cell to the 4-cell stage are commonly flushed from the oviduct and those from the 8-cell stage up to the hatched blastocyst from the uterine horn, respectively.
 
Embryo handling and manipulation
After flushing, embryos are collected from the flushing medium, put into an appropriate culture medium and morphologically examined in vitro under a stereo microscope. They are evaluated according to their morphological characteristics and classified into transferable or non-transferable embryos [4]. Following morphological evaluation embryos are stored for a short time, preferably less than 4 h, in a culture medium until they are transferred into recipients.

However at this time, i.e. immediately after recovering of embryos, all in vitro techniques related to embryo transfer can be adapted, as in vitro fertilization and cultivation, cryopreservation/vitrification, in vitro production of identical multiplets (bisection, proliferation of single blastomers), nuclear transfer (cloning), sexing and gene transfer. These techniques need, however, special handling and culture procedures.

Recipients
Selection of recipient gilts or sows has a major impact on embryo transfer results. Although prepuberal gilts can be used as recipient, cycling gilts and sows are privileged due to their established endocrine and uterine development. Recipients are synchronized to donor sows and treated in the same manner except for lower eCG application (750–850 IU) to avoid superovulation. There are also reports on asynchronous ET where the day of recipient's estrus cycle differs one day compared to the donor [39, 49]. However, the "mild" eCG stimulation of recipients create such hormonal milieu that the recipient is in accordance to that of the donor [9]. Therefore, recipients should not be asynchronous compared to the donor. Recipients do not underwent insemination. However, Martynenko and her group from Poltava [29, 30] performed an interesting experiment where recipients were inseminated post-ovulatory, i.e. 55–60 h after the onset of heat or about 15 h after ovulation, respectively. Embryos were transferred on day 7 using nonsurgical transcervical method. The pregnancy rate was 83% vs. 50% compared to control and 76% vs. 27% of the litter were from transplanted embryos. From this experiment is evident that support of the recipient's uterine environment for implantation can enhance ET outcome.

The breed of the recipients can influence the embryo transfer results. Meishan pigs could be used as recipients due to their higher placental efficiency [50]. The transfer of Mangalica embryos into Landrace recipients also doubled the common litter size of Mangalica [41].

Embryo transfer
Usually embryos are transferred surgically into recipients. This methods is available for several decades. The embryos, depended on the stage of development, are transferred either into the oviduct (one- to four-cell embryos) or into the tip of the uterine horn (8-cell embryo to blastocyst). Embryos are transferred in a small amount of culture medium by means of transfer pipettes or catheters. Minimal-invasive endoscopic transfer procedures were also developed and applied to minimize operation stress [3, 40, 45].

Surgical embryo transfer is one reason which minimize the application of this sophisticated method. Therefore, several attempts have been done to develop a nonsurgical transfer procedure [17, 19, 25, 52]. However, only after the development of a special catheter [13] there is a real chance to make the transcervical ET practicable. Nonsurgical deep-uterine embryo transfer has been successfully developed by the Spanish research group of the University of Murcia [27, 28]. Despite several limitations (i.e. use of pluriparous sows mainly, deposition into the uterine body, stage of embryo development) progress in nonsurgical embryo application is pointing the way for expanded application of ETS.

The transfer of 16–20 embryos seems to be optimal to achieve normal pregnancy rates [8]. On average, the pregnancy rate is about 70 and 56% after surgical and nonsurgical procedure (Tab. 2) and the litter size is about 7 piglets. However, a wide range of pregnancy from 17% with 2.4 piglets to 100% and litter size of 10.8 piglets is reported. Under optimal conditions, using high quality embryos, pregnancy rates and litter sizes can be obtained similar to results after artificial insemination.

STATE OF APPLICATION OF ETS

Factors affecting the application of ETS
Several factors may influence the success of ETS. Good quality embryos are one prerequisite for ET success, however, to state what a "good" embryo is, is still difficult. Currently, the morphological evaluation of embryos under the stereo-microscope is the most adapted method, but subjective. Several other quality assessment techniques have been applied or proposed such as cytogenetic analysis, application of microarray technology and analysis of protein expression and metabolomics. Although biochemical and molecular indicators are probably more precise and objective than the morphological criteria currently in use, all or most of the molecular methods are invasive. They result in the destruction or at least in destabilization of the cytoplasmic and biochemical ultrastructure of the gametes. At present, no single and reliable practicable assessment method compared to the morphological assessment is on hand. A feasible non-invasive method is to assess the quality of porcine embryos spectrophotometrically using lab-on-chip systems [2, 46, 48]. In a pilot study [Brüssow et al., unpublished data] porcine embryos at the morula stage (n = 161) were surgically recovered by flushing from the uterine horns of 18 gilts on day 5 of gestation. Embryos were analyzed by lab-on-chip measurement and were classified into two classes according to their spectral characteristics, i.e. absorbance rate (AR). Embryos with absorbance ratio higher than 1.1 were classified as Class 1, whereas those with lower AR were marked as Class 2. It is suggested that embryos with increased absorbance were characterized by more homogenous cytoplasm and proper blastomere distribution and therefore distinguished with increased developmental potential. In the present study, 77 embryos (48%) were classified as Class 1 and 84 (52%) as Class 2. Embryos of both classes were separately transferred to recipients (n = 4 per class). None of Class 2, but two recipients of Class 1 became pregnant and gave offspring (3 and 6 piglets born alive). Future research in this field could promote the non-invasive assessment of porcine embryos and validate morphological evaluation.

Superovulation with exogenous gonadotropins can effect embryo quality. Due to the "additional" source of gonadotropins a cohort of follicles will be recruited which normally would underwent atresia [31]. This increases the option that not fully developed oocytes are ovulated and further embryo development could be affected. A higher percentage of degenerated embryos, a lower number of blastocysts and a decreased pregnancy rate has been observed [20, 53].

The surgical and partly endoscopic embryo flushing techniques restrict the use of repeated embryo recovery in the same donor. James & Reeser [21] recovered surgically embryos 63 times from 13 purebred donor sows for an average of 4.8 times per donor. Recoveries were completed at three or six week intervals and an average of 15.6 transferable embryos were collected per surgery. Based on these limited observations it would appear that multiple surgical embryo recoveries may be completed on a specific donor at three and/or six week intervals without impairing future production. However, other loadable data has not published, yet.

Furthermore, a relatively high number of embryos has to be transferred (16–20 fresh or more than 40 vitrified embryos) to achieve "normal" litter size. This limits the effectiveness of the embryo transfer procedure, because of the number of embryos which can be recovered and that which should be transferred, the donor-recipient ratio is only 1:0.5–1:2. Finally, only 20% of the ovulated oocytes result finally in a piglet.

Additionally, ETS is an expensive process. Medical drugs, media and equipment influence the costs, too. Furthermore, trained specialists for the whole process of ETS are fundamental.

Current status and future application of ETS
Embryo transfer has a rather different importance in farm animals. Compared to cattle and sheep, embryo transfer in swine for commercial applications has been used only to a limited extend. This is primarily due to the high fecundity in pigs, the economic situation in pig production, the mainly surgical embryo collection and transfer techniques, the low donor-recipient ratio and the limitation to cryoconserve embryos and oocytes, i.e. to store embryos for a long time.

Although, ETS has been applied in several fields of swine production, there is only a little practical application, yet. ETS has been used to introduce new genetic material into closed herds and for extracting healthy stock from diseased source [12]. It has been used, but rather to a limited extend, for the export of embryos [22, 34, 51], for the exploitation of superior sows near end of useful reproductive life and for the preservation of pig breeds [5]. However, only the propagation of the endangered Mangalica swine breed was of practical success [40].

Embryo transfer is the general prerequisite for the practical application of reproductive in vitro techniques. There are manifold reports regarding in vitro production of embryos [1], the generation of multiplets (including clones) [8, 36, 42], the generation of sexed embryos/piglets and the production of transgenic pigs for gene-farming and [reviewed 35]. All these applications need embryo transfer techniques to generate offspring.

Successful long-time conservation of porcine embryos and oocytes (cryoconservation/ vitrification) [14, 32, 33, 47] would considerably improve the application of ETS. Despite of promising efforts these methods are still of low efficiency.

Successful in vitro production of porcine embryos could also benefit the application of ETS. The in vitro culture (IVC) system can influence embryo survival and embryos show restricted development after long-time IVC, yet. Problems include nuclear and cytoplasmic maturation, poor male pronuclear formation and polyspermy [1, 16, 26]. However, despite the problems, successful in vitro production of porcine embryos has improved and the transfer of embryos to recipients has resulted in acceptable pregnancy rates and litter sizes. In vitro production and long-time conservation of porcine oocytes and embryos are an essential tool for preservation of endangered pig breed. The establishment of gene banks needs these procedures and respective embryo transfer. The birth of piglets after the transfer of in vitro derived vitrified zygotes is promising for this reason [44].

SUMMARY

Embryo transfer in swine (ETS) is a sophisticated method including work steps of embryo recovery, embryo handling and transfer to recipients. Progress in several steps like optimized in vitro culture systems, cryopreservation, minimal-invasive recovery and non-surgical embryo application will promote further application of this biotechnique. Benefits of ETS will be the international exchange of porcine embryos of value breeds instead of live animals, the production of piglets for xenotransplantation and probably the conservation of endangered pig breeds. Nevertheless, the prospective application of ETS will not become the same significance compared to embryo transfer in other livestock species.

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Accepted for print: 20.09.2018

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Klaus-Peter Brüssow
Centre of Veterinary Sciences, Nicolaus Copernicus University, Toruń, Poland
87-100 Toruń
Poland
email: prof.bruessow@gmail.com

Jozsef Rátky
Department and Clinic of Reproduction, University of Veterinary Medicine, Budapest, Hungary
1400 Budapest
Hungary
email: jozsef.ratky@gmail.com

Paweł Antosik
Centre of Veterinary Sciences, Nicolaus Copernicus University, Toruń, Poland
87-100 Toruń
Poland
email: pantosik@umk.pl

Bartosz Kempisty
Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland
60-781 Poznań
Poland
email: bkempisty@ump.edu.pl

Jędrzej Maria Jaśkowski
Centre of Veterinary Sciences, Nicolaus Copernicus University, Toruń, Poland
87-100 Toruń
Poland
email: jedrzej.jaskowski@gmail.com

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