Effects of Donor Cell Passage Number, Lineage, and Sex on Cloned Embryo Transfer Efficiency in Critically Endangered Vietnamese Ỉ Pigs

1. Introduction

Pigs serve as essential livestock and represent valuable animal models in both biomedical and biological research. The strategic significance of agriculture and advancements in biomedicine underscore the need to conserve livestock diversity and safeguard porcine genetic resources [1]. In Vietnam, numerous indigenous pig breeds, including the Ỉ pig, have become extinct or are currently facing the threat of extinction [2]. Therefore, the development of in vitro gene banking is essential for safeguarding the genetic integrity of Ỉ pig breeds and ensuring their preservation in the face of epidemic threats.

Since the successful cloning of the sheep ‘Dolly’ in 1997, somatic cell nuclear transfer (SCNT) has emerged as a powerful biotechnological tool for replicating genetically selected individuals across various species [3]. Somatic cell nuclear transfer (SCNT) has found broad application across diverse domains, including the enhancement of livestock genetics, conservation efforts aimed at preserving endangered species, and the generation of transgenic animals for biomedical and therapeutic purposes [4].

Somatic cell nuclear transfer (SCNT) is a widely utilized technique for generating embryos, and has been shown to support full-term development following the transfer of embryos derived from cryopreserved somatic cells into recipient pigs [5]. Somatic cell nuclear transfer (SCNT) represents a promising strategy for generating piglets from rare or even extinct pig breeds. Despite the successful production of numerous piglets using this technique, the overall efficiency of porcine cloning remains relatively low [6]. The choice of donor cells play a critical role in determining the success rate of cloned pig production [7].

Donor cell passage number, cellular origin, and donor sex are recognized as potential determinants influencing the efficiency of cloned Ỉ pig embryo production [8]. In Vietnam, several studies have explored critical factors and proposed strategies to improve cloning outcomes, such as optimizing donor cell type and preparation [8], alongside advancements in oocyte and embryo culture systems [9]. Nevertheless, the specific effects of donor cell passage number, cellular origin, and donor sex on the efficiency of cloned Ỉ pig embryo transfer remain insufficiently examined. It is plausible that enhancing embryo transfer success rates could be achieved through the refinement of donor cell-related parameters, including passage number, cell line selection, and donor sex. Accordingly, the present study was designed to evaluate the impact of these donor cell characteristics on embryo transfer efficiency in the critically endangered Vietnamese Ỉ pig.

2. Materials and Methods

2.1. Ethics Statement

All the experimental procedures used in this study were performed following Vietnam legislation and Decision No. 2656/QĐ-BNN-KHCN of the Ministry of Agriculture and Rural Development in Vietnam on June 15.2021 (Currently, it is the Ministry of Agriculture and Environment).

2.2. Reagents and Chemicals

All reagents and chemicals used in this study were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Petri dishes used for cell culture are originated from Corning Inc. (Corning, NY, USA).

2.3. Oocyte Collection and In Vitro Maturation (IVM)

Oocyte collection and in vitro maturation (IVM) were performed following the protocol described by Van et al. (2021) [9]. Porcine ovaries from 6–8-month-old Landrace × Large White (LW) prepubertal gilts were obtained from a local slaughterhouse and transported to the laboratory within 3–5 hours in saline solution supplemented with 0.1 mg/ml streptomycin sulfate and 100 units/ml penicillin G potassium (Sigma-Aldrich) maintained at 35–37 °C. Upon arrival, the ovaries were washed three times with Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma-Aldrich Corp., St. Louis, MO, USA), also supplemented with the same concentrations of antibiotics, at 37 °C. Cumulus-oocyte complexes (COCs) were retrieved by scraping all visible antral follicles (≥2 mm in diameter) into TALP-HEPES medium, which consisted of 114 mM NaCl, 3.2 mM KCl, 2.0 mM CaCl₂·2H₂O, 0.5 mM MgCl₂·6H₂O, 10 mM sodium lactate, 0.1 mM sodium pyruvate, 2 mM NaHCO₃, 3 mg/ml bovine serum albumin, 10 mM HEPES, and antibiotics (100 units/ml penicillin G potassium and 0.1 mg/ml streptomycin sulfate; Sigma-Aldrich). The medium was dispensed into 60 mm Petri dishes (Falcon 351007, Thomas Scientific, NJ, USA), and COCs were collected under a stereomicroscope. Only oocytes exhibiting evenly granulated cytoplasm, no signs of lysis, and surrounded by at least three intact layers of cumulus cells were selected for further use.

Cumulus-oocyte complexes (COCs) were cultured in porcine oocyte medium (POM) as described by Yoshioka et al. (2008) [10], supplemented with 10 ng/ml epidermal growth factor (EGF; Sigma-Aldrich), 10 IU/ml equine chorionic gonadotropin (eCG; Serotropin, ASKA Pharmaceutical Co., Ltd., Tokyo, Japan), and 10 IU/ml human chorionic gonadotropin (hCG; Puberogen, Novartis Animal Health, Tokyo, Japan), following the protocol reported by Van et al. (2021) [9]. To synchronize oocyte maturation, the in vitro maturation (IVM) medium was further supplemented with 1 mM dibutyryl cyclic AMP (dbcAMP; Sigma) during the initial 22 hours of culture. IVM was conducted in 4-well culture dishes (Nunc MultiDishes, Thomas Scientific), with each well containing 500 µl of IVM medium overlaid with mineral oil (Sigma-Aldrich). Cultures were maintained for 22 hours in a controlled atmosphere of 5% CO₂, 5% O₂, and 90% N₂ at 39 °C. Subsequently, the COCs were transferred to maturation medium devoid of dbcAMP and cultured for an additional 22–24 hours under identical atmospheric conditions. Each well contained between 30 and 50 COCs.

2.4. Donor Cells Preparation

Donor cells were prepared following the protocol outlined by Van et al. (2021) [9]. Marginal ear tissue samples were collected via ear-punch biopsy from five Ỉ gilts (IDs: 6004, 9154, 9155, 9156, 9157) and one Ỉ boar (ID: 6002). The collected tissues were initially rinsed in 70% ethanol, followed by Dulbecco’s phosphate-buffered saline (DPBS; Sigma-Aldrich) supplemented with 100 IU/ml ampicillin and 100 µg/ml streptomycin sulfate (Sigma-Aldrich). The tissues were then minced into fragments approximately 1 mm³ in size. These fragments were seeded onto tissue culture flasks containing Dulbecco’s Modified Eagle Medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS), and incubated at 37 °C in a humidified atmosphere of 5% CO₂. Upon reaching ≥80% confluency, cells were harvested using 0.25% Trypsin-EDTA (Sigma-Aldrich) and subcultured until the third passage, as described by Freshney (2000) [11]. Cells at passage three were resuspended in a cryopreservation medium composed of 70% DMEM, 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich), and 20% FBS, at a final concentration of 3 × 10⁶ to 4 × 10⁶ viable cells/ml. The cell suspension was aliquoted into cryovials and held at 4 °C for 20–30 minutes to allow DMSO equilibration. Cryovials were then stored at −80 °C overnight before being transferred to liquid nitrogen for long-term preservation [12].

To assess the impact of donor cell passage number on embryo transfer efficiency in Ỉ pigs, donor cells at the 4th and 7th passages were cultured until reaching a minimum of 80% confluency and subsequently utilized for somatic cell nuclear transfer (SCNT). Prior to SCNT, cells underwent serum starvation in medium containing 0.2% fetal calf serum (FCS) for 48 hours to induce cell cycle synchronization. Approximately 30 minutes before nuclear transfer, donor cells were harvested using 0.25% (w/v) trypsin–0.02 mM EDTA (Sigma-Aldrich), followed by washing and resuspension in HEPES-buffered Tissue Culture Medium 199 (TCM-199).

2.5. SCNT and Embryo Culture

Somatic cell nuclear transfer (SCNT) embryos were generated using a zona-free protocol with minor modifications, based on previously established methods [13], [14], [15], [16]. Following 40–42 hours of in vitro maturation, cumulus cells were removed from oocytes by vortexing in TALP-HEPES medium containing 1 mg/ml hyaluronidase (Sigma-Aldrich). The TALP-HEPES medium consisted of 114 mM NaCl, 3.2 mM KCl, 2.0 mM CaCl₂·2H₂O, 0.5 mM MgCl₂·6H₂O, 10 mM sodium lactate, 0.1 mM sodium pyruvate, 2 mM NaHCO₃, 3 mg/ml bovine serum albumin, and 10 mM HEPES. Mature oocytes exhibiting the extrusion of the first polar body were selected for further processing.

To remove the zona pellucida, selected oocytes were incubated in 0.5% Pronase (Sigma-Aldrich) in TALP-HEPES for 3–6 minutes on a heated stage. Zona-free oocytes were then washed in TALP-HEPES supplemented with fetal bovine serum (FBS) and transferred to PZM3 medium [17] containing 2.5% FBS until enucleation. Prior to enucleation, oocytes were treated with 4 μM demecolcine (Sigma-Aldrich) in PZM3 supplemented with 2.5% FBS for 20–40 minutes to induce chromosome plate protrusion. Oocytes displaying protrusions were enucleated in TALP-HEPES medium supplemented with 7.5 µg/ml cytochalasin B (Sigma-Aldrich), using a blunt pipette and a closed-holding pipette to remove metaphase chromosomes.

Post-enucleation, zona-free cytoplasts were briefly washed in 300 μg/ml phytohemagglutinin (Sigma-Aldrich) in HEPES-buffered TCM-199 and immediately placed over a single donor cell resting at the bottom of a 100 µl TALP-HEPES droplet, facilitating attachment. The resulting couplets were incubated in fusion medium for 2–3 minutes and then transferred to a fusion chamber containing 2 ml of pre-warmed fusion medium composed of 0.3 mM mannitol, 0.05 mM CaCl₂·2H₂O, 0.1 mM MgSO₄·7H₂O, and 25 mg/ml polyvinyl alcohol (PVA). Fusion was achieved using a double direct current (DC) pulse of 70 V for 30 µsec with an electro-cell fusion system (LF 101, Nepa Gene Co., Ltd., Chiba, Japan).

Following fusion, couplets were cultured individually in PZM3 medium supplemented with 2.5% FBS at 38.5 °C under a humidified atmosphere of 5% CO₂ and 5% O₂. Fusion efficiency was assessed after 30–50 minutes; non-fused couplets were discarded. Successfully fused couplets were incubated for 2 hours to allow reprogramming, after which activation was performed using a single DC pulse of 65 V for 80 µsec. Activated embryos were subsequently cultured for 3 hours in PZM3 medium containing 7.5 µg/ml cytochalasin B under the same incubation conditions.

SCNT embryos were cultured using a modified Well-of-the-Well (WOW) system as described by Vajta et al. (2000) [18]. Microwells were manually created in 35-mm Petri dishes (Corning, USA) by pressing a heated steel needle into the dish surface. Each dish contained 20 microwells, which were covered with a 100 µl microdrop of PZM3 medium and overlaid with mineral oil. A single SCNT embryo was placed into each microwell. Embryo cultures were maintained at 38.5 °C in a humidified atmosphere of 5% CO₂ and 5% O₂. The day of activation was designated as Day 0. The PZM3 medium was not replaced throughout the culture period. Embryos at the morula and blastocyst stages were collected on Days 5, 6. On Day 5, the culture medium was supplemented with 10% (v/v) fetal bovine serum (FBS).

2.6. Embryo Transfer

Prepubertal gilts (Landrace × Yorkshire) weighing between 100 and 120 kg were utilized as surrogate recipients for somatic cell nuclear transfer (SCNT) embryos. Estrus synchronization was achieved through intramuscular administration of 1500 IU human chorionic gonadotropin (hCG; ZENOAQ, Japan), followed 72 hours later by 1000 IU equine chorionic gonadotropin (eCG; ZENOAQ, Japan). SCNT embryos at the compacted morula and blastocyst stages were transferred into the uterine horns of recipients on the fifth day post-standing estrus. Embryo transfer was performed surgically via a mid-ventral laparotomy under general anesthesia. The transfer medium consisted of NCSU-37 supplemented with 5 mg/ml bovine serum albumin (BSA) and 20 mM HEPES, excluding fetal bovine serum (FBS) [19]. Recipients were checked for pregnancy by trans-abdominal ultrasound examination 28 days after embryo transfer.

2.7. Experimental Design

Experiment 1. The effect of donor cell passage number on the efficiency of cloned Ỉ pig embryo transfer

This experiment was performed to assess the effect of donor cell passage number on the efficiency of cloned Ỉ pig embryo transfer. Ỉ pig fibroblasts of an individual (6004) at the 4^rd to 7^th passages were obtained and cultured as described above in DMEM medium supplemented either with 0.2% (v/v) fetal calf serum (FCS). Somatic cell nuclear transfer (SCNT) embryos were generated using Ỉ pig fibroblasts and cultured under serum-deprived conditions with cytoplasts derived from LW oocytes matured in a defined POM system. Embryos were maintained in PZM3 medium supplemented with fetal bovine serum (FBS) until Day 5, with Day 0 designated as the day of SCNT. Embryo transfer into surrogate mothers was performed at either the compacted morula stage on Day 5 or the blastocyst stage on Day 6, as previously described.

Experiment 2. The effect of sex of donor cells on the efficiency of cloned Ỉ pig embryo transfer

This experiment aimed to evaluate the impact of donor cell sex on the efficiency of cloned Ỉ pig embryo transfer. As previously outlined, LW oocytes obtained from a slaughterhouse were subjected to in vitro maturation (IVM) using POM medium. Female and male Ỉ pig fibroblast of 2 different individuals (9154 and 6002, respectively) selected from the cell passage associated with the highest pregnancy rate in Experiment 1 were introduced into enucleated mature oocytes. Embryos derived from either female or male donor cells were transferred into surrogate sows at two developmental stages: the compacted morula stage on Day 5 or the blastocyst stage on Day 6, following established protocols.

Experiment 3. The effect of cell lines on the efficiency of cloned Ỉ pig embryo transfer

This experiment aimed to evaluate the influence of cell lines on the efficiency of cloned Ỉ pig embryo transfer. Somatic cell nuclear transfer (SCNT) embryos were generated using three distinct cell lines of either female or male Ỉ fibroblasts, selected based on the highest pregnancy rates observed in Experiment 2, as previously described. Embryos derived from these donor cells were transferred into surrogate sows at two embryonic stages: the compacted morula stage on Day 5 and the blastocyst stage on Day 6, in accordance with established protocols.

2.8. Statistical Analysis

Data are were analyzed by ANOVA, followed by Tukey’s multiple comparisons test, using GraphPad Prism software (Version 7.02 for Windows, GraphPad Software, La Jolla, California, USA). P < 0.05 was defined as the significance level.

3. Results

3.1. Experiment 1. The Effect of Donor Cell Passage on the Efficiency of Cloned Ỉ Pig Embryo Transfer

The results of Experiment 1 are presented in Table 1. Ỉ pig fibroblasts from individual 6004 at the 5^th and 6^th passages exhibited significantly higher rates of pregnancy, farrowing, and piglet production compared to those at the 4^th and 7^th passages (Table 1, P < 0.05). There was no significant difference in the pregnancy, farrowing rates and piglet production between the 5^th and 6^th passages (Table 1, P > 0.05). Ỉ fibroblasts at the 5^th and 6^th passages are regarded as suitable for the transfer of cloned Ỉ pig embryos.

3.2. Experiment 2. The Effect of Sex of Donor Cells on the Efficiency of Cloned Ỉ Pig Embryo Transfer

Based on the findings from Experiment 1, Experiment 2 used male and female Ỉ pig fibroblasts derived from two distinct individuals 9154 (male) and 6002 (female) at the 5^th passage. The outcomes, summarized in Table 2, revealed no statistically significant differences in the pregnancy, farrowing rates, and piglet production between the male and female groups (Table 2, P > 0.05). Among the cloned Ỉ piglets that died within one week after delivery, the number of female piglets was higher (1/4 vs 0/4, P > 0.05, Table 2). In the both male and female groups, only a single pregnant recipient carried the pregnancy to term. The findings from Table 2 indicate that the sex of donor cells do not affect the cloned Ỉ pig production efficiency.

3.3. Experiment 3. The Effect of Cell Lines on the Efficiency of Cloned Ỉ Pig Embryo Transfer

Building upon the findings of Experiment 2, Experiment 3 employed fibroblast cells derived from three individual female Ỉ pigs (IDs: 6004, 9154, and 9157), all at the 5^th passage. The outcomes of this experiment are presented in Table 3. Statistical analysis revealed no significant differences in pregnancy rates, farrowing rates, or piglet yield among the three donor cell lines (Table 3, P > 0.05). The survival rates of live cloned Ỉ piglets at one week post-birth from three individual female Ỉ pigs 6004, 9154, and 9157 were 0.2%, 0.13%, and 0.3%, respectively; however, these differences were not statistically significant (P > 0.05). These results suggest that the donor cell lines does not influence the efficiency of cloned Ỉ pig production.

4. Discussion

The efficiency of somatic cell animal cloning is shaped by a multitude of interrelated factors, reflecting the inherent complexity of the process. This study focused on donor cell characteristics, including passage number, sex, and origin, to determine their mechanistic impact on cloning outcomes and thereby optimize the generation of cloned Ỉ pigs. The influence of cell passage number on the efficiency of cloned animal production continues to be a topic of scientific debate. Kubota et al. (2000) [20] reported that long-term culture of donor cells yielded better outcomes than short-term culture. In contrast, Rho et al. (2000) [21] found that donor cells at early passages were more effective than those at later passages. Zhang et al. (2009) [22] demonstrated that donor cells at 3^th to 6^th passages yielded significantly higher blastocyst formation rates compared to those at 1^th to 2^th and 7^th to 10^th passages. Van et al. (2024) [8] reported that using donor cells at the 5^th or 6^th passage significantly increased the blastocyst formation rate in cloned embryos. Additionally, Wells et al. (1999) [23] observed no significant differences in the developmental potential of somatic cell nuclear transfer (SCNT) embryos based on the number of donor cell passages.

Findings from Experiment 1 demonstrated that the passage number of donor cells significantly influenced the production efficiency of cloned Ỉ pigs and the use of donor cells at passage numbers less 5 or more than 6 did not result in the successful production of cloned Ỉ piglets. Our findings are consistent with those reported by Jin et al. (2019) [24]. Jin et al. (2019) [24] demonstrated that the cloning efficiency of piglets was higher when porcine fibroblasts at the 5^th or 6^th passage were used, compared to cells at earlier than the 5^th or beyond the 6^th passage. Notably, fibroblasts with more than 6^th passage failed to produce cloned piglets. Therefore, Jin et al. (2019) [24] limited the use of porcine fibroblasts more than the 6^th passage from use as donor cells in somatic cell nuclear transfer (SCNT) for cloned pig production. Contrary to our findings, Kubota et al. (2000) [20] and Miyoshi et al. (2003) [25] reported that the optimal range of donor cell passages for cloned bovine embryo production lies between the 10^th and 15^th passages. Notably, Miyoshi et al. (2003) [25] identified the 15^th passage as particularly suitable for cattle cloning. Discrepancies among studies may be attributed to variations in donor cell origin and laboratory-specific experimental protocols. In the current study, porcine fibroblasts were utilized, whereas Kubota et al. (2000) [20] and Miyoshi et al. (2003) [25] employed bovine fibroblasts.

Our results indicate that the use of long-term cultured senescent cells may reduce cloning efficiency, potentially posing a limiting factor in the application of somatic cell nuclear transfer (SCNT) in Ỉ pigs. Multiple studies have reported that increasing donor cell passage number induces epigenetic modifications including DNA methylation, histone alterations, and RNA regulatory changes such as m⁶A that compromise the efficiency of nuclear reprogramming following somatic cell nuclear transfer (SCNT) [26]. Prolonged in vitro culture of donor cells can result in cellular aging, the accumulation of genetic and epigenetic modifications, increasing oxidative stress, all of which may compromise the nuclear reprogramming of embryos produced through somatic cell nuclear transfer (SCNT) [27]. Additionally, extended culture duration may disrupt the regulation of imprinted genes, potentially contributing to abnormalities in embryonic and fetal development [28]. It could be one of the reasons explain why, in the present study, cloned Ỉ pigs were successfully generated only from donor cells at the 5^th or 6^th passage.

Subsequently, we evaluated the cloning efficiency of Ỉ piglets using Ỉ fibroblasts derived from both female and male donors. Based on the findings from Experiment 1, fibroblasts derived from two distinct individuals 9154 (male) and 6002 (female) at the 5^th passage were utilized. Although the female group exhibited lower survival and pregnancy rates compared to the male group, these differences were not statistically significant (0.3% vs 0.4% and 60% vs 70%, respectively, P > 0.05, Table 2). The results demonstrated that the sex of the donor cells does not influence the efficiency of cloned Ỉ pig production. This finding carries significant implications for the conservation of rare livestock breeds, demonstrating that cloning efficiency is unaffected by donor cell sex and thereby enabling the reproduction of both male and female offspring with comparable success.

The present findings align with those reported by Yoo et al. (2017) [29], yet stand in contrast to the observations of Liu et al. (2016) [30]. According to Yoo et al. (2017) [29], no significant differences in the pregnancy rates of cloned piglets derived from male versus female donor cells. Yoo et al. (2017) [29] reported that, despite the absence of significant differences in pregnancy and delivery rates between groups, cloned piglet production was greater when male fibroblast cells were used for reconstruction compared to female fibroblast cells. Meanwhile Liu et al. (2016) [30] demonstrated that embryos reconstructed using fibroblast cells derived from male Boer goats exhibited significantly higher survival rates and improved offspring quality compared to those reconstructed with cells from female goats. In this study, we only observed dead cloned Ỉ piglets in the female group, however, the efficiency of producing cloned piglets did not differ between the female and male groups (Table 2).

The developmental potential of SCNT embryos derived from male and female somatic cells remains insufficiently characterized [31]. The efficiency of producing cloned piglets from male and female fibroblasts range from 0.64% to 0.9% [31]. While the underlying reasons for the sex-specific differences in mortality are not yet understood, in our opinions, it remains necessary to conduct additional studies to enhance successful development to term and increase the survival of cloned Ỉ piglets.

Based on the results of Experiments 1 and 2, in Experiment 3 we used fibroblasts of three female Ỉ pigs (6004, 9154, 9157) at the 5^th passage for cloned Ỉ pig production. We assessed the impact of donor cell lines on the efficiency of cloned Ỉ pig embryo transfer. The impact of donor cell lines on the efficiency of cloned Ỉ pig embryo transfer. There was no significant difference in the pregnancy rates, farrowing rates, or piglet yield among the three cell lines (Table 3).

The generation of cloned piglets represents a highly intricate procedure, shaped by numerous variables that determine the efficiency of embryo transfer. Nevertheless, empirical data regarding the influence of distinct cell lines on transfer outcomes remain limited and lack consensus. The reason for this is unknown. The present results stand in contrast to the findings reported by previous studies of Kühholzer et al. (2001) [32]. In their study of nine donor cell lines derived from nine individual pigs, Kühholzer et al. (2001) [32] reported that only four of the cell lines successfully established pregnancies, and just one progressed to the 90th day of gestation. In our previous study [8], we optimized donor cell-related parameters influencing the efficiency of cloned pig embryo production, including fibroblast number, donor cell sex, and cell lineage prior to SCNT. The outcomes reported by Van et al. (2024) [8] were consistent with those observed in the present study. This factor may partly account for the discrepancy between the findings of Kühholzer et al. (2001) [32] and those observed in the present study. However, it will need further investigations to clarify this point.

5. Conclusion

In conclusion, the findings of this study suggest that donor cell passage plays a critical role in enhancing the efficiency of cloned Ỉ pig embryo transfer, whereas the sex and donor cell lines appear to have little or no impact on transfer efficiency of cloned Ỉ pig embryos.