Rate Of Embryonic Development Research Articles

Ten-year outcomes of patients who developed persistent azoospermia following chemotherapy associated with different oncological diagnoses: A retrospective cohort study from a different perspective.

This study evaluated the treatment procedures for chemotherapy (CT)-induced persistent azoospermia and their outcomes from a different perspective. In 63 patients (mean age: 30.16 ± 4.91 years) who had undergone CT 11 ± 5 years earlier, the semen volume, gonadotropins level, FSH level, genetics, micro-testicular sperm extraction (m-TESE) result, sperm DNA fragmentation index (SDFI), semen reactive oxidative stress (ROS) rate, duration of embryonic development, and pregnancy and baby take-home rates were examined. The correlations between the ROS rates and the SDFIs, m-TESE results, sperm motility, pathology scores, time-lapses, and baby take-home rates were evaluated. The semen volumes were 3.5 ± 1.1/ml. The FSH level following CT was 17.87 ± 5.80 mIU/ml. A sperm rate of 34.9% was found from the m-TESE result. The mean SDFI and ROS rate were 4 (<15-30>) and 1.29 ± 0.51, respectively. The time-lapse was calculated as 5h. Pregnancy and live birth were achieved at 20.63% and 12.7%, respectively. In the patients with a low ROS (≤1.42) and SDFI (≤15), the m-TESE success rate was high, the FSH value was low, the pathological score and fertilization rate were elevated, the embryonic cleavage period was normal, and the pregnancy and baby take-home rates were high. The sperms may be detected using m-TESE in patients who develop persistent azoospermia associated with CT due to different oncological diagnoses. Our study revealed that a low FSH value and normal ejaculatory ROS rates are positive predictive factors of sperm detection before m-TESE. The motility of the sperms detected after m-TESE and normal SDFI rates were found to be positive predictive criteria of high fertilization, good embryonic cleavage, pregnancy, and live birth.

Anaesthesia for assisted reproductive technology (ART): A narrative review

Assisted reproductive technology (ART) is used primarily to address the treatment of infertility which includes medical procedures such as in vitro fertilisation (IVF), intra-cytoplasmic sperm injection (ICSI), gamete intra-fallopian transfer (GIFT) or zygote intra-fallopian transfer (ZIFT). IVF has revolutionised infertility treatment and is nowadays widely accepted all over the world. The IVF is carried out as a daycare procedure and many anaesthetic regimens have been studied, tried and tested so far. An anaesthesiologist's role mainly comes into play during trans- vaginal oocyte retrieval and embryo transfer (ET) process of IVF. Various techniques of anaesthesia are practised which include general or regional anaesthesia, conscious sedation or monitored anaesthesia care, patient-controlled analgesia, acupuncture and transcutaneous electrical nerve stimulation (TENS). The anaesthetic management needs careful consideration of the effect of drugs on the maturation of oocytes or embryonic development, fertilisation and pregnancy rates. In view of the Coronavirus disease-19 (COVID-19) pandemic, ART clinics have been affected and due to the ambiguity of its effects on the reproductive outcome, anaesthesiologists need to be vigilant and cautious with anaesthetic management during pandemic times. This review includes a discussion of various anaesthetic options and agents along with their advantages or disadvantages if any. The literature sources for this review were obtained via PubMed, Medline, Cochrane Library and Google Scholar. The results of 82 out of 110 articles discussing different methods of anaesthesia for ART procedures over 25 years were compiled.

Treatment of mouse cumulus-oocyte complexes with L-carnitine during vitrification and in vitro maturation affects maturation and embryonic developmental rate after parthenogenetic activation.

The technique of oocyte vitrification remains a challenge in most animal species. The present study aimed to evaluate the effects of cumulus cell presence and L-carnitine (LC) treatment during vitrification of selected immature oocytes by brilliant cresyl blue (BCB) staining on maturation and embryonic developmental rate after parthenogenetic activation. Immature oocytes were obtained from C57BL/6 female mice ovaries and stained with BCB. The BCB+ cumulus-oocyte complexes (COCs) were then selected and random parts of COCs were denuded from cumulus cells (denuded oocytes: DOs). COCs and DOs were treated with/out LC (0.6mg/ml) during vitrification and in vitro maturation (IVM) procedures. A number of non-vitrified COCs were also treated with LC during the IVM process (fresh group). Maturation rate, intracellular glutathione (GSH) contents, and developmental competence of oocytes were also examined. The GSH levels in vitrified DOs+LC and vitrified COCs+LC groups were significantly higher (p<0.01) than untreated vitrified-warmed COCs and DOs. Maturation rate and blastocyst developmental rate were reduced after the vitrification-warming procedure compared with the fresh group. The vitrified COCs+LC group showed a higher percentage of mature oocytes and the ability to develop to blastocyst stage than the vitrified-warmed DOs group (p<0.01). These data indicated that the presence of cumulus cells around the competent oocyte and LC treatment during vitrification and IVM procedure could improve parthenogenetic developmental competence of vitrified-warmed oocytes by increasing GSH levels and accelerating oocyte maturation.

Zinc Chloride Supplemented during Ovarian Tissue Vitrification Improves In Vitro Follicle Development and Fertilization in Pigs

Ovarian tissue vitrification is a promising method to preserve follicles and gametes, but can be improved with mineral supplementation to the vitrification medium. The objective of this study was to determine the effects of supplementing 0.5 mg/mL zinc chloride to the media during cryopreservation of pig ovarian tissue. After thawing, the following criteria were evaluated: (1) follicular development and damage, (2) in vitro fertilization (IVF) characteristics, and (3) embryonic development. The number of damaged antral follicles (72.0 ± 3.8%) was less (p &lt; 0.05) in ovarian tissue vitrified in media supplemented with zinc chloride compared to those not supplemented with zinc chloride (86.7 ± 4.1%). Oocytes obtained from the antral follicles on ovarian tissue vitrified in media supplemented with zinc chloride had less (p &lt; 0.05) polyspermic penetration and higher (p &lt; 0.05) male pronuclear formation during IVF than oocytes obtained from ovarian tissue not supplemented with zinc chloride. There were no statistical differences in embryonic development rates. Based on these results, supplementing zinc chloride during the vitrification protocol improves follicular development and subsequent IVF in pigs.

Cryopreservation of embryos of humpback grouper (Cromileptes altivelis) using combinations of non-permeating cryoprotectants

In this study, Cromileptes altivelis embryos were successfully preserved using non-permeable cryoprotectants for the first time. We compared the toxicity of single non-permeable cryoprotectants and combination of non-permeable cryoprotectants on C. altivelis embryos. Five non-permeable cryoprotectants at 1 M (i.e., trehalose, sucrose, lactose, fructose, and glucose) were used to treat embryos in the somite, tail-bud, or heart-beating stages for 1 h. Survival rates and rates of normal embryonic development after treatment with trehalose, sucrose, or lactose were all greater than 86.97% and 86.01%, respectively. Levels of toxicity of trehalose, sucrose, and lactose were lower than those of fructose and glucose in C. altivelis embryos. Sucrose and trehalose were combined at different ratios (total concentration 1 M) and used to treat embryos for 1 h. Rates of survival and normal development for the somite, tail-bud, or heart-beating stages did not significantly differ among groups treated with sucrose + trehalose at ratios of 1:1 (S1T1), 1:3 (S1T3), and 3:1 (S3T1). Embryos treated with S1T1 had a little higher survival rate than those of the other treatment ratios(P > 0.05). Survival rates of embryos treated with T1S1, sucrose, and trehalose were 93.86 ± 4.17%, 91.58 ± 3.15%, and 92.79 ± 2.45%, respectively. S1T1 was less toxic than sucrose or trehalose alone under the same conditions. Various cryopreservation-related factors (e.g., the cryoprotectant solution, embryonic stage, equilibrium time, thawing temperature, and time) were screened. The optimal protocol was the treatment of embryos at the tail-bud and heart-beating stages with 1 M S1T1 for 2–10 min and thawing at 26 °C for 10 s after freezing. In total, 21 viable embryos were recovered after freezing and thawing, with a survival rate of 0.21–7.55% and maximum survival time of 5 days. Our results demonstrate that it is feasible to cryopreserve C. altivelis embryos using non-permeable cryoprotectants and provide a reference for the use of combinations of cryoprotectants for the species.

Rapid microgeographic evolution in response to climate change.

Environmental change is predicted to accelerate into the future and will exert strong selection pressure on biota. Although many species may be fated to extinction, others may survive through their capacity to evolve rapidly at highly localized (i.e., microgeographic) scales. Yet, even as new examples have been discovered, the limits to such evolutionary responses have not often been evaluated. One of the first examples of microgeographic variation involved pond populations of wood frogs (Rana sylvatica). Although separated by just tens to hundreds of meters, these populations exhibited countergradient variation in intrinsic embryonic development rates when reared in a common garden. We repeated this experiment 17 years (approximately six to nine generations) later and found that microgeographic variation persists in contemporary populations. Furthermore, we found that contemporary embryos have evolved to develop 14-19% faster than those in 2001. Structural equation models indicate that the predominant cause for this response is likely due to changes in climate over the intervening 17 years. Despite potential for rapid and fine-scale evolution, demographic declines in populations experiencing the greatest changes in climate and habitat imply a limit to the species' ability to mitigate extreme environmental change.

P–143 Improved embryonic development and utilization rates with EmbryoScope: A within-subject comparison versus a benchtop incubator

Abstract Study question In consecutive intracytoplasmic sperm injection (ICSI) cycles, is embryonic development in EmbryoScope better than the previous one obtained in a benchtop (G–185) incubator? Summary answer Embryonic development, and oocyte and embryo utilization rates (OUR and EUR) are significantly improved in the EmbryoScope, as compared to G–185. What is known already The time-lapse imaging (TLI) system, which allows a non-invasive continuous assessment of embryo morphokinetics parameters in a closed culture system has been developed, promising improved embryo development by reducing oscillations in pH, humidity and temperature. To investigate this hypothesis, one study has already compared embryonic development in a TLI versus a benchtop incubator. However, It has never been investigated whether embryonic development can be improved within-subject, by changing from benchtop incubator in the first intracytoplasmic sperm injection (ICSI) cycle to the EmbryoScope, a TLI incubator, in the following ICSI cycle, and that was the objective of the present study. Study design, size, duration This study had a retrospective within-subject design, in which each cycle served as its own control. Data were obtained via chart review of patients undergoing ICSI in a private university–affiliated IVF center that fulfilled the following criteria: second ICSI attempt in which embryos had been cultured in a TLI incubator system (TLI group, n = 71), preceded by a first ICSI attempt in which embryos had been cultured in a conventional incubator (Control group, n = 71). Participants/materials, setting, methods Embryonic development up to the fifth day of development, OUR (transferred embryos plus frozen embryos per retrieved oocytes) and EUR (transferred embryos plus frozen embryos per fertilized oocytes) were compared between the groups using generalized linear models followed by Bonferroni post hoc. The post hoc achieved power was 82.6%, considering the sample size, the effect size obtained for blastocyst development rate and 5% significance level. Main results and the role of chance There were significant differences in fertilization rate (76.0% ± 1.3 vs. 80.0% ± 1.4, p = 0.044, OR: 1.051, CI: 1.001 – 1.103), non-fertilization rate (14.8% ± 0.6 vs. 6.3% ± 0.4, p &amp;lt; 0.001, OR: 0.424, CI: 0.370 – 0.486), day–2 non-cleavage rate (3.8% ± 0.2 vs. 1.1% ± 0.1, p &amp;lt; 0.001, OR: 0.285, CI: 0.234 – 0.347), blastocyst development rate (40.9% ± 1.1 vs. 55.6% ± 1.3, p &amp;lt; 0.001, OR: 1.358, CI: 1.267 – 1.456), frozen blastocyst rate (31.8% ± 0.8 vs. 37.0% ± 0.9, p &amp;lt; 0.001, OR: 1.163, CI: 1. 085 – 1.248), OUR (40.7% ± 1.0 vs. 50.2% ± 1.1, p &amp;lt; 0.001, OR: 1.232, CI: 1.155 – 1.314), and EUR (52.4% ± 1.1 vs. 66.6% ± 1.2, p &amp;lt; 0.001, OR: 1.269, CI: 1.202 - 1.341), all in favor of TLI group. Pregnancy rate (30.2% vs. 30.8%, p = 0.940), implantation rate (24.6% ± 40.0 vs. 26.1% ± 41.6, p = 0.830), and miscarriage rate (21.1% vs. 15.0%, p = 0.622) were similar between Control and TLI groups, respectively. Limitations, reasons for caution (i) Different culture dishes were used in each system; (ii) it is not possible to confirm how much of the embryonic improvement was due to the culture conditions; (iii) the study design is not ideal for the comparison of clinical outcomes and, also, underpowered to do so. Wider implications of the findings: Even though the clinical outcomes were similar between the groups, the results may also lead to higher cumulative pregnancy outcomes following embryo thawing and transfer. Trial registration number Not applicable

Evaluation of the Effects of Human Bone Marrow Mesenchymal Stem Cells Conditioned Medium on Growth and Maturation of Mouse Ovarian Follicle after Vitrification

The aim of this research study is to evaluate the effect of human bone marrow mesenchymal stem cells conditioned medium (hBMSCs-CM) on growth and maturation of mouse ovarian follicle, and embryonic development after vitrification. The hBMSCs were cultured, and the derived CM was collected, concentrated, and stored. 14-day-old mice ovaries were collected and randomly divided into vitrified and non-vitrified groups. Then their isolated preantral follicles were cultured for 12 days in α-MEM supplemented with different concentrations of CM (2.5, 5, and 7.5%). Finally, the growth and diameter of follicles, maturation of oocytes, hormone level, and embryo developmental rate were assessed. The results showed the antrum formation, oocyte maturation, and hormone secretion were significantly higher in the presence of 7.5% CM (p < 0.001). In the vitrified group, the developmental rate of follicles was lower than the non-vitrified group, and the subgroup containing 7.5% CM showed better results than the 5%, and 2.5% CM subgroups. However, no changes in fertilization and embryonic development rates were observed. Supplementing follicle culture media with 7.5% CM could enhance follicle growth and oocyte maturation of follicles after vitrification.

Nest temperature assessment in an American crocodile (Crocodylus acutus) population on the central coast of Oaxaca, Mexico.

The temperatures at which eggs of crocodilians are incubated plays an important role in embryo survival, rate of embryonic development and sex definition. The aim of this study was to assess the nest temperatures of an American crocodile (Crocodylus acutus) population on the central coast of Oaxaca state in Mexico. The fieldwork was carried out from February to June 2018at Palmasola Lagoon, Oaxaca. Ten natural nests of C. acutus were carefully excavated to determine clutch size. When putting the eggs back in the nests, we placed a data logger in the center of the egg mass to determine the temperature parameters in the nest chamber environment, as well as the variation in temperature during the incubation period. All nests were revisited to count the number of hatched eggs (NHE) and to determine the hatching success (HS), along with the duration of the incubation period (IP). Hatching success was 89.04%. The mean clutch size in the American crocodile nests was 30.7±7.83 eggs (ranging from 17 to 46 eggs), and the mean incubation period was 77.6±5.89 days. The mean nest incubation temperature throughout the reproductive season was significantly different among nests. Based on the average temperature during the middle third of the incubation period, the nests should have produced both sexes, but with a higher proportion of males. This study tried to elucidate the impact of nest temperatures during the incubation period on embryo survival, as well as hatchling sex ratio in a local climate on the central coast of Oaxaca.

The rate of embryonic development and hatching in transgenic G3 mutiara catfish (Clarias gariepinus) eggs at room temperature

Mutiara catfish is one of the most important catfish strains developed in Indonesia. However, the growth stability of cultured non-transgenic Mutiara catfish fluctuates. This problem can be solved by developing transgenic Mutiara catfish (containing the African catfish growth hormone gene, CgGH) to improve the stability of fish growth. Controlled handling of eggs is carried out to reproduce this superior fish in each generation. The egg handling in a controlled system was performed to reproduce this superior fish in each generation. The G3 transgenic Mutiara catfish eggs production was carried out with three different broodstock pairs (pairs A, B and C). The results of observations of the rate of embryogenesis and hatching of the G3 eggs showed that eggs from pair A were faster than B and C. Transgenic catfish eggs hatch 1-1.5 hours faster than non-transgenic fish eggs at room temperature.

Controlled spawning and rearing of the sea cucumber, Parastichopus tremulus

AbstractThe red sea cucumber, Parastichopus tremulus, a cold water species with commercial potential, has recently attracted attention for wild harvest as well as for potential use in integrated multi‐trophic aquaculture in Scandinavian countries. Overharvesting has put natural stocks of sea cucumbers at risk in several countries. Our goal was to develop a rearing protocol for P. tremulus to enable sustainable production of this species. This study presents results from spawning and larval rearing conducted in both Norway (NO) and Sweden (SWE) during May–August 2019. We describe spawning induction and behavior, fertilization success, embryonic and auricularia larval development rate for this species under laboratory conditions. The larvae were fed a mixture of three species of live microalgae (SWE) and algal paste (NO). Larval development rate and survival were monitored at four different temperatures (7, 10, 13, and 16°C). Results showed faster development with increasing temperature. Daily food consumption rate was highest at the highest temperature. The combined effects of temperature and food availability on survival were investigated for the same four temperatures and three different feed concentrations. Only food availability affected the mortality rate, with the highest mortality in the low feeding regime of 1,000–2,000 cells ml−1 day−1.

Reproductive and embryological toxicity of lead acetate in male mice and their offspring and mitigation effects of quercetin

Exposure to heavy metals not only impacts on fertility in males, it may also affect the offspring. The aim of the present study was to examine the toxic effects of lead acetate on fertility in male mice and their offspring, and the potential effect of quercetin on mitigating the likely effects. Experimental mice were randomly divided into three groups and administered with (i) distilled water (control); (ii) lead acetate (150 mg/kg BW/day); (iii) lead acetate (150 mg/kg BW/day) with quercetin (75 mg/kg BW/day). Lead acetate administration in male mice adversely affected their fertility through changes in sperm motility, viability, morphology, maturity, membrane integrity, and intracellular reactive oxygen species (P < 0.05). Similar findings were observed in the offspring of the lead-treated male mice. Early embryonic development and implantation rate were also adversely influenced in both the sires and offspring when male mice were treated with lead acetate (P < 0.05). The data demonstrated that down-regulation of Cks2 (CDC28 protein kinase regulatory subunit-2) in sperm had an association with early embryonic development in lead acetate treated group. In conclusion, lead acetate administration adversely impacted on the fertility of the male mice and their male offspring fertility; on the other hand, paternal quercetin co-administration somewhat ameliorated the adverse effects of lead on male mice and their offspring.

Multigenerational exposure to warming and fishing causes recruitment collapse, but size diversity and periodic cooling can aid recovery

Global warming and fisheries harvest are significantly impacting wild fish stocks, yet their interactive influence on population resilience to stress remains unclear. We explored these interactive effects on early-life development and survival by experimentally manipulating the thermal and harvest regimes in 18 zebrafish (Danio rerio) populations over six consecutive generations. Warming advanced development rates across generations, but after three generations, it caused a sudden and large (30-50%) decline in recruitment. This warming impact was most severe in populations where size-selective harvesting reduced the average size of spawners. We then explored whether our observed recruitment decline could be explained by changes in egg size, early egg and larval survival, population sex ratio, and developmental costs. We found that it was most likely driven by temperature-induced shifts in embryonic development rate and fishing-induced male-biased sex ratios. Importantly, once harvest and warming were relaxed, recruitment rates rapidly recovered. Our study suggests that the effects of warming and fishing could have strong impacts on wild stock recruitment, but this may take several generations to manifest. However, resilience of wild populations may be higher if fishing preserves sufficient body size diversity, and windows of suitable temperature periodically occur.

Assessing the Viability of American Horseshoe Crab (Limulus polyphemus) Embryos in Salt Marsh and Sandy Beach Habitats.

AbstractFor animal embryos that develop externally, the physio-chemical environment can substantially affect offspring viability. In the case of the American horseshoe crab (Limulus polyphemus), sediment conditions along estuarine shorelines influence development rates and embryonic viability. Sandy beach habitats are considered to have optimal conditions for horseshoe crab embryonic development; however, spawning is often observed outside of these optimal habitats, in areas such as salt marshes, where reduced oxygen availability is thought to decrease the viability of eggs laid in these sediments. We excavated horseshoe crab eggs, embryos, and trilobites laid naturally in marsh and beach sediments in South Carolina to compare their development and viability between habitats. We found all developmental stages in both marsh and beach habitats. For two of three sampling areas, trilobites were more likely to be found at beaches than at marshes. Multivariate analyses demonstrate that the prevalence of early and middle developmental stages was similar between habitats but that beaches had a greater proportion of late-stage trilobites than marshes. The lower likelihood of finding trilobites at some marshes may reflect differences in spawning phenology between habitats or reduced rates of embryonic development in marshes compared to beaches, leading to potentially different developmental timelines rather than a true reduction in viability. Nevertheless, the substantial proportions of eggs laid in salt marshes that survive to the trilobite stage indicate that spawning in this habitat could represent a previously underappreciated source of recruitment for horseshoe crab populations that may need to be incorporated into population assessments.

Berberine regulates lipid metabolism via miR-192 in porcine oocytes matured in vitro.

BackgroundThe berberine (Ber) is an isoquinoline alkaloid compound extracted from Rhizoma coptidis and has the effect that reduces adipose. MicroRNA‐192 (miR‐192) is related to fat metabolism. However, the relevant mechanism of berberine on lipid metabolism during in vitro maturation (IVM) of porcine oocytes remains unclear.ObjectivesIn this study, we investigated the molecular mechanism by which berberine promotes the IVM and lipid metabolism of porcine oocytes via miR‐192.MethodsBer was added to IVM medium of porcine oocytes. MiR‐192 agomir, miR‐192 antagomir and negative control fragment were microinjected into the cytoplasm of oocytes without Ber. Rates of oocyte IVM and embryonic development in each group were observed. The content of lipid droplets in IVM oocytes in each group was analyzed by Nile red staining. Expression levels of miR‐192 and FABP3, SREBF1 and PPARG, were detected by qPCR and western blotting. The target genes of miR‐192 were determined by luciferase reporter assays.Results and ConclusionsWe found that Ber significantly increased the rate of oocytes IVM and blastocyst development, and decreased the area and numbers of lipid droplets in IVM oocytes. Ber significantly increased the expression of miR‐192 in IVM oocytes, and significantly decreased the expression of SREBF1 and PPARG, which were target genes of miR‐192. This study indicates that Ber promotes lipid metabolism in porcine oocytes by activating the expression of miR‐192 and down‐regulating SREBF1 and PPARG, thus, improving IVM of porcine oocytes.

Expression of obesity-related genes in human spermatozoa affects the outcomes of reproductive treatments

To study the abundance of obesity-related gene (ORG) mRNA in human spermatozoa and its association with sperm quality parameters, embryonic development, and pregnancy rates after assisted reproduction treatment (ART). Cross-sectional study of spermatozoa ORG mRNA expression, and sperm and embryonic development parameters of infertile couples attending a single ART center. University, in collaboration with a medically assisted reproduction center. One hundred six couples seeking fertility treatment and receiving ART. Expression of spermatozoa ORG mRNA was assessed by quantitative reverse transcription-polymerase chain reaction. Sperm and embryonic development parameters were measured by board-certified embryologists. Serum β-human chorionic gonadotropin levels and fetal heartbeat detection on ultrasound were used to document biochemical and clinical pregnancy, respectively. Correlations between the abundance of ORG transcripts in spermatozoa and sperm quality, embryonic development, and achievement of pregnancy. The abundance of spermatozoa FTO mRNA was positively correlated with total sperm count (r = 0.5030), fertilization rate (r = 0.4854), embryo cleavage rate (r = 0.5705), and high-quality embryo rate (r = 0.6982). The abundance of spermatozoa MC4R transcript was negatively correlated with sperm viability (r = -0.3111) and positively correlated with biochemical pregnancy (r = 0.4420). The abundance of MC4R and GNPDA2 transcripts was higher in spermatozoa of men with asthenozoospermia and teratozoospermia than in those with normozoospermia. To our knowledge, this is the first report showing that the abundance of MC4R and FTO transcripts in spermatozoa is associated with sperm and embryo quality parameters, as well as pregnancy rates. Overall, these results further support the view that male factors beyond classic sperm quality parameters, namely the abundance of ORG transcripts, also affect the outcome of ART.

Does parental heart rate affect embryonic heart rate during incubation? An experiment in Common Terns Sterna hirundo

Embryonic heart rate is a strong determinant of metabolic rate and the rate of embryonic development in oviparous species. In humans, embryonic heart rate is positively correlated with the heart rate of the mother. However, human embryos do not develop autonomously from the maternal circulatory system, making it impossible to separate maternal control from an independent embryonic adjustment of its heart rate to that of its mother. In birds, embryonic development does occur autonomously from the mother, while the embryo can auditively perceive the heart rate of the incubating parent. In this study, we used Common Terns (Sterna hirundo) to experimentally test whether the embryonic heart rate is affected by parental heart rate during incubation. We artificially incubated freshly laid eggs under standardized conditions and continuously exposed them to the sound of either a slow or fast parental heart rate throughout the first 18 days of embryonic development. At day 18 of embryonic development (a few days before hatching) there was no significant difference in the embryonic heart rate between both treatments. There was also no effect of treatment on either total duration of embryonic development or hatching success. We suggest that embryonic heart rate and development rate may not be affected by the heart rate of the parent because natural parental heart rates are very sensitive to external conditions that may fluctuate drastically, while embryos may need a relatively stable heart rate for optimal development.

The impact of fresh and frozen testicular tissue quality on embryological and clinical outcomes.

Our ability to predict the potential of testicular spermatozoa to support embryonic development is still limited. Although motility of testicular spermatozoa is associated with embryo development, the impact of morphology and the presence of spermatozoa in the testicular sample has not been previously researched. Moreover, while the majority of data indicate no effect of cryopreservation, there are studies reporting impaired clinical outcomes due to testicular cryopreservation. In a retrospective study, 118 ICSI-TESE cycles were analysed to study the impact of (a) total quality of testicular tissue, (b) testicular tissue cryopreservation and (c) presence/motility/morphology of testicular spermatozoa in fertilisation rate, embryonic development, clinical pregnancy (CPR), ongoing pregnancy (OPR) and live birth rate (LBR). Results showed that fertilisation rate was significantly affected by both total quality of testicular tissue (p<.05) and rare presence of spermatozoa (p<.01). Moreover, total tissue quality (p<.01), cryopreservation of low-quality samples (p<.01), absence of motile testicular spermatozoa (p<.01) and poor spermatozoa morphology (p<.05) had a negative impact on the number of good quality day 3 embryos. CPR, OPR or LBR was not affected by any parameters examined. Our data suggest that the quality of testicular tissue influences both fertilisation rate and embryo development. Moreover, cryopreservation of low-quality testicular samples has a negative impact on the number of available embryos for transfer.

Nuclear and cytoplasmic quality of oocytes derived from serum-free culture of secondary follicles in vitro.

In vitro culture of follicles is a promising technology to generate large quantities of mature oocytes and it could offer a novel option of assisted reproductive technologies. Here we described a 2-dimensional follicular serum-free culture system with 3-dimensional effect that can make secondary follicles develop into antral follicles (78.52%), generating developmentally mature oocytes in vitro (66.45%). The oocytes in this serum-free system completed the first meiosis; spindle assembly and chromosome congression in most oocytes matured from follicular culture were normal. However, these oocytes showed significantly lower activation and embryonic development rates, and their ability to produce Ca2+ oscillations was also lower in response to parthenogenetic activation, after which a 2-cell embryonic developmental block occurred. Oocytes matured from follicular culture displayed increased abnormal mitochondrial distribution and increased reactive oxygen species levels when compared to in vivo matured oocytes. These data are important for understanding the reasons for reduced developmental potential of oocytes matured from follicular culture, and for further improving the cultivation system.

Whole genome integrity and enhanced developmental potential in ram freeze-dried spermatozoa at mild sub-zero temperature

Freeze-dried spermatozoa typically shows a reduction in fertility primarily due to the DNA damage resulting from the sublimation process. In order to minimize the physical/mechanical damage resulting from lyophilization, here we focused on the freezing phase, comparing two cooling protocols: (i) rapid-freezing, where ram sperm sample is directly plunged into liquid nitrogen (LN-group), as currently done; (ii) slow-freezing, where the sample is progressively cooled to − 50 °C (SF-group). The spermatozoa dried in both conditions were analysed to assess residual water content by Thermal Gravimetric Analysis (TGA) and DNA integrity using Sperm Chromatin Structure Assay (SCSA). TGA revealed more than 90% of water subtraction in both groups. A minor DNA damage, Double-Strand Break (DSB) in particular, characterized by a lower degree of abnormal chromatin structure (Alpha-T), was detected in the SF-group, comparing to the LN-one. In accordance with the structural and DNA integrity data, spermatozoa from SF-group had the best embryonic development rates, comparing to LN-group: cleaved embryos [42/100 (42%) versus 19/75 (25.3%), P < 0.05, SL and LN respectively] and blastocyst formation [7/100 (7%) versus 2/75 (2.7%), P < 0.05, SF and LN respectively]. This data represents a significant technological advancement for the development of lyophilization as a valuable and cheaper alternative to deep-freezing in LN for ram semen.