ABSTRACT The aim of this study was to evaluate the effect of Insulin-Transferrin-Selenium (ITS) supplementation in embryo culture on the blastocyst rate and embryo quality. The bovine zygotes obtained by in vitro maturation (MIV) and fertilization (IVF) were transferred to Evolve medium in 4 groups (T1: Without ITS supplement; T2: ITS the first 48 h of culture (CIV1): T3: ITS after day 2 of culture (CIV2), T4: ITS during all culture time (CIV1+CIV2). All procedures were performed at 38.5 °C,5%CO2and90%relativehumidity. The cleavage and blastocyst rates were determined at 48 h and day 8 of culture. The total cell number and the production of reactive oxygen species (ROS) in the expanded blastocysts were determined by staining the nucleus with Hoechst and the oxidation of the fluorescent probe dichlorodihydrofluorescein diacetate (H2DCFDA). The T4 cultured embryos presented the highest rate of blastocysts (32.6%) and total cell number (145.6) compared to the control group without STI (26.2% and 98.6, respectively; p<0.05). Likewise, the production of ROS in T4 blastocysts was lower (p<0.05) than that obtained in the other study groups. The results show that the addition of the ITS supplement throughout the embryo culture time has beneficial effects on the percentage of blastocysts and embryo quality, assessed by the number of total cells and the production of ROS.

RESUMEN El estudio tuvo como objetivo evaluar el efecto del suplemento Insulina-Transferrina-Selenio (ITS) en el cultivo embrionario sobre la tasa de blastocistos y calidad embrionaria. Los cigotos de bovino obtenidos por maduración (MIV) y fertilización in vitro (FIV) fueron transferidos a medio Evolve en 4 grupos (T1: Sin suplemento ITS; T2: ITS las primeras 48 h de cultivo (CIV1): T3: ITS después del día 2 de cultivo (CIV2), T4: ITS todo el tiempo de cultivo (CIV1 + CIV2). Todos los procedimientos se realizaron a 38.5 °C, 5% de CO2 y 90% de humedad relativa. Las tasas de clivaje y de blastocistos se determinaron a las 48 h y día 8 de cultivo, respectivamente. El número de células totales y la producción de especies reactivas de oxígeno (EROs) en los blastocistos expandidos fueron determinados por tinción del núcleo con Hoechst y la oxidación de la sonda fluorescente diacetato diclorodihidrofluoresceína (H2DCFDA). Los embriones cultivados de T4 presentaron la mayor tasa de blastocistos (32.6%) y número de células totales (145.6) comparada al grupo control sin ITS (26.2% y 98.6, respectivamente; p<0.05). Asimismo, la producción de EROs en los blastocistos de T4 fue menor (p<0.05) que la obtenida en los demás grupos de estudio. Los resultados demuestran que la adición del suplemento ITS durante todo el tiempo de cultivo embrionario tiene efectos benéficos sobre el porcentaje de blastocistos y calidad embrionaria, valorada por el número de células totales y la producción de EROs.

ABSTRACT This experiment aimed to assess the effect of fetal calf serum (FCS) added to culture medium on day 5 to 7 after in vitro fertilization on production and subsequent cryotolerance of blastocysts from Ecuadorian Creole heifers. Immature cumulus-oocyte complexes (COCs) were collected by ovum pick-up (OPU) from 10 Creole heifers and from ovaries collected at abattoir in eight collection sessions. COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and culture (IVC). On day 1 after IVF, presumptive embryos from each oocyte source (OPU [O] and Abattoir [A]) were allocated randomly in two groups according to whether 2.5% (v/v) FCS was added to culture medium on day 5 after IVF: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+, and 4) A-FCS-. On day 7 after IVF, high quality embryos were vitrified. Cryotolerance of vitrified-warmed embryos was assessed according to blastocele re-expansion at 2 h and subsequent re-expansion and hatching at 24 and 48 h of re-incubation. Blastocysts rate on day 7 did not differ between oocyte source and FCS group. After vitrification/warming process, addition of FCS affected the re-expansion rate only at 2 h, irrespective of the oocyte source (p<0.05). Likewise, blastocysts hatching rate at 48 h of incubation was drastically affected only in OPU derived oocyte (p<0.01). Supplementation of FCS on day 5 after IVF did not improve blastocyst production and adversely affected the cryotolerance of in vitro bovine embryos derived from creole heifers raised on Ecuadorian Andean highlands.

RESUMEN El estudio tuvo como objetivo evaluar el efecto del suero fetal bovino (FCS) agregado al medio de cultivo los días 5 a 7 después de la fertilización in vitro sobre la producción y posterior criotolerancia de blastocistos de vaquillonas criollas ecuatorianas. Se recolectaron complejos cúmulo-ovocitos (COCs) inmaduros mediante aspiración intravaginal guiada por ultrasonido (OPU) de 10 vaquillonas criollas y de ovarios de matadero en ocho sesiones. Los COCs se sometieron a maduración in vitro (MIV), fecundación (FIV) y cultivo (IVC). El día 1 después de la FIV, los presuntos embriones de cada fuente de ovocitos (OPU [O] y Matadero [A]) se asignaron aleatoriamente en dos grupos según si se añadió FCS al 2.5% (v/v) al medio de cultivo el día 5 después de la FIV: 1) O-FCS+, 2) O-FCS-, 3) A-FCS+ y 4) A-FCS-. El día 7 después de la FIV se vitrificaron embriones de alta calidad. La criotolerancia de los embriones vitrificados-calentados se evaluó según la reexpansión del blastocele a las 2 h y la posterior reexpansión y eclosión a las 24 y 48 h de incubación. La tasa de blastocistos el día 7 no difirió entre la fuente de ovocitos y el grupo FCS. Después del proceso de vitrificación/ calentamiento, la adición de FCS solo afectó la tasa de reexpansión a las 2 h, independientemente de la fuente de ovocitos (p<0.05). Asimismo, la tasa de eclosión de los blastocistos a las 48 h de incubación se vio drásticamente afectada únicamente en los ovocitos derivados de OPU (p<0.01). En conclusión, la suplementación de FCS el día 5 después de la FIV no mejoró la producción de blastocistos y afectó negativamente la criotolerancia de embriones bovinos in vitro derivados de vaquillonas criollas criadas en los Andes ecuatorianos.

Abstract One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos. (IVP biotechnology development divisions hormones proteins factors nutrients PlateletRich Platelet Rich (PRP cells Therefore FBS (FBS IVM, IVM , (IVM) cumulusoocyte cumulus (COCs G 5 PRP) MIV SFB) SFB. . Subsequently bull respectively HSP70 HSP stress except group D7 D expected. expected expected) costeffective cost effective HSP7

Abstract The subcortical maternal complex, which consists of maternal-effect genes, plays a crucial role in the development of oocytes and preimplantation embryo until the activation of the zygote genome. One such gene, known as peptidyl-arginine deiminase VI (Padi6), is involved in the oocyte maturation, fertilization and embryonic development. However, the precise function of Padi6 gene in buffalo is still unclear and requires further investigation. In this study, the sequence, mRNA and protein expression patterns of Padi6 gene were analyzed in oocytes, preimplantation embryos and somatic tissues of buffalo. The coding sequence of gene was successfully cloned and characterized. Real-time quantitative PCR results indicated an absence of Padi6 transcripts in somatic tissues. Notably, the expression levels of Padi6 in oocytes showed an increased from the germinal vesicle stage to metaphase II stage, followed by a rapid decrease during the morula and blastocyst stages. Immunofluorescence analysis confirmed these findings, revealing a noticeable decline in protein expression levels. Our research provides the initial comprehensive expression profile of Padi6 in buffalo oocytes and preimplantation embryos, serving as a solid foundation for further investigations into the functionality of maternal-effect genes in buffalo. complex maternaleffect effect genome peptidylarginine peptidyl arginine Padi6, Padi , (Padi6) maturation However investigation study characterized Realtime Real time Notably stages findings (Padi6 (Padi

SUMMARY OBJECTIVE: The aim of this study was to compare endometrial thickness with the use of transdermal estrogen (gel) versus oral estrogen (pills) for endometrial preparation in the frozen embryo transfer cycle and serum estrogen concentrations during the preparation cycle, side effects, and chemical and clinical pregnancy rates. METHODS: This was a prospective, randomized controlled trial of women undergoing endometrial preparation for cryopreserved blastocyst transfer. A total of 88 women were randomized, of which 82 completed the study protocol. Of this group, 44 received 6 mg/day of estradiol valerate orally (pills group) and 38 received 4.5 mg/day of estradiol hemihydrate transdermally (gel group). Endometrial thickness was measured using transvaginal ultrasound between the 7 and 10th day of the cycle. Serum estradiol concentrations were measured on the day of initiating the cycle, on control transvaginal ultrasounds, and on the day of embryo transfer. Side effects were documented at each study visit. p<0.05 were adopted as statistically significant. The groups were compared using Student's t-test for continuous variables and chi-square or Fisher's exact test for categorical variables. RESULTS: There were no significant group differences (p>0.05) in endometrial thickness, biochemical and clinical pregnancy rates, miscarriage rate, blood estradiol concentrations, duration of estradiol administration, or cycle cancellation rates. CONCLUSION: Endometrial preparation with transdermal estrogen yielded similar reproductive outcomes to oral estrogen with fewer side effects. OBJECTIVE gel pills rates METHODS prospective 8 protocol 4 mgday mg 3 45 5 4. group. . th ultrasounds visit p005 p 0 05 p<0.0 Students Student s ttest t chisquare chi square Fishers Fisher RESULTS p>0.05 (p>0.05 rate administration CONCLUSION p00 p<0. p>0.0 (p>0.0 p0 p<0 p>0. (p>0. p< p>0 (p>0 p> (p> (p

Abstract In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of in vitro matured oocytes and blastocysts produced in vitro from buffalo crossbred (Bubalus bubalis), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to in vitro maturation to yield metaphase II oocytes (616) or followed in vitro fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three in vitro embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (BMP15, UCHL1, WEE1, NLRPs, KPNA7, ZP2, and ZP4) and blastocysts (APOA1, KRT18, ANXA2, S100A14, SLC34A2, PRSS8 and ANXA2) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future in vitro embryo production assays. technologies efficiency RNAseq RNA seq highthroughput high throughput analyses Bubalus bubalis, bubalis , bubalis) coexpression co Complexes slaughterhousederived slaughterhouse derived ovaries 616 (616 526. 526 . (526) 72%, 72 (72% 334 3 34 ±3.3 sd 21.3%, 213 21 (21.3% 418 4 18 ±4.1 41 7 grade 2 13976 13 976 13,97 62 8,649. 8649 8,649 8 649 (8,649) them 4,153 4153 153 (4,153 foldchange fold change 11 BMP15, BMP15 BMP (BMP15 UCHL1 UCHL WEE1 WEE NLRPs KPNA7 KPNA ZP2 ZP ZP4 APOA1, APOA1 APOA (APOA1 KRT18 KRT ANXA2 ANXA S100A14 SA S A SLC34A2 SLCA SLC PRSS Additionally 224 (224 2,200 2200 200 (2,200 components metabolism proliferation morphogenesis However bovine assays 61 (61 52 (526 72% (72 33 ±3. 21.3% (21.3 ±4. 1397 97 13,9 6 864 8,64 64 (8,649 4,15 415 15 (4,15 BMP1 (BMP1 (APOA KRT1 S100A1 SLC34A 22 (22 2,20 220 20 (2,20 (6 5 (52 (7 ±3 21.3 (21. ±4 139 9 13, 86 8,6 (8,64 4,1 (4,1 (BMP S100A (2 2,2 (2,2 ( (5 ± 21. (21 8, (8,6 4, (4, 2, (2, (8, (4 (8

Abstract The objective of the present study was to transpose sperm freezing methodology from domestic goat to the Tadjik markhor (Capra falconeri heptneri) and to address the feasibility to develop IVP and artificial insemination using such frozen semen. Semen of different adult markhor males were successfully recovered by electro-ejaculation and were then frozen using caprine methodology. Frozen semen showed good survival rates at thawing and good fertility rates were assessed in heterologous in vitro fertilization system with goat oocytes. LOPU/IVF was applied for Tadjik markhor females allowing the first successful blastocyst production in vitro. In an applied program, we also transposed successfully intrauterine AI method with frozen/thawed semen to the Tadjik markhor. Capra heptneri electroejaculation electro ejaculation oocytes LOPUIVF LOPU IVF program frozenthawed thawed

Abstract Embryo transfer in cattle is an increasingly important technique for cattle production. Full attainment of the benefits of the technology will depend on overcoming hurdles to optimal performance using embryos produced in vitro. Given its importance, embryo technology research should become a global research priority for animal reproduction science. Among the goals of that research should be developing methods to increase the proportion of oocytes becoming embryos through optimization of in vitro oocyte maturation and in vitro fertilization, producing an embryo competent to establish and maintain pregnancy after transfer, and increasing recipient fertility through selection, management and pharmacological manipulation. The embryo produced in vitro is susceptible to epigenetic reprogramming and methods should be found to minimize deleterious epigenetic change while altering the developmental program of the resultant calf to increase its health and productivity. There are widening opportunities to rethink the technological basis for much of the current practices for production and transfer of embryos because of explosive advances in fields of bioengineering such as microfluidics, three-dimensional printing of cell culture materials, organoid culture, live-cell imaging, and cryopreservation. importance science fertilization selection manipulation productivity microfluidics threedimensional three dimensional materials livecell live imaging cryopreservation

Resumen Se define a un embarazo ectópico cuando el blas tocisto en desarrollo se implanta fuera de la cavidad uterina. La localización más frecuente es en la trom pa, pero también puede ocurrir en el ovario, cérvix, cicatriz de cesárea, cuerno uterino (también mencio nado como intersticial en la literatura) o abdominal. Se estima que la incidencia de embarazo ectópico es aproximadamente el 2% de todos los embarazos, sien do la localización cornual solo el 2-4% de esos casos. El objetivo de este reporte es describir el caso de una paciente con sospecha de embarazo ectópico de loca lización incierta a quien se le realizó una laparoscopia exploradora por inicio de síntomas, evidenciando un embarazo ectópico cornual en el lecho de una salpin gectomía, producto de un embarazo ectópico tubario previo. Se realizó la cornuotomía con resección del mismo por laparoscopia y su evolución fue favorable con negativización de la subunidad beta. Este tipo de localización es rara y se asocia con grandes tasas de morbimortalidad materna. Representa un desafío diagnóstico y terapéutico para el ginecólogo que lo enfrenta.

Abstract Ectopic pregnancy occurs when a developing blastocyst implants at any site other than the uterine cavity. Ectopic pregnancy is most commonly found in the fallopian tube but may also occur in the cornua of the uterus (also found as interstitial in the literature), cervix, ovary, or abdominal cavity or in a cesarean scar. An estimated 2% of pregnan cies are ectopic, of which an interstitial pregnancy repre sents an extremely rare variant of ectopic pregnancy (EP), accounting for 2% to 4 % of all cases. The aim of this report is to describe the case of a patient with suspected ectopic pregnancy of uncertain location in which an exploratory laparoscopy was performed due to the onset of symptoms, discovering a cornual ectopic pregnancy in site of a past salpingectomy because of a tubal ectopic pregnancy. A cornuotomy by laparoscopy was performed to resect the cornual ectopic pregnancy. The patient had an unevent ful postoperative course, with negativization of human chorionic gonadotropin levels. This type of location is rare and is associated with high rates of maternal morbidity and mortality. It represents a diagnostic and therapeutic challenge for the gynecologist who faces it.

Abstract Despite relatively high maturation rate of in vitro matured oocytes in the dromedary camel, however, blastocyst production is very low after in vitro fertilization (IVF). Herein, the influences of oocyte collection method (follicular aspiration vs slicing; Experiment I), the addition of Insulin-like growth factor I (IGF-I) to the maturation medium (Experiment II) on in vitro maturation (IVM) of oocyte were investigated. Although the nuclear maturation did not differ regardless of collecting method, follicular aspiration led to lower degeneration rates than those in controls (P < 0.05). The percentages of oocytes at MII were greater in the presence of IGF-1 than in its absence (71.9% vs 48.4%, respectively, P<0.05). Additionally, the percentages of degenerated oocytes were higher in the control group compared to oocytes cultured in the presence of IGF-I (23.6% vs 10.4%, respectively, P<0.05). IGF-I treatment improved the quality of MII matured oocytes as evidenced by the decrease of cathepsin B (CTSB) activity, a marker of poor quality oocytes, when compared to control ones (P < 0.05). In conclusion, follicular aspiration decreased the degeneration rate; however, it had no effect on completion of maturation. IGF-I enhanced the IVM of oocyte and decreased degeneration rate. camel however IVF. IVF . (IVF) Herein slicing I, , I) Insulinlike Insulin like IGFI IGF (IGF-I II (IVM investigated P 0.05. 005 0.05 0 05 0.05) IGF1 1 IGF- 71.9% 719 71 9 (71.9 484 48 4 48.4% respectively P<0.05. P005 P<0.05 P<0.05) Additionally 23.6% 236 23 6 (23.6 104 10 10.4% CTSB (CTSB activity conclusion (IVF 00 0.0 71.9 7 (71. 48.4 P00 P<0.0 23.6 2 (23. 10.4 0. 71. (71 48. P0 P<0. 23. (23 10. (7 P<0 (2 ( P<

Abstract The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil. pickup pick up OPUIVF IVF (OPU-IVF superovulation (superovulation splitplot split plot design nonlactating non lactating andor SOVembryo 63day day interval 16 3 lutea cumulusoocyte cumulus oocyte COC (COC However 301, 301 (301 41.9% 419 41 9 609, 609 (609 202 33.1%. 331 33.1% . 33 1 33.1%) Also 49.6% 496 49 6 (49.6 118 11 8 11.8% 11. respectively 32.8% 328 (32.8 72 7 2 7.2% 7. 111, 111 (111 73 48, 48 (48 3. Overall counterparts Brazil 30 (30 41.9 4 60 (60 20 33.1 49.6 (49. 32.8 (32. (11 (4 (3 41. (6 33. 49. (49 32. (32 (1 (

Abstract Advancements in assisted reproduction (AR) methodologies have allowed significant improvements in live birth rates of women who otherwise would not be able to conceive. One of the tools that allowed this improvement is the possibility of embryo selection based on genetic status, performed via preimplantation genetic testing (PGT). Even though the widespread use of PGT from TE biopsy helped to decrease the interval from the beginning of the AR intervention to pregnancy, especially in older patients, in AR, there are still many concerns about the application of this invasive methodology in all cycles. Therefore, recently, researchers started to study the use of cell free DNA (cfDNA) released by the blastocyst in its culture medium to perform PGT, in a method called non-invasive PGT (niPGT). The development of a niPGT would bring the diagnostics power of conventional PGT, but with the advantage of being potentially less harmful to the embryo. Its implementation in clinical practice, however, is under heavy discussion since there are many unknowns about the technique, such as the origin of the cfDNA or if this genetic material is a true representative of the actual ploidy status of the embryo. Available data indicates that there is high correspondence between results observed in TE biopsies and the ones observed from cfDNA, but these results are still contradictory and highly debatable. In the present review, the advantages and disadvantages of niPGT are presented and discussed in relation to tradition TE biopsy-based PGT. Furthermore, there are also presented some other possible non-invasive tools that could be applied in the selection of the best embryo, such as quantification of other molecules as quality biomarkers, or the use artificial intelligence (AI) to identify the best embryos based on morphological and/or morphokitetic parameters. (AR conceive . (PGT) pregnancy patients cycles Therefore recently (cfDNA noninvasive non niPGT. (niPGT) practice however technique debatable review biopsybased Furthermore biomarkers AI (AI andor parameters (PGT (niPGT

Abstract The establishment and maintenance of a pregnancy that goes to term is sine qua non for the long-term sustainability of dairy and beef cattle operations. The oocyte plays a critical role in providing the factors necessary for preimplantation embryonic development. Furthermore, the female, or maternal, environment where oocytes and embryos develop is crucial for the establishment and maintenance of a pregnancy to term. During folliculogenesis, the oocyte must sequentially acquire meiotic and developmental competence, which are the results of a series of molecular events preparing the highly specialized gamete to return to totipotency after fertilization. Given that folliculogenesis is a lengthy process in the cow, the occurrence of disease, metabolic imbalances, heat stress, or other adverse events can make it challenging to maintain oocyte quality. Following fertilization, the newly formed embryo must execute a tightly planned program that includes global DNA remodeling, activation of the embryonic genome, and cell fate decisions to form a blastocyst within a few days and cell divisions. The increasing use of assisted reproductive technologies creates an additional layer of complexity to ensure the highest oocyte and embryo quality given that in vitro systems do not faithfully recreate the physiological maternal environment. In this review, we discuss cellular and molecular factors and events known to be crucial for proper oocyte development and maturation, as well as adverse events that may negatively affect the oocyte; and the importance of the uterine environment, including signaling proteins in the maternal-embryonic interactions that ensure proper embryo development. We also discuss the impact of assisted reproductive technologies in oocyte and embryo quality and developmental potential, and considerations when looking into the prospects for developing systems that allow for in vitro gametogenesis as a tool for assisted reproduction in cattle. longterm long operations Furthermore female competence fertilization cow disease imbalances stress remodeling genome divisions review maturation maternalembryonic potential

ABSTRACT The objective of this study was to identify differentially expressed genes (DEG) between females and males in early bovine embryos. Superovulated cattle (n = 4) in the age span of 16–18 months were artificially inseminated with semen from the same bull that has been proven to be fertile. Blastocysts were collected by routine non-surgical uterine flushing on day 7 after insemination. This study determines the sex of embryos using a micro-injection pipette to collect a few blastomeres through the Zona pellucida. The remaining blastomeres were used for single-cell RNA sequencing (scRNA-seq) analysis on the Illumina platform, followed by differential expression analysis, Gene Ontology (GO) function analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. The criteria for identifying DEG will be set at |log2(Fold change)| ≥ 1 and P<0.05. A total of 8004 DEG were detected in female (Denoted as BLX, n = 3) and male (Denoted as BLY, n = 3) blastocysts. Transcripts highly expressed in the female embryos were related to catalytic activity, nucleotide binding, and catabolic process, while transcripts highly expressed in the male embryos were linked to oxidative phosphorylation, mitochondrion, and the ribosome. Nine genes that may be involved in blastocyst growth and development were screened, suggesting that their differential expression may be responsible for the differences in the development of male and female embryos. This study provides important information on potential genes and pathways associated with differences in early female and male embryonic development. (DEG 4 1618 16 18 16–1 fertile nonsurgical non surgical insemination microinjection micro injection pellucida singlecell single cell scRNAseq scRNA seq (scRNA-seq platform GO (GO KEGG (KEGG |log2Fold log2Fold logFold |log2 Fold log2 log change| change | change) P005 P 0 05 P<0.05 800 Denoted BLX 3 BLY blastocysts activity binding process phosphorylation mitochondrion ribosome screened 161 16– |log P00 P<0.0 80 P0 P<0. 8 P<0 P<

Abstract GnRH analogues were widely used for controlld ovary stimulation, but their effects on oocyte quality remain contradictory. This study aimed to explore the influence of GnRH analogues on oocyte quality in mice. A total of 120 mice were randomly assigned to four groups:(i)GnRH-a+PMSG group; (ii) GnRH-ant+PMSG group; (iii) PMSG group; (iv) Control group. Ovaries were collected for quantitative real-time polymerase chain reaction (qRT-PCR) to assess GDF9 and BMP15 mRNA expression, and protein expression were evaluated by western blotting. Moreover, embryo developmental progress in vitro and implantation rate in vivo were recorded. Compared with control group, both GDF9 mRNA and protein expressions were strengthened in PMSG group, but reduced in the presence of GnRH-a or GnRH-ant. The GnRH-a group exhibited decreased BMP15 mRNA expression compared to PMSG group, while the GnRH-ant group did not show the same pattern. BMP15 protein expression were not statisticlly different among the four groups. Notably, there was no statistically difference in the expression of these two factors between GnRH-a and GnRH-ant groups. The percentage of zygotes progressing to the 2-cell stage and percentage of 2-cell advancing to the blastocyst stage were similar in the PMSG group and control group. However, both the GnRH-a and GnRH-ant groups showed decreased embryos development rates compared to other two groups. The embryonic implantation rate in control group (53.3%) was higher than that in the GnRH-a and GnRH-ant groups (33.3% and 30.8%, P<0.05). The difference between the PMSG (45.0%) and GnRHa group was statistically significant (P value of 0.023), but not between the PMSG and GnRH-ant group (P value of 0.486). No statistical difference was confirmed between GnRH-a and GnRH-ant groups. Our findings shed light on the safety of GnRH analogues in ovary stimulation, and highlight the need for further research to establish optimal and effective controlled ovary stimulation protocol. contradictory 12 groupsiGnRHa+PMSG groupsiGnRHaPMSG i a+PMSG a ii (ii GnRHant+PMSG GnRHantPMSG ant+PMSG ant iii (iii iv (iv realtime real time qRTPCR qRT PCR (qRT-PCR GDF BMP BMP1 blotting Moreover recorded GnRHant. GnRHant ant. pattern Notably 2cell cell 2 However 53.3% 533 53 3 (53.3% 33.3% 333 33 (33.3 308 30 8 30.8% P<0.05. P005 P P<0.05 . 0 05 P<0.05) 45.0% 450 45 (45.0% 0.023, 0023 0.023 , 023 0.023) 0.486. 0486 0.486 486 0.486) protocol 1 groupsiGnRHa aPMSG antPMSG 53.3 5 (53.3 33.3 (33. 30.8 P00 P<0.0 45.0 4 (45.0 002 0.02 02 048 0.48 48 53. (53. 33. (33 30. P0 P<0. 45. (45. 00 0.0 04 0.4 (53 (3 P<0 (45 0. (5 ( P< (4