Detection and Identification of Sarcocystis cruzi (Protozoa: Apicomplexa) by Molecular and Ultrastructural Studies in Naturally Infected Korean Cattle (Bos taurus coreanae) from Daejeon, Korea.

To survey the prevalence of Sarcocystis infections, 210 heart samples were collected from Korean native cattle (Bos taurus coreanae) at an abattoir in Daejeon Metropolitan City, Republic of Korea. Sarcocysts were detected form 31 specimens (14.8%) and identified as Sarcocystis cruzi via transmission electron microscopy. The wall of S. cruzi has flattened protrusions that did not contain fibrils or microfilaments. The protrusions arose irregularly from the base, contained a fine granular substance, lacked internal microfilaments, and measured approximately 0.21-1.25 µm in length and 0.05-0.07 µm in width. Sequence analysis revealed 99.5% homology to S. cruzi. This is the first report on the prevalence of S. cruzi in native cattle from the Republic of Korea.

Influences of somatic donor cell sex on in vitro and in vivo embryo development following somatic cell nuclear transfer in pigs.

OBJECTIVE: The present study investigates pre- and post-implantation developmental competence of nuclear-transferred porcine embryos derived from male and female fetal fibroblasts. METHODS: Male and female fetal fibroblasts were transferred to in vitro-matured enucleated oocytes and in vitro and in vivo developmental competence of reconstructed embryos was investigated. And, a total of 6,789 female fibroblast nuclear-transferred embryos were surgically transferred into 41 surrogate gilts and 4,746 male fibroblast nuclear-transferred embryos were surgically transferred into 25 surrogate gilts. RESULTS: The competence to develop into blastocysts was not significantly different between the sexes. The mean cell number of female and male cloned blastocysts obtained by in vivo culture (143.8±10.5 to 159.2±14.8) was higher than that of in vitro culture of somatic cell nuclear transfer (SCNT) groups (31.4±8.3 to 33.4±11.1). After embryo transfer, 5 pregnant gilts from each treatment delivered 15 female and 22 male piglets. The average birth weight of the cloned piglets, gestation length, and the postnatal survival rates were not significantly different (p<0.05) between sexes. CONCLUSION: The present study found that the sex difference of the nuclear donor does not affect the developmental rate of porcine SCNT embryos. Furthermore, postnatal survivability of the cloned piglets was not affected by the sex of the donor cell.

Differential gene-expression profiles from canine cumulus cells of ovulated versus in vitro-matured oocytes.

We compared the nuclear maturation status and gene-expression profiles of canine cumulus cells (CCs) derived from cumulus-oocyte complexes (COCs) that were spontaneously ovulated versus those that were matured in vitro. Cumulus-oocyte complexes were retrieved from uteri by surgical flushing (after spontaneous ovulation) or by ovariectomy follicle aspiration and in vitro maturation. The objective of Experiment 1 was to investigate the nuclear maturation status of in vivo- versus in vitro-matured oocytes. The objective of Experiment 2 was to compare gene-expression profiles of CCs derived from in vivo- versus in vitro-matured COCs. Genes analysed are related to cell maturation, development and apoptosis, including GDF9, MAPK1, PTX3, CX43, Bcl2 and BAX; mRNA expression for all of these genes, except for GDF9, differed (P<0.05) between in vivo- and in vitro-matured CCs. In conclusion, we found that gene-expression profiles are related to the quality of CCs and therefore posit that monitoring gene expression could be a useful strategy to guide attempts to improve in vitro culture systems.

Identification of bovine viral diarrhea virus infection in Saanen goats in the Republic of Korea.

Bovine viral diarrhea virus (BVDV) is one of the most important viral pathogens of livestock and causes substantial economic losses to the livestock industry worldwide. BVDV is not necessarily species specific and is known to infect domesticated and wild ruminants. In the present study, BVDV infection was identified in two Saanen goats from one farm, and two different viral subtypes were found, BVDV-1a and BVDV-2a. Each isolate was closely related to cattle isolates identified in the Republic of Korea. The two sequences obtained in this study were not consistent with border disease virus (BDV). The incidence of BVDV in this farm apparently occurred in the absence of contact with cattle and may be associated with grazing. This study demonstrates that BVDV infection may be possible to transmit among goats without exposure to cattle. Therefore, this result indicates that Saanen goats may act as natural reservoirs for BVDV. This is the first report of BVDV-1a infection in a Saanen goat.

Two cases of meningocele and meningoencephalocele in Jeju native pigs.

BACKGROUND: Meningocele and meningoencephalocele of the skull are congenital deformities. Various species, such as pigs, dogs, and cats, are susceptible to congenital meningocele and meningoencephalocele and the incidence is higher in large white and landrace pigs. CASE PRESENTATION: In this study, swelling was observed in the fontanel areas of the median planes of the skull cap in two female piglets of the same litter. Gross clinical examination, neurological examination, computed tomography (CT), and magnetic resonance imaging (MRI) were conducted on the symptomatic piglets. The gross clinical and neurological examinations revealed no specific findings, except for the swellings. According to the CT results, the length of the defect on the sagittal section of the skull was 4.7 mm in case 1 and 20.62 mm in case 2. Connected flow between the skull swellings and the cerebrospinal fluid (CSF) of the lateral ventricles was observed, and partial herniation was identified in case 2. On MRI, CSF with high T2 signals was identified in the arachnoid spaces between the cerebrum and the cerebellum in the two cases, which is consistent with intracranial hypertension. The size of the swelling formed in the parietal bones was 1.6 × 1.1 × 1.8 cm(3) (case 1) and 1.2 × 1.38 × 1.7 cm(3) (case 2). The increase in intracranial pressure was more obvious in case 2 than in case 1, and was accompanied by posterior displacements of the mesencephalon and cerebellum. CONCLUSIONS: Case 1 was diagnosed as meningocele resulting from meningeal herniation and case 2 was diagnosed as meningoencephalocele caused by brain tissue herniation.

Detection of Tick-Borne Pathogens in the Korean Water Deer (Hydropotes inermis argyropus) from Jeonbuk Province, Korea.

The objective of this study was to investigate the prevalence of tick-borne pathogens in the Korean water deer (Hydropotes inermis argyropus). Pathogens were identified using PCR which included Anaplasma, Ehrlichia, Rickettsia, and Theileria. Rickettsia was not detected, whereas Anaplasma, Ehrlichia, and Theileria infections were detected in 4, 2, and 8 animals, respectively. The most prevalent pathogen was Theileria. Of the 8 Theileria-positive animals, 2 were mixed-infected with 3 pathogens (Anaplasma, Ehrlichia, and Theileria) and another 2 animals showed mixed-infection with 2 pathogens (Anaplasma and Theileria). Sequencing analysis was used to verify the PCR results. The pathogens found in this study were identified as Anaplasma phagocytophilum, Ehrlichia canis, and Theileria sp. To the best of our knowledge, this is the first report identifying these 3 pathogens in the Korean water deer. Our results suggest that the Korean water deer may serve as a major reservoir for these tick-borne pathogens, leading to spread of tick-borne diseases to domestic animals, livestock, and humans. Further studies are needed to investigate their roles in this respect.

A Horsehair Worm, Gordius sp. (Nematomorpha: Gordiida), Passed in a Canine Feces.

Nematomorpha, horsehair or Gordian worms, include about 300 freshwater species in 22 genera (Gordiida) and 5 marine species in 1 marine genus (Nectonema). They are parasitic in arthropods during their juvenile stage. In the present study, the used gordian worm was found in the feces of a dog (5-month old, male) in July 2014. Following the worm analysis using light and scanning electron microscopes, the morphological classification was re-evaluated with molecular analysis. The worm was determined to be a male worm having a bi-lobed tail and had male gonads in cross sections. It was identified as Gordius sp. (Nematomorpha: Gordiidae) based on the characteristic morphologies of cross sections and areole on the cuticle. DNA analysis on 18S rRNA partial sequence arrangements was also carried out, and the gordiid worm was assumed to be close to the genus Gordius based on a phylogenic tree analysis.

Detection of Anaplasma sp. in Korean Native Goats (Capra aegagrus hircus) on Jeju Island, Korea.

Anaplasma species are obligate intracellular pathogens that can cause tick-borne diseases in mammalian hosts. To date, very few studies of their occurrence in Korean native goats (Capra aegagrus hircus) have been reported. In the present study, we investigated Anaplasma infection of Korean native goats on Jeju Island, Republic of Korea, and performed phylogenetic analysis based on the 16S rRNA gene sequences. Our results showed that Anaplasma infection was found mostly in adult female goats. The phylogenetic tree revealed that the 7 sequences identified in Korean native goats could belong to Anaplasma sp. and were distinct from A. marginale, A. centrale, and A. ovis. The results indicated that the sequences identified to belong to Anaplasma were closely related to sequences isolated from goats in China and were clustered within the same group. To our knowledge, this is the first study to detect Anaplasma sp. infection in Korean native goats.

Plum-Derived Exosome-like Nanovesicles Induce Differentiation of Osteoblasts and Reduction of Osteoclast Activation.

Osteoblasts and osteoclasts play crucial roles in bone formation and bone resorption. We found that plum-derived exosome-like nanovesicles (PENVs) suppressed osteoclast activation and modulated osteoblast differentiation. PENVs increased the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells and osteoblasts from mouse bone marrow cultures. Notably, PENVs elevated the expression of osteoblastic transcription factors and osteoblast differentiation marker proteins in MC3T3-E1 cells. Higher levels of phosphorylated BMP-2, p38, JNK, and smad1 proteins were detected in PENV-treated MC3T3-E1 cells. Additionally, the number of TRAP-positive cells was significantly decreased in PENV-treated osteoclasts isolated from osteoblasts from mouse bone marrow cultures. Importantly, osteoclastogenesis of marker proteins such as PPAR-gamma, NFATc1, and c-Fos were suppressed by treatment with PENVs (50 µg/mL). Taken together, these results demonstrate that PENVs can be used as therapeutic targets for treating bone-related diseases by improving osteoblast differentiation and inhibiting osteoclast activation for the first time.

Robust Three-Dimensional (3D) Expansion of Bovine Intestinal Organoids: An In Vitro Model as a Potential Alternative to an In Vivo System.

Intestinal organoids offer great promise for disease-modelling-based host-pathogen interactions and nutritional research for feed efficiency measurement in livestock and regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterisation and three-dimensional (3D) expansion of adult stem cells in livestock species compared to other species. Intestinal crypts derived from intestinal organoids under a 3D culture system from the small intestine in adult bovine were successfully established and characterised for functionality testing, including the cellular potentials and genetic properties based on immunohistochemistry, immunocytochemistry, epithelial barrier permeability assay, QuantSeq 3' mRNA-Seq. data and quantitative reverse transcription-polymerase chain reaction. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts, and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium, and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen and revealed that the genetic properties of bovine intestinal organoids were highly similar to those of in vivo. Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from the small intestine and robust 3D expansion of intestinal organoids in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo systems and will be facilitated as the practical model to replace animal experiments for various purposes in the fields of animal biotechnology.

Structural and functional characterization of recombinant human growth hormone isolated from transgenic pig milk.

This study aimed to establish and reproduce transgenic pigs expressing human growth hormone (hGH) in their milk. We also aimed to purify hGH from the milk, to characterize the purified protein, and to assess the potential of our model for mass production of therapeutic proteins using transgenic techniques. Using ~15.5 L transgenic pig milk, we obtained proteins with ≥ 99% purity after three pre-treatments and five column chromatography steps. To confirm the biosimilarity of our milk-derived purified recombinant hGH (CGH942) with commercially available somatropin (Genotropin), we performed spectroscopy, structural, and biological analyses. We observed no difference between the purified protein and Genotropin samples. Furthermore, rat models were used to assess growth promotion potential. Our results indicate that CGH942 promotes growth, by increasing bone development and body weight. Toxicity assessments revealed no abnormal findings after 4 weeks of continuous administration and 2 weeks of recovery. The no-observed-adverse-effect level for both males and females was determined to be 0.6 mg/kg/day. Thus, no toxicological differences were observed between commercially available somatropin and CGH942 obtained from transgenic pig milk. In conclusion, we describe a transgenic technique using pigs, providing a new platform to produce human therapeutic proteins.

Animal cloning by somatic cell nuclear transfer.

Animal cloning is becoming increasingly useful for its applications in biological inquiry and for its potential use in pharmaceutical, medical, and agricultural fields. Due to the complexity of the numerous steps required in reconstructing oocytes by nuclear transfer, detailed protocols are required to minimize the developmental damages inflicted during these manipulations and to standardize procedures across laboratories. Moreover, because oogenesis and early embryogenesis differ widely among mammalian species, it is essential that protocols be adapted according to each species concerned. Our objective here is to detail the protocols that have been most successful in producing laboratory and domestic animal clones.

In vitro differentiation of mesenchymal progenitor cells derived from porcine umbilical cord blood.

Mesenchymal stem/progenitor cells (MPCs) were isolated from porcine umbilical cord blood (UCB) and their morphology, proliferation, cell cycle status, cell-surface antigen profile and expression of hematopoietic cytokines were characterized. Their capacity to differentiate in vitro into osteocytes, adipocytes and chondrocytes was also evaluated. Primary cultures of adherent porcine MPCs (pMPCs) exhibited a typical fibroblast-like morphology with significant renewal capacity and proliferative ability. Subsequent robust cell growth was indicated by the high percentage of quiescent (G0/G1) cells. The cells expressed the mesenchymal surface markers, CD29, CD49b and CD105, but not the hematopoietic markers, CD45 and CD133 and synthesized hematopoietic cytokines. Over 21 days of induction, the cells differentiated into osteocytes adipocytes and chondrocytes. The expression of lineage specific genes was gradually upregulated during osteogenesis, adipogenesis and chondrogenesis. We conclude that porcine umbilical cord blood contains a population of MPCs capable of self-renewal and of differentiating in vitro into three classical mesenchymal lineages.

Rapid exacerbation of renal function after administration of hydroxyethyl starch in a dog.

Hydroxyethyl starches (HES) are commonly used synthetic colloidal solution in veterinary medicine. Despite of possible adverse effect to kidney injury in human, there is no report about nephrotoxic effects of HES in dogs. HES was administered to a Golden retriever (4-year-old, intact male) with ascites in order to increase plasma osmolality. Initially, the dog was mild azotemic, however, kidney function was rapidly deteriorated after several days of HES administration. Finally, histopathological examination revealed remarkable osmotic nephrosis. In the case reported herein, acute kidney injury was remarkably developed after HES administration. Clinical and histopathologic findings of acute kidney injury support nephrotoxic effects of HES to a dog.

Seasonal changes in hemograms and Theileria orientalis infection rates among Holstein cattle pastured in the mountains in the Republic of Korea.

In the current study, we compared seasonal changes in complete blood counts (CBCs) and rates of infection with a tick-borne pathogen between Holstein cattle housed indoors and those maintained outside on pasture. There were differences in white blood cell (WBC) parameters, but the changes were not associated with seasons or the housing type. Analysis of red blood cell (RBC) parameters showed lower values in August and November versus March, and in the cattle maintained on pasture versus the housed cattle. In comparison with the RBC count of the housed cattle in March (10.1M/µL), the RBC counts of the pastured cattle were significantly lower in August (7.8M/µL; p<0.01) and November (7.5M/µL; p<0.01). The hematocrit (HCT) also showed a decrease in March (33.5%), August (30.0%, p<0.01) and November (28.5%, p<0.01). According to PCR analysis, the Theileria infection rate among the pastured cattle in March was only 11%, but this rate increased to 22% and 60% in August and November, respectively. The RBC count (7.4M/µL) and HCT (27.7%) values in Theileria-positive pastured cattle in November showed a dramatic decrease compared to those of cattle examined in March. Phylogenetic analysis revealed that these Theileria isolates correspond to T. orientalis. These results suggest that a remarkable increase in tick infestation in mountainous areas in the summer may cause increased rates of infection with T. orientalis, leading to significant changes in the RBC profile after grazing. Therefore, these hematological changes may be associated with T. orientalis infection caused by tick-biting; thus, additional studies on the pathogenicity of T. orientalis are needed.

The effects of artificial activation timing on the development of SCNT-derived embryos and newborn piglets.

This study investigated the effects of two different activation regimens on the developmental potential of somatic cell nuclear transfer (SCNT) embryos and postnatal survivability of the cloned piglets. In vitro matured oocytes were enucleated and reconstructed with porcine fetal fibroblasts. On the basis of the activation regimen used, the reconstructed porcine embryos were allocated into two groups: Group 1-simultaneous electrical pulses and activation group (SFA group); and Group 2-electrical fusion without calcium followed by electrical pulses with calcium after colcemid and cytochalasin B treatment for 5h (DA group). Embryonic development in both SFA and DA groups was determined at day 6 of culture in NSCU-23 medium. To investigate the post-implantation development after the two activation methods, embryos were cultured for 1 day and then transferred into the oviducts of estrus-synchronized recipients. DA group had significantly (p<0.05) higher cleavage rates than SFA group. However, the developmental rate to the blastocyst stage and the mean cell number of blastocysts did not differ (p>0.05) between SFA and DA groups. Moreover, the pregnancy rate of SFA group was not significantly different compared to DA group. A total of 20 cloned piglets (SFA group-8 live piglets, DA group-11 live piglets and one stillborn) were obtained in the present study. The birth weight of the cloned piglets (live births) did not differ (p>0.05) between the two groups. Furthermore, no difference was observed in the postnatal survival rates of the cloned piglets obtained using two different activation regimens. These results suggest that the timing of artificial activation and additional chemical treatments do not affect the developmental rate of porcine SCNT embryos. Remarkably, the pregnancy rate and postnatal survivability of the cloned piglets did not vary between SFA and DA groups.

Neurogenic and cardiomyogenic differentiation of mesenchymal stem cells isolated from minipig bone marrow.

The present study investigated the potential of minipig bone marrow-mesenchymal stem cells (BM-MSCs) to differentiate in vitro into neuron- and cardiomyocyte-like cells. Isolated BM-MSCs exhibited a fibroblast-like morphology, expressed CD29, CD44 and CD90, and differentiated into osteocytes, adipocytes and chondrocytes. Upon induction in two different neuronal specific media, most of BM-MSCs acquired the distinctive morphological features and positively stained for nestin, neurofilament-M (NF-M), neuronal nuclei (NeuN), ß-tubulin, galactocerebroside (Gal-C) and glial fibrillary acidic protein (GFAP). Expression of nestin, GFAP and NF-M was further demonstrated by RT-PCR and RT-qPCR. Following cardiomyogenic induction, MSCs exhibited a stick-like morphology with extended cytoplasmic processes, and formed cluster-like structures. The expression of cardiac specific markers α-smooth muscle actin, cardiac troponin T, desmin and α-cardiac actin was positive for immunofluorescence staining, and further confirmed by RT-PCR and RT-qPCR. In conclusion, our results showed the in vitro differentiation ability of porcine BM-MSCs into neuron-like and cardiomyocyte-like cells.

Electrical activation enhances pre-implantation embryo development following sperm injection into in vitro matured pig oocytes.

The objective of this study was to evaluate the effect of electrical stimulation (EST) on pronuclear formation, chromosomal constitution, and developmental capability among in vitro matured pig oocytes following intracytoplasmic sperm injection (ICSI). After ICSI, the oocytes were randomly distributed and cultured into 3 groups: the EST activated ICSI group, non-activation ICSI group, and in vitro fertilization (IVF) group. The proportion of oocytes in which 2 pronuclei were formed in ICSI groups was significantly higher in the former groups than in the IVF group (96.2 and 93.5 vs. 64.5%, respectively, P<0.05). The cleavage rate was significantly higher in EST activated ICSI group (78.6%) than in the IVF and non-activated ICSI groups (51.8 and 46.0%, respectively, P<0.05), as was the proportion of oocytes that developed to the blastocyst stage at day 7 (18.9 vs. 11.6 and 9.1%, respectively, P<0.05). Diploid blastocysts were observed in 52.4, 63.0, and 65.2% of oocytes in the IVF, activated, and non-activated ICSI groups, respectively. Eight out of 23 gilts (34.8%) were confirmed to be pregnant in activated ICSI groups, but none of these pregnancies were carried to term. These results show that oocyte activation after ICSI is effective in elevating the cleavage rate and blastocyst development, while ensuring normal chromosome composition. Further research is needed to determine the pregnancy maintenance requirements for ICSI-embryos in pigs.

Efficiency of gene transfection into donor cells for nuclear transfer of bovine embryos.

The production of transgenic (TG) animals by somatic cell nuclear transfer (SCNT) has proven to be a more efficient method than other methods, such as gene injection or sperm mediation. The present study was intended to evaluate the efficiency of gene transfection by Effectene (Qiagen, Inc.), a lipid-based reagent compared to electroporation in fetal-derived fibroblast cells (FFC), cumulus-derived fibroblast cells (CFC), and adult ear skin-derived fibroblast cells (AEFC). Parameters compared were factors such as chromosome abnormality, gene expression, and the incidence of apoptosis. Further, the TG embryos with transfected donor cells generated by electroporation or Effectene were compared to IVF and SCNT embryos in terms of rates of cleavage, blastocyst formation, and blastocyst cell number. Most of the cells (>80%) at confluence were at G0/G1 and considered to be suitable nuclear donors for cloning. Transfection with a plasmid containing the enhanced green fluorescent protein (pEGFP-N1) gene into FFC did not increase the incidence of chromosomal abnormalities. The rates of apoptosis in different cell types transfected with pEGFP-N1 were 3.3%-5.0%, and the values did not differ among groups. In addition, the rates of apoptosis in various cells between 5-7 and 20-22 cell passages did not differ. However, the efficiency of gene transfecton into FFC by Effectene reagent (14.2 +/- 1.7) was significantly (P < 0.05) higher than that obtained by electroporation (5.1 +/- 1.0). Among various cell types, the efficiency of gene transfection by Effectene and eletroporation of FFC (14.2 +/- 1.7 and 5.1 +/- 1.0, respectively) was significantly (P < 0.05) higher than transfection of CFC and AEFC by either method (9.4 +/- 1.5 and 3.3 +/- 0.8, 8.8 +/- 0.7, and 2.1 +/- 0.4, respectively). In TG embryos produced by SCNT with electroporation and Effectene, the rates of cleavage and blastocyst formation were significantly lower (P < 0.05) than those of IVF controls, but rates did not differ between SCNT and TG embryos. Similarly, significantly higher (P < 0.05) total cell numbers in day-8 blastocysts were observed in IVF controls than those in SCNT and TG embryos, but did not differ between SCNT and TG (136 vs. approximately 110, respectively). The results demonstrated that, though there were no difference in the rates of chromosomal aneuploidy and the incidence of apoptosis among various cell types, transfected with or without pEGFP-N1, FFC were the cell type most effectively transfected and Effectene was a suitable agent for transfection.

Microtubulin configuration and mitochondrial distribution after ultra-rapid cooling of bovine oocytes.

Considerable attention has been focused on the cryopreservation of mammalian oocytes, as a consequence of poor development of cryopreserved bovine oocytes in vitro, in order to enhance the application of genetic engineering. Experiments were carried out to evaluate the viability and ultra-structural changes of bovine oocytes cryopreserved by ultra rapid cooling methods. Oocytes that had been allowed to mature for 22 hr were exposed to a mixture of cryoprotectants (3.2 M ethylene glycol, 2.36 M dimethyl sulfoxide (DMSO), 0.6 M sucrose), and were cryopreserved by very rapid cooling either within glass capillaries or as droplets on copper electron microscope grids. After being warmed, the oocytes were cultured in in vitro maturation (IVM) medium for an additional 2 hr. Viability was assessed by determining the development rate after fertilization with frozen-thawed semen from which motile sperm had been recovered using a Percoll density gradient, and by immunochemical evaluation of microtubule and mitochondrial morphology. Cleavage and development rates were significantly (P < 0.05) lower in oocytes cryopreserved by vitrification than in in vitro fertilization (IVF) control group, but did not differ in the open-pulled glass (OPG) or copper grid (CG) groups. In most oocytes cryopreserved by vitrification, the microtubules were partially or completely broken. Similarly mitochondria appeared to be abnormal compared to that of unfrozen oocytes. Oocytes cultured in IVM medium supplemented with both cytochalasin B (a protein synthesis inhibitor) and 2-mercaptoethanol (an antioxidant) showed less damage to microtubules, but not to mitochondria after cryopreservation. In conclusion, this study showed that bovine oocytes can be cryopreserved by vitrification within small droplets using CGs. While damage to microtubules and mitochondria may be involved in reduced viability, supplementation of IVM medium with cytochalasin B appears to enhance stabilization of microtubules during oocyte cryopreservation.