Abstract
Background/Aim: Microminipigs have been maintained in small populations of closed colonies, involving risks of inbreeding depression and genetic drift. In order to avoid these risks, we assessed the applicability of cloning technology. Materials and Methods: Male and female clones were produced from a stock of cryopreserved somatic cells, obtaining offspring by means of natural mating. Phenotypic and genotypic characteristics of original microminipigs, clones and their offspring were analyzed and recorded. Results and Conclusion: Clones presented characteristics similar to those of the cell-stock data. Although the body weight of clones tended to be heavier than that of the cell-stock data, body weights of their offspring were similar to those of previous reports. Thus, cloned microminipigs have the potential to be a valuable genetic resource for reproduction and breeding. Our proposed methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity.
- Miniature pig
- experimental animal
- SCNT
- clone
- offspring
- full-sib
- cryopreservation
- risk
- inbreeding
- genetic drift
Microminipigs were produced as experimental animals by Fuji Micra Inc. in Shizuoka, Japan (1). At 6 months, their body weight reaches about 10 kg (2), which is less than that of conventional miniature pig breeds, such as the Göttingen miniature pig (1). Some Japanese researchers have already used them in pharmaceutical and toxicological studies (3-8).
Microminipigs resulted from breeding between pot-bellied pigs and other types of miniature pigs (1), which might have favoured genetic diversity. In fact, Ando et al. (9) described 11 haplotypes of swine leukocyte antigen (SLA) class I and II in a resource herd of microminipigs. These pigs have been suggested as a potential genetic resource to develop animals for experimental use.
Microminipigs have been maintained in small populations, increasing the risk for inbreeding depression and progressive genetic drift. With a constant genetic background, even though microminipigs may have a population with relevant genetic diversity, they cannot be supplied in large numbers for commercial use.
On the other hand, few reports have described successful cloning of pigs from somatic cells (10-12). Therefore, microminipig cloning may be used to preserve an invaluable genetic resource and provide a large number of full-sib groups from multiple cloned-parents.
In this study, we collected and cryopreserved somatic cells from individual microminipigs; we recorded some of their particular phenotypic characteristics and determined their genotypes associated with coat-colour genotypes and SLA haplotypes. Subsequently, we produced pig clones and enable their natural mating, registering the same characteristics explored in the original sample of microminipigs (i.e. phenotypic characteristics, coat colour genotypes, SLA haplotypes), in addition to biochemical parameters. Based on this, we assessed the applicability of the cloning technology for microminipig reproduction and breeding.
Materials and Methods
Characterization of microminipigs and cell stock. Sixty microminipigs were used in this study, obtained from a closed herd produced by Fuji Micra Inc. (Shizuoka, Japan). We recorded their weights at birth, coat colour and snout characteristics; snouts were classified as either straight (Figure 1A and C) or undershot-jaws snouts (Figure 1B).
Somatic cells from microminipigs were obtained by means of ear biopsy. After quick rinses in 75% ethanol and PBS, ear tissues were minced into pieces of 2-3 mm. Six to eight ear samples of individual pigs were cultured with 10 μl Cellmatrix Type I-P drop (Nitta Gelatin Inc., Osaka, Japan) in 35-mm cell-culture dishes and incubated in 2 ml of Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Paisley, UK) with 10% embryonic stem cell foetal bovine serum (ES Cell FBS; Invitrogen, Grand lsland, NY, USA) and antibiotics. Dishes were then placed in a humidified incubator at 37°C with 5% CO2, changing the culture medium every other day. Fibroblast outgrowth was collected from the dishes with 0.25% trypsin-EDTA and passaged at 1:2 split ratios. Somatic cells obtained at passages 1 to 3 were frozen in CELLBANKER (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan).
Genotype analysis of extension locus and dominant white locus. To detect mutations in the Extension (E) locus, which encodes the melanocortin receptor 1 (MC1R) (13) and in the Dominant white (W) locus, associated with the Kit genes (14), we used polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) method, a technique previously described by Okumura (15).
Haplotype analysis of the SLA. Alike previous studies (16), SLA haplotypes were explored. The amplification of 11 microsatellite markers was performed via PCR using selected primers (Slams064, Slams073, SlamsA00, SlamsA16, SlamsA05, SlamsA14, SlamsA13, Slams044, Slams043, Slams095 and Slams047).
Somatic cell cloning. From the somatic cells of donor microminipigs, we selected homozygous SLA haplotype and silver coat colour from one male and one female. Ovaries from prepubertal gilts were obtained at a local abattoir. Cumulus-oocyte complexes (COCs) were collected from follicles (3-6 mm in diameter) in Medium 199 (Sigma-Aldrich, St. Louis, MO, USA) with 1 mg/ml BSA, 20 mM Hepes and 100 U/ml penicillin G potassium. About 50 COCs were cultured in 500 μl of maturation medium HP-POM (Research Institute for the Functional Peptides, Yamagata, Japan), adding 1 mM dibutyryl cAMP, 10 IU/ml equine chorionic gonadotropin (eCG) and 10 IU/ml human chorionic gonadotropin (hCG), in four-well dishes for 20-22 h. The COCs were subsequently cultured for 24 h with the same maturation medium (HP-POM) but without the dibutyryl cAMP and the hormones. This latter cultivation was carried out at 39°C under conditions of 5% O2, 5% CO2 and 90% N2. Matured oocytes with the first polar body were cultured in PZM5 (Research Institute for the Functional Peptides) supplemented with 0.1 μg/ml demecolcine, 35 mM sucrose, 25 mM Hepes and 5% FBS for 10-30 min. A first polar body and cytoplasm, presumptively containing nucleus, were pushed out through a slit on the zona pellucida using a fine glass needle. Fibroblasts synchronized by serum starvation for 72 h were used as nuclear donors. A single donor cell was inserted into the perivitelline space of an enucleated oocyte. Donor cell-oocyte complexes were then placed in a 0.28 M mannitol solution containing 100 μM MgSO4 and 0.01% (w/v) polyvinyl alcohol (PVA) and held between two electrode needles. Membrane fusion was induced by applying a single direct current pulse (200 V/mm, 20 μs) and a pre- and post-pulse alternating current field of 3 V and 1 MHz for 3 s, respectively. Enucleated oocytes, reconstructed with fibroblasts, were then activated by a single direct current pulse of 150 kV/cm for 99 μs in 0.28 M mannitol solution (50 μM CaCl2, 100 μM MgSO4 and 0.01% (w/v) PVA). Activated oocytes were cultured in PZM5 supplemented with 4 μg/ml cytochalasin B and 1 mM valproic acid ((VPA); Wako, Osaka, Japan) under paraffin oil in a plastic Petri dish for 2 h at 39°C, with an atmosphere of 5% CO2 and humidified air. The embryos were subsequently cultured in PZM5 with 1 mM VPA for 18 h and, again, cultured in PZM5 without the additive agent until transfer. On day two, after nuclear transfer, the reconstructed embryos were transferred to the oviducts of prepubertal gilts, which weighted 100 to 120 kg. Recipient gilts were Duroc, Large White or crossbreeds. All recipients were in oestrus, induced by an intramuscular injection of 1,000 IU eCG and 750 IU hCG.
Offspring of the cloned microminipigs. Offspring was obtained by natural mating of cloned microminipigs (5 females, 5 males). We recorded their coat colour and snout characteristics. Genotype and haplotype analysis were performed using the aforementioned methods. For biochemical analysis of the offspring, serum samples were obtained via jugular venipuncture of 6-month-old microminipigs; 13 blood parameters were analysed in the Health Science Research Institute (Shizuoka, Japan).
Management of cloned microminipigs and their offspring. Cloned microminipigs and their offspring were housed individually or in groups in pens of at least 0.54 m2/head, at temperatures ranging from 16°C to 36°C (30°C to 36°C until they reached the age of 2 weeks), with humidity ranging from 60% to 90% and in constant 12/12 light/dark cycles. The animals were weaned at the age of 4 weeks and body weights were measured once a week (0-26 weeks of age). Feeding management was set according to their age: 4- to 7-week-old animals were provided with 3-6% of their body weight of a weaning diet (>81.0% total digestible nutrients (TDN), >18.0% crude protein (CP); JA Higashinihon Kumiai Shiryou Co., Ltd, Gunma, Japan); animals between 8 and 12 weeks of age were provided with 3-6% of their body weight of a piglet diet (>77.0% TDN, >16.0% CP, JA Higashinihon Kumiai Shiryou Co., Ltd); and, finally, pigs aged 13 weeks and above were provided with 2-5% of their body weight of a laboratory miniature pig diet (mean=318 kcal/100 g, mean=15.7% CP; Oriental Yeast Co., Ltd., Tokyo, Japan). They had free access to tap water at all ages. All procedures were approved by the Animal Care and Use Committee of Shizuoka Prefectural Research Institute of Animal Industry, Swine and Poultry Research Centre, which comply with the Guidelines for Proper Conduct of Animal Experiments from the Science Council of Japan (2006).
Results
Characterization of microminipigs and cell stock. From the 60 microminipigs included in this study, most of them presented straight snouts (70.0%) and only a 30.0% presented undershot-jaw snouts. We recorded three coat colours: 33.3% of the microminipigs were described as white, 23.3% as black and 43.3% presented a silver coat colour.
Genotype analysis of Extension locus and Dominant white locus. Considering the genotypes associated with coat colour, all microminipigs studied presented an E/E genotype in the Extension locus. Microminipigs with black or silver coat colour had an i/i genotype in the Dominant white locus and white coloured microminipigs presented I/i or I/I genotypes.
Haplotype analysis of the SLA. Based on the combination of the 11 microsatellite markers for the SLA locus, we described nine SLA haplotypes, classified from A to I (Table I). Nine (15.0%) microminipigs showed homozygous genotype (A/A) and 32 (53.3%) showed heterozygous genotype (A/-) for haplotype A, totalling in a frequency of 41.7% for the haplotype A. On the other hand, homozygous genotypes of C and D summed 1.7%, with haplotype frequencies of 26.7% and 6.7%, respectively. Haplotype frequencies of B, E, F, G, H and I are 5.8%, 1.7%, 3.3%, 10.8%, 1.7% and 1.7%, respectively.
Microminipig clones. Five clones were reproduced using the cryopreserved donor somatic cell-stock from one of the microminipig males (Figure 1A). All male microminipig clones presented a silver coat and straight snout, similar to the characteristics registered in their microminipig donors. Their SLA haplotype was A/A, the Extension locus E/E and the Dominant white locus i/i, identical to the genotypes registered in their microminipig donors. The average body weight at birth of male clones was 594 g, which is 1.5-times heavier than that of the data from the cell-stock (400 g) (Table II).
Five clones were reproduced using the cryopreserved donor somatic cell-stock from one of the microminipig females (Figure 1B). All female microminipig clones presented a silver coat and undershot-jaw snout, similar to the characteristics from the cell-stock data. Their SLA haplotype was A/A, the Extension locus E/E and the Dominant white locus i/i, identical to the genotypes from the microminipig cell-stock data. The average body weight at birth of the female clones was 549 g, which is 1.2-times heavier than that of their microminipig cell-stock data (460 g) (Table II). We did not observe any external abnormality in any of the microminipig clones.
Offspring derived from microminipig clones. Sperm mobility of the cloned males (>6 months old) was above 70%. Cloned females presented clear signs of oestrus after 8 months of age. The behavioural oestrus continued for 2.16±0.47 days and the length of oestrous cycle was estimated to be 21.9±0.3 days. By natural mating of five male and five female clones, we obtained 50 males and 55 females in 21 parturitions. Their gestation period was 112.8±1.7 days; total and alive litter sizes were 6.39±0.99 and 4.57±1.26, respectively. Offspring presented silver or black coat colour, straight snout and no observable abnormalities (Figure 1C). Their SLA haplotype was A/A, the Extension locus E/E and the Dominant white locus i/i.
The body weight at birth of the offspring was significantly lower compared with that of male (p<0.01) and female clones (p<0.05). The body weight of the offspring at 6 months was significantly lower than that in male clones (p<0.01). The difference of that with female clones was not statistically significant (Table II). Concerning blood biochemical analysis, we randomly chose seven males and nine females (Table III). Most of the parameters observed showed lesser differences between male and female offsprings, except for total cholesterol, triglycerides, calcium and potassium, that showed higher levels in females.
Discussion
There are two main strategies to preserve pig genetic resources. The first one is the maintenance of live animals, which is the most certain method, but requires high costs associated with space, facilities and management. The second strategy is the cryopreservation of germ cells and embryos. Currently, preservation of frozen semen is the most popular and useful method to conserve swine genetic resources. The cryopreservation of oocytes or embryos is still being studied, exploring the potential applications for pig reproduction (17, 18). These aforementioned two strategies are not adequate for the maintenance of genetic resources from a limited population, even though it presents significant genetic diversity. In order to avoid inbreeding depression and to ensure the phenotype and genotype traits of the offspring, here we proposed a new method for the production of clonal microminipigs as experimental animals. This new method consists in the cryopreservation of somatic cells, with the registration of the corresponding phenotype and genotype, followed by the production of offspring from somatic-cell cloned parents. This method can preserve the genetic resource of microminipigs per se.
In order to confirm the diversity of the original microminipigs, we observed phenotypic characteristics and genotypes associated with SLA and coat colours. The phenotypic characteristics, such as coat colour and snout type, differed among individuals; genotypes also presented relevant diversity. SLA is one of the histocompatibility antigens and has important roles associated with sensitivity to various infectious pathogens. In our study, we described nine SLA haplotypes (Table I), similar to a previous study that described 11 SLA haplotypes (9). Concerning coat-colour genotypes, the original microminipigs presented two types of Dominant white locus. Thus, the studied cell-stock of original microminipigs showed relevant genetic diversity and turned out to be an important genetic resource.
Phenotypic characteristics and genetic outcomes of cloned microminipigs reflected those of the donor microminipigs, as previously described in a study with Landrace pigs (19). On the other hand, the body weights at birth of cloned microminipigs were heavier compared with that from the donor microminipigs (Table II). Although we did not measure the body weights of the donor pigs at the age of 6 months, we assume that they would be lower compared with that in clones, considering that Kawaguchi et al. (2) reported the average body weight of 6-month-old pigs kept at the Fuji Micra Inc.to be below 10 kg. The explanation for this body weight differences between microminipig clones and donors could be related with several factors. Uterine, environmental and/or epigenetic factors can produce different maternal effects on genetically identical animals (20). Previous studies have reported that miniature pig clones present greater body weights at birth when the recipients are conventional breeds (21, 22). In these cases, the differences in body weights could be attributed to the different body sizes (21) or small litter sizes (22) of the recipients.
Based on natural mating, we obtained 105 individuals, which presented no apparent abnormalities. The body weights of the offspring produced via natural mating in our study were similar to those reported by Kawaguchi et al. (2). In cloned mice, it was suggested that epigenetic effects tended to disappear in the offspring produced by natural mating of the clones (23). This seems to be replicated in our outcomes; in the cloned-microminipig offspring we also described a normal growth performance, which had also been reported previously for swine species (24-27). Finally, as shown in Table III, the serum biochemical parameters of the offspring were similar to that from previously described parameters of microminipigs (2).
In conclusion, cloned microminipigs have the potential to be valuable genetic resources for reproduction and breeding. By applying the presented methodology, particular desired characteristics from microminipigs can be selected and propagated from a variety of heterogeneous populations. Furthermore, this methodology might be useful to provide a large number of animals with adequate quality from a limited population with sufficient genetic diversity.
Acknowledgements
The Authors gratefully acknowledge Drs. Takato Nakamura, Kimio Bamba and Makoto Mori in Shizuoka University and Dr. Naohiko Okumura for their valuable suggestions in preparing this manuscript. They appreciate the technical support of Ms. Yukiko Otsu (Shizuoka Perfectural Toubu Public Center) during early days of the study. They are also indebted to all staffs in Shizuoka Prefectural Research Institute of Animal Industry, Swine and Poultry Research Center for taking good care of animals.
- Received June 20, 2016.
- Revision received July 11, 2016.
- Accepted July 12, 2016.
- Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved