The objectives of the present studies were to investigate the developmental capacity of dromedary camel oocytes selected by brilliant cresyl blue (BCB) staining and to investigate the expression of select transcripts in germinal vesicle (GV) stage oocytes. These transcripts included BMP15 and GDF9 as important transcripts for folliculogenesis and oocyte development, Zar1 and Mater as maternal transcripts required for embryonic development, Cyclin B1 and CDK1 as cell cycle regulators and Oct4 and STAT3 as transcription factors. Dromedary camel oocytes were retrieved from ovaries collected at a local slaughterhouse. After exposure to BCB staining, cumulus–oocyte complexes (COCs) from BCB+, BCB− and control (selected based on morphological criteria) groups were subjected to in vitro maturation, in vitro fertilization and in vitro culture. For gene expression studies, after BCB staining cumulus cells were stripped off and the completely denuded GV stage oocytes were used for RT-PCR analysis of selected transcripts. BCB+ oocytes showed higher maturation, and fertilization rates compared with BCB− and control groups. Indices of early embryonic development, namely, cleavage at 48 hours post insemination (hpi), and development to morula at day 5 and day 7 blastocyst rates were also significantly higher in the BCB+ group. RT-PCR revealed a higher expression of BMP15, GDF9, Zar1, Mater, Cyclin B1, CDK1, OCT4 and STAT3 in good quality oocytes that stained positively for BCB (BCB+). Collectively, results provide novel information about the use of BCB screening for selecting good quality oocytes to improve in vitro embryo production in the dromedary camel.

Our objective was to assess the effect of benchtop incubators with low oxygen concentrations on the clinical and embryological parameters of our patients. We conducted a prospective, randomized, opened controlled trial on infertile patients in stimulated cycles. In total, 738 infertile patients were assessed for eligibility and, after final exclusions, 230 patients were allocated either to a 5% O2 group (benchtop incubator) or a 20% O2 group (classic incubator). Finally, 198 patients in the 5% O2 group and 195 in the 20% O2 group were analysed. The outcomes measured were fertilization rate, clinical pregnancy rate, and live birth rate. The primary outcome – live birth rate per all transfers – did not show any improvement in the 5% oxygen group over the 20% oxygen group (25.3% versus 22.6%, P=0.531), but the number of day 5 blastocysts was significantly higher (P=0.009). Fertilization rate did not show any beneficial effect of reduced oxygen (5%) (73.4%±22.4% versus 74.6%±24.0%, P=0.606) per all transfers but there was statistically significant difference in the day 5 SET subgroup (85.3±15.1 versus 75.1±17.5; P=0.004). Clinical pregnancy rate showed results in favour of the 5% oxygen group for all subgroups (day 3: 23.7% versus 21.1%, P=0.701; day 5 SET: 35.0% versus 30.6%. P=0.569) but showed statistical significance only in the day 5 SET subgroup (51.1% versus 29.8%; P=0.038). Culturing of embryos in benchtop incubators under low oxygen produced more blastocysts and therefore was a better alternative for embryo selection, which resulted in higher pregnancy rates. To achieve higher live birth rates, embryo quality is not the only factor.

To explore whether different polyvinylpyrrolidone (PVP) concentrations affect the results of intracytoplasmic sperm injection (ICSI), a prospective study was conducted for 194 couples undergoing 210 ICSI therapy cycles. These cycles were divided into three groups (10, 7 and 5% groups) using the corresponding concentration of PVP for sperm immobilization. The main outcome measures were analyzed. Results indicated that, with a decrease in PVP concentrations, all of the main outcome measures increased. In particular, the high-quality cleavage embryo rate in the 7% group was significantly lower than in the 5% group (P < 0.01), and the cleavage, high-quality cleavage embryo, and high-quality blastocyst rates in the 5% group were significantly higher than those in the 10% group (all P < 0.001). For high-/intermediate-quality semen, all of the main outcome measures were significantly increased with 5% PVP. For the poor-quality semen, only the high-quality cleavage embryo and high-quality blastocyst rates were significantly higher in the 5% group. Therefore, lowering PVP concentrations greatly promoted the development of embryos in ICSI cycles, with an optimal concentration of 5% for ICSI.

Nutrition influences the microenvironment in the proximity of oocyte and affects early embryonic development. Elevated blood urea nitrogen, even in healthy dairy cows, is associated with reduced fertility and there is high correlation between blood urea levels and follicular fluid urea levels. Using a docking calculation (in silico), urea showed a favorable binding activity towards the ZP-N domain of ZP3, that of ZP2, and towards the predicted full-length sperm receptor ZP3. Supplementation of oocyte maturation medium with nutrition-related levels of urea (20 or 40 mg/dl as seen in healthy dairy cows fed on low or high dietary protein, respectively) dose-dependently increased: (i) the proportion of oocytes that remained uncleaved; and (ii) oocyte degeneration; and reduced cleavage, blastocyst and hatching rates. High levels of urea induced shrinkage in oocytes, visualised using scanning electron microscopy. Urea downregulated NANOG while dose-dependently upregulating OCT4, DNMT1, and BCL2 expression. Urea at 20 mg/dl induced BAX expression. Using mathematical modelling, the rate of oocyte degeneration was sensitive to urea levels; while cleavage, blastocyst and hatching rates exhibited negative sensitivity. The present data imply a novel role for urea in reducing oocyte competence and changing gene expression in the resultant embryos.

The aim of our study was to examine whether: (1) the exposure of bovine embryos to the BHV-1 virus in vitro can compromise their further development and alter the ultrastructural morphology of cellular organelles; (2) whether the zona pellucida (ZP) can be a barrier protecting embryos against infection; and (3) whether washing with trypsin after viral exposure can prevent virus penetration inside the embryo and subsequent virus-induced damages. The embryos were recovered from superovulated Holstein-Friesian donor cows on day 6 of the estrous cycle. Only compact morulas or early blastocysts were selected for experiments with virus incubation. We used the embryos either with intact ZP (either with or without trypsin washing) or embryos in which the ZP barrier was avoided by using the microinjection of a BHV-1 suspension under the ZP. ZP-intact embryos (n = 153) were exposed to BHV-1 at 106.16 TCID50/ml for 60 min, then washed in trypsin according to IETS guidelines and postincubated in synthetic oviduct fluid (SOF) medium for 48 h. Some of the embryos (n = 36) were microinjected with 20 pl of BHV-1 suspension under the ZP, the embryos were washed in SOF medium and cultured for 48 h. Embryo development was evaluated by morphological inspection, the presence of viral particles was determined both immunocytochemically, using fluorescent anti-IBR–FITC conjugate and by transmission electron microscopy (TEM) on the basis of the ultrastructure of the cellular organelles.

It was found that BHV-1 exposure impairs embryo development to higher preimplantation stages independent of the presence of the ZP or the trypsin treatment step, as most of the embryos were arrested at the morula stage when compared with the control. Immunofluorescence analysis confirmed the presence of BHV-1 particles in about 75% of embryos that were passed through the trypsin treatment and in all the BHV-1-microinjected embryos. Ultrastructural analysis, using TEM, revealed the presence of virus-like particles inside the BHV-1-exposed embryos, where the trypsin washing step was omitted. Conversely, in trypsin-treated BHV-1-exposed embryos, TEM detected only the envelope-free virus-like particles adhered to pores of the ZP. The embryos that were microinjected with BHV-1 suspension showed the presence of BHV-1 particles, as well as ultrastructural alterations in cell organelles. Taken together these findings may suggest that BHV-1 infection compromises preimplantation development of bovine embryos in vitro and therefore the ZP may not be enough on its own to prevent virus-induced damage, unless it is not accompanied with trypsin washing.

Using the whole-cell voltage-clamp technique to clamp sea urchin oocytes we show that the fertilising spermatozoon triggers an inward current of −521 ± 56.7 pA (n = 8) at activation. Simultaneously, the plasma membrane depolarises and the conductance increases from 23.4 ± 1.4 to 40.6 ± 1.2 nS (n = 8). The I/V curve for the peak activation current is linear and the current reverses between 0 and + 20 mV, suggesting a non-specific ion current. Since injection of inositol triphosphate induced an inward current of −1062 ± 314 pA (n = 4), and the current was inhibited by preloading oocytes with the calcium chelator BAPTA, the non-specific activation current in sea urchin appears to be calcium dependent.

We previously showed in organ culture of testes fragments from Cynops pyrrhogaster that mammalian folicle-stimulating hormone (FSH) stimulates secondary spermatogonia to differentiate into primary spermatocytes. In this report, we demonstrate in organ culture that FSH stimulates DNA synthesis in secondary spermatogonia and Sertoli cells: the numbers of secondary spermatogonia and Sertoli cells incorporating 5-bromo-2′ -deoxyuridine (BrdU)throughout the culture period in the presence of FSH were 3-5 times those incorporating BrdU in the absence of FSH. Moreover, addition of FSH, induced after a day a remarkable increase in the number of spermatogonia and Sertoli cells incorporating BrdU. The above results indicate that FSH stimulates and induces DNA synthesis in spermatogonia and Sertoli cells. Most of the spermatogonia within a cyst were labelled simultaneously and at the same density, indicating that they underwent synchronous DNA synthesis, whereas all the Sertoli cells within a cyst were not labelled simultaneously, indicating that they synthesised DNA asynchronously. When testes fragments pulse-labelled with Brdu were cultured in FSH for 14 days, the secondary spermatogonia differentiated into primary spermatocytes, whereas in the absence of FSH they failed to differentiate and most died by the 7th day. The above results together show that FSH is required for the proliferation of both secondary spermatogonia and Sertoli cells as well as the differentiation of secondary spermatogonia into primary spermatocytes.

In non-human primates, it is difficult to collect sufficient numbers of oocytes for producing identical embryos by somatic cell nuclear transfer (SCNT). Because of this factor, inter-species SCNT (iSCNT) using heterospecific oocytes is an attractive alternative approach. The objective of this study was to produce iSCNT-derived blastocysts using enucleated cow (Bos taurus) metaphase II oocytes and adult rhesus monkey (Macaca mulatta) fibroblasts. Ear skin tissue from a 6-year-old male rhesus monkey was collected by biopsy and fibroblasts were isolated. Immature cumulus–oocyte complexes from cow ovaries were collected and matured in vitro in Medium 199. The enucleated oocytes were reconstructed with rhesus monkey fibroblasts and iSCNT embryos were cultured in modified synthetic oviduct fluid in an atmosphere of 5–5.5% CO2 under various conditions (37–39 °C and 5–20% O2) to examine the effects of in vitro culture conditions. Most embryos were arrested at the 8- or 16-cell stage and only three blastocysts were derived in this way using iSCNT from a total of 1153 cultured activated embryos (0.26% production rate). Two of the three blastocysts were used for counting nuclear numbers using bisbenzimide staining, which were 51 and 24. The other iSCNT-derived blastocyst was used to analyse mitochondrial DNA (mtDNA) by PCR, and both rhesus monkey and cow mtDNA were detected. Although the development rate was extremely low, this study established that iSCNT using two phylogenetically distant species, including a primate, could produce blastocysts. With improvements in the development rate, it may be possible to produce rhesus monkey iSCNT-derived embryonic stem cell lines for studies on primate nucleus and cow mitochondria interaction mechanisms.

Ovarian stimulation with exogenous follicle stimulating hormone (FSH) has been used to increase the number of viable oocytes for laparoscopic oocyte recovery (LOR) in goats. The aim of this study was to evaluate the effect of two FSH protocols for ovarian stimulation in goats on the expression pattern of epidermal growth factor (EGF) receptor (EGFR) in cumulus–oocyte complexes (COCs) recovered by LOR. After real-time qRT-PCR analysis, expression profiles of morphologically graded COCs were compared prior to and after in vitro maturation (IVM) on a FSH protocol basis. The use of a protocol with higher number of FSH injections at a shorter interval resulted in GI/GII COCs with a higher level of EGFR expression in cumulus cells, but not in the oocyte, which was correlated with an elevated meiotic competence following IVM. Based on the maturation profile and EGFR expression patterns observed between groups, the morphological selection of COCs prior to IVM was not a good predictor of oocyte meiotic competence. Therefore, EGFR may be a good candidate marker for indirect prediction of goat oocyte quality. The IVM process of goat COCs increased the EGFR expression in oocytes and cumulus cells, which seemed to be strongly associated with the resumption of meiosis. In summary, differential EGFR expression in goat cumulus cells was associated with the in vivo prematuration process, and in turn, the upregulation in the entire COC was associated with IVM. Cause-and-effect relationships between such increased expression levels, particularly in the oocyte, and oocyte competence itself still need to be further investigated.

The role of mitogen-activated protein (MAP) kinase in mouse egg activation induced by protein kinase inhibitors and a protein tyrosine kinase (PTK) inhibitor was investigated. Separated egg proteins were first probed with anti-Active MAP kinase antibody and then re-probed with anti-ERK2 antibody. Staurosporine and Ro-31-8220, at concentrations that normally inhibit protein kinase C, did not affect egg activation or MAP kinase activity, while higher dosages caused egg activation. Staurosporine at 2 μM induced the metaphase–interphase transition without emission of the second polar body (PB2), while Ro-31-8220 at 40 μM induced PB2 emission, first cleavage, and then the transition to interphase. Half the eggs were also activated by the PTK inhibitor genistein. In each treatment, the proportion of eggs that entered interphase was well correlated with the degree of MAP kinase inactivation. Artificial activation of this kinase by okadaic acid overcame the interphase transition. These data suggest that protein kinase inhibitors and a protein tyrosine kinase inhibitor induce the interphase transition by inactivating MAP kinase in mouse eggs.

Acrosin, an acrosomal serine protease, has been associated with binding of spermatozoa and their penetration through the zona pellucida. This study was aimed at determining whether the remaining proacrosin/acrosin system on rabbit perivitelline spermatozoa still has proteolytic activity and whether this activity is involved in further penetration of unfertilised rabbit eggs. Eight hundred and sixty-five rabbit perivitelline spermatozoa were evaluated by the gelatin-substrate film technique for the detection of acrosin on individual spermatozoan. Fifteen per cent of the studied spermatozoa showed small digestion halos on the gelatin film. The proteolytic activity of rabbit perivitelline spermatozoa was inhibited in the presence of 1 mg/ml of soybean trypsin inhibitor (SBTI) or with 20 μg/ml of a mixture of the monoclonal anti-proacrosin/acrosin antibody. In vitro fertilisation occurred in 21.8% of rabbit oocytes co-incubated with perivitelline spermatozoa and was completely inhibited when oocytes were incubated with 600 μg/ml of a mixture of three anti-acrosin monoclonal antibodies (ACRO-A8C10, ACRO-C2B10 and ACRO-C5F10). Inseminations in the presence of anti-cholera monoclonal antibody (irrelevant to spermatozoa) resulted in 17.6% fertilisation. These results support the idea that the residual proacrosin/acrosin system in perivitelline spermatozoa might be involved in spermatozoal binding and/or second penetration through the zona pellucida.

To determine the effects of sperm DNA fragmentation (SDF) on embryo morphokinetic parameters, cleavage patterns and embryo quality, this retrospective study analyzed 151 intracytoplasmic sperm injection (ICSI) cycles (1152 embryos collected) between November 2016 and June 2019. SDF was assessed using sperm chromatin dispersion. The cycles were divided into two groups based on the SDF rate: SDF < 15% (n = 114) and SDF ≥ 15% (n = 37). The embryo morphokinetic parameters, cleavage patterns, and embryo quality were compared between the two groups. The morphokinetic parameters tPNf, t2, t3, t4, t5, t6, and t8 were achieved significantly earlier in the SDF < 15% group compared with in the SDF ≥ 15% group. The fertilization and 2PN rates seemed to be significantly higher in the SDF < 15% group compared with in the SDF ≥ 15% group, while the abnormal cleavage rates were similar. However, a significantly higher rate of chaotic cleavage (CC) was observed in the SDF ≥ 15% group. The D3 high-quality embryo and available embryo rates were similar between the two groups. The blastocyst formation, high-quality blastocyst, and available blastocyst rates in the SDF < 15% group were significantly higher than those in the SDF ≥ 15% group. With an increase in SDF level, the chemical pregnancy, clinical pregnancy and implantation rates tended to decrease, while the miscarriage rate increased. This study demonstrated that SDF ≥ 15% reduces the fertilization rate of ICSI cycles and affects certain morphokinetic parameters. A higher SDF level can also induce a higher rate of CC, with subsequent decreases in the blastocyst formation rate and blastocyst quality.

During oocyte maturation, the cumulus–oocyte complexes (COCs) expand dramatically. This phenomenon, which is known as cumulus expansion, is the result of the synthesis and accumulation of hyaluronan in the extracellular space between cumulus cells. The purpose of this study was to investigate the effect of 6-diazo-5-oxo-l-norleucine (DON), an inhibitor of hyaluronan synthesis, on cumulus expansion during in vitro porcine oocyte maturation and hyaluronan accumulation within COCs. Further, this study aimed to examine the influence of hyaluronan accumulation within COCs on the rate of oocyte maturation. Cumulus expansion was observed during in vitro maturation. However, the addition of DON to the maturation medium significantly inhibited cumulus expansion. The total inhibition of hyaluronan accumulation within COCs was observed with the use of confocal microscopy. Moreover, a positive correlation between the area of cumulus expansion and the rate of oocyte maturation was observed. These results demonstrate that the hyaluronan accumulation within the COCs during oocyte maturation affects oocyte maturation. On the basis of these results, we propose that hyaluronan accumulation within the COCs during cumulus expansion is a necessary step in the porcine oocyte maturation process.

The aim of this work was to evaluate the effect of cryopreservation protocols on subsequent development of in vitro produced bovine embryos under different culture conditions. Expanded in vitro produced blastocysts (n = 600) harvested on days 7–9 were submitted to controlled freezing [slow freezing group: 10% ethylene glycol (EG) for 10 min and 1.2°C/min cryopreservation]; quick-freezing [rapid freezing group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s]; or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. Control group embryos were not exposed to cryoprotectant or cryopreservation protocols and the hatching rate was evaluated on day 12 post-insemination. In order to evaluate development, frozen–thawed embryos were subjected to granulosa cell co-culture in TCM199 or SOFaa for 4 days. Data were analyzed by PROC MIXED model using SAS Systems for Windows®. Values were significant at p < 0.05. The hatching rate of the control group was 46.09%. In embryos cultured in TCM199, slow freezing and vitrification group hatching rates were 44.65 ± 5.94% and 9.43 ± 6.77%, respectively. In embryos cultured in SOFaa, slow freezing and vitrification groups showed hatching rates of 11.65 ± 3.37 and 8.67 ± 4.47%, respectively. In contrast, the rapid freezing group embryos did not hatch, regardless of culture medium. The slow freezing group showed higher hatching rates than other cryopreservation groups. Under such conditions, controlled freezing (1.2°C/min) can be an alternative to cryopreservation of in vitro produced bovine embryos.

While the freezing techniques of mammal embryos have been providing promising results, the cryopreservation of teleostean eggs and embryos have remained unsuccessful up to now. Therefore, this work aimed to develop a procedure of cryogenic preservation of embryos of Prochilodus lineatus and to observe, at both structural and ultrastructural levels, the morphological alterations that took place after the application of freezing/thawing techniques. The embryos at the morula stage could not tolerate exposure to the cryoprotectants ethylene glycol monomethyl ether, propylene glycol monomethyl ether, methanol, dimethyl sulphoxide and propylene glycol, presenting 100% of mortality. Embryos at the 4- to 6-somites stage tolerated exposure to propylene glycol and dimethyl sulphoxide, and the results revealed no significant differences (α = 0.05) regarding survival from both treatments. None of the freezing, thawing and hydration protocols was effective on preserving embryo viability. The ultrastructural analyses of frozen and thawed embryos showed that cells from ectoderm, somites, notochord and endoderm were structurally intact, with well preserved nuclei and mitochondria. The yolk globules were able to tolerate the freezing process, but the yolk syncytial layer was unorganized, displaying an electron-dense and compacted appearance, collapsed reticules, nuclei with modified chromatin and ruptures on the plasmatic membrane at the contact zone with endoderm. It might be concluded that the procedures tested for freezing were unable to avoid the formation of intracellular ice crystals, leading to drastic morphological modifications and making P. lineatus embryos unviable.

Spermatogenesis is a highly co-ordinated and complex process. In vitro propagation of spermatogonial stem cells (SSCs) could provide an avenue in which to undertake in vivo studies of spermatogenesis. Very little information is known about the SSC biology of teleosts. In this study, collagenase-treated testicular cells of farmed catfish (Clarias batrachus, popularly known as magur) were purified by Ficoll gradient centrifugation followed by magnetic activated cell sorting using Thy1.2 (CD90.2) antibody to enrich for the spermatogonial cell population. The sorted spermatogonial cells were counted and gave ~3 × 106 cells from 6 × 106 pre-sorted cells. The purified cells were cultured in vitro for >2 months in L-15 medium containing fetal bovine serum (10%), carp serum (1%) and other supplements. Microscopic observations depicted typical morphological SSC features, bearing a larger nuclear compartment (with visible perinuclear bodies) within a thin rim of cytoplasm. Cells proliferated in vitro forming clumps/colonies. mRNA expression profiling by qPCR documented that proliferating cells were Plzf + and Pou2+, indicative of stem cells. From 60 days onwards of cultivation, the self-renewing population differentiated to produce spermatids (~6 × 107 on day 75). In vitro-produced sperm (2260 sperm/SSC) were free swimming in medium and hence motile (non-progressive) in nature. Of those, 2% were capable of fertilizing and generated healthy diploid fingerlings. Our documented evidence provides the basis for producing fertile magur sperm in vitro from cultured magur SSCs. Our established techniques of SSC propagation and in vitro sperm production together should trigger future in vivo experiments towards basic and applied biology research.

Sea urchin spermatozoa were injected into mature mouse oocytes to determine whether they can activate mouse oocytes and, if so, how they behave within the oocyte cytoplasm of such a distant species. While injection of a single spermatozoon into each oocyte did not activate any of the oocytes, injection of 10 spermatozoa activated about 20%. Within the cytoplasm of unactivated oocytes, sperm heads commonly transformed into chromosome-like structures. When a single spermatozoon was injected, and oocytes were then activated by Sr2+, about 30% of the activated oocytes had both female (mouse) and male (sea urchin) pronuclei when examined 8 h after sperm injection. These results indicated that sperm-borne oocyte activating factor(s) and the cytoplasmic factors controlling the development of the sperm pronucleus are not strictly species-specific.

While the first attempt to cool and preserve stallion semen was done 200 years ago (by Spallazani) and the first pregnancy achieved with frozen-thawes stallion semen almost 40 years ago (Barker & Gandier, 1957), frozen stallion semen has not been used widely, mainly because of its association with low fertility.

In marine animals with external fertilization, gametes are released into seawater where fertilization and embryo development occur. Consequently, pollutants introduced into the marine environment by human activities may affect gametes and embryos. These xenobiotics can alter cell physiology with consequent reduction of fertilization success. Here the adverse effects on the reproductive processes of the marine invertebrate Ciona intestinalis (ascidian) of different xenobiotics: lead, zinc, an organic tin compound and a phenylurea herbicide were evaluated. By using the electrophysiological technique of whole-cell voltage clamping, the effects of these compounds on the mature oocyte plasma membrane electrical properties and the electrical events of fertilization were tested by calculating the concentration that induced 50% normal larval formation (EC50). The results demonstrated that sodium currents in mature oocytes were reduced in a concentration-dependent manner by all tested xenobiotics, with the lowest EC50 value for lead. In contrast, fertilization current frequencies were differently affected by zinc and organic tin compound. Toxicity tests on gametes demonstrated that sperm fertilizing capability and fertilization oocyte competence were not altered by xenobiotics, whereas fertilization was inhibited in zinc solution and underwent a reduction in organic tin compound solution (EC50 value of 1.7 µM). Furthermore, fertilized oocytes resulted in a low percentage of normal larvae with an EC50 value of 0.90 µM. This study shows that reproductive processes of ascidians are highly sensitive to xenobiotics suggesting that they may be considered a reliable biomarker and that ascidians are suitable model organisms to assess marine environmental quality.

This study was performed to improve production efficiency at the level of recipient pig and donor nuclei of transgenic cloned pigs used for xenotransplantation. To generate transgenic pigs, human endothelial protein C receptor (hEPCR) and human thrombomodulin (hTM) genes were introduced using the F2A expression vector into GalT–/–/hCD55+ porcine neonatal ear fibroblasts used as donor cells and cloned embryos were transferred to the sows and gilts. Cloned fetal kidney cells were also used as donor cells for recloning to increase production efficiency. Pregnancy and parturition rates after embryo transfer and preimplantation developmental competence were compared between cloned embryos derived from adult and fetal cells. Significantly higher parturition rates were shown in the group of sows (50.0 vs. 4.1%), natural oestrus (20.8 vs. 0%), and ovulated ovary (16.7 vs. 5.6%) compared with gilt, induced and non-ovulated, respectively (P < 0.05). When using gilts as recipients, final parturitions occurred in only the fetal cell groups and significantly higher blastocyst rates (15.1% vs. 21.3%) were seen (P < 0.05). Additionally, gene expression levels related to pluripotency were significantly higher in the fetal cell group (P < 0.05). In conclusion, sows can be recommended as recipients due to their higher efficiency in the generation of transgenic cloned pigs and cloned fetal cells also can be recommended as donor cells through correct nuclear reprogramming.