Embryo Production Research Articles

Effect of Hormones, Season and Lactation on Follicular Size, Oocyte Competency and Embryo Production in Sahiwal Cattle

Background: Transvaginal ultrasound-guided ovum pick-up (OPU) and in vitro embryo production (IVEP) techniques are widely used for enhancing reproductive efficiency. The number and quality of embryos have been reported to be influenced by various factors like source of hormones, season, lactation in cattle. Methods: Twenty-one multiparous, normal cyclic Sahiwal cows (aged: 6-8 years; lactating: n-11 and non-lactating: n-10) were selected randomly and the experiment was conducted during hot (April to September) and cold (October to March) seasons. All the donor cows were administered 10 µg GnRH at random stage of oestrous cycle followed by FSH (Folltropin-V™ or Stimufol) at the rate of 100, 60 and 40 mg after 48, 60 and 72 h of GnRH administration. After 24 hours of coasting period, follicular aspiration was performed by stabilizing the targeted follicles with the help of ultrasonography. The procedures of COCs grading, in vitro maturation, fertilization, followed by culture were performed as per the standard protocols. Results: The hormonal treatment was effective in improving the oocytes’ developmental competency and in vitro embryo production; with no statistical differences between the two sources. The follicle size distribution revealed a lower (P less than 0.05) medium-sized and small-sized follicular proportion during the hot season with a higher average count of dominant (large-sized) follicles. While, good quality (A+B+C) oocytes, matured and fertilized oocytes were significantly higher (P less than 0.05) in non-lactating donors, especially among cool-season collections.

Progesterone supplemented uterine epithelial cell co-culture improves in vitro quality embryo production in buffalo

Objectives: The present study was conducted to study the effect of progesterone and uterine luminal epithelial cells monolayer on blastocyst development and hatching rate. Material and Methods: The isolation, culture, and characterization of slaughterhouse-derived uterine epithelial cells were done using standard protocol. The steroid hormones estrogen and progesterone were supplemented in embryo developmental media (EDM), and day 04 embryos were cultured in different groups as progesterone supplementation (T1), co-cultured with epithelial cell monolayer (T2), co-cultured with progesterone supplemented epithelial cell monolayer (T3), or without any treatment (control). Finally, the effect of different treatments was analyzed in terms of blastocyst and hatching rate. Results: The isolated epithelial cells depicted compact cuboidal or columnar morphology at the confluence. Immunocytochemical localization and polymerase chain reaction study revealed positive expression of cytokeratin and absence of vimentin. Significantly higher blastocyst and hatching rates were noted in the T3 group, followed by T2, T1, and control groups. Conclusion: The present study revealed improved in vitro embryo production after co-culturing embryos with progesterone-supplemented uterine epithelial cells in buffalo.

Complications in equine transvaginal ultrasound‒guided follicular aspiration

Transvaginal ultrasound-guided follicular aspiration has revolutionised equine assisted reproduction, particularly in in vitro embryo production. This literature review examines the complications associated with transvaginal ultrasound-guided follicular aspiration in mares and its potential impact on future fertility. While transvaginal ultrasound-guided follicular aspiration is generally safe, it has inherent risks, ranging from mild rectal bleeding to severe complications such as peritonitis and acute haemorrhage. Factors influencing complication rates include veterinarian experience, mare anatomy and procedural technique. Despite concerns, studies suggest that repeated transvaginal ultrasound-guided follicular aspiration does not significantly impair future fertility, with normal pregnancy rates observed in mares undergoing artificial insemination or embryo transfer after transvaginal ultrasound-guided follicular aspiration. However, further research is needed to fully understand the long-term effects on ovarian function and oocyte quality. Veterinary practitioners must prioritise appropriate training and procedural precautions to minimise the risk of complications and safeguard mare welfare and fertility in the expanding field of equine assisted reproduction.

Curcumin enhances developmental competence and ameliorates heat stress in in vitro buffalo (Bubalus bubalis) embryos

Background and Aim: Buffalo is the principal dairy animal and plays a major role in the economic growth of the dairy industry, contributing nearly 50% of the country’s milk production. The Buffalo core body temperature is typically 38.5°C, but it can rise to 41.5°C in the summer, causing heat stress, which leads to the generation of reactive oxygen species or oxidative stress and affects the reproductive physiology of animals. Curcumin acts as an antioxidant, improves cellular development, and combats the effect of heat stress on in vitro-produced embryos. This study aimed to examine the impact of curcumin on developmental competence and the expression of important genes under normal and heat-stressed conditions during in vitro embryo production in buffalo. Materials and Methods: Group-1: All embryo production steps (i.e., in vitro maturation [IVM], in vitro fertilization [IVF], and in vitro culture [IVC]) were conducted at 38.5°C. The presumed zygotes were cultured in media supplemented with different concentrations of curcumin, that is, 0 μM, 5 μM, and 10 μM of curcumin. Group-2: All embryo production steps (i.e., IVM, IVF, and IVC) were carried out at 38.5°C. The presumed zygotes were cultured in media supplemented with different concentrations of curcumin, that is, 0 μM, 5 μM, and 10 μM of curcumin, but the early cleaved embryos were exposed to heat stress (39.5°C) for 2 h after 48 h of IVF and then cultured at 38.5°C for embryo production. Results: Blastocyst production was 16.63 ± 1.49%, 21.46 ± 0.67%, and 6.50 ± 1.17% at control, 5 μM and 10 μM of curcumin at 38.5°C, respectively, whereas at 39.5°C, it was 8.59 ± 1.20%, 15.21 ± 1.31%, and 3.03 ± 1.20% at control, 5 μM and 10 μM curcumin, respectively. The blastocyst rate was found to be significantly higher (p < 0.05) at 5 μM curcumin compared with the control or 10 μM at 38.5°C and 39.5°C. The antioxidant, antiapoptotic, and pluripotency-related genes exhibited higher (p < 0.05) expression in the presence of 5 μM curcumin compared to 10 μM or control at both temperatures. Conclusion: Curcumin supplementation in embryo culture media effectively enhances embryo production in vitro and mitigates the adverse effects of heat stress. Keywords: antioxidant, buffalo, curcumin, embryo, heat stress.

Compatibility of dry incubator on in vitro production of bovine embryos

Embryo culture is crucial to achieve successful outcomes in in vitro production-embryo transfer for cattle. This study explored the innovative use of dry incubators for bovine embryo culture, building on their advantages in human medicine, such as reduced contamination risk, stable temperature control, and lower gas consumption. In this study, we examined changes in osmotic pressure, the in vitro developmental potential of IVP embryos including the cleavage rate, blastocyst development rate, blastocyst diameter, and blastocyst cell number, morphokinetics, and the transcriptional profile of the blastocysts between humidified and dry incubators. Our research demonstrates the feasibility of this approach, showing that although the osmotic pressure gradually increases over the culture period (on day 8: 271.7 vs. 299.0, respectively; P < 0.09), it did not negatively affect the blastocyst formation rate (62.4% vs. 69.8%) and the morphological quality of blastocysts (diameter: 237.4 vs. 242.8, total cell number: 189.2 vs. 242.8). Embryos cultured in dry incubators exhibited morphokinetics comparable to those cultured in conventional humidified incubators. Furthermore, RNA-seq revealed that while a few genes showed changes, the transcriptomic profiles of blastocysts cultured in dry incubators were largely similar to those of blastocysts cultured in humidified incubators. These findings highlight the considerable potential of dry incubators for the in vitro production of bovine embryos.

IVF with frozen-thawed sperm after prolonged capacitation yields comparable results to ICSI in horses: A morphokinetics study

Intracytoplasmic sperm injection (ICSI) is the current clinical practice for the in vitro production of equine embryos. The use of conventional fertilization methods such as in vitro fertilization (IVF), has historically been associated with poor success in horses. However, recent improvements have led to better outcomes with IVF, though only when using fresh semen, which limits its use in clinical practice. IVF remains in its infancy in equine reproduction, and several unknowns remain about the technique. One significant gap in knowledge concerns the morphokinetics of IVF embryos and how they differ from their ICSI counterparts. To address this, we performed IVF using frozen-thawed sperm from five different stallions following sperm selection and a prolonged capacitation period of 10 h, on a total of 109 oocytes. We then analyzed the cleavage rate (cleaved/initial oocytes), blastocyst rate (blastocyst/initial zygotes), and blastocyst development (blastocyst/cleaved zygotes) of the IVF cycles, and compared them with those of the clinical ICSI cycles during the same period. We also evaluated time-lapse images of the developed embryos to assess developmental time points such as time to morula compaction and blastocyst expansion, as well as morula and blastocyst sizes. Overall, developmental rates were not different between IVF and ICSI cycles (blastocyst rate 41.1 % IVF and 41.8 % ICSI, p > 0.05). However, development proceeded faster in IVF cycles (blastocyst expansion IVF 155.5 ± 18.5 h; ICSI 167.2 ± 19.6 h; p < 0.05) and IVF embryos were also larger (blastocyst area IVF 22608 ± 2857 μm2; ICSI 20806 ± 1505 μm2; p < 0.05). The faster development and larger size might suggest a more advanced developmental stage. The implications of these findings need to be further evaluated to assess their association with pregnancy potential. The successful developmental rates achieved in IVF cycles demonstrate the potential of this technique for clinical application, although the amount of frozen-thawed semen required is significantly higher in IVF than in ICSI, which is an important consideration for mare and stallion owners. Nonetheless, the use of frozen-thawed semen in equine IVF, coupled with comparable blastocyst rate, presents promising potential for broader clinical adoption of the IVF technique.

Feline cumulus cells and oocytes show massive accumulation of lipid droplets and upregulation of PLIN2 expression after in vitro maturation

This study investigates lipid content and expression of genes involved in lipid metabolism in feline cumulus cells and oocytes before and after in vitro maturation (IVM). Domestic cats represent valuable models in reproductive research, yet the efficiency of in vitro embryo production remains suboptimal, with contributing factors still under investigation. We characterized lipid droplets (LDs) in oocytes collected from adult queens, both before and after IVM, using confocal microscopy with Bodipy 493/503 staining. We also quantified the expression of four genes involved in regulation of lipid metabolism. Our findings revealed a substantial accumulation of LDs and a significant upregulation of PLIN2 expression in both cumulus cells and oocytes following IVM. The number and total area of LDs and fluorescence intensity increased markedly in oocytes at the MII stage, while the average LD diameter decreased. Similarly, cumulus cells showed an increase in number and total area of LDs post-IVM, suggesting their involvement in oocyte maturation by modulating lipid homeostasis. This study presents the first detailed characterization of lipid droplet dynamics in feline oocytes and cumulus cells, providing insights into metabolic processes during IVM, which are critical for optimizing assisted reproductive technologies in felids.

Effect of the addition of IGF-1 during in vitro culture on the embryonic development speed from different crossbreed bovine embryos.

Supplementation with insulin-like growth factor type 1 (IGF-1) during in vitro culture of bovine embryos has yielded mixed results, likely due to genetic variability among embryos. This work aimed to evaluate the effect of IGF-1 at two concentrations on the development speed embryos from primary F1 crossbreeds used in dual-purpose cattle farming in the Colombian low tropics. Specifically, we investigated the influence of IGF-1 and embryo breed on the blastocyst formation rate. Oocytes were sourced from non-pregnant cows: Bos taurus indicus (20 Brahman and 14 Gyr) and Bos taurus taurus (12 Holstein and 28 Romosinuano). Oocytes were fertilized with semen from specific bulls (Recoil for Holstein, Gabinete for Gyr, and UBER POI 1490 for Brahman). The resulting embryos from each crossbreed group were randomly distributed in three different cultured media with 50 ng/mL IGF-1, 100 ng/mL IGF-1, or no IGF-1 (control) for 7 days. Results showed that 50 ng/mL IGF-1 significantly increased embryo production by day 6 (25.9%±14.56%) compared to control (20.5%±11.84%) and 100 ng/mL IGF-1 (23.0%±9.54%) (p < 0.05). By day 7, both 50 ng/mL (42.6%±26.55%) and 100 ng/mL (49.7%±21.98%) IGF-1 groups exhibited significantly higher production rates compared to the control group (p < 0.001). The embryo breed also influenced development, with Gyr-Holstein (GxH) crossbreeds showing the highest production rates (p < 0.001). In conclusion, IGF-1 supplementation enhances in vitro embryo production, with the effect influenced by both breed and IGF-1 concentration. These findings suggest that breed-specific optimization of IGF-1 conditions is necessary to maximize embryonic development outcomes.

Production of Microspore-Derived Plants by Anther Culture of Cyclamen coum

Cyclamen is one of the most important ornamental crops sold worldwide as a potted flower for winter production. Cyclamen species take up a wide swathe of habitats across Turkey. Ten wild Cyclamen species grow naturally in Turkey, some of them endemic. This study aimed to produce haploid plants of C. coum using anther culture. The microspore developmental stage was evaluated by staining anther with acetocarmine (%2), and then the stage was correlated with bud size. It was determined that the buds between 7.64-8.23 mm had the appropriate bud size for the late uninuclear stage. Anthers were cultured in B5 medium containing different levels of NAA (0.1, 1, 2 mg/L), 2,4-D (0.1, 1, 2 mg/L), and kinetin (0, 1 mg/L), 90 g/L sucrose and 3 g/L gelrite for haploid embryo production. Anthers were kept at 4°C for 4 days after culture. The explants were incubated at 24°C in a completely dark condition until the embryo was formed, then embryos were transferred to hormone-free media in 8:16 hours (light: dark) photoperiod. The experiment was carried out for two years. In the first year, 12 different media were examined in view of regeneration and the experiments were continued with selected 7 media in the second year. The highest callus regeneration rates were %5.71 and 14.5% and the highest embryo induction rates varied between 8.57% and 4.0% in the first and second year respectively. Embryo/callus formation was observed in 7 of a total of 12 different media tested for haploid plant production, and the best media were kinetin (1 mg/L) + NAA (1, 2 mg/L) and kinetin (1 mg/L) +2,4-D (2 mg/L). Our findings indicated that cold pre-treated anther explants collected at appropriate flower bud size resulted in embryo production Additionally, B5 medium supplemented with NAA and kinetin ensured successful embryo regeneration from anther explants in wild C. coum.

Injectable progesterone in 13-month-old prepubertal Nellore heifers: effects on in vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of injectable progesterone administration before ovum pick-up (OPU) on in vitro embryo production (IVEP) and pregnancy rate in prepubertal Nellore heifers. Twenty-three Nellore females, 13.04 ± 0.8 months of age, were randomly assigned to two groups: control group (no progesterone application) and progesterone group (intramuscular administration of 1 mL [150mg/mL] progesterone 7 days before OPU). The second OPU was performed after 28 days (crossover between groups). After OPU, oocytes were selected for IVEP and a sample was fixed in 4% paraformaldehyde. Viable oocytes were subjected to immunofluorescence staining for IGFBP2 and caspase-3. For each oocyte, one image was generated per channel (A555 and A488 filters) and analyzed with the ImageJ program for the quantification of fluorescence intensity (in pixels). The produced embryos were transferred to multiparous Nellore cows and pregnancy was diagnosed by ultrasound 30 days after transfer. Data were analyzed using the MIXED and GLIMMIX procedure of SAS®. Treatment with injectable progesterone had no effect on the number or size of the antral follicles of heifers. No differences between the control and progesterone groups were observed for total number of aspirated oocytes (P = 0.78), viable oocytes (P = 0.80), cleavage rate (P = 0.60), or blastocyst rate (P = 0.56). There were no differences in pixel intensity between the two groups, nor in the levels of IGFBP2 or caspase-3. However, a difference was observed in the pregnancy rate of embryos produced from heifers of the progesterone group (42.1%; 32/76) when compared to the rate of embryos produced from control heifers (28.4%; 23/81) (P = 0.02). The use of injectable progesterone before OPU in prepubertal Nellore heifers does not improve IVEP but increases the pregnancy rate after transfer.

Increased in vitro production of bovine embryos resulting from oocyte maturation in the presence of triciribine, a specific inhibitor of AKT

The aim of this study was to evaluate the effect of different concentrations of triciribine, a selective Akt inhibitor, on various aspects of oocyte maturation and on the IVF of bovine embryos. Cumulus-oocyte complexes (COCs) were matured in vitro in medium supplemented with: 0 (control), 1, 5, 10, and 20 μM of triciribine. The nuclear maturation was assessed by staining with acetic orcein, while the cytoplasmic maturation was evaluated by mitochondrial (MitoTracker® Red CMXRos) and lipid droplets distribution (LipidTOX). COCs were fertilized in vitro and cultured for nine days. Cleavage rates, blastocyst production, and hatching rates were determined on days three, seven, and nine of in vitro culture, respectively. Oocytes from COCs treated with 1 μM of triciribine were stained at 3, 6, and 9 h of IVM to determine the inhibitor's involvement in germinal vesicle breakdown. Analysis of variance (ANOVA) of the data was performed and the means were compared using the SNK test at a 5 % significance level. Exposure of COCs to 1, 5, and 10 μM of triciribine did not alter the number of matured oocytes (P < 0.05), a concentration of 20 μM reduced the number of oocytes in MII with a consequent increase in oocytes in MI (P < 0.05). This concentration markedly reduced the number of oocytes with peripheral cortical granules and the rates of cleavage and blastocysts (P < 0.05). On the other hand, when COCs were matured in the presence of 1 μM, there was an increase in the blastocyst rate (P < 0.05), but without altering the timing of meiosis resumption (P < 0.05). It is concluded that the Akt pathway participates in the nuclear and cytoplasmic events of in vitro maturation of bovine oocytes, but through mechanisms that do not interfere with germinal vesicle breakdown. Modulation of Akt activity in bovine COCs during IVM with 1 μM of triciribine increases the in vitro production of bovine embryos.

Embryo production by Holstein heifers superovulated with a recombinant long-acting follicle stimulating hormone analog.

This study aimed to evaluate embryo yield in Holstein heifers superovulated with a single injection of recombinant, long-acting human follicle-stimulating hormone (rFSH) versus multiple injections of pituitary-derived follicle-stimulating hormone (FSH). In Experiment 1, heifers were assigned randomly to one of 4 experimental groups: Control (280mg of pituitary derived FSH; six injections of 40mg and two injections of 20mg, each ~12h apart, n = 16); rFSH1 (50 μg of FSH analog protein, n = 16); rFSH2 (75 μg of FSH analog protein, n = 16) or; rFSH3 (100 μg of FSH analog protein, n = 16). The rFSH was administered as a single injection. Estrous cycles were presynchronized with gonadorelin acetate (GnRH) and an intra-vaginal progesterone insert (CIDR) on d -8, followed by cloprostenol sodium (PGF2α) on d -3 and d -2 with removal of the CIDR, and GnRH on d 0. On d 5, ovarian follicles ≥ 5mm in diameter were ablated by transvaginal ultrasound-guided aspiration. On d 6.5, heifers received a CIDR and the rFSH or the first injection of pituitary-derived FSH. On d 9, heifers received two injections of PGF2α, 12h apart. On d 10.5, the CIDR was removed, and on d 11, heifers received a GnRH injection. Heifers were AI-inseminated 12- and 24-hours post-GnRH injection, and uterine contents were flushed trans-cervically on day 18 (7 d after the GnRH injection). Ovarian follicles ≥ 5mm and corpora lutea were counted via ultrasound on days 5, 9, and 18. In Experiment 1, group did not affect (P = 0.52) the number of follicles ≥ 5mm (Control = 15.9 ± 1.2; rFSH1 = 17.5 ± 1.3; rFSH2 = 17.1 ± 1.3; rFHS 3 = 18.6 ± 1.4 follicles) or the number of corpora lutea (P = 0.96) on d 9 (Control = 1.1 ± 0.3; rFSH1 = 1.1 ± 0.3; rFSH2 = 1.1 ± 0.3; rFSH3 = 0.9 ± 0.2). Furthermore, there was no effect (P = 0.28) of rFSH dose on freezable embryos (grade 1 and 2 embryos) collected on d 18 (Control = 4.7 ± 1.1; rFSH1 = 4.7 ± 1.2; rFSH2 = 4.4 ± 1.1; rFSH3 = 2.6 ± 0.7 embryos). In Experiment 2, Control (n=8) and rFSH1 (n=16) groups were repeated in 3 replicates using the same protocols as Experiment 1. Consequently, Results showed that rFSH produced fewer total number of ova/embryos (Control = 9.9 ± 1.5 vs. rFHS1 = 5.9 ± 0.9, P = 0.04) and fewer freezable embryos (Control = 5.3 ± 1.0 vs. rFSH1 = 1.4 ± 0.3, P < 0.01). In conclusion, the single rFSH injection effectively induced superovulation; however, its repeated use reduced embryo production.

Ин витро култивиране на говежди ембриони

Embryo cultivation is the longest step during the in vitro embryo production process. In this study, the effect of adding 5% FCS or 3 mg/ml BSA to SOF and KSOM in vitro culture media was investigated. When adding FCS in SOF medium, a better effect on separation was observed (P &lt; 0.05), and the percentage of blastocysts was higher - 58.6%. When BSA was added to SOF medium, the percentage of blastocysts was 40.1% - significantly lower. When cultured in KSOM medium with the addition of FCS, the division rate was lower than when cultured in SOF+FCS and the proportion of blastocysts was also slightly lower. When BSA was added to the KSOM medium, the percentage of blastocysts was almost as in the SOF+BSA group. Аt the conclusion of the conducted experiment, the addition of FCS in KSOM and SOF media is beneficial for in vitro cultivation, resulting in a higher percentage of blastocysts.

Oviduct epithelial spheroids during in vitro culture of bovine embryos mitigate oxidative stress, improve blastocyst quality and change the embryonic transcriptome

BackgroundIn vitro embryo production is increasingly used for genetic improvement in cattle but bypasses the oviduct environment and exposes the embryos to oxidative stress with deleterious effects on further development. Here we aimed to examine the effect of oviduct epithelial spheroids (OES) on embryo development and quality in terms of morphology and gene expression during two co-culture times (4 days: up to embryonic genome activation at 8–16 cell stage vs. 7 days: up to blastocyst stage) and under two oxygen levels (5% vs. 20%).MethodsBovine presumptive zygotes produced by in vitro fertilization (day 0) using in-vitro matured oocytes were cultured in droplets of synthetic oviductal fluid (SOF) medium with or without (controls) OES for 4 or 7 days under 5% or 20% oxygen (4 treated and 2 control groups). Cleavage rates were evaluated on day 2 and blastocyst rates on days 7–8. Expanded blastocysts on days 7–8 were evaluated for total cell numbers and gene expression analysis by RNA-sequencing.ResultsUnder 20% oxygen, blastocyst rates and total cell numbers were significantly higher in the presence of OES for 4 and 7 days compared to controls (P < 0.05), with no difference according to the co-culture time. Under 5% oxygen, the presence of OES did not affect blastocyst rates but increased the number of cells per blastocyst after 7 days of co-culture (P < 0.05). Both oxygen level and OES co-culture had a significant impact on the embryonic transcriptome. The highest number of differentially expressed genes (DEGs) was identified after 7 days of co-culture under 20% oxygen. DEGs were involved in a wide range of functions, including lipid metabolism, membrane organization, response to external signals, early embryo development, and transport of small molecules among the most significantly impacted.ConclusionOES had beneficial effects on embryo development and quality under both 5% and 20% oxygen, mitigating oxidative stress. Stronger effects on embryo quality and transcriptome were obtained after 7 than 4 days of co-culture. This study shows the impact of OES on embryo development and reveals potential molecular targets of OES-embryo dialog involved in response to stress and early embryonic development.

Putrescine supplementation improves the developmental competence of in vitro produced bovine embryos

The aim of this study was to investigate the effect of putrescine, anti-apoptotic, antioxidant, and a cell proliferation stimulant, on embryo development and quality by supplementing it to in vitro culture medium. In this study, oocytes were obtained from the ovaries of Holstein cattle. Following maturation and fertilization, the presumptive zygotes were randomly assigned to two groups. The first group (Putrescine, n = 435) was supplemented with putrescine at a concentration of 0.5 mM to in vitro culture. The second group (n = 407) was maintained under standard culture conditions without any supplementations to the medium. Following the determination of the developmental stages of the embryos, only those in the blastocyst stage were subjected to differential staining and the cell numbers of the embryos were determined. Moreover, the TUNEL assay was employed to ascertain the extent of cell death and the apoptotic index in the embryos. Additionally, the levels of ROS were determined in the embryos. Furthermore, gene expression analyses were conducted on blastocyst-stage embryos to ascertain the potential of putrescine supplementation in embryo development along specific pathways. Following in vitro culture, the blastocyst formation rate was 44.37 % in the putrescine group and 32.97 % in the control group (P < 0.05). The counts of ICM (60.60 ± 15.79 vs 50.73 ± 16.74), TE (117.70 ± 23.67 vs 94.0 ± 22.46), and TCC (178.30 ± 26.15 vs 144.73 ± 26.86) were found to be statistically higher in blastocysts developing after putrescine supplementation compared to the control group. Furthermore, the number of apoptotic cells (7.69 ± 2.17 vs 9.96 ± 3.99) and the apoptotic index (5.07 % vs 8.01 %) were found to be lower in the putrescine group in comparison to the control group. Nevertheless, it was established that the ROS level in the control group was approximately two-fold higher than in the putrescine group (P < 0.05). The findings also revealed that putrescine up-regulated the gene expression of SOD, GPX4, CAT, BCL2, NANOG and GATA3 while simultaneously down-regulating the BAX expression level. In conclusion, the supplementation of putrescine to the culture medium during in vitro bovine embryo production was found to contribute to the improvement of embryo quality and early embryonic development.

The Immediate Partial Removal of Cumulus-Oocyte Complexes: A Refined Approach for Rapid Observation of In Vitro Fertilization.

Despite the rapid advancements in clinical and laboratory technologies for in vitro fertilization (IVF), a significant proportion (10%-15%) of patients continue to experience fertilization disorders, leading to low fertilization rates and the production of nonviable embryos. Short-term fertilization involves the early removal of granulosa cells to observe the extrusion of the second polar body, enabling the assessment of fertilization and early remedial measures to address low fertilization rates and complete fertilization failure. However, the observation of short-term fertilization in IVF is impeded by challenges such as excessively large unprocessed clusters of cumulus-oocyte complexes and adhesion between eggs, necessitating complex external procedures for granulosa cell removal, and prolonged observation duration. To tackle this issue, this study proposes a method of immediate partial removal of granulosa cells post egg retrieval. This approach streamlines subsequent stages of short-term fertilization and traditional fertilization monitoring, reduces the time needed for oocyte extrusion, minimizes the likelihood of external environmental impacts on the oocytes, and decreases the risk of missing the critical fertilization observation window. Consequently, the study yields novel clinical evidence aimed at enhancing the operational efficiency of embryonic laboratories in IVF.

Supplementation of spermidine enhances the quality of postovulatory aged porcine oocytes

BackgroundSpermidine (SPD) is an intermediate compound in the polyamine metabolism which takes critical part in a variety of cellular processes. In particular, it has been reported to exert anti-aging effects, suppress the age-related diseases, and extend lifespan across species. However, whether it has the favorable influence on the quality of postovulatory aged oocytes remains elusive.MethodsImmunostaining and fluorescence intensity measurement were used to evaluate the effects of postovulatory aging and SPD supplementation on the oocyte fragmentation, spindle/chromosome structure, actin polymerization, dynamics of cortical granules (CGs) and ovastacin, mitochondrial distribution and function, as well as autophagy levels. In addition, in vitro sperm binding assay and in vitro fertilization (IVF) experiment were applied to assess the impacts of postovulatory aging and SPD supplementation on the sperm binding ability and fertilization capacity of oocytes.ResultsHere, we showed that supplementation of SPD during postovulatory aging could relieve the deterioration of porcine oocytes. Specifically, we found that postovulatory aging impaired the oocyte quality by damaging the morphological integrity of oocytes, maintenance of spindle/chromosome structure, and dynamics of actin cytoskeleton. Postovulatory aging also weakened the sperm binding ability and fertilization capacity of oocytes by compromising the distribution pattern of CGs and their content ovastacin. Notably, supplementation of SPD attenuated these defects in postovulatory aged porcine oocytes via strengthening mitochondrial function, eliminating excessive reactive oxygen species (ROS), inhibiting apoptosis, and enhancing autophagy levels.ConclusionAltogether, our findings demonstrate that SPD supplementation is a feasible approach to ameliorate the quality of postovulatory aged oocytes, which can be potentially applied to the human assisted reproductive technology (ART) and in vitro production of animal embryos.

In vitro maturation of bovine oocytes: the role of ROS, oxidative stress, and the impact of natural antioxidant supplementation

According to available data in the literature, Brazil has one of the largest cattle herds in the world and is one of the leading embryo producers, with a clear predominance of embryos produced in vitro. These data influence the ongoing pursuit of optimizing the results obtained in IVEP (in vitro embryo production). In this technique, in vitro maturation (IVM) is such a crucial stage that it justifies studies involving components that, when added to the media, can lead to improved cleavage rates and blastocyst development. It is well known that oocyte quality is influenced by oxidative stress, and in this context, the inclusion of antioxidants in the IVM medium may enhance these outcomes. Thus, the present study aimed to review oocyte maturation, the impact of oxidative stress, and the addition of antioxidants, such as Moringa oleifera, to the IVM media.

Reproductive Performance of Ongole Grade, Simmental, and Limousin Cattle Given Superovulation Treatment

Reproductive technologies have the potential to enhance livestock productivity, with superovulation being one such method aimed at increasing the number of ovulated oocytes per estrous cycle through hormonal stimulation. This study aimed to evaluate the reproductive performance of three cattle breeds—Ongole Grade (OG) (n=20), Simmental (n=18), and Limousin (n=20)—subjected to superovulation treatment. The cattle, aged 2.5 to 4 years with a body condition score of 3.5 to 4, were intensively managed. Superovulation was induced using CIDR (Controlled Internal Drug Release) for synchronization, followed by follicle-stimulating hormone (FSH) administration. Reproductive performance data, including age at first estrus, number of corpora lutea (CL), and embryo production, were collected over three estrous cycles. A quasi-experimental design was employed with breed as the primary treatment factor, and data were analyzed using analysis of variance (ANOVA). The results indicated that breed had a significant effect (P0.05) on the age of first estrus, with OG cattle reaching first estrus at 621.3±93.3 days, Simmental at 522.4±75.5 days, and Limousin at 570.4±88.1 days. However, no significant differences (P0.05) were observed among the breeds in terms of the number of CL, collected embryos, or transferable embryos across the superovulation cycles.

Advanced assisted reproduction technologies in endangered mammalian species.

A new synergistic approach of classical conservation strategies combined with advanced assisted reproduction technologies (aART) allows for protection and rescue of endangered keystone species at the brink of extinction, which can help to safeguard complex ecosystems. Reproduction biology and management in mammal species is not only challenging in regards to their diverging sizes, anatomy, and often unknown physiology; it also requires customized training or chemical restraint protocols for safe handling. Besides these general challenges, there are several new assisted reproduction techniques (ART) specifically tailored to critically endangered mammals. The current portfolio of ART in these mammalian taxa is ranging from sexual cycle characterization and manipulation, semen collection and cryopreservation, artificial insemination, biobanking of living cells, oocyte collection, invitro fertilization (IVF), and embryo production, embryo transfer as well as stem cell-derived invitro gametogenesis for generating gametes in culture. The article covers advanced assisted reproduction technologies (aART), success and challenges, as well as ethical implications.