Assisted reproductive technology is today considered a safe and reliable medical intervention, with healthy live births a reality for many IVF and ICSI treatment cycles. However, there are increasing numbers of published reports describing epigenetic/imprinting anomalies in children born as a result of these procedures. These anomalies have been attributed to methylation errors in embryo chromatin remodelling during in vitro culture. Here we re-visit three concepts: (1) the so-called ‘in vitro toxicity’ of ‘essential amino acids’ before the maternal to zygotic transition period; (2) the effect of hyperstimulation (controlled ovarian hyperstimulation) on homocysteine in the oocyte environment and the effect on methylation in the absence of essential amino acids; and (3) the fact/postulate that during the early stages of development the embryo undergoes a ‘global’ demethylation. Methylation processes require efficient protection against oxidative stress, which jeopardizes the correct acquisition of methylation marks as well as subsequent methylation maintenance. The universal precursor of methylation [by S-adenosyl methionine (SAM)], methionine, ‘an essential amino acid’, should be present in the culture. Polyamines, regulators of methylation, require SAM and arginine for their syntheses. Cystine, another ‘semi-essential amino acid’, is the precursor of the universal protective antioxidant molecule: glutathione. It protects methylation marks against some undue DNA demethylation processes through ten-eleven translocation (TET), after formation of hydroxymethyl cytosine. Early embryos are unable to convert homocysteine to cysteine as the cystathionine β-synthase pathway is not active. In this way, cysteine is a ‘real essential amino acid’. Most IVF culture medium do not maintain methylation/epigenetic processes, even in mouse assays. Essential amino acids should be present in human IVF medium to maintain adequate epigenetic marking in preimplantation embryos. Furthermore, morphological and morphometric data need to be re-evaluated, taking into account the basic biochemical processes involved in early life.

In the last decades, the universal use of mobile phones has contributed to radiofrequency electromagnetic radiation environmental pollution. The steady growth in mobile phone usage has raised concerns about the effects of phone radiation on male reproductive health. Epidemiological studies report a sharp decline in sperm counts in developing countries, and worldwide with c. 14% of couples having difficulties to conceive, many of which are attributed to a male infertility factor. Environment and lifestyle factors are known to contribute to male infertility. Exposure to heat, radiation, or radioactivity might induce damage to biological tissue organs, including the testis. Given the ubiquitous use of mobile phones, the potential adverse effects of the resulting environmental radiation needs to be elucidated further. It seems to be an apparent relationship between the increased exposure to mobile phone radiofrequency and sperm quality decline, but the evidence is not conclusive. Our review summarizes the evidence concerning the possible adverse effects of cell phone radiation on the male reproductive system, with a focus on sperm quality. Also, we critically analyze the effects of elevated testicular temperature and oxidative stress on male fertility and how these factors could interfere with the physiological activities of the testis.

There has been no report on the outcome of vitrified blastocyst transfer from a vitrified oocyte injected with immotile testicular spermatozoa with only multiple morphological abnormalities of the sperm flagella (MMAF). A couple diagnosed with MMAF returned to the clinic to attempt pregnancy using their vitrified oocytes. Testicular spermatozoa were injected intracytoplasmically, and the following intracytoplasmic sperm injection results were observed. In the second cycle, surplus vitrified oocytes and testicular retrieved sperm were used, but no pregnancy ensued. In the third cycle, a surplus vitrified blastocyst was transferred, and a healthy female child was delivered, with a birth weight of 3050 g and a birth length of 53 cm. In this report we describe a successful pregnancy achieved in a patient presenting MMAF. The successful pregnancy was obtained from vitrified oocytes microinjected with testicular retrieved sperm in a vitrified blastocyst transfer.

Cell phones operate with a wide range of frequency bands and emit radiofrequency-electromagnetic radiation (RF-EMR). Concern on the possible health hazards of RF-EMR has been growing in many countries because these RF-EMR pulses may be absorbed into the body cells, directly affecting them. There are some in vitro and in vivo animal studies related to the consequences of RF-EMR exposure from cell phones on embryo development and offspring. In addition, some studies have revealed that RF-EMR from cellular phone may lead to decrease in the rates of fertilization and embryo development, as well as the risk of the developmental anomalies, other studies have reported that it does not interfere with in vitro fertilization or intracytoplasmic sperm injection success rates, or the chromosomal aberration rate. Of course, it is unethical to study the effect of waves generated from cell phones on the forming human embryos. Conversely, other mammals have many similarities to humans in terms of anatomy, physiology and genetics. Therefore, in this review we focused on the existing literature evaluating the potential effects of RF-EMR on mammalian embryonic and fetal development.

Long non-coding RNAs (lncRNAs) exert vital functions in the occurrence and development of various tumours. The aim of this study was to examine the regulatory effect and underlying molecular mechanism of lncRNA small nucleolar RNA host gene 14 (SNHG14) on the proliferation, invasion and migration of thyroid tumour cells. The expression of SNHG14 in thyroid tumour cell lines was determined using qRT-PCR. CCK-8 and western blot were used to detect the effects of SNHG14 on proliferation and apoptosis of thyroid tumour cells. The effect of SNHG14 on the migration and invasion of thyroid tumour cells was analyzed using immunofluorescence, wound-healing and transwell assays. A targeting relationship between SNHG14 and miR-93-5p was determined using bioinformatics software and luciferase reporter assays. In addition, CCK-8, immunofluorescence, wound-healing and transwell assays were applied to demonstrate that SNHG14 promoted the proliferation, migration and invasion of thyroid tumour cells by targeting miR-93-5p. The biological function of SNHG14 in vivo was explored through a xenograft model and immunohistochemistry. SNHG14 was upregulated in thyroid tumour cells compared with normal cells. Downregulation of SNHG14 effectively reduced the proliferation, migration and invasion of TPC-1 cells, and induced cell apoptosis. Moreover, SNHG14 directly targeted miR-93-5p and there was a negative correlation between them. Further functional experiments illustrated that miR-93-5p overexpression dramatically reversed the promoting role of SNHG14 in proliferation, migration and invasion of TPC-1 cells. Our results demonstrated that SNHG14 promotes the proliferation, invasion and migration of thyroid tumour cells by downregulating miR-93-5p.

The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300–400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus–oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

Sperm DNA fragmentation can be produced in one (ssSDF) or both (dsSDF) DNA strands, linked to difficulties in naturally achieving a pregnancy and recurrent miscarriages, respectively. The techniques more frequently used to select sperm require centrifugation, which may induce sperm DNA fragmentation (SDF). The objective of this study was to assess whether the microfluidic-based device FertileChip® (now ZyMot®ICSI) can diminish the proportion of sperm with dsSDF. First, in a blinded split pilot study, the semen of nine patients diagnosed with ≥60% dsSDF, was divided into three aliquots: not processed, processed with FertileChip®, and processed with swim up. The three aliquots were all analyzed using neutral COMET for the detection of dsSDF, resulting in a reduction of 46% (P < 0.001) with FertileChip® (dsSDF: 34.9%) compared with the ejaculate and the swim up (dsSDF: 65%). Thereafter, the FertileChip® was introduced into clinical practice and a cohort of 163 consecutive ICSI cycles of patients diagnosed with ≥60% dsSDF was analyzed. Fertilization rate was 75.41%. Pregnancy rates after the first embryo transfer were 53.2% (biochemical), 37.8% (clinical), 34% (ongoing) and the live birth rate was 28.8%. Cumulative pregnancy rates after one (65.4% of patients), two (27.6% of patients) or three (6.4% of patients) transfers were 66% (biochemical), 56.4% (clinical), 53.4% (ongoing) and the live birth rate was 42%. The selection of spermatozoa using Fertile Chip® significantly diminishes the percentage of dsSDF, compared with either the fresh ejaculate or after swim up. Its applicability in ICSI cycles of patients with high dsSDF resulted in good laboratory and clinical outcomes.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at −80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

In this work, the presence of calcium-dependent calcium channels and their receptors (RyR) has been investigated in Paracentrotus lividus eggs and early embryos, from unfertilized egg to four-blastomere stages. Electrophysiological recordings of RyR single-channel current fluctuations showed that RyRs are functional during the first developmental events with a maximum at zygote stage, c. 40 min after fertilization, corresponding to the first cleavage. The nature of vertebrate-like RyRs active at this stage was established by specific activation/blockade experiments.

The finding of conjoined oocytes is a rare occurrence that accounts for only 0.3% of all human retrieved oocytes. This phenomenon is quite different from that of a traditional single oocyte emanating from one follicle, and may result in dizygotic twins and mosaicism. Given the insufficient evidence on how to approach conjoined oocytes, their fate is variable among different in vitro fertilization (IVF) centres. In this observational report, we propose a new protocol for the use of these conjoined oocytes using intracytoplasmic sperm injection (ICSI), laser-cutting technique and next-generation sequencing (NGS). The first case report demonstrates that conjoined oocytes can penetrate their shared zona pellucida (ZP) at Day 6. The second case is that of a 25-year-old female patient who underwent a successful embryo transfer cycle after removal of one oocyte in which a pair of conjoined human oocytes underwent ICSI, laser-cutting separation and NGS testing. The patient achieved pregnancy and gave birth to single healthy female originally derived from conjoined oocytes. This case provided a means through which normal pregnancy may be achieved from conjoined oocytes using laser-cutting separation techniques. The protocol described may be especially beneficial to patients with a limited number of oocytes.

The Dazl (deleted in azoospermia-like) gene encodes an RNA-binding protein containing an RNA recognition motif (RRM) and a DAZ motif. Dazl is essential for gametogenesis in vertebrates. In this study, we report the cloning of Dazl cDNA from Cynops cyanurus. Ccdazl mRNA showed a germline-specific expression pattern as expected. Ccdazl expression gradually decreased during oogenesis, suggesting that it may be involved in oocyte development. Phylogenetic analysis revealed that the Ccdazl protein shares conserved motifs/domains with Dazl proteins from other species. Cloning of Ccdazl provides a new tool to carry out comparative studies of germ cell development in amphibians.

Toll-like receptor 4 (TLR4) is best known for its role in bacteria-produced lipopolysaccharide recognition. Regarding female reproduction, TLR4 is expressed by murine cumulus cells and participates in ovulation and in cumulus–oocyte complex (COC) expansion, maternal–fetal interaction and preterm labour. Despite these facts, the role of TLR4 in ovarian physiology is not fully understood. Therefore, the aim of the present study was to investigate the effects of TLR4 genetic ablation on mice folliculogenesis and female fertility, through analysis of reproductive crosses, ovarian responsiveness and follicular quantification in TLR4−/− (n = 94) and C57BL/6 mice [wild type (WT), n = 102]. TLR4-deficient pairs showed a reduced number of pups per litter (P = 0.037) compared with WT. TLR4−/− mice presented more primordial, primary, secondary and antral follicles (P < 0.001), however there was no difference in estrous cyclicity (P > 0.05). A lower (P = 0.006) number of COC was recovered from TLR4−/− mice oviducts after superovulation, and in heterozygous pairs, TLR4−/− females also showed a reduction in the pregnancy rate and in the number of fetuses per uterus (P = 0.007) when compared with WT. Altogether, these data suggest that TLR4 plays a role in the regulation of murine folliculogenesis and in determining ovarian endowment. TLR4 deficiency may affect ovulation and pregnancy rates, potentially decreasing fertility, therefore the potential side effects of its blockade have to be carefully investigated.

Congenital domestic absence of vas deferens (CBAVD) is a common factor in male infertility, and percutaneous epididymal sperm aspiration (PESA) combined with intracytoplasmic sperm injection (ICSI) is a primary clinical treatment, but the effect of the sperm obtained on pregnancy outcome remains to be explored. This study aimed to investigate the relationship between sperm motility with clinical outcome of PESA–ICSI in infertile males with CBAVD. A cohort of 110 couples was enrolled. In total, 76 infertile males were included in the high motility group, while the remaining 34 males were placed in the low motility group. Clinical pregnancy, embryo implantation rate and live birth rate were included as the primary outcome. After all follow-ups, we found that the high motility group achieved higher normal fertilization rates, cleavage rates, transplantable embryo rates and high-quality embryo rates than those in low motility group (normal fertilization rate, 78.2 ± 11.7% vs. 70.5 ± 10.2%, P = 0.003; cleavage rate, 97.1 ± 2.9% vs. 92.3 ± 3.0%, P = 0.000; transplantable embryo rate, 66.8 ± 14.9% vs. 58.6 ± 12.6%, P = 0.009 and high-quality embryo rate, 49.9 ± 10.5% vs. 40.5 ± 11.2%, P = 0.000). Additionally, compared with the low motility group, the clinical pregnancy rates, embryo implantation rates, and live birth rates in the high motility group were significantly increased (pregnancy rate, 61.8% vs. 26.5%, P = 0.009; embryo implantation rate, 36.5% vs. 18.0%, P = 0.044; live birth rate, 55.3% vs. 17.6%, P = 0.000). We concluded that the motility of sperm obtained by PESA affected the clinical outcome of ICSI in infertile males with CBAVD.

The Hippo pathway is involved in the proliferation of intrafollicular cells and in early embryonic development, mainly because effectors of this pathway are key transcription regulators of genes such as CTGF and CYR61, which are involved in cell proliferation. Recent studies by our group found that fibroblast growth factor 18 (FGF18) is present in the fallopian tube during early embryonic development, leading to the hypothesis that FGF18 may have a role during embryonic development. Therefore, the aim of the following study was to determine whether FGF18 modulates the expression of Hippo pathway target genes, CTGF and CYR61, during oocyte maturation and early embryonic development. Three experiments were carried out, with in vitro maturation (IVM) of cumulus–oocyte complexes (COCs) and embryo culture. In experiment one, FGF18 (100 ng/ml) induced an increase (P < 0.05) in CTGF gene expression at 12 h post-exposure. In experiment two, FGF18 (100 ng/ml) induced a reduction (P < 0.05) in CTGF expression at 3 h post-exposure. In the third experiment, day 7 embryos exposed to FGF18 during oocyte IVM expressed greater CTGF mRNA abundance, whereas FGF18 exposure during embryo in vitro embryo culture did not alter CTGF expression in comparison with untreated controls. The preliminary data presented here show that FGF18 modulates CTGF expression in critical periods of oocyte nuclear maturation, cumulus expansion and early embryonic development in cattle.

The present work evaluated animal models comprising direct and maternal effects to estimate (co)variance components and genetic parameters of growth rates and Kleiber ratio in Harnali sheep. The information on pedigree and targeted traits of 1862 lambs born to 144 sires and 591 dams was collected for the period from 1998 to 2018. The traits studied were average daily gain from birth to 3 months of age (ADG1), 3 months to 6 months of age (ADG2), and 6 months to 12 months of age (ADG3) and their corresponding Kleiber ratios as KR1, KR2 and KR3, respectively. The statistical methods included the general linear model for analyzing the effects of fixed factors and animal models for deriving variance components for targeted traits. According to best model evaluated on the basis of likelihood ratio test, the estimated direct heritability was low in magnitude and ranged from 0.04 to 0.14. Direct heritability estimates for ADG1, ADG2, ADG3, KR1, KR2 and KR3 were 0.06, 0.14, 0.05, 0.04, 0.11 and 0.05, respectively. The maternal genetic effects contributed (4–7%) significantly for ADG1, KR1 and KR2 traits. The genetic correlations ranged from −0.35 ± 0.11 (ADG1-KR2) to 0.98 ± 0.01 (ADG2-KR2 and ADG3-KR3) and phenotypic correlations ranged from −0.36 ± 0.02 to 0.98 ± 0.01 for ADG1-KR2 and ADG2-KR2, respectively. The significant maternal effects along with low levels of direct effects for average daily gain and Kleiber ratio at different age group should be considered while setting selection and managerial strategies to achieve anticipated growth rates in Harnali sheep.

Oxidative stress causes several diseases and dysfunctions in cells, including oocytes. Clearly, oxidative stress influences oocyte quality during in vitro maturation and fertilization. Here we tested the ability of coenzyme Q10 (CoQ10) to reduce reactive oxygen species (ROS) and improve mouse oocyte quality during in vitro culture. Treatment with 50 μM CoQ10 efficiently reduced ROS levels in oocytes cultured in vitro. The fertilizable form of an oocyte usually contains a cortical granule-free domain (CGFD). CoQ10 enhanced the ratio of CGFD–oocytes from 35% to 45%. However, the hardening of the zona pellucida in oocytes was not affected by CoQ10 treatment. The in vitro maturation capacity of oocytes, which was determined by the first polar body extrusion, was enhanced from 48.9% to 75.7% by the addition of CoQ10 to the culture medium. During the parthenogenesis process, the number of two-cell embryos was increased by CoQ10 from 43.5% to 67.3%. Additionally, treatment with CoQ10 increased the expression of Bcl2 and Sirt1 in cumulus cells. These results suggested that CoQ10 had a positive effect on ROS reduction, maturation rate and two-cell embryo formation in mouse oocyte culture.

Lipid accumulation occurs in cultured embryos and is associated with reduced cryotolerance. Here we report the use of a multiple pathway lipid modulator cocktail (l-carnitine, linoleic acid and forskolin) to improve cryosurvival. First, we stained oocytes and embryos with Oil Red to examine the time course of lipid accumulation during in vitro fertilization (IVF) and embryo culture. Then we evaluated the effects of the lipid modulators cocktail on lipid content, developmental rates and survival after vitrification. In our conditions, lipid accumulation was detected (P < 0.05) at the end of in vitro maturation (IVM) and after 4 days of embryo culture (D4-D5). In experiment 1, we used lipid modulator cocktail during IVM. Reduced (P < 0.05) lipid accumulation was detected in oocytes (Control: 49.9 ± 1.6, Lip. Mod. IVM: 45.0 ± 1.8) but no changes were present at blastocyst stage (Control: 62.4 ± 2.6, Lip. Mod. IVM: 66.8 ± 2.7). Treated oocytes presented decreased (P < 0.05) blastocyst rates and lower (P < 0.05) re-expansion after vitrification. In experiment 2, lipid modulators cocktail was used during embryo culture (from D4–D7 or D6–D7). Treatment had an effect on lipid metabolism, as lipid content was increased (P < 0.05) in D7 blastocysts in treated groups (Control: 52.7 ± 3.1a, D4: 65.9 ± 2.6b, D6: 78.1 ± 2.7b). However, no effect was present for cleavage, blastocyst and cryosurvival rates. No difference was detected in mean cell number comparing the three groups (Control: 78.9 ± 9.6, D4: 82.6 ± 16.5, D6: 68.3 ± 7.8), but apoptosis rate was increased (P < 0.05) in vitrified-warmed blastocysts from treated groups (Control: 14.77*, D4: 22.28, D6: 22.22). We concluded that the combined use of lipid modulators was efficient to promote changes in lipid content of oocytes and embryos in bovine, but those changes did not reflect positively on embryo development or cryosurvival.

It has previously been reported that in ex vivo planar explants prepared from Xenopus laevis embryos, the intracellular pH (pHi) increases in cells of the dorsal ectoderm from stage 10.5 to 11.5 (i.e. 11–12.5 hpf). It was proposed that such increases (potentially due to H+ being extruded, sequestered, or buffered in some manner), play a role in regulating neural induction. Here, we used an extracellular ion-selective electrode to non-invasively measure H+ fluxes at eight locations around the equatorial circumference of intact X. laevis embryos between stages 9–12 (˜7–13.25 hpf). We showed that at stages 9–11, there was a small H+ efflux recorded from all the measuring positions. At stage 12 there was a small, but significant, increase in the efflux of H+ from most locations, but the efflux from the dorsal side of the embryo was significantly greater than from the other positions. Embryos were also treated from stages 9–12 with bafilomycin A1, to block the activity of the ATP-driven H+ pump. By stage 22 (24 hpf), these embryos displayed retarded development, arresting before the end of gastrulation and therefore did not display the usual anterior and neural structures, which were observed in the solvent-control embryos. In addition, expression of the early neural gene, Zic3, was absent in treated embryos compared with the solvent controls. Together, our new in vivo data corroborated and extended the earlier explant-derived report describing changes in pHi that were suggested to play a role during neural induction in X. laevis embryos.

The evaluation of early reproduction traits in Beetal goat was performed for possible effects of genetic and non-genetic factors on litter size at birth (LSB), litter size at weaning (LSW), litter weight at birth (LWB), litter weight at weaning (LWW) and age at first kidding (AFK). The data records consisted of information of pedigree and targeted traits pertained to 223 does born to 25 sires and 122 dams between the years 2004 to 2019. A general linear model was used for assessment of non-genetic factors such as period of birth, type of birth and dam’s weight at kidding on studied traits. Genetic evaluation of targeted traits was done to estimate variance components and genetic parameters under dyadic mixed modelling. The estimates of least-square means for LSB, LSW, LWB, LWW and AFK were observed as 1.27 ± 0.03, 1.25 ± 0.03, 3.24 ± 0.07 kg, 13.08 ± 0.30 kg and 27.56 ± 0.58 months, respectively. Only the period of birth showed significant (P < 0.05) effects for targeted traits in this study. The estimates of direct heritability for LSB, LSW, LWB, LWW and AFK were low in magnitude as 0.08, 0.03, 0.10, 0.03 and 0.06, respectively. The moderate to high genetic and phenotypic correlations among litter traits indicate simultaneous improvement for these traits. It was concluded that low ranged direct heritability estimates for targeted traits indicated modest scope for genetic improvement of reproductive efficiency in Beetal goat through selection and, therefore, adoption of improved managerial practices is necessary to improve reproductive efficiency of Beetal goat.

The aim of this study was to investigate mitochondrial ND5 gene polymorphisms and their relationship with in vitro maturation (IVM) and in vitro culture (IVC) of Sanjabi sheep. Blood and ovarian samples of adult ewes were obtained from a local slaughterhouse. For each ovarian sample, cumulus–oocyte complexes larger than 3 mm in diameter were aspirated from follicles, and their IVM and IVC rates were recorded. A 666-bp fragment of the ND5 gene was amplified using the polymerase chain reaction. The samples were genotyped using a modified single-stranded conformation polymorphism (SSCP) method, and an association study was conducted with IVM and IVC rates. Six different SSCP patterns, designated A, B, C, D, E and F with respective frequencies of 8, 47, 4, 4, 32 and 5%, respectively, were observed. According to the results of association analysis, there was no significant association between the ND1 gene polymorphisms and the IVM and IVC rates (P > 0.05).