Multilayer perceptron and support vector regression models for feline parturition date prediction.

A crucial challenge in feline obstetric care is the accurate prediction of the parturition date during late pregnancy. The classic simple linear regression (SLR) model, which employed the fetal biparietal diameter (BPD) as the single input feature, was frequently applied for such prediction with limited accuracy. Since Multilayer Perceptron (MLP) and Support Vector Regression (SVR) are now two of the most potent scientific regression models, this study, for the first time, introduced such models as the new promising tools for feline parturition date prediction. The following features were candidate inputs for our models: biparietal diameter (BPD), litter size, and maternal weight. We observed and compared the performance results for each model. As the best-performed model, MLP delivered the highest coefficient score (0.972 ± 0.006), lowest mean absolute error score (1.110 ± 0.060), and lowest mean squared error score (1.540 ± 0.141), respectively. For the first time in this study, BPD, litter size, and maternal weight were considered the essential features for the innovative MLP and SVR modeling. With the optimized model parameters and the described analytical platform, further verification of these advanced models in feline obstetric practices is feasible.

Differential transcript usage across mammalian oocytes at the germinal vesicle and metaphase II stages.

Ongoing progress in mRNA-Sequencing technologies has significantly contributed to the refinement of assisted reproductive technologies. However, the prior investigations have predominantly concentrated on alterations in overall gene expression levels, thereby leaving a considerable gap in our understanding of the influence of transcript isoform expression on fundamental cellular mechanisms of oocytes. Given the efficacy of differential transcript usage (DTU) analysis to address such knowledge, we conducted comprehensive DTU analysis utilizing mRNA-Seq datasets of germinal vesicle (GV) and metaphase II (MII) oocytes across six mammalian species from the SRA database, including cow, donkey, horse, human, mouse, and pig. To further illuminate the roles of these genes, we also conducted a rigorous Gene Ontology (GO) term enrichment analysis. While the DTU analysis of each species exhibited several genes with alterations in their transcript isoform usage, referred to as DTU genes, this study focused on only ten cross-species DTU genes sharing among a minimum of five distinct species (FDR≤0.05). These cross-species DTU genes were as follows: ABCF1, CDC6, CFAP36, CNOT10, DNM3, IWS1, NBN, NDEL1, RAD50 and ZCCHC17. GO term enrichment analysis unveiled the alignment of these cross-species DTU gene functions with RNA and cell-cycle control mechanisms across diverse mammalian species, thereby suggesting their vital roles during oocyte maturation. Further exploration of the transcript isoforms of these genes hence bore the potential to uncover novel transcript isoform markers for future reproductive technologies in both human and animal contexts.

Analysis of occurrence and risk factors associated with pet rabbits' tumors in Central Thailand.

Rabbit oncology is gaining more attention as more pet rabbits are surviving beyond their normal lifespans. Due to the limited epidemiological information on pet rabbits' tumors in Thailand, this study aimed to report the prevalence and the potential risk factors associated with tumors in pet rabbits in Thailand. From 2018 to 2022, 93 tissue biopsies from tumor-suspected lesions on pet rabbits were gathered from animal hospitals in Bangkok and Chonburi provinces, Thailand. According to histopathology confirmation, tumors and tumor-like lesions were diagnosed. In this study, the overall tumors were 67.74% (n=63) out of the submitted cases (n=93). The most commonly affected organ systems were reproduction (65.08%) and integumentary (22.22%). Rabbits older than 5 years were 3.85 times more likely to have reproductive tumors than younger rabbits (95% confidence interval (CI): 1.45-10.27, P≤0.01), and the most frequently occurring tumor type was uterine adenocarcinoma. Furthermore, male rabbits had a 17.02 times higher probability of developing cutaneous tumors than female rabbits (95% CI: 4.19-69.11, P≤0.001), and the most frequently occurring tumor type was soft tissue sarcoma. The results of this study thus suggested that the age and sex of the rabbits were potential risk factors for tumor development in Thailand. The knowledge gained from our study also provided the recommendation for owners to monitor their rabbits' health annually, particularly after late middle age, and rendered guidance for tumor detection in practical clinics.

Cross-species analysis of differential transcript usage in humans and chickens with fatty liver disease.

Background and Aim: Fatty liver disease is a common condition, characterized by excess fat accumulation in the liver. It can contribute to more severe liver-related health issues, making it a critical concern in avian and human medicine. Apart from modifying the gene expression of liver cells, the disease also alters the expression of specific transcript isoforms, which might serve as new biological markers for both species. This study aimed to identify cross-species genes displaying differential expressions in their transcript isoforms in humans and chickens with fatty liver disease. Materials and Methods: We performed differential gene expression and differential transcript usage (DTU) analyses on messenger RNA datasets from the livers of both chickens and humans with fatty liver disease. Using appropriate cross-species gene identification methods, we reviewed the acquired candidate genes and their transcript isoforms to determine their potential role in fatty liver disease's pathogenesis. Results: We identified seven genes - ALG5, BRD7, DIABLO, RSU1, SFXN5, STIMATE, TJP3, and VDAC2 - and their corresponding transcript isoforms as potential candidates (false discovery rate ≤0.05). Our findings showed that these genes most likely contribute to fatty disease development and progression. Conclusion: This study successfully identified novel human-chicken DTU genes in fatty liver disease. Further research is encouraged to verify the functions and regulations of these transcript isoforms as potential diagnostic markers for fatty liver disease in humans and chickens.

A novel cross-species differential tumor classification method based on exosome-derived microRNA biomarkers established by human-dog lymphoid and mammary tumor cell lines' transcription profiles.

Background and Aim: Exosome-derived microRNA (miRNA) has been widely studied as a non-invasive candidate biomarker for tumor diagnosis in humans and dogs. Its application, however, was primarily focused on intraspecies usage for individual tumor type diagnosis. This study aimed to gain insight into its application as a cross-species differential tumor diagnostic tool; we demonstrated the process of identifying and using exosome-derived miRNA as biomarkers for the classification of lymphoid and mammary tumor cell lines in humans and dogs. Materials and Methods: Exosome-derived miRNA sequencing data from B-cell lymphoid tumor cell lines (n=13), mammary tumor cell lines (n=8), and normal mammary epithelium cultures (n=4) were pre-processed in humans and dogs. F-test and rank product (RP) analyses were used to select candidate miRNA orthologs for tumor cell line classification. The classification was carried out using an optimized support vector machine (SVM) with various kernel classifiers, including linear SVM, polynomial SVM, and radial basis function SVM. The receiver operating characteristic and precision-recall curves were used to assess the performance of all models. Results: MIR10B, MIR21, and MIR30E were chosen as the candidate orthologs from a total of 236 human-dog miRNA orthologs (p≤0.01, F-test score ≥10, and RP score ≤10). Their use of polynomial SVM provided the best performance in classifying samples from various tumor cell lines and normal epithelial culture. Conclusion: The study successfully demonstrated a method for identifying and utilizing candidate human-dog exosome-derived miRNA orthologs for differential tumor cell line classification. Such findings shed light on a novel non-invasive tumor diagnostic tool that could be used in both human and veterinary medicine in the future.

The reversible effects of gossypol toxicity on male pigeons' reproductive performance.

Background and Aim: Gossypol, a cotton seed derivative, is well known for its reversible antifertility in male reproduction across species. Its antifertility and reversibility effects on male reproductive function vary among species in dose-and time-dependent manners. In this study, the antifertility potential of gossypol in pigeons was evaluated for the first time to determine whether it might be used as a dietary supplement for pigeon population control. Materials and Methods: Male pigeons were assigned into three experimental groups: The gossypol-treated group (n = 12), the sham control group (n = 6), and the negative control group (n = 6). There were two experimental periods: A gossypol-feeding period of 28 days and a gossypol-free period of 28 days. During the gossypol-feeding period, birds in the gossypol-treated group were fed 4 mg of gossypol extract per day. Birds in the sham control group were fed 0.5 mL of mixed ethanol and sunflower oil, while those in the negative control group were fed 0.5 mL of phosphate buffer saline. After the gossypol-feeding phase was completed, all remaining pigeons in all groups continued to receive their regular diet for an additional 28 days (gossypol-free phase). The body weight and semen quality of the birds in the experimental groups were compared to evaluate gossypol's antifertility effect. Results: In the gossypol-treated group as compared to the control groups, the percentages of sperm motility and viability were significantly lower at 21 days, and the percentage of normal sperm morphology was significantly lower at 28 days during the gossypol-feeding period. After gossypol withdrawal, these antifertility effects were resumed and reached a comparable semen quality to the control groups within 14 days. Conclusion: Gossypol supplementation (4 mg/day for 28 days) could lower male pigeons' reproductive performance in terms of sperm motility, viability, and sperm morphology. Such infertility was, however, reversible within 14 days after gossypol withdrawal without any side effects on the pigeons, suggesting its application as a safe contraceptive feeding for male pigeons.

Seasonal effect on semen availability and quality of racing pigeon in Thailand.

BACKGROUND AND AIM: Seasonal variations among geographical regions could influence pigeon semen quality differently. This study aimed to determine the seasonal effect on semen availability and quality of racing pigeons in Thailand to understand and improve breeding management in the country. MATERIALS AND METHODS: Semen was collected from six fertile captive pigeons once a week during summer (March-June), monsoon (July-October), and winter (November-February) during 2019-2020. The success rate of semen collection and semen quality was determined in each season - by which changes in average temperature, humidity, and photoperiod were obtained. RESULTS: Comparable success rates of semen collection were acquired among different seasons, while varied semen qualities were revealed. The percentages of total motility and progressive motility score of sperm were significantly lowest in summer (66.35±3.40 and 3.88±0.15, respectively) compared to monsoon (85.45±2.91 and 4.67±0.10, respectively) and winter (79.29±1.96 and 4.37±0.10, respectively), while its concentration (×109 sperm/mL) and outputs (×106 sperm) were significantly highest in winter (7.62±0.54 and 91.44±10.83, respectively) compared to summer (4.23±0.41 and 48.45±6.35, respectively) and monsoon (3.57±0.30 and 51.45±7.21, respectively). Besides, semen samples collected from birds housing at an average temperature of <29.5°C demonstrated better sperm motility sperm concentration and total sperm counts than those from at a higher temperature. CONCLUSION: Winter was regarded as the best season contributing the best semen quality, while summer was the worst. Due to the fluctuation of temperature during summer and winter, the seasonal temperature was implied as the major factor contributing to changes in sperm quality of racing pigeons in Thailand.

Support vector regression algorithm modeling to predict the parturition date of small - to medium-sized dogs using maternal weight and fetal biparietal diameter.

BACKGROUND AND AIM: Fetal biparietal diameter (BPD) is a feasible parameter to predict canine parturition date due to its inverted correlation with days before parturition (DBP). Although such a relationship is generally described using a simple linear regression (SLR) model, the imprecision of this model in predicting the parturition date in small- to medium-sized dogs is a common problem among veterinarian practitioners. Support vector regression (SVR) is a useful machine learning model for prediction. This study aimed to compare the accuracy of SVR with that of SLR in predicting DBP. MATERIALS AND METHODS: After measuring 101 BPDs in 35 small- to medium-sized pregnant bitches, we fitted the data to the routine SLR model and the SVR model using three different kernel functions, radial basis function SVR, linear SVR, and polynomial SVR. The predicted DBP acquired from each model was further utilized for calculating the coefficient of determination (R2), mean absolute error, and mean squared error scores for determining the prediction accuracy. RESULTS: All SVR models were more accurate than the SLR model at predicting DBP. The linear and polynomial SVRs were identified as the two most accurate models (p<0.01). CONCLUSION: With available machine learning software, linear and polynomial SVRs can be applied to predicting DBP in small- to medium-sized pregnant bitches.

Differential DNA methylation analysis across the promoter regions using methylated DNA immunoprecipitation sequencing profiling of porcine loin muscle.

BACKGROUND AND AIM: Pork leanness and marbling are among the essential traits of consumer preference. To acquire knowledge about universal epigenetic regulations for improving breed selection, a meta-analysis of methylated DNA immunoprecipitation sequencing (MeDIP-seq) profiling data of mixed loin muscle types was performed in this study. MATERIALS AND METHODS: MeDIP-seq profiling datasets of longissimus dorsi muscle and psoas major muscles from male and female pigs of Landrace and Tibetan breeds were preprocessed and aligned to the porcine genome. Analysis of differential methylated DNA regions (DMRs) between the breeds was performed by focusing on transcription start sites (TSSs) of known genes (-20,000-3000 bases from TSS). All associated genes were further reviewed for their functions and predicted for transcription factors (TF) possibly associated with their TSSs. RESULTS: When the methylation levels of DMRs in TSS regions of Landrace breed were compared to those of Tibetan breed, 10 DMRs were hypomethylated (Landrace < Tibetan), and 19 DMRs were hypermethylated (Landrace > Tibetan), accordingly (p≤0.001). According to the reviews about gene functions, all associated genes were pieces of evidence for their roles in a variety of muscle and lipid metabolisms. Prediction of the binding TFs revealed the six most abundant binding TFs to such DMRs-associated TSS (p≤0.0001) as follows: ZNF384, Foxd3, IRF1, KLF9, EWSR1-FLI1, HES5, and TFAP2A. CONCLUSION: Common DMRs-associated TSS between the lean-type and the marbled-type loin muscles were identified in this study. Interestingly, the genes associated with such regions were strongly evidenced for their possible roles on the muscle trait characteristics by which further novel research topics could be focused on them in the future.

Novel classifier orthologs of bovine and human oocytes matured in different melatonin environments.

It has been demonstrated that melatonin influences the developmental competence of both in vivo and in vitro matured oocytes. It modulates oocyte-specific gene expression patterns among mammalian species. Due to differences among study systems, the identification of the classifier orthologs-the homologous genes related among mammals that could universally categorize oocytes matured in environments with varied melatonin levels is still limitedly studied. To gain insight into such orthologs, cross-species transcription profiling meta-analysis of in vitro matured bovine oocytes and in vivo matured human oocytes in low and high melatonin environments was demonstrated in the current study. RNA-Seq data of bovine and human oocytes were retrieved from the Sequence Read Archive database and pre-processed. The used datasets of bovine oocytes obtained from culturing in the absence of melatonin and human oocytes from old patients were regarded as oocytes in the low melatonin environment (Low). Datasets from bovine oocytes cultured in 10-9 M melatonin and human oocytes from young patients were considered as oocytes in the high melatonin environment (High). Candidate orthologs differentially expressed between Low and High melatonin environments were selected by a linear model, and were further verified by Zero-inflated regression analysis. Support Vector Machine (SVM) was applied to determine the potentials of the verified orthologs as classifiers of melatonin environments. According to the acquired results, linear model analysis identified 284 candidate orthologs differentially expressed between Low and High melatonin environments. Among them, only 15 candidate orthologs were verified by Zero-inflated regression analysis (FDR ≤ 0.05). Utilization of the verified orthologs as classifiers in SVM resulted in the precise classification of oocyte learning datasets according to their melatonin environments (Misclassification rates < 0.18, area under curves > 0.9). In conclusion, the cross-species RNA-Seq meta-analysis to identify novel classifier orthologs of matured oocytes under different melatonin environments was successfully demonstrated in this study-delivering candidate orthologs for future studies at biological levels. Such verified orthologs might provide valuable evidence about melatonin sufficiency in target oocytes-by which, the decision on melatonin supplementation could be implied.

Prenatal diagnosis of foetal hydrocephalus and suspected X-linked recessive inheritance of cleft lip in a Chihuahua.

A 3.5-year-old, 2.9 kg, multiparous Chihuahua presented with abdominal distension; pregnancy was diagnosed. On Day 7 before parturition, prenatal sonograms showed anechoic bilateral dilated cerebral lateral ventricles, suggesting fluid-filled regions (ventriculomegaly) in one foetus. A Caesarean section was performed and the male newborn had an abnormally enlarged dome-shaped head and a cleft lip, and died 6 days after birth. According to the family pedigree, the X-linked recessive inheritance of an orofacial cleft from the unaffected mother was suggested. This report clearly demonstrates that canine foetal ventriculomegaly (hydrocephalus) can be diagnosed in utero. For dog breeds predisposed to congenital ventriculomegaly, early detection is important for the prediction of perinatal survival and adequate supportive care can be applied at delivery.

An innovative approach to predict immune-associated genes mutually targeted by cow and human milk microRNAs expression profiles.

AIM: Milk is rich in miRNAs - the endogenous small non-coding RNA responsible for gene post-transcriptional silencing. Milk miRNAs were previously evidenced to affect consumer's immune response. While most studies relied on a few well-characterized milk miRNAs to relate their immunoregulatory roles on target genes among mammals, this study introduced a procedure to predict the target genes based on overall milk miRNA expression profiles - the miRNome data of cow and human. MATERIALS AND METHODS: Cow and human milk miRNome expression datasets of cow and human milk lipids at 2, 4, and 6 months of lactation periods were preprocessed and predicted for their target genes using TargetScanHuman. Enrichment analysis was performed using target genes to extract the immune-associated gene ontology (GO) terms shared between the two species. The genes within these terms with more than 50 different miRNAs of each species targeting were selected and reviewed for their immunological functions. RESULTS: A total of 146 and 129 miRNAs were identified in cow and human milk with several miRNAs reproduced from other previous reports. Enrichment analysis revealed nine immune-related GO terms shared between cow and human (adjusted p≤0.01). There were 14 genes related to these terms with more than 50 miRNA genes of each species targeting them. These genes were evidenced for their major roles in lymphocyte stimulation and differentiation. CONCLUSION: A novel procedure to determine mutual immune-associated genes targeted by milk miRNAs was demonstrated using cow and human milk miRNome data. As far as we know, this was the 1st time that milk miRNA target genes had been identified based on such cross-species approach. Hopefully, the introduced strategy should hereby facilitate a variety of cross-species miRNA studies in the future.

A demonstration of the H3 trimethylation ChIP-seq analysis of galline follicular mesenchymal cells and male germ cells.

OBJECTIVE: Trimethylation of histone 3 (H3) at 4th lysine N-termini (H3K4me3) in gene promoter region was the universal marker of active genes specific to cell lineage. On the contrary, coexistence of trimethylation at 27th lysine (H3K27me3) in the same loci-the bivalent H3K4m3/H3K27me3 was known to suspend the gene transcription in germ cells, and could also be inherited to the developed stem cell. In galline species, throughout example of H3K4m3 and H3K27me3 ChIP-seq analysis was still not provided. We therefore designed and demonstrated such procedures using ChIP-seq and mRNA-seq data of chicken follicular mesenchymal cells and male germ cells. METHODS: Analytical workflow was designed and provided in this study. ChIP-seq and RNA-seq datasets of follicular mesenchymal cells and male germ cells were acquired and properly preprocessed. Peak calling by Model-based analysis of ChIP-seq 2 was performed to identify H3K4m3 or H3K27me3 enriched regions (Fold-change≥2, FDR≤0.01) in gene promoter regions. Integrative genomics viewer was utilized for cellular retinoic acid binding protein 1 (CRABP1), growth differentiation factor 10 (GDF10), and gremlin 1 (GREM1) gene explorations. RESULTS: The acquired results indicated that follicular mesenchymal cells and germ cells shared several unique gene promoter regions enriched with H3K4me3 (5,704 peaks) and also unique regions of bivalent H3K4m3/H3K27me3 shared between all cell types and germ cells (1,909 peaks). Subsequent observation of follicular mesenchyme-specific genes-CRABP1, GDF10, and GREM1 correctly revealed vigorous transcriptions of these genes in follicular mesenchymal cells. As expected, bivalent H3K4m3/H3K27me3 pattern was manifested in gene promoter regions of germ cells, and thus suspended their transcriptions. CONCLUSION: According the results, an example of chicken H3K4m3/H3K27me3 ChIP-seq data analysis was successfully demonstrated in this study. Hopefully, the provided methodology should hereby be useful for galline ChIP-seq data analysis in the future.

Epidermal growth factor improves developmental competence and embryonic quality of singly cultured domestic cat embryos.

This study examined the influence of EGF on the expression of EGF receptors (EGFR) and developmental competence of embryos cultured individually versus those cultured in groups. Cat oocytes were in vitro matured and fertilized (IVM/IVF), and cleaved embryos were randomly assigned to one of seven culture conditions: one group each in which embryos were subjected to group culture supplemented with or without 5 ng/ml EGF and five groups in which embryos were subjected to single-embryo culture supplemented with EGF (0, 5, 25, 50 or 100 ng/ml). Morulae, blastocysts and hatching blastocysts were assessed at days 5 and 7; post IVF, respectively, and total blastocyst cell numbers were assessed at day 7. Relative mRNA expressions of EGFR of 2-4-cell embryos, 8-16-cell embryos, morulae and blastocysts cultured in groups or singly with or without EGF supplementation were examined. OCT3/4 and Ki67 in blastocysts derived from the group or single-embryo culture systems with or without EGF supplementation were localized. A higher rate of embryos cultured in groups developed to blastocysts than individually incubated cohorts. Although EGF increased blastocyst formation in the single-embryo culture system, EGF did not affect embryo development in group culture. Expression levels of EGFR decreased in morulae and blastocysts cultured with EGF. An increased ratio of Ki67-positive cells to the total number of cells in the blastocyst was observed in singly cultured embryos in the presence of EGF. However, EGF did not affect the expression of OCT3/4. These findings indicate that EGF enhanced developmental competence of cat embryos cultured singly by stimulating cell proliferation and modulating the EGFR expression at various developmental stages.

Insulin-like growth factor-1 (IGF-1) enhances developmental competence of cat embryos cultured singly by modulating the expression of its receptor (IGF-1R) and reducing developmental block.

OBJECTIVE: The aim of this study is to determine the effects of insulin-like growth factor-1 (IGF-1) and the mRNA expression of IGF-1 receptor (IGF-1R) during the in vitro development of cat embryos cultured in groups versus singly. METHODS: Cumulus-oocyte complexes (COCs) were matured and fertilized in vitro with frozen-thawed semen. Cleaved embryos (48h post-fertilization) were randomly assigned to one of the following treatments: 1) group embryo culture without IGF-1 (10 embryos per 50µl droplet), 2) single-embryo culture without IGF-1, and 3) to 6) single-embryo culture (50µl droplet per embryo) supplemented with different concentrations of IGF-1 (5, 25, 50 and 100ng/ml, respectively). During in vitro culture, the embryos were analyzed for development to the morula, blastocyst and hatching blastocyst stage. Relative mRNA expression of IGF-1R was also examined by qPCR at the morula and blastocyst stages. In addition, the mRNA expression of IGF-1R in morula-stage embryos treated with IGF-1 was determined. The influence of IGF-1 to preimplantation embryo development was then explored by co-incubation with 0.5µM IGF-1R inhibitor (Picropodophyllin; PPP). RESULTS: Group embryo culture led to a significantly higher blastocyst development rate compared with single-embryo culture (P<0.05). The poor development of singly cultured embryos coincided with the significantly lower IGF-1R expression in morulae than in group-cultured morulae. IGF-1 (25 or 50ng/ml) supplementation significantly improved the blastocyst formation rate of single embryos to a level similar to group culture by promoting the morula-to-blastocyst transition. IGF-1 supplementation (25 or 50ng/ml) of singly cultured embryos upregulated the expression of IGF-1R mRNA in morula-stage embryos to the same level as that observed in group-cultured embryos (without IGF-1). The beneficial effects of IGF-1 on singly cultured embryo were (P<0.05) suppressed by PPP even in the group culture embryo without growth factor supplementation. CONCLUSION: IGF-1 supplementation improves the developmental competence of feline embryos cultured individually and also increases IGF-1R gene expression to levels similar to group-cultured embryos.

In vitro culture of feline embryos increases stress-induced heat shock protein 70 and apoptotic related genes.

Developmental competence and quality of in vitro produced embryos has been demonstrated to be lower than in vivo derived embryos. This study aimed specifically to determine the effects of in vitro culture of feline embryos using various culture densities on developmental competence and expression of stress- and apoptotic-related genes in terms of heat shock protein 70 (HSP70) and apoptotic-related (BAX and BCL-2) gene expressions. In experiment 1, we characterized the inducible form of a feline-specific HSP70 mRNA sequence, as it has not been previously reported. The primers for feline HSP70 mRNA were synthesized and tested on heat-treated cat fibroblasts. In experiment 2, feline embryos were cultured at different culture densities (embryo:culture volume; 1:1.25, 1:5 and 1:20). The developmental competence was determined along with HSP70, BAX and BCL-2 transcript abundances using quantitative RT-PCR. In vivo derived embryos were used as a control group. A partial cat HSP70 mRNA sequence (190 bp) was characterized and exhibited high nucleotide identity (93 to 96%) with other species. Cleaved embryos cultured at high density (1:1.25) developed to blastocysts at a lower rate than those generated from lower densities. Irrespective of the culture densities used, in vitro cultured blastocysts showed increased levels of HSP70 and BAX transcripts compared with in vivo counterparts. Blastocysts derived from the highest culture density (1:1.25) showed higher levels of upregulation of HSP70 and BAX transcripts than those cultured at lower culture densities (1:5 and 1:20). In conclusion, increased levels of pro-apoptotic (BAX) and stress-response (HSP70) transcripts correlated with developmental incompetence of embryos cultured at high embryonic density, indicating that stress accumulated during in vitro embryo culture affected the fate for embryo development and quality.