Abstract
Abstract
The low efficiency in obtaining piglets after production of cloned embryos was challenged in two steps—first by performing in vitro culture for 5–6 days after cloning to obtain later-stage embryos for more precise selection for transfer, and second by reducing the number of embryos transferred per recipient sow. The data set consisted of combined results from a 4-year period where cloning was performed to produce piglets that were transgenic for important human diseases. For this, different transgenes and cell types were used, and the cloning work was performed by several persons using oocytes from different pig breeds, but following a standardized and optimized protocol. Results showed that in vitro culture is possible with a relatively stable rate of transferable embryos around 41% and a pregnancy rate around 90%. Furthermore, a reduction from around 80 embryos to 40 embryos transferred per recipient was possible without changing the efficiency of around 14% (piglets born out of embryos transferred). It was concluded that this approach can increase the efficiency in obtaining piglets by means of in vitro culture and selection of high-quality embryos with subsequent transfer into more recipients. Such changes can also reduce the need for personnel, time, and material when working with this technology.
Introduction
S
After the laboratory work, cloned embryos are transferred to recipients, and different approaches have been used to increase efficiencies in this step (e.g., recipient parity and transfer method; Schmidt et al., 2010). Furthermore, the number of cloned embryos transferred to each recipient has been an issue, and a linear relation between number of embryos transferred and piglets born has been demonstrated in some studies (Schmidt et al., 2010), but not in others (Kurome et al., 2013). However, the overall purpose at transfer is to ascertain a pregnancy that will result in the birth of piglets. To obtain a large litter size is not a target in itself with transgenic, cloned piglets, because transgenic piglets will often serve as founder animals that are bred naturally to obtain the next generation.
With in vivo–derived embryos, pregnancies are obtained by transferring around 20–25 embryos to one recipient; however, with cloned embryos, the numbers are typically 100–200 per recipient (Liu et al., 2014), and even up to 428 embryos to one recipient have been described (Li et al., 2013). These numbers are so high because most groups perform the transfer on days 0–3 after cloning, and, at that early stage, the embryos have not yet shown much of their developmental capacities, so any thorough selection of the best-quality embryos is not possible. However, it should be a point of concern to transfer such high embryo numbers, not only from a biological point of view when considering if and how such many embryos can establish a robust pregnancy, but also from a practical point when considering the heavy workload it is to produce so many cloned embryos.
An obvious solution would be to culture the embryos in vitro for 5–6 days to demonstrate that they can reach the morula–blastocyst stages. However, only a few groups apart from ours (e.g., Vajta and Callesen, 2012; Liu et al., 2014) have done so on cloned embryos (e.g., Lagutina et al., 2006; Yamanaka et al., 2009; Lee et al., 2013) because of a concern about a negative influence of such culture period on cloned embryos (e.g., Niemann et al., 2010; Kurome et al., 2013). Furthermore, such later-stage embryos allow for a more precise evaluation and thus selection of embryos for transfer, which may result in a higher pregnancy result. This could make transfer of fewer embryos a realistic approach, meaning that the cloned embryos are divided into more recipients; however, this has not been tested.
The aim of this experiment was, therefore, to demonstrate the ability to perform 5- to 6-day in vitro culture on cloned pig embryos with stable and robust results and to evaluate the effect of reducing the numbers transferred of cloned day-5- to day-6-old pig embryos. Preliminary results were presented earlier (Callesen et al., 2014).
Materials and Methods
Since 2006, our group has worked in pig with cloning and transfer of embryos to produce transgenic piglets as animal models for important human diseases. The first years were used to establish and stabilize the procedure (Vajta and Callesen, 2012; Callesen et al., 2014), but the protocol reported here has been used for the last 4 years. The procedure has been described in detail previously (e.g., Liu et al., 2013), but in brief was as described below. All chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated.
Collection of oocytes
Ovaries from slaughtered Large White (LW) gilts and sows were transported to the laboratory at 30–33°C. Cumulus–oocyte complexes aspirated from 2- to 6-mm follicles were selected for further use if they had a homogeneously looking ooplasm and three to four layers of cumulus cells.
In vitro maturation
Oocytes were cultured for 42–46 h in groups of ∼50 per well in four-well dishes (Nunc, Roskilde, Denmark) in bicarbonate-buffered Tissue Culture Medium (TCM-199) supplemented with 10% (vol/vol) cattle serum (CS; Danish Technical University, Frederiksberg C, Denmark), 10% (vol/vol) pig follicular fluid, 10 IU/mL pregnant mare's serum gonadotropin (PMSG), 5 IU/mL human chorionic gonadotropin (hCG; Suigonan Vet, Intervet, Ballerup, Denmark), 0.04 mg/mL glutamine, and 0.15 mg/mL gentamicin at 38.5°C with 5% CO2 in air with maximum humidity.
Somatic cell nuclear transfer
Cloning was performed on day 0 using handmade cloning (HMC; Kragh et al., 2004). After partial digestion of the zona pellucida with 3.3 mg/mL Pronase, oriented bisection of matured oocytes was performed manually under stereomicroscope with a microblade (AB Technology, Pullman, WA, USA). Donor cells were fibroblasts isolated from a 40-day-old male LW fetus or from an ear biopsy taken from newborn Yucatan or Göttingen minipigs and were cultured as previously described (Kragh et al., 2009). Some of the cell lines were made transgenic for different gene types (e.g., al-Mashhadi et al., 2013; Kragh et al., 2009; Staunstrup et al., 2012). Each ooplasm without nucleus was fused with one cell in fusion medium [0.3 M mannitol, 0.1 mM MgSO4 and 0.01% (wt/vol) polyvinyl alcohol (PVA)] in a fusion chamber (BTX Microslide 0.5-mm fusion chamber, model 450; BTX, San Diego, CA, USA) using a single direct current (DC) impulse of 2.0 kV/cm for 9 μsec. One hour later, each ooplasm–cell pair was fused with another ooplasm in activation medium (fusion medium supplemented with 0.1 mM CaCl2) by a single DC pulse of 0.86 kV/cm for 80 μsec. After incubation in porcine zygote medium-3 (PZM-3; Yoshioka et al., 2002) supplemented with 5 μg/mL cytochalasin B and 10 μg/mL cycloheximide for 4 h or 2 mM 6-dimethylaminopurine (DMAP) for 3 h, the reconstructed embryos were cultured individually in microwells [well-of-the wells (WOWs); Vajta et al., 2000] made in four-well dishes filled with PZM-3 medium in an atmosphere of 5% CO2, 5% O2, and 90% N2 with maximum humidity. Developmental rates [percent reconstructed embryos developed into morulae/blastocysts (day 5/6)] were determined.
Embryo transfer and birth of piglets
Morulae and blastocysts of transferable quality were collected on days 5 and 6 to be surgically transferred to LW sows 4 or 5 days after onset of estrus, which was observed 4–6 days after weaning (Schmidt et al., 2010). Pregnancies were diagnosed by ultrasonography around day 28. Piglets were delivered by natural birth ∼24 h after induction with prostaglandin around day 116. The number of live piglets was assessed on the first day after birth.
Statistical analyses
All statistical analyses were performed with software of R (v. 2.15.2). Frequencies were analyzed by chi-squared test, and mean values by one-way analysis of variance (ANOVA), and Tukey's Honestly Significant Difference test (Tukey HSD). In all comparisons, differences with p<0.05 were considered statistically significant.
Experimental design
Data were collected from experimental cloning rounds performed in 2010–2013. In each round, three to five people did the actual cloning work using one donor cell type (transgenic or not). Cloning was performed over 2 consecutive days to produce enough day-5 and day-6 embryos of transferable quality to use one to three recipient sows on one transfer day.
Experiment 1: Evaluating effect of in vitro culture of cloned embryos
This was tested on: (1) In vitro development, using cloning rounds where detailed registrations of the cloning work were available from at least two of the cloning persons; (2) in vivo development, using all rounds where embryos were transferred to recipients that completed their pregnancy period.
Experiment 2: Reducing number of cloned embryos transferred to recipients
This was tested as part of experiment 1, i.e., when cloning was done with one particular nontransgenic donor cell line. At the day of transfer, the number of embryos transferred to one recipient was randomly selected to be either 30–49, 50–69, or 70–90.
Results
In total, 52 experimental rounds were performed using donor cells that were either nontransgenic (43% of the rounds) or transgenic with one of nine different gene constructs.
In experiment 1, a total of 15,093 reconstructed embryos were produced in 233 person-days (i.e., total number of cloning days for all persons doing cloning), and after 5–6 days of in vitro culture 6362 (41%) were of transferable quality. Of these embryos, 83±28 [mean±standard deviation (SD)] were transferred to 105 recipients on 52 transfer days, and 94 (90%) became pregnant, but 10 (10%) later aborted. At term, on average 7.2 piglets were born from the 84 recipients. Table 1 presents the results for each of the 4 years in the experimental period.
No significant differences (p<0.05) were found between years in any of the rows.
See Materials and Methods for details.
SEM, standard error of the mean.
In experiment 2, a total of 1272 cloned embryos were transferred to 26 recipients, and 21 (81%) gave birth to in average 7.1 piglets (14% of embryos transferred). Table 2 presents the results for each of the three experimental groups.
Mean per recipient±SEM.
Mean of recipients giving birth±SEM.
Indicates significant difference (p<0.05) in the same row.
SEM, standard error of the mean.
Discussion
This work showed two things: (1) In vitro culture of cloned embryos is possible with quite stable and satisfying overall results over a longer period and with various types of cells and with different persons doing the cloning work. (2) Transfer of fewer day-5 to day-6 cloned embryos can be performed with the same efficiency in obtaining piglets. This naturally results in fewer piglets per recipient, but the lower numbers did not hinder the successful completion of the pregnancy. Together, this can be a way to increase the efficiency in obtaining piglets by dividing the produced cloned embryos into more recipients.
Further work is needed to fully exploit the possibility to reduce numbers of cloned embryos for transfer. The basis is the simple morphological evaluation of embryonic quality on the transfer day, and the limitations in this traditional evaluation are well known, so additional measures should be considered related to key events during the cloning procedure (cell behavior, cloning quality, early embryo development, etc.). This would also bring in the quality of the donor cell line that can be of very varying viability. Our results were obtained using only one nontransgenic cell line that works well in our system. However, for other cell types, the situation could be different with lower developmental rates. Further work is also needed to better understand the recipient's reaction to transfer of embryos, i.e., how few embryos can be transferred and still result in a clear and robust pregnancy signal to the recipient.
In conclusion, a reasonably high and stable developmental rate was obtained after 5–6 days of in vitro culture of cloned pig embryos, resulting in high and robust pregnancy results. This could be used to reduce the number of embryos transferred without decreasing the overall efficiency of piglets born per embryo transferred.
Footnotes
Acknowledgments
The authors thank J. Adamsen, R. Kristensen, A.M. Pedersen, B. Synnestvedt, and K. Villemoes for very qualified technical assistance.
This work was supported financially by the DAGMAR project (Danish National Research Infrastructures Program, 2136-08-0007) and the “Pigs and Health” project (Danish National Advanced Technology Foundation, 013-2006-2). The pigs were housed and handled according to Danish regulations on genetically modified animals, and experiments were approved by the Danish Animal Experiments Inspectorate (2004/561-925 and -1733).
Author Disclosure Statement
The authors declare that there are no conflicts of interest.