In Vitro Embryo Production Research Articles

Metabolic signature of spent culture media shows lipid metabolism as a determinant of pregnancy outcomes.

In the present study, we investigated the spent culture media of in vitro produced (IVP) bovine embryos which did (group Pregnant) or did not (group Non-pregnant) establish pregnancy after transfer. For that purpose, IVP embryos on D5 were transferred to individual droplets for the last 48h of culture. Embryos at the blastocyst stage were then transferred to synchronized recipients, while respective culture media drops were collected and evaluated individually. The list of metabolites present in spent culture media was obtained by electrospray ionization mass spectrometry (ESI-MS) and analysed with Metaboanalyst® to characterize the metabolic profile of each group. The spectrometric analysis showed that pathways related to lipid metabolism, particularly fatty acids degradation via beta-oxidation, were more present in the Pregnant group whereas no significant pathway was identified in the group Non-pregnant. By using this method, we were able to identify a metabolic signature in culture media that allows for a better comprehension of preferential metabolic routes taken by the most viable embryos. These findings offer great insights into the biochemistry of embryo development and reveal a potential target for the development of better-quality IVP systems, as well as tools to identify bovine embryos with greater chances to establish and maintain pregnancy.

The impact of various antioxidant supplementation on ram's sperm quality, fertilization, and early embryo development, in vitro

The in vitro embryo production (IVEP) is very stressful for gametes. Gametes are subjected during in vitro manipulation to many different types of stress; oxidative stress is the most prominent one, which will cause damage or alter the genetic material of the sperm and reduce the quality of the oocytes, and has a crucial impact on the possibility of developing embryos even after implantation. This study aimed to determine the influence of antioxidants on the achievement of In vitro culture (IVC) and sperm's ability to adhere to and penetrate further into In vitro maturated oocytes. For this purpose, we have incubated ram sperm using four different treatments in terms of antioxidants: melatonin, cysteamine, vitamin C, and vitamin E. They were incubated by the standard methods of maturation and capacitation of sperm. The oocytes were fertilized by spermatozoa that had been capacitated with two groups of fertilization media, the first group containing melatonin and the second group containing cysteamine. Compared with other groups, sperms treated with melatonin demonstrated hyperactivity, and the fertilization rate was significantly increased. As for the IVF medium containing melatonin, it was superior to cysteamine in embryo development rates. In conclusion, melatonin could be a promising tool for improving sperm competence for fertilizing oocytes and embryo development in sheep.

Assessment of ova collection with or without centrifugation after ovarian slicing for In vitro fertilization of slaughterhouse specimens of Iraqi Awassi ewes

This study aims to determine the most appropriate method for oocyte collection based on their quality and quantity by slicing with or without centrifugation and testify its ability for in vitro embryo production (IVP). The ovum was collected in the Artificial Insemination Laboratory, College of Veterinary Medicine, University of Mosul, Mosul city between 1/9/2020 to 1/11/2020. A total twenty females Genitalia slaughtered at local abattoir were collected. In vitro fertilization was done according to private lab protocols (Soran laboratory related to Soran Private Hospital for fertility disease and embryo transfer) in Irbil city/Iraq. Results of the study show no significant changes between the two methods in the extraction of Grade A oocytes. However, the slicing method, which was 33.60% of ova recovery than centrifugation ORC techniques 22.73%, ORCs method in presence Grade C oocyte higher than ORCs method which were 29.09 and 20.00%, respectively also results of in vitro fertilization show that slicing method produces embryo in high percentage 45.4% than ORCs method 25.0%. We can conclude that the best method for oocyte collection was the slicing method and this method results in the best embryo production during in vitro fertilization compared with the ORCs method.

β-Mercaptoethanol in culture medium improves cryotolerance of in vitro-produced bovine embryos.

The objective of this study was to investigate the effects of adding β-mercaptoethanol (βME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 μM βME for 72 h. Embryos cultured in 100 μM βME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) μM βME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 μM βME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 μM βME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-βME, control IVC and βME-supplemented PWC; (iii) βME-CTRL, βME-supplemented IVC and control PWC; or (iv) βME-βME, βME-supplemented IVC and βME-supplemented PWC. βME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in βME-CTRL (84.0%) and βME-βME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-βME (73.1%). Hatching rates were higher in CTRL-βME (58.1%) and βME-βME (63.8%) than in CTRL-CTRL (36.6%) and βME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in βME-βME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding βME to the IVC medium reduced development but increased cryotolerance, whereas adding βME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

Importance of Antioxidant Supplementation during In Vitro Maturation of Mammalian Oocytes.

Simple SummaryIn vitro embryo production (IVEP) is a technology that is widely used in the field of reproductive biology. The first and most important step of IVEP is termed in vitro maturation or IVM, in which the oocyte is allowed to mature in a synthetic medium under controlled laboratory settings. The quality of in vitro matured oocytes is still low compared to that of those matured inside an animal’s body. One of the key elements that was found to affect oocyte quality is the accumulation of large amounts of reactive oxygen species (ROS) within oocytes during IVM. The addition of antioxidants to oocyte IVM media is one of the most effective methods for preventing ROS accumulation. In this article, we highlight the latest events showing how ROS harms mammalian oocytes. We also discuss the possible impacts of antioxidant supplementation on the IVM rate and oocyte quality. The in vitro embryo production (IVEP) technique is widely used in the field of reproductive biology. In vitro maturation (IVM) is the first and most critical step of IVEP, during which, the oocyte is matured in an artificial maturation medium under strict laboratory conditions. Despite all of the progress in the field of IVEP, the quality of in vitro matured oocytes remains inferior to that of those matured in vivo. The accumulation of substantial amounts of reactive oxygen species (ROS) within oocytes during IVM has been regarded as one of the main factors altering oocyte quality. One of the most promising approaches to overcome ROS accumulation within oocytes is the supplementation of oocyte IVM medium with antioxidants. In this article, we discuss recent advancements depicting the adverse effects of ROS on mammalian oocytes. We also discuss the potential use of antioxidants and their effect on both oocyte quality and IVM rate.

Characterization of sperm cell membrane charge and selection of high-quality sperm using microfluidics in stallions

Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there is no information available about approximate charges or ZP in equine sperm. Therefore, in this study we aimed to characterize equine sperm ZP and identify its associations with known measures of sperm quality. Additionally, we aimed to complete a comprehensive comparison of conventional sperm selection techniques as compared to the novel method of microfluidic sorting. Ejaculates (n = 22) were partitioned into fresh (∼23 °C, 0 h; n = 12) and cooled (∼4 °C, 24 h; n = 10) groups, and processed by swim up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and microfluidic chip (MF) sorting. Motility, progressive motility, cell viability, normal morphology, and ZP were evaluated for both unprocessed fractions and post-selected fractions. The ZP of both fresh and cooled samples was net negative and also correlated with motility and progressive motility for both fresh and cooled samples (P < 0.05). The ZP of cooled samples was also correlated with viability (P < 0.05). Among the compared methods of sperm selection, MF was highly effective in selecting high quality sperm as determined by the measured parameters. Percent motility, progressive motility, normal morphology, and viability of MF selected sperm were of higher quality than sperm selected by SU, and of similar to DG-SU and DGC without the use of potentially harmful centrifugation steps. Correlations between ZP, motility, and viability parameters may indicate a role of external charge on the motility and survival of sperm within the female reproductive tract. In conclusion, we identified an average net negative ZP on equine sperm and correlations between ZP and other measures of sperm quality, as well as having identified MF as a novel effective method of equine sperm selection for IVP.

Vitrification and in-straw warming do not affect pregnancy rates of biopsied bovine embryos

In the cattle industry, in vivo or in vitro embryo production combined with genotyping and cryopreservation technologies allows the selection and conservation of embryos carrying genes for desirable traits. This study aimed to assess the efficiency of a vitrification method suitable for in-straw warming of biopsied in vivo derived (IVD) bovine embryos. Three experiments were carried out using two methodologies: the Cryotop®, the gold standard vitrification and 3-step warming methodology, or the VitTrans, a vitrification/in-straw 1-step warming method that enables direct embryo transfer to the uterus. In experiment 1, intact and biopsied in vitro produced (IVP) day 7 expanded blastocysts were vitrified using the Cryotop® and warmed in 1- or 3-steps. No differences in survival rates were recorded at 24 h after warming for intact or biopsied IVP blastocysts irrespective of the warming procedure. In experiment 2, the effect of the time from trophectoderm (TE) biopsy to vitrification/in-straw warming on post-warming survival rate was assessed. No significant differences in survival were observed when blastocysts were vitrified/in-straw warmed immediately after biopsy or after 3 h in culture when compared to intact blastocysts. In experiment 3, IVD embryos were vitrified 3 h after biopsy using the Cryotop® or the VitTrans method and pregnancy rates were assessed at day 60 after transfer. Fresh, biopsied embryos served as control. Similar pregnancy rates were observed when IVD biopsied embryos were transferred fresh or vitrified/warmed by the Cryotop® or VitTrans method. No significant effect of the embryo quality or developmental stage was detected on the percentage of pregnant recipients when IVD biopsied embryos were transferred fresh or after vitrification. While fresh female IVD embryos produced significantly higher pregnancy rates than male embryos, there were no differences in pregnancy rates when male or female vitrified/warmed embryos were transferred. About 81% from the biopsies analyzed successfully determined the embryo sex, confirming that DNA was there, and it was efficiently amplified. To conclude, our findings indicate that both vitrification methodologies produced similar post-warming outcomes for both intact and biopsied IVP embryos. Besides, vitrification/in-straw warming of biopsied IVD bovine embryos did not affect the viability to originate pregnancy, being a useful option for their direct transfer in field conditions.

Fluazuron Influences the in vitro Production Embryos of Wagyu Cow

Imported breeds raised in tropical countries demand greater use of chemical control of ectoparasites, however, this practice can compromise the results of biotechnologies of reproduction. Thus, the objective of this study was to evaluate the presence of chemical residues of tick or their metabolites in the blood and in the follicular fluid and to verify the in vitro embryo production (IVEP) of Wagyu cows, raised in a tropical country, submitted to fluazuron based tick treatment. Twenty adult Wagyu bovine females were used, donors of oocytes, divided into 2 groups: G1 - animals that were not submitted to tick control and G2, animals that were submitted to chemical control of ticks with fluazuron based product (2.5 mg kg-1 of body weight). After application (D0), all cows were submitted to estrous synchronization and, in four moments (D12, D33, D54 and D75) the aspirations of the follicular fluid from the dominant follicles were performed, the oocytes were collected for IVEP and were collected blood samples for extraction and analysis of the presence of chemical residues, using GC-MS. Plasma residues of fluazuron were detected up to 54 days after application of the tick, but no residues were detected in the follicular fluid. Group 1 had a higher number of total and viable oocytes (p&lt;0.0001), however, the viability rate and the blastocyst rate was lower (p&lt;0.0001), showing that the use of ticks compromised IVEP.

Follicular fluid steroid hormones and in vitro embryo development in Duroc and Landrace pigs

The Duroc sire line has a smaller litter size compared to the Landrace dam line and we have previously observed fewer surface follicles on Duroc ovaries one day after weaning. In that same study, a broader cumulus expansion and faster nuclear maturation were observed for Duroc oocytes at 20 h of in vitro maturation (IVM), while Landrace oocytes showed more advanced stages of cortical granule distributions. However, no differences between breeds were observed after the final IVM period. The aim of this study was to assess subsequent in vitro embryo production (IVP) in Duroc and Landrace. Furthermore, follicle diameter and steroid hormone levels in follicular fluid (FF) were measured to study possible relation to oocyte developmental competence. Follicular phase sow ovaries were collected one day after weaning and follicle size of the 10 largest follicles were measured per ovary before aspiration. Cumulus-oocyte complexes (COCs) were matured in vitro, and cumulus expansion was analysed by assessing individual COC areas at 0 and 20 h. Fertilization of Duroc and Landrace oocytes was performed with sperm from both a Duroc and a Landrace boar. A larger follicle diameter was observed for Landrace animals (5.7 vs. 4.8 mm, P < 0.0001) and individual COC area was additionally larger at 0 h after aspiration (P < 0.0001) compared to Duroc. Contrary, cumulus expansion from 0 to 20 h of maturation was broader for Duroc oocytes than for Landrace (407 ± 67% vs. 319 ± 31%, P < 0.0001). After fertilization, cleavage rate was higher for Duroc oocytes, and the highest blastocyst yield was obtained for Duroc oocytes fertilized with the Landrace sperm. Steroid hormone analysis of the follicular fluid showed differences in the pathways between breeds with a higher total level of estrogens (P = 0.01) and aromatase products/substrates ratio (P < 0.01) in Landrace compared to Duroc. In conclusion, results suggest that Duroc oocytes have a better in vitro oocyte developmental competence when cultured under the same in vitro conditions and breed differences in steroidogenesis were found in the early follicular phase.

Bioactive supplements influencing bovine in vitro embryo development.

Ovum pickup and in vitro production (IVP) of bovine embryos are replacing traditional multiple ovulation embryo transfer (MOET) as the primary means for generating transferable embryos from genetically elite sires and dams. However, inefficiencies in the IVP process limit the opportunities to produce large numbers of transferable embryos. Also, the post-transfer competency of IVP embryos is inferior to embryos produced by artificial insemination or MOET. Numerous maternal, paternal, embryonic, and culture-related factors can have adverse effects on IVP success. This review will explore the various efforts made on describing how IVP embryo development and post-transfer competency may be improved by supplementing hormones, growth factors, cytokines, steroids and other bioactive factors found in the oviduct and uterus during early pregnancy. More than 40 of these factors, collectively termed as embryokines, are reviewed here. Several embryokines contain abilities to promote embryo development, including improving embryo survivability, improving blastomere cell numbers, and altering the distribution of blastomere cell types in blastocysts. A select few embryokines also can benefit pregnancy retention after IVP embryo transfer and improve neonatal calf health and performance, although very few embryokine-supplemented embryo transfer studies have been completed. Also, supplementing several embryokines at the same time holds promise for improving IVP embryo development and competency. However, more work is needed to explore the post-transfer consequences of adding these putative embryokines for any adverse outcomes, such as large offspring syndrome and poor postnatal health, and to specify the specific embryokine combinations that will best represent the ideal conditions found in the oviduct and uterus.

Effects of single or serial embryo splitting on the development and morphokinetics of in vitro produced bovine embryos

Embryo splitting can be used in cattle in vitro production (IVP) to improve embryo availability and to increase selection intensity. Despite this widespread utility, a comparative investigation of the viability of IVP embryos split at Day 2 (2-cell stage), Day 3 (8-cell stage), and blastocyst stage has not been undertaken. Similarly, the suitability of splitting Day 3 embryos with atypical numbers of blastomeres, and the feasibility of serial-splitting cleavage stage embryos, have not been investigated in cattle. Here, we demonstrate that the strategy most likely to produce the greatest output of viable embryos is the splitting of Day 3 embryos into four parts, regardless of whether embryos with exactly eight cells or an atypical number of blastomeres are used. This approach was found to produce 1.8 blastocysts per zygote on average compared to just 0.4 blastocysts per zygote for non-split controls. Single-splitting was also found to be superior to serial-splitting which, whilst feasible, impaired embryo viability as judged by cell number at day 7 post-insemination. Interestingly, zygotes (≥2 cells) split once on either Day 2 or Day 3 post-insemination, whilst resulting in smaller blastocysts than control embryos, displayed higher cell counts than expected at the blastocyst stage, suggesting a compensatory mechanism might be at play. Indeed, time-lapse imagery revealed that zygotes split at 2-cells reached the compact morula and expanded blastocyst stages earlier than either those split at Day 3 or non-split controls. Developmental events between splits originating from the same progenitor appeared well synchronized only up to the third cleavage division.

Effect of Nellore (Bos indicus) donor age on in-vitro embryo production and pregnancy rate.

The aim of this study was to evaluate the effect of age of Nellore (Bos indicus) donors on the efficiency of in vitro embryo production (IVEP) and pregnancy rate. Thirty-six donors, including 11 female calves (13 ± 0.61 months), 17 prepubertal heifers (25 ± 0.78 months) and 8 cows (83 ± 28 months), were submitted to 3 procedures of ovum pickup (OPU) on random days of the estrous cycle at intervals of 21 days. Caspase-3 and IGFBP2 were quantified in oocytes and blastocysts for the evaluation of oocyte and embryo quality. The produced embryos were vitrified (n=445) and transferred to synchronized recipients. Cows produced a larger number of follicles (cows: 54.5 ± 6.2; calves: 20.0 ± 0.57; prepubertal heifers: 20.8 ± 0.46), total oocytes (cows: 45.97 ± 7.22; calves: 28.93 ± 6.14; prepubertal heifers: 27.21 ± 4.94) and cleaved oocytes (cows: 21.14 ± 4.22; calves: 13.09 ± 3.72; prepubertal heifers: 12.4 ± 3.19). The cleavage rate was similar between age categories; however, cows tended (p < 0.07) to produce a larger number of blastocysts (9.74 ± 2.26) per OPU than calves (5.57 ± 1.99) and prepubertal heifers tended to have a higher blastocyst yield (35.4%) than calves (27.1%) (p < .07). The expression levels of IGFBP2 and caspase-3 were higher in oocytes derived from calves compared to the other two categories. The pregnancy rate was higher in calves (43.1%) and cows (40.4%) than in prepubertal heifers (33.8%) (p=.03). Despite the larger numbers of follicles and viable oocytes in cows, the blastocyst production results and pregnancy rates obtained indicate that the use of young females as oocyte donors in IVEP is feasible and may contribute to reduce the generation interval.

Synchronization of follicle wave emergence before ovarian superstimulation with FSH and ovum pick-up improves in vitro embryo production in pregnant heifers

The objective of the present study was to evaluate effects of synchronization of timing of follicle wave emergence, before ovarian superstimulation and ovum pick-up (OPU), on ovarian response and embryo production in pregnant heifers. Pregnant (47–69 days of gestation) Holstein heifers (n = 64), 19.0 ± 0.3 months of age, were assigned in a completely randomized design to one of two groups: synchronization of follicular wave emergence using follicle ablation (Synchronized) or untreated control (Non-synchronized). Superstimulatory treatments consisting of 160 mg (280 IU) of porcine follicle stimulating hormone (p-FSH), administered in four decreasing dose treatments 12 h apart, were initiated 36 h after follicle ablation or at random stages of the follicular wave in heifers of the Synchronized and Non-synchronized group, respectively. Ovum pick-up was performed in all heifers 40 h after the last p-FSH administration and retrieved cumulus oocyte complexes (COCs) were subjected to in vitro embryo production (IVEP) procedures. Ultrasonography was performed immediately before OPU to determine number and size of ovarian follicles. Differences in treatment responses between groups were evaluated using generalized linear mixed models. Total number of follicles at the time of OPU was not different between treatment groups (P = 0.61), however, the number of small follicles (<6 mm) was greater (P = 0.05) in heifers of the Non-synchronized group, whereas number of medium size follicles (6–10 mm) tended (P = 0.09) to be greater in heifers of the Synchronized group. Heifers in the Synchronized group had a greater (P = 0.01) percentage of medium sized follicles and a lesser (P = 0.01) percentage of smaller sized follicles than heifers in the Non-synchronized group. There were no differences (P > 0.15) in total number of recovered COCs, or number of viable COCs between groups. Cleavage percentage (84.5% and 72.8%) and blastocyst percentage (48.2% and 33.4%) were greater (P < 0.01) in heifers of the Synchronized than Non-synchronized group, respectively. As a result, mean number of blastocysts per OPU/heifer was greater (P = 0.006) in the heifers of the Synchronized (8.9 ± 1.0) than the Non-synchronized (5.5 ± 0.9) group. In conclusion, synchronizing the time of follicle wave emergence in pregnant heifers, prior to ovarian superstimulation with FSH and OPU results in a greater superstimulatory response and oocyte competence leading to greater embryo production.

Efficacy of the novel microfluidic chip as a sperm selection method for equine ICSI

Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte quality and competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is essential to employ superior methods of selecting high quality sperm to maximize IVP outcomes. Clinical sperm selection techniques for stallions are generally limited to swim-up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and variations of these methods, despite the rapidly expanding use of novel techniques in humans and other species. One novel method of sperm selection is a microfluidic (MF) chip, which utilizes natural rheotactic behaviors of sperm to select for a highly motile and viable population of cells that would be optimal for IVP. However, no comprehensive comparison between MF and the conventional methods of DGC and DG-SU has been performed for stallions. Thus, we aimed to compare conventional equine sperm selection techniques to the novel method of MF sorting. Ejaculates (n = 22) were partitioned into fresh (∼23°C, 0 h; n = 12) and cooled (∼4°C, 24 h; n = 10) groups, and processed by wash-only, SU, DGC, DG-SU, and MF (ZyMōt™ Multi, 850µL, Sperm Separation Device™, DxNow, Gaithersburg, MD). Sperm motility, progressive motility, viability, and normal morphology,parameters used as basic estimates of sperm quality, were evaluated for both the unprocessed fraction and post-selected fractions. Among the compared methods of sperm selection, MF was user-friendly and effective in selecting high quality sperm as determined by individual parameters and by a summative sperm quality index (SQI) that combined individual factors. MF selected sperm with greater percentages of motility, progressive motility, viability, and normal morphology than unprocessed, wash-only, and SU-selected groups (P < 0.0001). Additionally, sperm selected by MF had numerically greater motility, progressive motility, and SQI, in comparison to DGC and DG-SU groups (P > 0.05). Cell viability and morphology parameters were comparable among MF, DGC, and DG-SU. Thus, MF-selected sperm not only exhibited numerically higher quality than DG-SU and DGC-selected sperm, but also the method did not use potentially harmful centrifugation steps. Therefore, MF may pose additional advantages over conventional methods. In conclusion, to select for superior sperm within a total ejaculate, implementation of MF technologies could be of interest to equine ICSI laboratories. This study was funded by the Theriogenology Foundation and the Center for Equine Health at UC Davis.

Management of monozygotic twins in the mare

In the horse, only a small number of monozygotic twins and triplets have been reported and commonly, the outcome is poor. As in man, an association between the occurrence of monozygotic twins and the use of assisted reproductive technologies (ART) has been suggested. In the mare, transfer of in vitro produced (IVP) embryos appears to increase the incidence of monozygotic twinning (1.6%; 4/254) compared to transfer of in vivo embryos (0%; 0/413) (Dijkstra et al. Equine Vet J. 2020; 52: 258-261). Due to the type of placental arrangement, these monozygotic twin pregnancies are difficult to reduce to a singleton using reduction techniques commonly applied to dizygotic twins. In this report, 8 cases of monozygotic twins are presented, all of which resulted from transfer of a single IVP embryo. Seven were presumed to be monochorionic diamniotic/diallantoic and one dichorionic diamniotic/diallantoic, based on membrane classification by ultrasound and/or post-expulsion examination. Similar to previously described cases, twin pregnancy only became apparent at 25 days of gestation, when two embryos with heartbeat were visualized. Depending on gestational stage, different twin reduction techniques were applied. In one case, only referred at day 40 of gestation andclassified as dichorionic diamniotic/diallantoic, a transvaginal ultrasound guided aspiration (TUGA) was performed. Reduction of two other twins was performed by per rectum compression of the fetal thorax at day 65 of gestation. In two other cases, cranio-cervical dislocation was performed at day 75-80 of gestation. Finally, transcutaneous ultrasound guided twin reduction was attempted at day 120 of gestation in one case. However, only one of the 6 treated monozygotic twins was successfully reduced, resulting in a live foal born following thorax compression at 65 days of gestation; all other cases resulted in death of both fetuses. In addition, two remaining cases (2/8) lost their pregnancy spontaneously at around 42 days and 8 months of gestation, whereby in the latter the presence of twins was not established at previous ultrasound examinations. Based on these limited cases, only one monozygotic twin was successfully reduced, using the thoracic compression method at day 65 of gestation. These results highlight the need for further investigation into the best reduction method, depending on the gestational stage and on how much the demise of one fetus affects the survival of its co- twin, which may depend on placental arrangement.

Embryo biotechnologies in sheep: Achievements and new improvements.

To date, large-scale use of multiple ovulation and embryo transfer (MOET) programmes in ovine species is limited due to unpredictable results and high costs of hormonal stimulation and treatment. Therefore, even if considered reliable, they are not fully applicable in large-scale systems. More recently, the new prospects offered by in vitro embryo production (IVEP) through collection of oocytes post-mortem or by repeated ovum pick-up from live females suggested an alternative to MOET programmes and may be more extensively used, moving from the exclusive research in the laboratory to field application. The possibility to perform oocytes recovery from juvenile lambs to obtain embryos (JIVET) offers the great advantage to significantly reduce the generation interval, speeding the rate of genetic improvement. Although in the past decades several studies implemented novel protocols to enhance embryo production in sheep, the conditions of every single stage of IVEP can significantly affect embryo yield and successful transfer into the recipients. Moreover, the recent progresses on embryo production and freezing technologies might allow wider propagation of valuable genes in small ruminants populations and may be used for constitution of flocks without risks of disease. In addition, they can give a substantial contribution in preserving endangered breeds. The new era of gene editing might offer innovative perspectives in sheep breeding, but the application of such novel techniques implies involvement of specialized operators and is limited by relatively high costs for embryo manipulation and molecular biology analysis.

Success rate in a clinical equine in vitro embryo production program

In vitro embryo production (IVEP) via Ovum Pick-Up (OPU) and Intracytoplasmic Sperm Injection (ICSI) has become a popular breeding technique in Warmblood mares because of the high success rate and several practical advantages. IVEP offers a solution for a variety of reproductive issues including, but not limited to, sub-fertility in stallions or mares, poor quality or scarce frozen semen, difficulty in synchronizing donor and recipient mares, and inefficient use of recipient mares. In 515 OPU-ICSI sessions performed in our facility in 2021, a mean of 25.9 antral follicles were aspirated yielding an average 13.8 immature oocytes, which were shipped overnight to a specialized ICSI laboratory (Avantea). One or more blastocysts (range: 0–13 blastocysts) were produced from 78% of procedures with a mean of 2.12 blastocysts per session; the likelihood of pregnancy after transfer of a cryopreserved thawed IVP blastocysts in 2021 (n = 781) was 77.7%. Several donor mare, recipient mare, stallion and embryonic factors influence the likelihood of producing an in vitro blastocyst or achieving pregnancy. Approximately 60% of the transferred IVP blastocysts yield a foal; moreover, neither gestation length nor the health of foals is noticeably influenced by IVEP. On the other hand, a skewed sex ratio towards colts is apparent among IVEP foals resulting from day 7 but not day 8 embryos, suggesting that male embryos develop more rapidly in vitro. Although serious complications after OPU are uncommon, owners should be aware of their existence, because some complications can be life-threating.

Effects of Heat-Stress on Oocyte Number and Quality and In Vitro Embryo Production in Holstein Heifers

Background: This study aimed to determine the effects of environmental temperature on the number and quality of oocytes and embryo production rates obtained by performing ovum pick up (OPU). Heat stress leads to long-term, short-term, visible, and invisible effects in dairy cows. Its effects on reproduction are evident in all stages, from oocyte development to birth. Disturbance in ovarian follicle development, follicular dominance deficiency, anoestrus, polyspermia, embryoniclosses, decreased fetal growth, and abortion are some examples of responses to these effects. The aim of the present study was aimed to determine the effects of ambient temperature on oocyte quality and number and embryo production rates.Materials, Methods &amp; Results: The animal material used in this study comprised 10 Holstein heifers. At the beginning of the study, the heifers were 13-15 months old. OPU was performed at different times of the year, and weather conditions were recorded. Grouping according to ambient temperature was done as &lt; 10°C (group 1), 10-25°C (group 2), and &gt; 25°C (group 3). The veterinary ultrasonography device and a set of compatible intravaginal OPU probe, catheter, and aspiration device were used for OPU application. All antral follicles with diameters of 2-8 mm in the ovaries were aspirated. The aspirated follicle fluids were examined under a stereo microscope, and the cumulus-oocyte complexes (COC) were collected and classified according to their structure. A, B, and C-quality oocytes were included in the in vitro embryo production process. After performing 69 OPUs on random days of the cycle, the number of oocytes per OPU was found to be 8.72, 6.32, and 6.85 in groups 1, 2, and 3, respectively (P &lt; 0.05). The number of viable oocytes per OPU was 6.83, 4.64, and 4.65 in groups 1, 2, and 3, respectively (P &lt; 0.05). The statistical difference between the first group and the other groups was significant for cleavage and blastocyst counts (P &lt; 0.05).Discussion: All the negative effects of heat stress on animals resulted from the increased body temperature. Reproductive performance is adversely affected by high temperatures and humidity during periods of high ambient temperatures. Metabolic heat is released, and the heat load increases due to the metabolism of nutrients in cattle. Internal body temperature is regulated via the dissipation of metabolic heat to the environment. The amount of heat dissipated via conduction andconvection depends on the unit body weight, surface area, skin and coat color, difference in temperature gradient of the animal and ambient temperature, and humidity. In the present study, it was determined that the blastocyst development rates of the oocytes obtained in the warm season (&gt;25°C [group 3]) were lower than those of the other groups. It was concluded that this may be because the oocytes developed under chronic heat stress in the animals, and several cycles were required to enhance oocyte quality and developmental potential. Additional studies are needed to investigate the response of oocytes obtained with OPU to heat stress during embryonic developmental stages and to determine the sensitivity and effects of embryonic tissue damage according to developmental stages. Based on the results of the present study, it was concludedthat performing OPU and in vitro embryo production (IVEP) when the ambient temperature is close to the thermoneutral limits may increase the blastocyst development rates. Keywords: blastocyst, heat stress, heifer, in vitro embryo production, oocyte quality, ovum pick-up.

Antioxidant effect of bioactive compounds isolated from Syzygium aromaticum essential oil on the in vitro developmental potential of bovine oocytes

Syzygium aromaticum essential oil (EOSA) is an interesting antioxidant supplement for improving bovine in vitro embryo production (IVEP). Therefore, identifying the bioactives in EOSA is essential for increasing embryonic development. In this study, we compared the effect of EOSA to that of its bioactive compounds on nuclear and cytoplasmic maturation, bioenergetic/oxidative stress parameters, and preimplantation embryonic development of bovine oocytes. Viable oocytes (n = 2,272) were matured under six conditions: control (without antioxidant), cysteamine (100 μM CYS), EOSA20 (20 μg/mL EOSA), eugenol (83 μM EUG), β-caryophyllene (19 μM β-CA), and acetyl eugenol (12 μM ACT). In the first experiment, oocytes were evaluated to determine their in vitro maturation (IVM) according to the expansion and viability of cumulus cell layer, presence of the first polar body (1 PB), metaphase II (MII), and mitochondrial distribution. In the second experiment, oocytes were evaluated to determine their bioenergetic/oxidative status based on the levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial membrane potential (ΔΨm). In the third experiment, mature oocytes were parthenogenetically activated (PA), and the embryos were cultured. In the first experiment, no difference was observed in the percentage of MII among groups containing antioxidants (P > 0.05). However, EOSA20, EUG, and CYS improved the percentage of 1 PB oocytes (P < 0.05). Moreover, EUG improved the viability and expansion of cumulus cell layers (P < 0.05). Additionally, cytoplasmic maturation was higher in the EUG and ACT, and mitochondrial aggregation patterns were higher in the EUG, CYS, and ACT than in the other groups (P < 0.05). In the second experiment, ROS levels were reduced in oocytes matured with EUG and CYS (P < 0.05) and GSH levels increased in the same groups (P < 0.05). Oocytes treated with EOSA20, EUG, ACT, and CYS had lower ΔΨm than control. In the third experiment, EUG, EOSA20, and CYS resulted in higher cleavage rates, and EUG and β-CA resulted in more structures with ≥ 8 cells than the other components (P < 0.05). EUG caused a higher percentage of blastocysts than the other components (P < 0.05). EUG, EOSA20, and CYS improved embryo quality (P < 0.05). In conclusion, EUG is the EOSA bioactive compound with the greatest potential for application as an antioxidant and can be commercially applied for IVM of bovine oocytes to result in higher reproducibility and efficiency for IVEP.

Effect of two 'progressively motile sperm-oocyte' ratios on porcine in vitro fertilization and embryo development.

Sperm motility and viability of cryopreserved semen vary between boars and straws, which influences the outcomes of in vitro embryo production (IVEP). However, progressive motility is usually not considered during IVEP. The aim of this study was to assess fertilization with a 500:1 and 250:1 'progressively motile sperm to oocyte' ratio on IVEP outcomes using semen from three Duroc and three Landrace boars. Frozen-thawed sperm was centrifuged through a 45/90% Percoll® density gradient and sperm quality parameters were assessed. In vitro matured oocytes were fertilized at the two ratios, a portion was stained 10-12 h after start of fertilization to analyze fertilization and polyspermy, while the remaining zygotes were cultured up to day 7. The 500:1 ratio resulted in a higher fertilization and blastocyst yield on day 6 compared with the 250:1 ratio, but no effect of ratio was observed for polyspermy, cleavage rate or blastocyst cell number. Individual differences between boars were observed for fertilization, cleavage and blastocyst rates, but not for the other IVEP outcomes. In conclusion, a higher fertilization and blastocyst yield was obtained with the 500:1 ratio compared with the 250:1 ratio, while polyspermy level was consistent across ratios. Differences in IVEP outcomes were still observed between the individual boars although adjusted for progressive motility. Promising blastocyst yields and high total blastocyst cell counts were obtained with sperm from both breeds.