Maturation Medium Research Articles

Effect of putrescine supplementation to in vitro maturation medium on embryo development and quality in cattle

This study aimed to investigate the effect of putrescine supplementation to maturation medium during in vitro embryo production in cattle on maturation and embryo development/quality. Oocytes obtained from the ovaries of Holstein cattle were used in the study. Obtained cumulus-oocyte complexes were evaluated according to morphological structure, cytoplasmic features, and cumulus cell number, and only Category-I ones were used in the study. Before the in vitro maturation step, oocytes were randomly divided into two groups. In the first group (Putrescine group, n = 159), 0.5 mM putrescine was added to the maturation medium before in vitro maturation. No addition was applied to the maturation medium of the second group (Control group, n = 149). Cumulus expansion degrees of oocytes following maturation (Grade I: poor, Grade II: partial, and Grade III: complete) were determined. In addition, the meiosis of oocytes after maturation was evaluated by differential staining. Then the oocytes were left for fertilization with sperm and finally, possible zygotes were transferred to the culture medium. After determining the developmental stages and quality of the embryos after in vitro culture, only the embryos at the blastocyst stage were stained with the differential staining method to determine the cell numbers. When the cumulus expansion degrees of the groups were evaluated, the Grade III cumulus expansion rate in the putrescine group was higher than the control group (74.21% and 60.4%; respectively) and the Grade I expansion rate (11.95% and 26.17%; respectively) was found lower (p < .05). When the resumption of meiosis was evaluated according to the cumulus expansion degrees, it was determined that the rate of resumption of meiosis increased as the cumulus expansion increased. In addition, the cleavage rates of oocytes and reaching the blastocyst in the putrescine group were found to be higher than in the control group (p < .05). Moreover, inner cell mass, trophectoderm cells, and total cell counts were found to be higher in blastocysts obtained after the putrescine supplementation to the maturation medium compared to the control group (p < .05). As a result, it was determined that the putrescine supplementation to the maturation medium during in vitro embryo production in cattle increased the degree of cumulus expansion and the rate of resumption of meiosis. In addition, putrescine supplementation was thought to increase the rate of reaching the blastocyst of oocytes due to better cell development in embryos.

Effects of insulin-like growth factor 1, leukemia inhibitory factor, and basic fibroblast growth factor on goat oocyte maturation and early embryonic development in vitro

In this study, we investigated the effects of insulin-like growth factor 1 (IGF-1), leukemia inhibitory factor (LIF), and basic fibroblast growth factor (bFGF) on the in vitro maturation of goat oocytes and early embryonic development of in vitro-fertilized (IVF) oocytes. We also aimed to prepare an efficient in vitro maturation culture system for goat oocytes as a technical basis for improving both the quality of goat oocytes and the efficiency of embryo production in vitro. The experimental results revealed that the addition of bFGF (40 ng/mL), LIF (20 ng/mL), and IGF-1 (20 ng/mL) to the maturation medium improved the maturation rate of goat oocytes. Compared with the control group, the in vitro maturation rate of oocytes (83.0% vs. 70.93%, P < 0.05) and the development rate of IVF blastocysts (45.88% vs. 35.81%, P < 0.05) in the bFGF, LIF, and IGF-1 (FLI) group were significantly increased. The total number of developing blastocysts (114.40 vs. 83.20, P < 0.05) and the ratio of inner cell mass cells (39.50 vs. 25.33, P < 0.05) were also significantly higher in the FLI group than in the control group. Evaluation of maternal gene expression revealed that FLI could significantly increase the expression levels of DNMT1, ZAR1, and NLRP5 and cumulus cell expansion genes PTX3, TNFAIP6, PTGS2, and HAS2 in mature oocytes (P < 0.01). We indirectly proved that bFGF, LIF, and IGF-1 support goat oocyte maturation and early embryonic development. In summary, this study established a reliable and efficient in vitro goat oocyte maturation culture system using bFGF, LIF, and IGF-1. The results of this study can serve as a technical basis for improving the quality of goat oocytes matured in vitro, potential of early embryonic development, IVF of goat embryos, and in vitro production of other livestock.

Inhibition of protein degradation increases the Bt protein concentration in Bt cotton

Bacillus thuringiensis (Bt) cotton production is challenged by two main problems, i.e., the low concentration of Bt protein at the boll setting stage and the lowest insect resistance in bolls among all the cotton plant’s organs. Therefore, increasing the Bt protein concentration at the boll stage, especially in bolls, has become the main goal for increasing insect resistance in cotton. In this study, two protein degradation inhibitors (ethylene diamine tetra acetic acid (EDTA) and leupeptin) were sprayed on the bolls, subtending leaves, and whole cotton plants at the peak flowering stage of two Bt cultivars (medium maturation Sikang 1 (SK1) and early maturation Zhongmian 425 (ZM425) in 2019 and 2020. The Bt protein content and protein degradation metabolism were assessed. The results showed that the Bt protein concentrations were enhanced by 21.3 to 38.8% and 25.0 to 38.6% in the treated bolls of SK1 and ZM425 respectively, while they were decreased in the subtending leaves of these treated bolls. In the treated leaves, the Bt protein concentrations increased by 7.6 to 23.5% and 11.2 to 14.9% in SK1 and ZM425, respectively. The combined application of EDTA and leupeptin to the whole cotton plant increased the Bt protein concentrations in both bolls and subtending leaves. The Bt protein concentrations in bolls were higher, increasing by 22.5 to 31.0% and 19.6 to 32.5% for SK1 and ZM425, respectively. The organs treated with EDTA or/and leupeptin showed reduced free amino acid contents, protease and peptidase activities and significant enhancements in soluble protein contents. These results indicated that inhibiting protein degradation could improve the protein content, thus increasing the Bt protein concentrations in the bolls or/and leaves of cotton plants. Therefore, the increase in the Bt protein concentration without yield reduction suggested that these two protein degradation inhibitors may be applicable for improving insect resistance in cotton production.

The Effects of Duration of the In Vitro Maturation Process on the Maturation Level and Apoptosis of Kacang Goat

The success of in vitro fertilization and embryo culture depends on the success of in vitro maturation. However, standard culture conditions usually increase reactive oxygen species (ROS), which have been implicated as a major cause for reduced embryonicdevelopment. It is well-known that higher than physiological levels of ROS trigger granulosa cell apoptosis and thereby reduce the transfer of nutrients and survival factors to oocytes, leading to apoptosis. This study aimed to determine the optimal timing ofoocyte maturation and its relationship to DNA fragmentation. Ovaries were collected from a slaughterhouse and the follicles aspirated. The cumulus oocyte complexes were divided into groups and transferred to a maturation medium, where they were maintained for 18 hours (P1), 22 hours (P2) and 24 hours (P3) to evaluated maturation rate. Matured oocytes were characterized as oocytes that reached the MII stage. Matured oocytes were counterstained with terminal deoxynucleotidyl transferase nick-end labelling (TUNEL). The results showed that the maturity rate of Kacang goat oocytes reached 46% after 18 hours, 77% after 22 hours, and 63% after 24 hours. However, the results showed that the expression of DNA fragmentation in P2 (2.4 ± 0.89) were significantly different from P1 (5.4 ± 2.61) and P3 (9.0 ± 2.12). In conclusion, the optimal timing of in vitro maturation of Kacang goat oocytes is 22 hours.

P-118 Are cumulus cells required during in vitro maturation of human oocytes at the germinal vesicle stage? a systematic review

Abstract Study question Does the presence of cumulus cells affect the in vitro maturation (IVM) rate of human oocytes at the germinal vesicle (GV) stage obtained in vivo? Summary answer The oocyte maturation and fertilization rates are higher in the presence of cumulus cells (CC) comparing with their absence in the culture system. What is known already According to the ESHRE guidelines IVM is currently not considered experimental, but nevertheless it is not very often used in reproductive medicine clinics. Accumulated data of more than 30 years of existence of IVM method can present to us how effective is the level of oocytes maturation in vitro obtained by aspiration follicles in vivo. Some studies reported that oocytes were matured after denudation, and in others they are cultured in cumulus oocyte complexes or in the presence of CC. Therefore, a comparison of oocyte maturation at the GV stage with and without CC was performed in this study. Study design, size, duration We searched Medline and Embase up to January 10, 2023. Full texts of classical articles, clinical studies, clinical trials, clinical trial protocols, comparative studies, controlled clinical trials, multicentre studies, randomized controlled trials were included Participants/materials, setting, methods Participants were women undergoing infertility treatment with IVM. All oocytes from the included studies were obtained by follicular aspiration in vivo. Main results and the role of chance A preliminary screening of the word combination “human oocyte in vitro maturation” yielded 355 results, from which 42 were original publications with full-text. Selected papers included a full description of the stimulation protocol, maturation medium, and the group of immature oocytes consisted of gametes only at the GV stage. A total of 24,649 GV oocytes were examined for IVM rate. The average of oocyte maturity was 61.5 ± 10.7%. However, the distribution of oocyte maturation data depending on the presence or absents of CC in the culture media. The maturation rate was higher in oocytes with cumulus cells than in denuded oocytes without cumulus (64.7% vs 53.5%, respectively, p˂0.05). The fertilization rate had the same tendency (69.2% vs. 59.5%, p˂0.05). However, there was no significant difference between the groups in embryo development up to the cleavage stage (74.9% vs 76.5%, p &amp;gt; 0.05). Unfortunately, there are very only a few studies that provide data on the more advanced stages of embryo development as a blastocyst, which does not allow a large sample to be made for any IVM method. Limitations, reasons for caution This study does not take into account the influence of stimulation protocols and the composition of the maturation medium, which may in turn also affect the results of oocyte IVM. Wider implications of the findings The obtained data can be used for comparison with new maturation protocols that will be aimed at increasing the maturation rates, in addition, these data are important for comparing the efficacy of maturation obtained from ovaries outside the body for the purpose of preserving fertility. Trial registration number not applicable

P-209 Implication of delta opioid receptor in the in vitro maturation of oocytes and its effects on subsequent fertilization and embryo development in mice

Abstract Study question Could opiods modulate in vitro maturation of oocyte and improve fertilization rates? Summary answer Delta opioid receptor was present in mice oocytes, changing its expression pattern depending on the maturation stage and helping oocytes to reach blastocyst stage. What is known already The molecular mechanisms responsible for oocyte maturation are not completely understood and different molecules have been reported to be implicated as modulators of this process. The endogenous opioid peptides could be interesting candidates because they are involved in the regulation of reproductive physiology at multiple sites. When those receptors are activated, the adenylyl cyclase is inhibited, thus reducing cyclic AMP and blocking PKA. Moreover, opioids are also able to activate proteins involved in maturation. There is evidence suggesting a role for opioids during oocyte maturation because the signaling exerted by opioids is similar to that necessary for the meiosis resumption. Study design, size, duration Immature cumulus–oocyte complexes (COCs) from female 8 to 10 week old WT (C57BL/6xCBA) were retrieved after EGF stimulation. Mouse in vitro maturation culture was performed. In vitro grown oocytes, their corresponding cumulus (CC) and granulosa cells (GC) were classified at germinal vesicle stage (GV), at metaphase 1 (MI) and at metaphase 2 (MII). Participants/materials, setting, methods The presence of DOR was analysed in granulosa cells and oocytes at each stage of maturation by qRT-PCR and immunocytochemistry. AKT and MAPK signalling pathway (ERK 1/2) was studied by immunocytochemistry. Similarly, COCs were matured in vitro in the absence and presence of DPDPE and naltrindole at various concentrations. After IVM, in vitro fertilisation (IVF) was performed and development was observed until the blastocyst stage was reached, analysing fertilisation and embryo production percentages (p &amp;lt; 0.05). Main results and the role of chance Our findings showed that OPRD1 was present in mice oocytes and granulosa cells, changing its expression pattern depending on the maturation stage. The evaluation of RT-PCR examination and immunohistochemical (IHC) staining revealed the presence of the transcript for Oprd1 gene in the different stages of the mouse oocyte's maturation, as well as in the granulosa cells at the moment of the extraction from the ovary. The obtained transcript was compared with the transcript for Oprd1 of mouse cerebral cortex as a positive control. The expected 482 bp fragment was detected in the germinal vesicle stage and in granulosa cells, whereas in MI and MII stage the signal was lower. Immunofluorescence analysis revealed that OPRD1 protein was present in mouse oocytes but, its localization differed at the different stages of the maturation process. Furthermore, the selective delta opioid agonist DPDPE, modulating PI3K/Akt and MAPK pathways, helped oocytes to reach blastocyst stage. Finally, we examined that the observed effect of DPDPE during oocyte maturation in the in vitro fertilization and subsequent embryo development could be blocked by the opioid antagonist naltrindole. Limitations, reasons for caution Although we have obtained higher rates of blastocysts, the quality of these embryos still needs to be analysed and transferred to recipient females to evaluate the offspring. Wider implications of the findings Our study has clinical implications for the improvement of oocyte in vitro maturation techniques (IVM). OPRD1 could be a possible therapeutic target for in vitro maturation culture medium, as it could improve the blastocyst rates obtained in the actual reproduction assisted techniques. Trial registration number not applicable

Effect of a low ethanol concentration during in vitro maturation of bovine oocytes and subsequent embryo development

The present study investigated the effects of low ethanol exposure on bovine oocytes. Cumulus-oocyte complexes (COCs) were aspirated for the antral follicles of slaughterhouse-derived ovaries. These COCs were incubated in maturation medium containing 0, 0.1, and 0.2% ethanol for 21 h and subjected to fertilization and in vitro development, and then the rates of nuclear maturation, mitochondrial DNA copy number (Mt-cn) and protein (TOMM40), ATP content and lipid content in oocyte, fertilization, and blastulation were examined. Furthermore, COCs were incubated with 0 or 0.1% ethanol and then mitochondrial membrane potential (MMP) and the glucose consumption of COCs was determined. In addition, gene expression in oocytes was examined by RNA sequencing. Ethanol (0.1 and 0.2%) increased Mt-cn and Mt-protein levels whereas 0.2% ethanol increased the blastulation rate and ATP content in oocytes and decreased lipid content in oocytes. Ethanol (0.1%) increased MMP in oocytes and decreased glucose consumption of COCs. Eight stage embryos derived from 0.1% ethanol treated oocytes had higher levels of trimethyl-H3K9 compared with that of nontreated counterpart. RNA sequencing revealed that differentially expressed genes were associated with glycolysis/gluconeogenesis, carbon metabolism, sphingolipid metabolism, amino acid metabolism, and fatty acid degradation pathways. In conclusion, even 0.1% concentrations of ethanol during in vitro maturation considerably affects oocyte metabolism and histone configuration of embryos.

Does the addition of follicular fluid in the in vitro maturation medium increase the oocyte maturation and embryo production in alpacas?

In alpacas (Vicugna pacos), the high cost of in vitro embryo production is also a consequence of the use of several substances in the culture medium. In addition, embryo production rates in this species are still considered low. Thus, in attempt to reduce the cost and to improve the in vitro embryo production rates, this study evaluates the effect of adding follicular fluid (FF) in the in vitro maturation medium on oocyte maturation and subsequent embryo production. After ovary collection at the local slaughterhouse, the oocytes were recovered, selected, and allocated in experimental groups: standard maturation medium (G1) and simplified medium added by 10% FF (G2). The FF was acquired from follicles between 7- and 12-mm diameter. The cumulus cell expansion and the embryo production rates were analyzed by chi-square with p < 0.05. No differences (p > 0.05) were observed in maturation rate between G1 (66.36%) and G2 (63.12%) groups. Likewise, no significant difference (p > 0.05) was verified between G1 and G2 for morula (40.85 vs 38.45%), blastocyst (7.01 vs 6.93%), and total number of embryos (47.87 vs 45.38%). In conclusion, it was possible to simplify the medium used for in vitro maturation of alpaca oocytes resulting in embryo production rates similar to the standard medium.

Porcine Follicular Fluid-Derived Exosome: The Pivotal Material for Porcine Oocyte Maturation in Lipid Antioxidant Activity.

Several studies have examined exosomes derived from porcine follicular fluid (FF), but few have reported their application in controlled experiments. The main concern in the field of embryology may be that controlled conditions, such as using a defined medium intermittently, cause poor results in mammalian oocyte maturation and embryo development. The first reason is the absence of the FF, which copes with the majority of the processes emerging in oocytes and embryos. Therefore, we added exosomes derived from porcine FF to the maturation medium of porcine oocytes. For morphological assessment, cumulus cell expansion and subsequent embryonic development were evaluated. Moreover, several stainings, such as glutathione (GSH) and reactive oxygen species (ROS), fatty acid, ATP, and mitochondrial activity, as well as evaluations of gene expression and protein analysis, were used for the functional verification of exosomes. When the oocytes were treated with exosomes, the lipid metabolism and cell survival of the oocytes were fully recovered, as well as morphological evaluations compared to the porcine FF-excluded defined medium. Therefore, controlled experiments may produce reliable data if the exosomes are treated with the desired amounts, and we suggest applying FF-derived exosomes to promote experimental data when performing controlled experiments in embryology.

Beneficial Effects of Catalpol Supplementation during In Vitro Maturation of Porcine Cumulus-Oocyte Complexes.

Oxidative stress degrades oocytes during in vitro maturation (IVM). Catalpol, a well-known iridoid glycoside, exhibits antioxidant, anti-inflammatory, and antihyperglycemic effects. In this study, catalpol supplementation was tested on porcine oocyte IVM and its mechanisms. Corticalgranule (GC) distribution, mitochondrial function, antioxidant capacity, DNA damage degree, and real-time quantitative polymerase chain reaction were used to confirm the effects of 10 μmol/L catalpol in the maturation medium during IVM. Catalpol treatment significantly increased the first-pole rate and cytoplasmic maturation in mature oocytes. It also increased oocyte glutathione (GSH), mitochondrial membrane potential and blastocyst cell number. However, DNA damage as well as reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Mitochondrial membrane potential and blastocyst cell number were also increased. Thus, the supplementation of 10 μmol/L catalpol in the IVM medium improves porcine oocyte maturation and embryonic development.

Does using microfluidic sperm sorting chips in bovine IVEP affect blastocyst development?

This study aimed to determine the effect of microfluidic sperm sorting chip on embryo development and quality in the sperm treatment step in in vitro embryo production in cattle. Only A-quality oocytes obtained from the ovaries of Holstein cattle were included in the study. These oocytes were first placed in in vitro maturation medium, and matured oocytes were randomly divided into two groups at the 24th hour of maturation. Oocytes in the first group with the Microfluidic Sperm Sorting Chip (MFSC, n = 154) were put into a fertilization medium with spermatozoa prepared with microfluidic sperm sorting device. Oocytes in the second group (Con, n = 169) were fertilized with spermatozoa prepared by the routine sperm treatment step of the commercial company. The rate of cleavage (85.71% vs. 76.33%, respectively) and of reaching the blastocyst (44.15% vs. 32.54%, respectively) in the MFSC group were higher than the control group. In addition, it was determined that the numbers of ICM (45.8 ± 2.04 vs. 39.2 ± 1.85, respectively), TE (122.13 ± 2.19 vs. 115.0 ± 2.61, respectively), TC (167.93 ± 2.89 vs. 154.2 ± 2.62, respectively) increased in the MFSC group compared to the control group. A statistically significant difference was found in the number of cells with apoptosis per embryo (5.14 ± 0.77 vs. 11.91 ± 0.79) and apoptotic index rates (3.06 ± 0.47 vs. 7.72 ± 0.55%) of the MFSC and Con groups. As a result, we concluded that using microfluidic sperm sorting chips during sperm treatment in bovine IVEP increases the rate of reaching the blastocyst, embryo development, and quality and reduces the possibility of apoptosis in developing blastocysts. For this reason, it is also thought that the use of microfluidic sperm sorting devices during sperm treatment in bovine IVEP may be a new alternative in this field.

Effect of melatonin in maturation medium on equine oocyte maturation, cleavage, and blastocyst production following ICSI

Melatonin has been shown to have beneficial effects in in vitro oocyte maturation and embryo production through antioxidant activity but also through melatonin receptors on oocytes and cumulus cells as well. The purpose of this study was to investigate the effects of melatonin in maturation medium on equine oocyte maturation, cleavage, and blastocyst production with maturation performed in a reduced oxygen atmosphere. Oocytes were recovered via trans-vaginal follicular aspiration and divided randomly into three groups each with 5 replicates. The oocytes were placed in maturation medium containing no added melatonin (Control), 10−9 M melatonin (Group 1), or 10−7 M melatonin (Group 2) in an atmosphere of 5% CO2, 5% O2, and 90% N2. ICSI was performed on metaphase II oocytes after 28-30 hours of maturation. Presumptive zygotes were observed on day 2 post-injection for cleavageand on days 5 through 10 for blastocyst development. Results: No significant difference (p>.01) was determined for maturation to metaphase II, 59/86 (68.6%), 63/88 (71.5%), 66/89 (74.2%) for Control Group, Group 1, and Group 2 respectively. No significant difference (p>.01) was determined for cleavage 43/59 (72.9%), 43/63 (68.2%), 44/66 (66.7%) for Control Group, Group 1, and Group 2 respectively. No significant difference (p>.01) was determined for blastocyst formation 19/59 (32.2%), 25/63 (39.6%), 26/66 (39.4%) for Control Group, Group 1, and Group 2 respectively. While there was a slight trend towards an increase in blastocyst production with the addition of melatonin to maturation medium under reduced oxygen concentration, the difference was not statistically significant and further work would be required to evaluate its effect.

Blastocyst production using a new equine FSH for in vitro maturation

Currently, the majority of the equine ART laboratories collect germinal vesicle (GV)-stage oocytes to perform ICSI, and the gonadotrophins used for in vitro maturation are mainly porcine, bovine, or human. In this study, the effect of two different concentrations of equine FSH (eFSH) in the maturation medium was evaluated. Cumulus-oocytes complexes (COCs) were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to the groups. The control group was our standard maturation medium (BMM - TCM199-Earle's salts, 10% FBS, and additives) with human gonadotrophins added (Lazzari.et.al. Journal of Equine Veterinary Science. 2020; 89:103097); experimental groups were composed of BMM with eFSH added (0.01 and 0.05 IU/mL). After holding, COCs were transferred to maturation medium and cultured for 36h. ICSI was performed, and presumptive zygotes were cultured. The cleavage rate and number of cells were evaluated on D3, blastocyst formation and embryo quality, classified on A-B-C grade scale (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351), were evaluated from D6-10. Differences were analyzed by Fisher's Exact Test (significance level P<0.05). There was no difference (P>0.05) in degenerated, GV-stage, or MI oocytes after maturation for control, 0.01, and 0.05, respectively. The number of oocytes that reached the (meiosis) MII-stage was lower (P<0.05) for 0.01 U/mL (56.5%, 35/62)than the control (73.2%, 52/71), and also tended to be lower (P=0.0985) compared to 0.05 U/mL (71.2%, 47/66). There was no difference in cleavage or cell number among groups (Table 1). Group 0.01 U/mL had a higher (P<0.05) blastocyst rate (45.7%, 16/35) than control and 0.05 U/mL groups (28.8%,15/52; 25.5%, 12/47; respectively). No difference was found in embryo quality among groups. Although the 0.01 U/mL group showed a lower MII rate, it also showed numerically less 2-cell stage embryos, more 4-8 and 8-16 cells stage embryos, and more cleaved embryos. Additionally, the 0.01 U/mL group tended to have more high-quality embryos (grade A) than the control group and showed a higher total blastocyst rate compared to the other groups. Additional studies are required to establish an ideal maturation protocol using this new equine FSH.

Factors affecting post-mortem production of equine ICSI blastocysts

In vitro fertilization of equine oocytes by intracytoplasmic sperm injection (ICSI) has enabled post-mortem production of equine embryos. This procedure makes it possible to secure one last offspring from mares that must be suddenly euthanized. Currently, little is known about how clinical factors influence the outcome. The aim of this study was to evaluate the effect of the following clinical factors on the chance of a successful session (≥1 embryo) and blastocyst rates after post-mortem ICSI: Mare age, number of collected oocytes per mare, location of euthanasia (at our clinic/in the field), duration of ovary transport, method of ovary removal (during anaesthesia, after pentobarbital, after bolt gun), reason for euthanasia (acute life-threatening conditions requiring immediate euthanasia vs. non-acute conditions, where euthanasia could be postponed). The study was performed retrospectively and included ovaries from 65 euthanized mares in the period between 2019-2022. The mares, mainly Warmbloods, were aged between 1 to 26 years old (average 14.4 years old). If euthanasia was performed in the field, the ovaries were transported to our clinic (0.5 to 14 hours) in sterile saline at 22-32⁰C. The oocytes were collected by scraping each follicle. After overnight holding, the oocytes were matured for 30 hours. The mature metaphase II oocytes were inseminated by ICSI with the desired semen and after7-10 days of culture, blastocysts were vitrified. In total, 842 oocytes were collected from 65 mares (range: 1 to 47 oocytes per mare, average = 13), 744 oocytes were placed in maturation medium and the remaining degenerated oocytes were discarded. A total of 65% (481/744) of the oocytes reached the MII stage and were injected, and 11.4% (55/481) of these reached the blastocyst stage within 10 days after ICSI. The proportion of successful sessions was 37%. The factors that were shown to affect the chance of a successful session, were the number of collected oocytes per mare and the reason for euthanasia (acute vs. non-acute; multiple logistic regression, p < 0.05). The other examined factors did not affect the chance of a successful session (multiple logistic regression, p > 0.05). When comparing mares euthanized for non-acute reasons to mares euthanized for acute reasons, there was a higher chance of a successful session (50%, 16/38 vs. 20.7%, 6/29, respectively, p = 0.02, Fisher's exact test) and a higher blastocyst rate (16.2%, 44/272 vs. 5.3%, 11/209, p = 0.0001, Fisher's exact test) in mares euthanized for non-acute reasons. This study reports that post-mortem, in vitro embryo production can be performed on mares euthanized both in the field and at the ICSI clinic, but with a lower chance of a successful session for mares euthanized due to acute life-threatening conditions.

Effect of pre-maturation oocyte timing on equine ICSI embryo development rates

Within the last 5 years, client adoption of ovum-pick-up (OPU) paired with equine intracytoplasmic sperm injection (ICSI) and in vitro embryo production (IVP) in Brazil has been rapid. Equine ICSI has facilitated the use of expensive semen be it imported, rare, poor-quality or low motility for IVP. A pre-maturation step occurs between OPU on the farm and transport to the lab. Oocytes are kept in 5 ml vials of pre-maturation medium, held at 22°C until placement in maturation medium. Oocytes in pre-maturation medium are kept at this low temperature to inhibit the onset of germinal vesicle breakdown and the onset of oocyte maturation. If maturation begins during transport, oocyte maturation rates and oocyte quality would be severely reduced. Recently, (Merlo, B et al 2022 Anim Reprod Sci 107071) reported different rates of equine ICSI embryo development based on timing of oocyte pre-maturation and maturation. In our IVP system, oocyte maturation time is tightly controlled between 28 and 32 hours, so we decided to evaluatethe timing of pre-maturation since it can vary between 0 and 24 hours. This variation is based on the time required for OPU completion by multiple teams, aspirating on the same day in different locations. We tested the potential impact of oocyte pre-maturation in 3-time intervals 0-8, 9-16 and 17-24 hours, in the 2021-2022 breeding season. The average blastocyst formation rate based on total oocytes collected was 17%. Embryo development from oocytes aspirated in the 3 pre-maturation time intervals was: 0-8 hours, 522 embryos produced from 3413 oocytes aspirated (15.3%); 9-16 hours, 471 embryos produced from 2667 oocytes aspirated (17.7%) and 17-24 hours, 3931 embryos produced from 22,896 oocytes aspirated (17.2%). There were no significant differences observed (Mann-Whitney test p >0.05), demonstrating that up to 24 hours, the holding time of pre-maturation oocytes, held at 22.5°C had no significant impact on embryo development in our equine ICSI embryo production system.

Influence of secretome obtained from preovulatory follicular fluid on energy metabolism and meiotic competence of equine cumulus–oocyte complexes

Secretome is composed by proteins and microvesicles secreted by the granulosa cells that line the follicular wall. These bioactive compounds promote follicular growth and oocyte maturation, influencing oocyte metabolism. Hence, we hypothesized that addition of secretome from preovulatory follicular fluid (FF) to maturation medium may influence the meiotic competence and metabolism of cumulus-oocyte-complexes (COCs) obtained post mortem. FF was retrieved from preovulatory follicles (2 mares) after 32h of hCG administration. Three ml of FF from both mares were pooled, diluted 1:1 in PBS and centrifuged (4000g, 1h at 4°C) using a 10K Amicon® Ultra-15 Centrifugal Filter Unit and protein concentration was measured. A total of 186 COCs were retrieved from post mortem ovaries and were subjected to in vitro maturation (IVM) in TCM-199 medium, 10% of FBS and 5 mU/ml of FSH (Control; 59 oocytes) or with 20 µg/ml of secretome (Secretome20; 66 oocytes) or with 40 µg/ml of secretome (Secretome40; 61 oocytes) for 28 h in 5% CO2/95% air at 38.2°C. Oocytes were denuded, fixed in 4% formaldehyde and stained with Hoechst 33342 to assess chromatin conformation by fluorescence microscopy. Also, selected metabolites of the maturation medium prior to andafter IVM were assessed by magnetic proton nuclear resonance spectroscopy. After IVM, a trend towards glucose consumption and pyruvic acid production was observed compared to their controls prior IVM; a significant increase in lactic acid production in all treatments was observed (Table 1; p < 0.05). Our results show a tendency for quicker meiosis resumption in COCs incubated with Secretome40 (Table 2). In conclusion, secretome obtained from preovulatory follicular fluid added during IVM does not change COCs’ energy metabolism nor maturation rate of equine cumulus-oocyte complexes in our in vitro culture system. Funding: MCCIN/AEI (PID2020-112723RB-I00; RYC-2017-21545; RYC2020-028915-I), Junta de Extremadura-FEDER (IB20005).

Case Report: Presence of volatile organic compounds (VOCs) in the OPU-oocyte searching room and its effect on equine in vitro embryo production

Volatile organic compounds (VOCs) are hydrocarbon-based organic compounds with known detrimental effects on mammalian in vitro embryo development. Only strict air quality monitoring guarantees optimal culture conditions. Our VOC-free laboratory receives bovine and equine oocytes collected by OPU from nine different stations, to produce bovine and equine embryos in vitro by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) respectively. During fourteen consecutive equine OPU sessions, the mean blastocyst rate obtained from the oocytes collected at one of the stations were the lowest (10.4 %), when compared to the others (31.8 %). The quality of the air was suspected and passive absorption badges were placed for 5 days near where oocytes were searched for and further processed before transport (Old Room). The badges were returned for chromatographic analysis and the results indicated a poor air quality. Alcohol compounds exceeded by two-fold the recommended threshold for offices and living rooms (Ethanol: 335 ppb and Isopropanol: 129 ppb). The oocyte searching room was then moved to a different location within the station (New Room), to allow restricted access and improved air quality conditions. In vitro embryo production rates were monitored for anothereleven OPU sessions after moving the oocyte searching room to the new location. The OPU, ICSI and in vitro culture (IVC) conditions remained the same throughout the experiment. Briefly, oocytes were collected by ovum pick up (OPU) and transported to the laboratory overnight in 5 ml tubes, containing in vitro maturation (IVM) medium (IVF-Bioscience, UK) in a portable incubator at 38.2 ˚C and 5 % CO2. After 32 h of IVM, ICSI was performed on all MII oocytes and injected oocytes were cultured for nine days in Global medium (Cooper Surgical, Denmark), at 38˚C, 5 % CO2 and 5 % O2. The oocyte maturation, cleavage and blastocyst rates obtained from oocytes selected and packed in the Old vs. New Room were analyzed by ANOVA and compared by Fisher's exact test. There was no significant difference between the maturation (51.06 % vs. 48.94 %) and cleavage rates (68.75 % vs. 82.61 %) using oocytes selected and packed in the Old vs. New Room respectively. Blastocyst rates significantly improved after moving the oocyte search room to the new location: 10.42 % (5/48) for Old Room vs. 39.13 % (9/23) for New Room (P<0.01). The present work demonstrates the importance of controlling the conditions in the OPU oocyte collection room, with evidence suggesting an effect of VOCs on in vitro embryo development.

Pre-maturation of oocytes for equine cloning using cAMP modulators

The asynchrony of cytoplasmic and nuclear maturation in cumulus oocyte complexes (COCs) due to decreased concentrations of cyclic adenosine phosphate (cAMP) has been shown to result in decreased oocyte developmental competence (Rose et al., Theriogenology. 2013;79: 142–148). The aim of this study was to evaluate the effect of pre-maturation treatment with cAMP modulators, previously shown to delay spontaneous nuclear maturation, on the developmental competence of oocytes used for equine cloning. Equine ovaries were obtained from a slaughterhouse and processed within 1-2h. The COCs were washed three times with H-SOF medium and transferred to cryovials filled with a 1:1 mixture of DMEM-F12 and TCM-199 containing 10% FCS and kept at 20-22°C. Groups of COCs were either untreated (control) or treated with 50 mM forskolin (FSK) and 100 mM 3-isobutyl-1-methylxanthine (IBMX) for 4 or 18h. Immature oocytes from all three groups were washed 3 times with H-SOF medium, then transferred to maturation medium, and incubated for 18h at 38.5°C in a humidified atmosphere of 5% CO2 in air. A total of 715 oocytes were collected in 6 replicates (191 oocytes inthe 4h FSK-IBMX group, 239 in the 18h FSK-IBMX group, and 285 oocytes in the control). The methods used for in vitro maturation (IVM), SCNT, and embryo culture were identical for all three groups. Six different fibroblast cell lines were used to provide the donor nuclei. Meiotic maturation, fusion, cleavage, and embryonic development in vitro were all evaluated. Data were analyzed using the Genstat 18th Edition statistical software package. While the meiotic maturation and cleavage rates were similar for the three groups, the proportion of fused couplets that developed to the blastocyst stage was lower for the 4 FSK-IBMX group (15.4 ± 5.7%) compared to the control group (25.6 ± 6.4%; P<0.05). Interestingly, the fusion rates for oocytes of the 4 and 18h FSK-IBMX groups (90.3 ± 4.7% and 94.3 ± 2.6%, respectively) were greater than that for oocytes of the control group (79.1 ± 2.1%; P<0.05). As a result, the pre-maturation treatment did not reduce the blastocyst yield from couplets reconstructed. Further studies are needed to determine the impact of such cAMP modulating treatments on embryos’ in vivo viability following transfer to recipient mares.

Gene expression by equine oocytes and cumulus cells, a window into the unknown

In vitro maturation (IVM) of oocytes (OC) is a critical step for the in vitro production of equine embryos. However, the regulatory mechanisms, biological processes, cellular components, and molecular functions involved in IVM are not entirely understood. We previously analyzed the gene expression dynamics of gonadotropin receptors in cumulus cells (CC) during IVM, demonstrating an upregulation of the follicle-stimulating hormone receptor gene (FSHR) and downregulation of the luteinizing hormone choriogonadotropin receptor gene (LHCGR) in mature compared to immature CC (de la Fuente.et.al. Journal of Equine Veterinary Science 2022; 113). Moreover, we found that CC from in vivo maturing cumulus-oocyte complexes (COC) obtained from dominant gonadotropin-stimulated follicles (DF-CC) had a lower expression of FSHR and a higher expression of LHCGR compared to their in vitro counterparts. However, the effect of IVM medium composition on the expression of gonadotropin receptors has not been investigated. Thus, the aim of this study was to determine differences in the gene expression of OC and CC following IVM of three reported IVM media: A) EQ-IVM (IVF-Bioscience) a commercially available medium, B) DMEM/F12-based medium (Meyers.et.al. Reproduction, Fertility and Development. 2019; 31:1874-1884.) and C) M199-based medium (Choi.et.al. Theriogenology. 2002; 58:771-774) and their difference with DF-CC samples. Extracted total RNA from OC and CC was sequenced and raw reads were trimmed (trimmgalore) and mapped (STAR) to the current equine referencegenome. Mapped reads were quantified (featurecount) and compared between groups, using the DESeq2 package (FDR cutoff 0.1; Min (log2) fold change 1.5). Overall, there was a mapping rate of >85%, and an average of 14,000 expressed genes in OC and CC samples with ∼68% categorized as protein-coding genes. Based on gene expression dis-similarities, OC and CC samples were partially clustered according to their respective maturation media, but all samples showed a different gene expression profile compared to DF-CC samples. Comparing the gene expression of CC between the A and B media, we found 607 differentially expressed genes (DEGs), 1905 DEGs between A and C, and 957 DEGs between B and C. All three media showed a similar DEGs number when compared to DF-CC samples (∼10K DEGs). FSHR showed a higher expression in all IVM samples compared to DF-CC. However, LHCGR presented a lower expression only in two of the IVM media used (B & C) compared to DF-CC, showing no significant expression difference in the commercial IVM media tested. These data highlight the dynamics of gonadotropin receptors during IVM, which may contribute to the understanding of the physiology of oocyte maturation and allow optimization of IVM conditions in horses. Funding: The Theriogenology Foundation, the Population Health and Reproduction Department Seed Grant, and the Center for Equine Health.

Shale oil enrichment evaluation and production law in Gulong Sag, Songliao Basin, NE China

Based on the results of drilling, tests and simulation experiments, the shales of the Cretaceous Qingshankou Formation in the Gulong Sag of the Songliao Basin are discussed with respect to hydrocarbon generation evolution, shale oil occurrence, and pore/fracture evolution mechanism. In conjunction with a substantial amount of oil testing and production data, the Gulong shale oil enrichment layers are evaluated and the production behaviors and decline law are analyzed. The results are drawn in four aspects. First, the Gulong shales are in the stage of extensive hydrocarbon expulsion when Ro is 1.0%–1.2%, with the peak hydrocarbon expulsion efficiency of 49.5% approximately. In the low–medium maturity stage, shale oil migrates from kerogen to rocks and organic pores/fractures. In the medium–high maturity stage, shale oil transforms from adsorbed state to free state. Second, the clay mineral intergranular pores/fractures, dissolution pores, and organic pores make up the majority of the pore structure. During the transformation, clay minerals undergo significant intergranular pore/fracture development between the minerals such as illite and illite/smectite mixed layer. A network of pores/fractures is formed by organic matter cracking. Third, free hydrocarbon content, effective porosity, total porosity, and brittle mineral content are the core indicators for the evaluation of shale oil enrichment layers. Class-I layers are defined as free hydrocarbon content equal or greater than 6.0 mg/g, effective porosity equal or greater than 3.5%, total porosity equal or greater than 8.0%, and brittle mineral content equal or greater than 50%. It is believed that the favourable oil layers are Q2–Q3 and Q8–Q9. Fourth, the horizontal wells in the core area of the light oil zone exhibit a high cumulative production in the first year, and present a hyperbolic production decline pattern, with the decline index of 0.85–0.95, the first-year decline rate of 14.5%–26.5%, and the single-well estimated ultimate recovery (EUR) greater than 2.0×104 t. In practical exploration and production, more efforts will be devoted to the clarification of hydrocarbon generation and expulsion mechanisms, accurate testing of porosity and hydrocarbon content/phase of shale under formation conditions, precise delineation of the boundary of enrichment area, relationship between mechanical properties and stimulated reservoir volume, and enhanced oil recovery, in order to improve the EUR and achieve a large-scale, efficient development of shale oil.