Heat stress can impair semen quality in imported bull sires. Recent research has shown the potential of Lasia spinosa Thw. (LST) to improve semen quality in boars, so we aimed to investigate the effect of LST supplementation on semen quality in subfertile bulls, focusing on semen characteristics in both fresh and frozen-thawed ejaculates as well as the transcriptomic changes in testicular tissues. Six imported beef bull sires presenting poor semen quality for > 5 months at the Bull Semen Production Center were included in the experiment. They received daily supplementation with LST (5 g/100 kg body weight). Semen and serum testosterone evaluations were performed weekly for 24 weeks. For histologic and transcriptomic evaluations, testicular biopsies were performed on three LST-supplemented bulls, pre- and post-supplementation, and three control bulls. We observed significant improvements in movement kinetics, viability, acrosome integrity, membrane integrity, and sperm head morphology in LST-supplemented bulls. Frozen-thawed semen presented significant enhancements in parameters such as motility and mitochondrial activity. Serum testosterone significantly increased after supplementation, correlating significantly with sperm motility and mitochondrial activity. Transcriptomic analysis identified 16 downregulated genes in subfertile bulls; LST supplementation induced upregulation of 11 genes related to germ cell structure, spermatogenesis, and sperm quality, including CNFN, KRT17, KRT1, KRT19, DSC3, KLK8, SPRR4, SPRR2E-like, Serpin B4-like, and WFDC18-like. Our findings highlight the potential of LST supplementation as a promising intervention for enhancing reproductive performance in subfertile bulls.

Poor semen quality is a significant issue among imported purebred boars in Thailand, affecting both semen quality itself and reducing productivity at AI stations. The present study investigated the effects of dietary supplementation with Lasia spinosa Thw. (LST) on improving boar semen quality. A group of boars experiencing poor semen quality were monitored, and three were selected due to their persistent low semen profiles for the supplementation experiment. Dried LST powder was prepared in small bags at a dosage of 4 g per 100 kg body weight and added to the regular feed for daily oral administration over an 8-week experiment period. Semen was collected from the boars one week before treatment initiation and at the end of the supplementation period. Each sample was assessed for volume, sperm concentration, motility, viability, acrosome integrity, mitochondrial activity, and morphology. Dietary supplementation with LST significantly improved semen volume (P = 0.03), sperm motility (P = 0.03), viability (P = 0.03), and acrosome integrity (P = 0.03). The percentage of normal sperm head morphology remained unchanged. However, both major and minor sperm defects were significantly reduced (P = 0.03). In conclusion, LST powder supplementation enhances semen quality in subfertile boars and is a feasible intervention for swine farms.

The abdominal testes of Asian elephants show normal spermatogenesis. Heat shock in cryptorchid testes elevates heat shock factor (HSF) expression, leading to germ cell apoptosis, while increased heat shock proteins (HSPs) levels provide protection. To investigate how heat shock affects elephant spermatogenic cells, focusing on heat shock-related molecules and the cell death mechanism, immunohistochemistry and TUNEL staining were employed to assess the immunoexpression of several heat shock-related molecules and the status of apoptosis in elephant fibroblasts (EF) induced by heat shock stimulus. Additionally, the immunoexpression of heat shock-related molecules and cell proliferation status in the elephant spermatogenic cells. Our finding indicated that heat shock-induced HSF1 immunoexpression in EF leads to apoptosis mediated by T-cell death-associated gene 51 (TDAG51) while also upregulating HSP70 to protect damaged cells. In elephant spermatogenic cells, immunostaining revealed a predominance of proliferating cell nuclear antigen (PCNA)-positive cells with minimal TDAG51- and TUNEL-positive cells, suggesting active proliferation and apoptosis suppression during normal spermatogenesis in the abdominal testis. Interestingly, spermatogonia co-immunoexpressed HSF1 and HSP90, potentially reducing apoptosis through protective mechanisms different from those observed in other mammals. Spermatogenic cells did not show immunolocalisation of HSP70, and hence, it may not contribute to protecting the spermatogonia from heat shock because the transcriptional activity of HSF1 is suppressed by HSP90A binding. This study provides insight into the specific heat shock response and defence mechanisms in elephant spermatogenic cells and may contribute to our understanding of species-specific adaptation to environmental stresses of the testis.

Lyophilisation is an alternative method for sperm preservation. The aim of this study was to evaluate the effects of freeze-thawing (F/T) and freeze-drying (F/D) on the quality of epididymal goat sperm. Sperm from each region of the epididymis (caput, corpus and cauda) were collected and evaluated for the expression of phospholipase C zeta (PLC-ζ), protamine 1 (PRM1), transition protein 1 (TNP1) and 2 (TNP2). The effects of F/T and F/D on sperm quality in terms of PLC-ζ expression, chromatin stability (Chromomycin A3; CMA3) and DNA integrity were examined. The fertilising ability after intracytoplasmic sperm injection (ICSI) was also tested. Fresh sperm existed PLC-ζ, PRM1, TNP1 and TNP2, irrespective of the regions of the epididymis. However, different patterns of PLC-ζ expression were found. Although PRM1, TNP1, TNP2 were still expressed after F/T or F/D, only F/T could preserve the presence of PLC-ζ. For fresh sperm, caput epididymal sperm had the lowest evidence of chromatin stability when compared to sperm harvested from other regions of the epididymis. The F/T and F/D further increased the numbers of CMA3-positive sperm (P < 0.001). In all cases, no CMA3 staining was observed in caudal epididymal sperm. The caudal epididymal sperm had significantly greater proportions of sperm with intact DNA compared with caput and corpus epididymal sperm, especially when F/T and F/D were performed. The fertilisation rates of F/D sperm tended to decrease when compared with F/T sperm (4.2 ± 3.2 vs. 13.6 ± 9.0, P = 0.08). It is concluded that the sperm recovered from the caudal epididymis is suitable for freezing and lyophilisation. However, poor fertilisation rates of F/D sperm were coincidently observed, with a deficit demonstration of PLC-ζ.

The aim of this study was to optimise the ovarian responses using different FSH (follicle stimulating hormone) treatments in goats. Group 1 (control), the ovaries were stimulated with a total dose of 200 mg FSH per doe. The FSH injections were performed at 12-h intervals starting on day 9 after progesterone implantation. The FSH doses were decreased to 160 mg/doe and 130 mg/doe for Groups 2 and 3, respectively. The numbers of small, medium and large follicles were determined by real-time B mode ultrasonography. Ovum pick-up (OPU) was performed by laparoscopy under general anesthesia at 96, 42 and 36 h after the initial dose of FSH for Group 1, 2 and 3, respectively. The numbers of recovered cumulus-oocyte-complexes (COCs) and their quality were examined. The COCs were further matured and fertilised in vitro. It was found that the numbers of small- and medium-sizes follicles in Group 2 and 3 were significantly higher than those in Group 1, whereas the numbers of large follicles in Group 1 were significantly higher than those in the other groups (p < 0.05). There was no significant difference in oocyte recovery rates and the quality of COCs among groups (p > 0.05). However, the low-dose FSH (Group 3) tended to enhance oocyte recovery rates and oocyte quality compared to Group 1 (p = 0.07 and 0.06, respectively). Although the meiotic competence of recovered oocytes from Group 3 was similar to that of Group 1, cleavage rates were significantly greater than those in Group 1 (52.30 vs. 30.15%, respectively). In conclusion, ovarian stimulation with a low dose of FSH, related to the interval time between the first FSH injection and the OPU, improved oocyte recovery rates and the developmental competence of goat oocytes.

Worldwide heat stress (HS) conditions have a negative impact on dairy cow fertility. However, understanding of the effect of heat stress on endometrial functions is still unclear. The present study aimed to investigate the effects of differential heat exposure conditions on the immune response and prostaglandin biosynthesis of bovine endometrium challenged with bacterial lipopolysaccharide (LPS). Cultures of endometrial cells were grown to confluence at 37 °C (control) and 40.4 °C for 24 h after confluence (short-term heat exposure) and 40.4 °C for 8 days from the beginning of the culture (long-term heat exposure), prior to a challenge by 100 ng/mL LPS for 12 h. LPS altered ALOX12, IL8, IL1B, S100A8, PTGES and AKR1B1 expressions, as well as secretory IL8 and PGF2α. Short-term heat exposure decreased S100A8, IL8 and PGF2α compared with the control temperature, while long-term heat exposure decreased S100A8 and PGF2α. In contrast, HSPA5 expression was not altered by heat exposure or LPS. Indeed, the short-term heat treatment was insufficient for accomplishing the responses of the endometrium to LPS treatment for IL8, S100A8 and PTGES expressions when compared with other temperature conditions. Our findings showed that heat exposure could compromise endometrium immune response and prostaglandin biosynthesis in different ways based on elevated temperature duration, which could reduce subsequent fertility.

Overall efficiency of cell reprogramming for porcine fibroblasts into induced pluripotent stem cells (iPSCs) is currently poor, and few cell lines have been established. This study examined gene expression during early phase of cellular reprogramming in the relationship to the iPSC colony morphology and in vitro pluripotent characteristics. Fibroblasts were reprogrammed with OCT4, SOX2, KLF4 and c-MYC. Two different colony morphologies referred to either compact (n = 10) or loose (n = 10) colonies were further examined for proliferative activity, gene expression and in vitro pluripotency. A total of 1,697 iPSC-like colonies (2.34%) were observed after gene transduction. The compact colonies contained with tightly packed cells with a distinct-clear border between the colony and feeder cells, while loose colonies demonstrated irregular colony boundary. For quantitative expression of genes responsible for early phase cell reprogramming, the Dppa2 and EpCAM were significantly upregulated while NR0B1 was downregulated in compact colonies compared with loose phenotype (p < .05). Higher proportion of compact iPSC phenotype (5 of 10, 50%) could be maintained in undifferentiated state for more than 50 passages compared unfavourably with loose morphology (3 of 10, 30%). All iPS cell lines obtained from these two types of colony morphologies expressed pluripotent genes and proteins (OCT4, NANOG and E-cadherin). In addition, they could aggregate and form three-dimensional structure of embryoid bodies. However, only compact iPSC colonies differentiated into three germ layers. Molecular signature of early phase of cell reprogramming coupled with primary colony morphology reflected the in vitro pluripotency of porcine iPSCs. These findings can be simply applied for pre-screening selection of the porcine iPSC cell line.

Background: Cryptorchid testes in most mammals show the arrested spermatogenesis except a few mammals including elephants because of the heat. Induced stress by heat shock in the cryptorchid testes usually eliminates the germ cells by apoptosis or to protect the germ cells from the heat shock by an increase in the expression of heat shock factors (HSFs) and heat shock proteins (HSPs). Previously we showed that elephant spermatogenic cells conserve the same pathway to eliminate the defected germ cells as in the other animals although the ratio of apoptosis was low and they did not maintain the same pathway to protect cells through Hsp70 as shown in the other animals.

Although the interspecies hybridization of bovids, such as cattle-yak (Bos taurus x Bos grunniens), has heterosis benefits, the infertility of hybrid males affects the maintenance of dominant traits in subsequent generations. To achieve reproductive capacity, male germ cell development requires coordinated changes in gene expression, including DNA methylation and generalized histone modifications. Although gene expression-related mechanisms underlying hybrid male sterility have been investigated recently, information on the cell types and stage-specific controls remains limited. Here, we used immunohistochemistry and image analyses to evaluate the 5-methylcytosine (5MC) and acetyl-histone H3 Lys9 (AcK9) expression in all spermatogonia and testicular somatic cell types to determine their roles in cattle-yak spermatogenesis. Testicular tissues from yak (1-3 years old) and backcrossed hybrids (2 years old) were used. In yak, the AcK9 expression levels increased in all cell types during maturation, but the 5MC expression levels did not change until reaching 3 years when they increased in all testicular cell types, except spermatogonia. Cattle-yak hybrids showed higher 5MC expression levels and different AcK9 expression levels in all cell types compared to the same-aged yak. These results suggested that both gene modulation by AcK9 and constant levels of DNA methylation are required for spermatogenesis during maturation in yak. Therefore, inappropriate expression levels of both AcK9 and DNA methylation might be the major factors for disruption of normal germ cell development in cattle-yak. Additionally, various modulations occurred depending on the cell type. Further experiments are needed to identify the stage-specific gene expression modulations in each cell type in yak and cattle-yak to potentially solve the infertility issue in crossbreeding.

The Malayan tapir is a large endangered herbivore native to South-east Asia with fewer than 2500 animals remaining in the wild. Although a small number of animals (183 animals held by 60 institutions) are managed in zoos and breeding centres, there is limited information on the fundamental reproductive biology of this species. The purpose of this present study was to evaluate the associations of reproductive protein biomarkers (CRISP2 and CRISP3) in the seminal plasma and spermatozoa with reproductive characteristics in male Malayan tapirs. Ejaculates were collected from zoo-housed animals by electroejaculation and assessed for sperm motility and quality traits. Seminal plasma and sperm pellets were analysed for CRISP protein expression by immunoblotting. The reproductive tract of a single animal was also analysed for CRISP2 and CRISP3 protein expression and localization by immunohistochemistry. Our results showed that both CRISP2 and CRISP3 are expressed in the seminal plasma and spermatozoa derived from Malayan tapirs. CRISP expression was positively correlated with semen quality, especially ejaculate volume, number of motile sperm, and acrosomal integrity. In addition, CRISP2 and CRISP3 protein expression were slightly high in males that had recently sired an offspring. The results suggest that CRISP proteins may serve as biomarkers for ejaculate quality and fertility in male Malayan tapirs. These findings may have significant implications for planning future breeding and re-introduction efforts for this species.

One of the causes of infertility in boars is nutritional deficiency. Interestingly, zinc has been used for improving semen quality in boars. Thus, the objective of this study was to investigate the effect of zinc as feed supplementation in order to improve semen quality in infertile boars. Three purebred Pietrain boars with a poor semen quality history were selected for the experiment. The semen samples were collected once a week for 4 weeks before the treatment period and the semen evaluated immediately. Fifteen grams (a total concentration of 150 ppm) of zinc were supplemented daily in the normal feed. The testing period was 16 consecutive weeks. The semen samples were evaluated for volume, concentration, motility (by computer-assisted sperm assay), sperm viability (by SYBR-green staining), acrosome integrity (by FITC-PNA staining), mitochondrial activity (by JC-1 staining) and sperm morphology (by Giemsa's staining for sperm head and formal saline fixation for sperm tail). The semen samples were also preserved in commercial semen extender and stored at 16 °C for three days after ejaculation. The results showed that zinc supplementation significantly improved acrosome integrity (40.38 ± 7.13% and 70.56 ± 21.36%, respectively) and sperm viability (66.38 ± 5.46% and 79.66 ± 4.11%, respectively) at the ejaculation date and also significantly enhanced acrosome integrity after three days preservation (27.84 ± 7.78% and 67.36 ± 22.43%, respectively). In addition, we also found an increasing trend in acrosome integrity, sperm viability and progressive motility after 4 to 8 weeks of zinc supplementation. This study revealed that daily zinc addition can improve semen quality in infertile boars and can be used on farms to improve semen quality.

We previously demonstrated that the histone deacetylase inhibitor, trichostatin A (TSA), improves the development of cat-cow interspecies somatic cell nuclear transfer (iSCNT) embryos. In this study, we investigated if 50 nM TSA treatment for 24 h would improve the rate of embryo development to the blastocyst stage in cat-pig iSCNT embryos, compared with cat-cow iSCNT embryos under same treatment. Despite the SCNT technique (Piezo technique) differing from our previous study (conventional technique), we confirmed that TSA supplementation at 50 nM improved the development of cat-cow iSCNT blastocysts. Porcine oocytes showed the capability to support cat iSCNT embryo development up to the blastocyst stage without TSA treatment although bovine oocytes did not. The blastocyst developmental rate of TSA treated cat-pig iSCNT embryos was significantly lower than that of the non-treated groups (0.68 and 7.95%, respectively, p < 0.05). We used in vitro-fertilized (IVF) porcine embryos to compare the effects of acetylation levels on H3K9 between cat-pig iSCNT embryos and naturally fertilized embryos. We found no differences in acetylation levels of H3K9 between the TSA treated and non-treated iSCNT groups, and the levels were lower than that of IVF embryos. In conclusion, the effects of TSA treatment on iSCNT embryo production were found to be species-specific and dependent on individual characteristics.

The aim of this study was to investigate the effects of encapsulation with different divalent cations and temperatures on the quality of ram spermatozoa during cold storage. Experiment 1: diluted semen was allocated randomly into three groups: control, encapsulation with calcium (Ca 2+ ) alginate and encapsulation with barium (Ba 2+ ) alginate. The samples were stored at two different temperatures (4 °C or 16 °C) for 24 h and subsequently examined for spermatozoa quality. Experiment 2: determination of sperm functionality by means of sperm binding was performed. Spermatozoa with and without Ba 2+ alginate were cooled for 24 h and then used for sperm binding assay. Experiment 1: the progressive motility of spermatozoa in Ba 2+ alginate stored at 16 °C was better than that stored at 4 °C (P = 0.042). Encapsulation with Ba 2+ alginate and storage at 16 °C significantly improved progressive motility when compared to Ca 2+ alginate ( P < 0.001). The motility characteristics of curve velocity, straight linear velocity and amplitude lateral head displacement of Ba 2+ alginate at 16 °C were higher than Ca 2+ alginate (P = 0.045, P = 0.005 and P = 0.013, respectively). A low storage temperature (4 °C and 16 °C) did not markedly decrease the viability or acrosome integrity of spermatozoa, irrespective the type of crosslinking. Although the spermatozoa released from Ba 2+ alginate were motile and could bind to the zona pellucida, the numbers of bound spermatozoa were significantly lower than in the control group ( P < 0.001, Experiment 2). In conclusion, Ba 2+ alginate is preferable to Ca 2+ alginate for the encapsulation of ram spermatozoa with cold storage at 16 °C.

The aim of the present study was to determine the most suitable embryonic stage and embryo freezing technique for commercial implementation of frozen embryo trading by small-scale sheep producers. There was a 2 x 2 factorial design utilized for conducing the study consisting of two embryo stages (2 - 8 cells or morula/blastocyst) and two cryopreservation protocols (vitrification or slow-freezing). For the in vivo produced embryos, there were treatments of crossbred donor ewes to induce superovulation. Embryos were recovered surgically on either Day 2 or 5.5 after estrous onset. The embryos were cryopreserved using either a vitrification or slow-freezing method before there was transfer to recipients. Ovarian response, embryo survival and lambing outcomes were analyzed. There were no differences in number of recovered and fertilized embryos at the two embryonic developmental stages. There were no effects of embryonic stages and cryopreservation methods on pregnancy rate, twinning rate, fetal birth weights and lamb weight at 1 month of age. When there was use of vitrified embryos for transfers, there was a greater lamb weight at 2 months of age (8.38 +/- 0.20 compared with 7.78 +/- 0.21 kg; P = 0.044) than when there was transfer of embryos cryopreserved using slow freezing procedures. Considering economic and practical benefits to small-scale sheep farms, morula/blastocyst stage-embryo collection and transfer into the uterus is more efficacious than transferring 2 - 8 cells embryos into the oviduct. Results of this study may contribute to the genetic improvement in the flocks of small-scale sheep producers.

The Activin/Nodal signaling pathway is necessary for promoting the self-renewal and pluripotency in several species. However, these effects in porcine stem cells have yet to be examined. We hypothesized that Activin/Nodal signaling could be an important factor for maintenance of porcine induced pluripotent stem cells (iPSCs). The effects of activin A on pluripotency of porcine iPSCs derived from Sertoli cells were examined. Two iPS cell lines were cultured with different culture conditions (control, activin A, TGF-beta 1 inhibitor and activin A combined with TGF-beta 1 inhibitor). The Sertoli iPSCs treated with different conditions were compared for morphology, alkaline phosphatase (AP) activity, OCT4 protein expression and quantitative expression of genes (endogenous pluripotency genes and cell differentiated genes). This study revealed that the activin A treated iPSCs increased intensity of OCT4 protein expression. Furthermore, expressions of the pluripotent genes including OCT4 and NANOG were significantly upregulated in the activin A treated group (P < 0.05). In contrast, the selective TGF-beta 1 inhibitor adversely affected to iPSCs morphology and patterns of the AP and OCT4 protein expression. The TGF-beta 1 inhibitor also resulted in significant upregulation and downregulation for DES (mesoderm differentiation) and GATA 6 (endoderm differentiation), respectively (P < 0.05) when compared with other groups. In conclusion, activin A significantly upregulated the endogenous expression of OCT4 and NANOG pluripotent genes, thereby improving networks of the pluripotency in porcine iPSCs.

At weaning, 150 primiparous sows were assigned sequentially to receive 400 IU equine chorionic gonadotropin (eCG) with 200 IU human chorionic gonadotropin (hCG) (Gn600), or 400 IU eCG with 400 IU hCG (Gn800), or served as untreated controls. Compared with control and Gn600, the Gn800 had increased estrus response by day 7 and reduced wean-to-estrus intervals (P < 0.05). Compared with controls, both Gn600 and Gn800 increased numbers of large follicles (≥6 mm) at estrus detection (P < 0.05) and the subsequent farrowing rates (P < 0.05). Litter size was unaffected. These data demonstrate the efficacy of supplemental hCG for alleviating effects of seasonal infertility in primiparous sows.

Cardiac progenitors are a promising cell source for treating myocardial infarction. In this study, we improved the protocol for cardiac differentiation of rabbit induced pluripotent stem cells (iPSC) using Rho-associated protein kinase (ROCK) inhibitor (Y-27632), low O-2 tension (hypoxia) and melatonin treatments. In experiment 1, the rabbit iPSCs were differentiated into cardiac cell fate via embryoid body (EB) formation with or without ROCK inhibitor. EB diameters were measured on day 3 of differentiation. In experiment 2, the EBs were plated on gelatin-coated dishes and further cultured in different oxygen tensions, hypoxia (5% oxygen) and normoxia (20% oxygen). The plated EBs were examined for proliferative activity and the production of reactive oxygen species (ROS). Experiment 3 studied the effects of oxygen tensions and melatonin on the differentiation of cardiac cell fate in terms of cardiac progenitor gene expression (NKX2.5) and FLK1 positive cells. ROCK inhibitor significantly improved EB formation by mean of increased EB diameter (P<0.05) compared with the control. Hypoxia also significantly increased the numbers of newly DNA synthetic cells indicating greater proliferative activity when compared with normoxia (P<0.05). The melanin treatment during iPSCs differentiation significantly decreased ROS production only in hypoxia (P<0.05). In addition, the combination of hypoxic condition and melatonin treatment significantly upregulated a NKX2.5 cardiac progenitor gene and FLK1 positive cells compared with the controls and normoxia-melatonin treatment (P<0.05). It is concluded that an optimizing condition using a combination of ROCK inhibitor, hypoxic condition and melatonin improved differentiation of rabbit iPSCs towards cardiac progenitor cells.

This study aimed to establish a business model of reproductive biotechnology in small ruminant. Sets of questionnaires were administered to interview 115 farmers from different regions in Thailand. Data including age, educational background, farm size and type, source of replacement breeders, breeding technology, farm problems and attitude toward the technology were collected. Logistic regression analysis along with neutral network analysis was used to identify factors associated technology interest with P < 0.05. Results showed that among 115 participants, 72.2% were interested in reproductive biotechnology. In univariate logistic regression analysis, farmer's age [OR=0.96, 95% CI (0.93,1.00)], educational background [OR=1.17, 95% CI (0.13,0.80)], production problems [OR=1.28, 95% CI (1.12,1.478)] and marketing problems [OR=1.40, 95% CI (1.18,1.67)] were independently associated with technology interest (P < 0.05). Similar to the neural network analysis, farmer's age, overall farm problems, marketing problems, production problems and educational background were the primary factors influencing technology interest of farmers. Next, the data from 18 semi-structured interviews were interpreted to establish suggested business models of reproductive biotechnology package. This indicated that a single business model could not fit the expectation of all farmers. Thus, six business models were established and 2 models were initially implemented in 7 farms with a moderate successful rate. In conclusion, the implement of reproductive biotechnology in small ruminant farm should be addressed to the young farmers with high education to improve the animal genetic value and sustain their livelihoods. However, the different farm managements play a key role in the success of these business models.

Assisted reproductive technologies including superovulation, laparoscopic AI (LAI), and embryo transfer (ET) are important tools for genetic improvement in the sheep industry. The present study aimed to determine the effects of embryo stage and cryopreservation method on field trial outcomes of embryo transfer on small-scale farms in Thailand. Black Dorper ewes (n = 16) were used as donors and mixed breed ewes (n = 21) were used as recipients. Donors were superovulated as previously described (Tríbulo et al. 2012 Theriogenology 77, 1679-1685, 10.1016/j.theriogenology.2011.12.013) and inseminated by LAI within 22 to 24 h after standing heat (Day 0). Donors females were flushed on Day 2 to recover 2- to 8-cell embryos (n = 8) or on Day 6 to recover blastocyst-stage embryos (n = 8). Recovered embryos were randomly cryopreserved by either slow freezing or vitrification methods. Into 21 recipients was inserted an intravaginal device impregnated with CIDR® that was left in place for 10 days. Those received 300 IU of pregnant mare serum gonadotropin (PMSG) on Day 9 and randomly assigned to receive embryos on either Day 2 or 6 after oestrus. Two- to 8-cell embryos were thawed and transferred into the ipsilateral oviduct (n = 1-5 embryos/recipient) on Day 2. Five recipients received 15 slow-frozen embryos and 5 received 13 vitrified embryos. Blastocyst stage embryos were thawed and transferred into the ipsilateral uterine horn (n = 1-4 embryos/recipient) on Day 6. Five recipients received 11 slow-frozen embryos and 4 received 7 embryos. Pregnancy diagnosis was determined by ultrasonography 45 days after embryo transfer. Pregnancy rate was calculated as the proportion of ewes with at least one pregnancy out of the total number of ewes that received embryos. Chi-squared analysis was used to determine the effects of embryo freezing technique and embryo stage on pregnancy rate (SAS 9.2, SAS Institute Inc., Cary, NC, USA). All donor ewes responded to the superovulation program (donor had 3 to 6 corpora lutea). The mean number of viable embryos recovered was 4.3 ± 2.4 and 3.1 ± 3.7 for Day 2 and Day 6, respectively. Nineteen of the 21 recipient ewes responded to the synchronization program and received embryos in the study. There was no effect (P > 0.05) of embryo stage (5/10 = 50% v. 3/9 = 33.3% for 2- to 8-cell v. blastocyst, respectively) or cryopreservation method (4/10 = 40% v. 4/9 = 44.4% for slow-freezing and vitrification, respectively) on pregnancy rate following embryo transfer. Results of the present study suggest that similar pregnancy rates following embryo transfer in sheep under tropical conditions in Thailand can be obtained using either 2- to 8-cell embryos or blastocyst-stage embryos and with embryos that have been cryopreserved by slow-freezing or vitrification. Further research with larger numbers of animals is necessary to confirm the preliminary results of the present study.

The establishment of porcine induced pluripotent stem cells (piPSC) is important in the field of human biomedical regenerative medicine. The pig model is a more representative model than current rodent models because it better mimics human physiology in different organ systems. The piPSC can be traditionally generated by reprogramming somatic cells using 4 transcription factors (4TF: OCT4, SOX2, KLF4, and c-MYC), similarly in human. However, it is difficult to maintain the pluripotent state of reprogrammed cells and they exhibit poor differentiation capacity. Hence, the 4TF may be not enough to reprogram porcine somatic cells. This study aimed to establish piPSC by adding LIN28 (referred to as 5TF) to the traditional 4TF, via retroviral vector. Here, we report the successful establishment of 3 piPSC lines by using the 5TF. All 5TF-piPSC lines exhibited a normal karyotype (38, XY) and a typical mouse embryonic stem cell (ESC) morphology, including tightly packed and dome-like shape, even after they were propagated over 40 passages. All 5TF-piPSC lines were positive for alkaline phosphatase staining and expressed high levels of ESC-like markers (OCT4, SOX2, NANOG, and SSEA-1). Importantly, the 5TF-piPSC lines showed pluripotent capacity, as evidenced by differentiation into 3 germ layers in vitro following cystic embryoid body formation, as well as by efficiently forming teratomas containing all 3 embryonic germ layers in vivo. Moreover, the 5TF-piPSC lines showed spontaneously contractile cardiomyocytes and expressed cardiomyocyte markers (cardiac troponin T) during spontaneous cardiac differentiation using cell aggregation into spherical-like structures referred to as embryoid bodies. Thus, the addition of LIN28 TF promoted long-term pluripotency of piPSC and enhanced the ability to differentiate towards 3 embryonic germ layers and cardiac lineage. This research project is supported by grants from the Mahidol University, Thailand.