The success of an assisted reproduction cycle should be the achievement of a healthy singleton live birth following the replacement of one embryo. Therefore, one of the most critical points for embryologists has been the selection criteria and how to choose the best embryo to transfer with high implantation potential. In this vein, morphological evaluation has been historically the method applied. However, this practice relies on a limited number of single observations and is associated with high operator variability. Recently, a major innovation in embryo culture has been the introduction of a new type of incubator with integrated time-lapse monitoring, which enables the embryologist to analyze the dynamic events of embryo development, from fertilization to blastocyst formation. This novel practice is quickly growing and has been implemented in many IVF clinics worldwide. Therefore, the main aim of this review is to illustrate the benefits of time-lapse technology in a modern embryology laboratory. In particular, we discuss the blastocyst collapse(s) event and morphometric blastocyst assessment and analyse their association with embryo viability and implantation potential.

We present a commentary on the article published in the Zygote FirstView: ‘Importance of real-time measurement of sperm head morphology in intracytoplasmic sperm injection’ by Fumiaki Itoi and colleagues. We comment on the importance of providing the microscope setup details whenever sperm morphology visualization is discussed. The claim of ×6000–10,000 magnification is misleading as such levels of magnification are impossible to achieve.

The aim of the present study was to evaluate the physiological and morphological parameters of pregnant does for early prediction of prenatal litter size. In total, 33 does were screened using ultrasonography and further categorized into three groups based on does bearing twins (n = 12), a single fetus (n = 12), or non-pregnant does (n = 9). The rectal temperature °F (RT) and respiration rate (RR) as physiological parameters, while abdominal girth in cm (AG) and udder circumference in cm (UC) as morphological parameters were recorded at different gestation times, i.e. 118, 125, 132 and 140 days. In addition to this, age (years) and weight at service (kg) were also used. The statistical analyses included analysis of variance (ANOVA) and linear discriminant analysis (LDA). The results indicated that groups had significant (P < 0.05) differences among morphological parameters at each gestation time, with higher AG and UC in does bearing twins followed by a single fetus and non-pregnant does. However, both physiological parameters were non-significantly (P > 0.05) associated with litter size groups. It was also revealed that the studied parameters showed increasing trends over gestation time in single and twin fetus categories, but they were on par among non-pregnant does. The results of the LDA revealed that estimated function based on age, weight at service, RR, RT, AG and UC had greater (ranging from 75.00 to 91.70%) accuracy, sensitivity and specificity at different gestation times. It was concluded that using an estimated function, future pregnant does may be identified in advance for single or twin litter size, with greater accuracy.

Regarding the low number of embryos that reach the blastocyst stage when cultured in vitro, this study aimed to evaluate the effects of quercetin on pre-implantation mouse (Mus musculus) embryos obtained using in vitro fertilization, especially during the passage from morula to blastocyst. Furthermore, we studied whether quercetin also affected the expression of hypoxia-inducible factor 1α (HIF-1α). The culture medium for the embryos was supplemented with quercetin, for long or short periods of time, and then the development potential, total cell number, apoptosis rates and expression of HIF-1α were studied to determine the effect of quercetin. Embryos failed to develop when cultured for long periods of time with quercetin, implying the possible toxic effects of this, alternatively antioxidant, compound. However, a short culture from morula to blastocyst significantly improved the development potential of in vitro produced embryos, increasing the final total cell number and reducing the apoptosis rate, observing similar results to those embryos cultured in low-oxygen concentrations or developed in utero. Furthermore, in embryos treated with quercetin for 2 or 4 h we found an increase in HIF-1α compared with untreated embryos. This work could imply a way to use quercetin in fertility clinics to improve the production of healthy blastocysts and, consequently, increase the success rates in assisted reproduction techniques.

Polycystic ovary syndrome is an endocrine disorder commonly found among females of reproductive age. Different factors have been correlated with this syndrome, although the aetiology of the disease is still unrecognized with both environmental and hereditary factors leading to the progression. Hormonal effects of the AKT pathway have made it an interesting study unit for PCOS cases. The aim of this study was to investigate the expression patterns of genes involved in the AKT pathway, including IRS1, IRS2, AKT1 and AKT2. In total, 13 human oocytes were collected for this study at the meiosis II stage, in which seven of them were collected from individuals with polycystic ovaries and the rest formed the control group of individuals with no signs of polycystic ovaries. RNA was extracted from oocytes and then the RNA was converted into cDNA for the real-time PCR process. Expression levels of four genes in the AKT pathway, in addition to housekeeping gene (ACTB), were evaluated. Expression levels of each gene were quantified using real-time PCR and statistical analysis was performed. The results of this study showed that there was no significant correlation between the expression of genes in oocyte samples obtained from patients with polycystic ovaries and the control group. This study is the first to evaluate the expression levels of genes involved in the AKT pathway in human oocyte samples. Therefore, it provides crucial information to form the basis of further studies.

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen–thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 μg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

This study is a comparative analysis of the biochemical, hormonal, and mineral compositions of follicular fluid in preovulatory and cystic follicles of water buffalo (Bubalus bubalis). In total, reproductive tracts from 215 buffalo along with intact ovaries were collected randomly from an abattoir. The incidence of cystic conditions found in this study was 3.72% (8/215), involving the right ovary in 62.5% of instances and the left ovary in 37.5% of instances during the non-breeding season. Follicular fluid was aspirated from preovulatory follicles (12–15 mm diameter, oestrogen-active, follicular phase or stage IV corpus luteum on one of the two ovaries, n = 10) and cystic follicles (at least 20 mm diameter, no corpus luteum on any one of the two ovaries, n = 8). The follicular fluid samples were assayed for biochemical components (uric acid, creatinine, blood urea nitrogen, cholesterol, total protein, glucose, ascorbic acid, and alkaline phosphatase), hormones (progesterone, estradiol, and insulin), and minerals (calcium, magnesium, phosphorus, copper, zinc, and cobalt). Cystic follicles had greater (P < 0.05) concentrations of creatinine, blood urea nitrogen, cholesterol, progesterone, copper, zinc, and cobalt, and lesser (P < 0.05) concentrations of uric acid, glucose, ascorbic acid, estradiol, insulin, calcium, magnesium, and phosphorus compared with preovulatory follicles. These results indicated the marked differences in follicular fluid composition between preovulatory and cystic follicles in buffalo. Some of the changes were indicative of oxidative stress and disturbed steroidogenesis, two important mechanisms shown to be associated with cystic ovarian disease in various species. Further studies are warranted to investigate whether these differences are directly or indirectly involved in the formation of cystic follicles or are mere manifestations of the condition.

Here we report a quantitative analysis of human metaphase II (MII) oocytes from a 22-year-old oocyte donor, retrieved after ovarian-controlled hyperstimulation. Five surplus donor oocytes were processed for transmission electron microscopy (TEM), and a stereological analysis was used to quantify the distribution of organelles, using the point-counting technique with an adequate stereological grid. Comparisons between means of the relative volumes (Vv) occupied by organelles in the three oocyte regions, cortex (C), subcortex (SC) and inner cytoplasm (IC), followed the Kruskal–Wallis test and Mann–Whitney U-test with Bonferroni correction. Life cell imaging and TEM analysis confirmed donor oocyte nuclear maturity. Results showed that the most abundant organelles were smooth endoplasmic reticulum (SER) elements (26.8%) and mitochondria (5.49%). Significant differences between oocyte regions were found for lysosomes (P = 0.003), cortical vesicles (P = 0.002) and large SER vesicles (P = 0.009). These results were quantitatively compared with previous results using prophase I (GV) and metaphase I (MI) immature oocytes. In donor MII oocytes there was a normal presence of cortical vesicles, SER tubules, SER small, medium and large vesicles, lysosomes and mitochondria. However, donor MII oocytes displayed signs of cytoplasmic immaturity, namely the presence of dictyosomes, present in GV oocytes and rare in MI oocytes, of SER very large vesicles, characteristic of GV oocytes, and the rarity of SER tubular aggregates. Results therefore indicate that the criterion of nuclear maturity used for donor oocyte selection does not always correspond to cytoplasmic maturity, which can partially explain implantation failures with the use of donor oocytes.

Oocyte-mediated somatic cell haploidization is a process in which a diploid cell halves its chromosomal content by segregating its homologue within the ooplasm. Replacing the donor oocyte nucleus with a patient’s female diploid somatic nucleus can generate patient-genotyped oocytes. Insemination of these resulting constructs enables their activation and induces a reductive meiotic division, haploidizing the diploid female donor cell that can subsequently support syngamy with the male genome and create a zygote. So far, experimental data for this method have been limited and have not consistently proven the generation of chromosomally normal embryos. Overall, we achieved reconstruction of murine oocytes with a micromanipulation survival rate of 56.5%, and a correct haploidization and fertilization rate of 31.2%, resulting in a 12.7% blastocyst rate. Time-lapse analysis revealed that reconstructed embryos underwent a timely polar body extrusion and pronuclear appearance followed by a satisfactory embryonic cleavage, comparable with the control. Whole genome sequencing of the analyzed embryos indicated that 27.3% (6/22) were properly diploid. Our findings suggest that diploid cell haploidization may be a feasible technique for creating functional gametes in mammals.

Spermatogonial stem cells (SSCs) are the basis of male spermatogenesis and fertility. SSCs are distinguished by their ability to self-renew and differentiate into spermatozoa throughout the male reproductive life and pass genetic information to the next generation. Immunohistochemistry (IHC), immunocytochemistry (ICC) and Fluidigm reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze the expression of PLZF and VASA in mice testis tissue. In this experimental study, whereas undifferentiated spermatogonial cells sharply expressed PLZF, other types of germ cells located in the seminiferous tubule were negative for this marker. Conversely, the germ cells near the basal membrane of the seminiferous tubule showed VASA expression, whereas the undifferentiated germ cells located on the basal membrane were negative. The ICC analysis indicated higher expression of PLZF in the isolated undifferentiated cells compared with differentiated germ cells. Fluidigm real-time RT-PCR results demonstrated a significant expression (P < 0.05) of VASA in the SSCs compared with differentiated cells and also showed expression of PLZF in undifferentiated spermatogonia. These results clearly proved the role of PLZF as a specific marker for SSCs, and can be beneficial for advanced research on in vitro differentiation of SSCs to functional sperms.

Smoking has dangerous and sometimes irreversible effects on various body tissues, including the reproductive system. We conducted this research to determine the in vivo effects of cigarette smoke condensate (CSC) on reproduction in mice. In this experimental in vivo study, 32 male and female NMRI mice were divided into four groups. The mice were injected with CSC (CSC-1R3F) for 28 days. The mice were mated 1 day after the last injection and observed daily for 1 week for the presence of a vaginal plug to track mating. We evaluated mating success rate, and sperm and oocyte quality, pregnancy outcome, childbearing status, and in vitro fertilization (IVF). The results showed a decrease in successful mating in female mice that received the CSC injections. CSC significantly influenced the number of offspring born to males. When the CSC was injected into male mice, there was a significant increase in the number of offspring compared with the group in which only the females received CSC injections. According to the results, there was a negative effect of CSC on morphological parameters in male and female mice. Also, successful IVF after exposure to CSC was significantly decreased in the female mice treated group. The results indicated that CSC significantly affected the number of offspring and fecundity success in females.

This study aimed at the performance evaluation of a closed flock of Marwari sheep and also to study the effect of accumulated inbreeding on the growth traits using a linear mixed model methodology. The data generated for 39 years (1981 to 2020) on Marwari sheep maintained at ICAR-Central Sheep & Wool Research Institute, Arid Region Campus (CSWRI, ARC), Bikaner, Rajasthan, India were used for analysis on growth traits. The overall least-squares means (LSM) of live weights at birth (BWT), weaning (3MWT), 6 months (6MWT), 9 months (9MWT) and 12 months (12MWT) were observed to be 3.02 ± 0.01, 14.30 ± 0.04, 20.12 ± 0.05, 23.68 ± 0.06 and 26.39 ± 0.07 kg, respectively. Overall LSM for average daily gain from birth to 3 months (ADG1), 3 to 6 months (ADG2) and 6 to 12 months (ADG3) were 125.44 ± 0.40, 67.37 ± 0.40 and 35.83 ± 0.29 g/day, respectively. Kleiber ratio (KR) from birth to 3 months (KR1), 3 to 6 months (KR2), and 6 to 12 months (KR3) were 16.78 ± 0.02, 6.58 ± 0.04 and 3.05 ± 0.02, respectively. Results revealed a 4.36, 25.83, 36.33, 31.50 and 28.99% improvement in the live weights since the inception of the improvement programme. This is also reflected by a significant effect of sire on all the growth traits. The estimate of inbreeding in the flock was 1.55%. Highly inbred animals were 5.13% (>5% Fi). The study revealed the non-significant effect of inbreeding level on all growth traits except for BWT and KR3. For BWT, inbreeding classes had variation; however, a negative effect was not seen. The inbreeding class (>5% Fi) was reduced by 0.05 units for KR3 as against its preceding class. Dam’s age at lambing and weight influenced the birth weight and subsequent weights. The study concluded that the selection programme of Marwari sheep is in the right direction; however, regular monitoring of inbreeding is necessary and factors affecting growth must be monitored to attain better growth rates in the nucleus.

Cell-free DNA (cf-DNA) is defined as DNA fragments that are released into the body fluids from apoptosis or necrosis cells, including follicular fluid (FF), which can affect the microenvironment of the oocyte associated with infertility. We aimed to investigate a relationship between apoptosis of cumulus cells (CCs) and cf-DNA levels in FF and clinical outcomes of women undergoing intracytoplasmic sperm injection (ICSI). Therefore, 82 FF samples were collected, and the corresponding CCs were isolated for ICSI procedures. FF cf-DNA concentration was quantified using ALU-quantitative polymerase chain reaction (PCR), and CCs DNA fragmentation index (DFI) was evaluated by the terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labelling (TUNEL) method. We found that cf-DNA and DFI levels were significantly higher in FF and CCs samples related to the age of women ≥37 years compared with the age of women < 37 years. Moreover, in older and younger women, FF cf-DNA and CCs DFI levels were significantly lower when the anti-Müllerian hormone (AMH) level was > 1.1 ng/ml compared with when AMH ≤ 1.1 ng/ml. In addition, patients with a low number of retrieved oocytes ≤ 6 had significantly higher levels of CCs DFI and FF cf-DNA than women with a higher number of retrieved oocytes > 6. Additionally, we observed that higher levels of cf-DNA and DFI were associated with poor oocyte maturity and poor embryo quality. Finally, cf-DNA and DFI levels were significantly lower in pregnant women than in non-pregnant ones. We conclude that DFI and cf-DNA levels in the oocyte microenvironment could have potential use in evaluating oocyte and embryo developmental competence.