Comparison of ICSI, IVF, and in vivo derived embryos to produce CRISPR-Cas9 gene-edited pigs for xenotransplantation.

Genome editing in pigs for xenotransplantation has seen significant advances in recent years. This study compared three methodologies to generate gene-edited embryos, including co-injection of sperm together with the CRISPR-Cas9 system into oocytes, named ICSI-MGE (mediated gene editing); microinjection of CRISPR-Cas9 components into oocytes followed by in vitro fertilization (IVF), and microinjection of in vivo fertilized zygotes with the CRISPR-Cas9 system. Our goal was to knock-out (KO) porcine genes involved in the biosynthesis of xenoantigens responsible for the hyperacute rejection of interspecific xenografts, namely GGTA1, CMAH, and ß4GalNT2. Additionally, we attempted to KO the growth hormone receptor (GHR) gene with the aim of limiting the growth of porcine organs to a size that is physiologically suitable for human transplantation. Embryo development, pregnancy, and gene editing rates were evaluated. We found an efficient mutation of the GGTA1 gene following ICSI-MGE, comparable to the results obtained through the microinjection of oocytes followed by IVF. ICSI-MGE also showed higher rates of biallelic mutations compared to the other techniques. Five healthy piglets were born from in vivo-derived embryos, all of them exhibiting biallelic mutations in the GGTA1 gene, with three displaying mutations in the GHR gene. No mutations were observed in the CMAH and ß4GalNT2 genes. In conclusion, in vitro methodologies showed high rates of gene-edited embryos. Specifically, ICSI-MGE proved to be an efficient technique for obtaining homozygous biallelic mutated embryos. Lastly, only live births were obtained from in vivo-derived embryos showing efficient multiple gene editing for GGTA1 and GHR.

Effect of chemical activators after intracytoplasmic sperm injection (ICSI) on embryo development in alpacas.

Low motility and low sperm concentration are characteristics of alpaca semen. Thus, the intracytoplasmic sperm injection (ICSI) technique represents an alternative to improve the reproductive capacity of the male. However, the effect of post-ICSI activation in alpaca is not yet known. The aim of the present study was to compare the effect of chemical activators on alpaca embryo development after ICSI. Alpaca ovaries were collected from a local slaughterhouse and transported to the laboratory. Category I, II and III oocytes were matured for 30 h at 38.5 °C. After ICSI, injected oocytes were randomly divided and activated as follows: i) 5 µM ionomycin for 5 min, ii) 7% ethanol for 4 min, iii) 5 µM ionomycin for 5 min, window period 3 h plus 7% ethanol for 4 min, iv) 5 µM ionomycin for 5 min, window period 3 h, a second ionomycin treatment for 5 min, followed by 1.9 mM 6-DMAP for 3 h, v) 10 mM SrCl2 for 3 h. Culture was carried out for 5 days in SOFaa at 38.5 °C. The cleavage rate was the lowest in the SrCl2 group, morula development was the lowest in the SrCl2 and without activation groups, and blastocyst stage was not different between groups (P<0.05). The rates with SrCl2 were lower in total embryos produced, whereas in transferable embryos they were lower with 2Io/6-DMAP and with SrCl2 (P<0.05). In conclusion, alpaca oocyte activation is more efficient with ionomycin and ethanol to produce transferable embryos.

The ability of donkey sperm to induce oocyte activation and mule embryo development after ICSI.

Members of the Equus genus exhibit a fascinating capacity for hybridization, giving rise to healthy offspring. Mules, resulting from the mating of a mare with a jack, represent the most prevalent equid hybrid, serving diverse roles in our society. While in vitro embryo production, particularly through Intracytoplasmic Sperm Injection (ICSI), has rapidly gained significance in domestic horses, the in vitro production in other equids remains largely unexplored. Utilizing donkey sperm for fertilizing horse oocytes not only addresses this gap but also provides an opportunity to investigate donkey sperm's fertilization capability in vitro to further improve donkey ICSI. In this work, we initially studied the localization of donkey sperm Phospholipase C zeta (PLCζ) and assessed the sperm's capacity to induce pronuclear formation and maternal SMARCA4 recruitment upon injection into pig oocytes through ICSI. Subsequently, we investigated the injection of donkey sperm into horse oocytes, evaluating in vitro production up to the blastocyst stage using sperm from different jacks, including frozen and refrigerated samples. Distinct patterns of PLCζ localization were observed for donkey sperm cells compared to their horse counterparts. Additionally, donkey sperm exhibits a reduced ability to induce porcine oocyte activation. However, when injected into horse oocytes, donkey sperm demonstrated sufficient capability to induce oocyte activation as no discernible differences in cleavage or blastocyst rates are observed between in vitro produced mules and horse ICSI embryos. Our study not only delineates PLCζ localization in donkey sperm but also suggests potential differences in the ability to induce oocyte activation in pigs compared to horses while observing no distinctions in pronuclear recruitment of SMARCA4. Interestingly, donkey sperm remains sufficiently capable of inducing horse oocyte activation for in vitro mule blastocyst production.

Use of confocal microscopy and intracytoplasmic sperm injection (ICSI) to assess viability of equine oocytes from young and old mares after vitrification.

BACKGROUND: The impact of vitrification on oocyte developmental competence as a function of donor age remains an important issue in assisted reproductive technologies (ARTs). METHODS: Equine germinal vesicle (GV) or metaphase II (M(II) oocytes were vitrified using the Cryotop® method. Spindle organization and chromosome alignment were evaluated from confocal imaging data sets of in vivo (IVO) or in vitro (IVM) matured oocytes subjected to vitrification or not. Intracytoplasmic sperm injection (ICSI) from the same groups was used to assess developmental potential. RESULTS: An increase in chromosome misalignment was observed in spindles from older mares when compared to those of younger mares (P < 0.05). When MII oocytes subjected to vitrification were examined following warming, there was no difference in the percentage of oocytes displaying chromosome misalignment. Next, GV oocytes, collected from the ovaries of younger and older mares, were compared between fresh IVM and IVM following vitrification and warming. For nonvitrified samples, an age difference was again noted for spindle organization and chromosome alignment, with a higher (P < 0.05) percentage of normal bipolar meiotic spindles with aligned chromosomes observed in nonvitrified oocytes from young versus older mares. Vitrification led to a reduction of spindle length (P < 0.05) for oocytes from old mares, whether vitrified at GV or MII stages, whereas this effect was not observed in oocytes from young mares except those vitrified at GV and subjected to IVM. Oocyte developmental potential after vitrification was evaluated after ICSI of vitrified and warmed MII or GV oocytes from young mares. From 25 MII oocytes, 18 oocytes were injected with sperm, and six blastocysts were produced, which, upon transfer to mares' uteri, resulted in four pregnancies. Immature (GV) oocytes collected from live mares were also vitrified, warmed, and matured in vitro before ICSI. In this group, nonvitrified, control, and vitrified oocytes did not differ (P > 0.05) with respect to the incidence of maturation to MII, cleavage after ICSI, or blastocyst development. CONCLUSION: These findings demonstrate an effect of maternal age in an equine model at the level of meiotic spindle integrity and chromosome positioning that is influenced by both the meiotic stage at which oocytes are vitrified and whether meiotic maturation occurred in vivo or in vitro.

Standardization of a Sex-Sorting Protocol for Stallion Spermatozoa by Means of Absolute RT-qPCR.

Sperm sexing is a technology that can generate great economic benefits in the animal production sector. Techniques such as sex-sorting promise over 90% accuracy in sperm sexing. However, for the correct standardization of the technique, some laboratory methodologies are required. The present manuscript describes in detail a standardized equine sperm sex-sorting protocol using an absolute qPCR-based methodology. Furthermore, the results of absolute qPCR were implemented and validated by generating equine/bovine heterologous embryos by intracytoplasmic sperm injection (ICSI) of presumably sexed equine spermatozoa into bovine oocytes using a piezoelectric system (Piezo-ICSI). Our results indicated that equine sex-sorting spermatozoa had a 97% and 94% certainty for X and Y sperm, respectively, while presumptive female and male equine/bovine hybrid embryos, generated by Piezo-ICSI, had an accuracy of 92% with respect to the desired sex. Therefore, it is concluded that the presented methodology is a reliable, cost-effective, and relatively simple option for standardizing sex-sorting of equine spermatozoa. This is supported by the results of the correct sexing of Piezo-ICSI heterologous embryos generated with the sexed spermatozoa, validating the correct sexing and viability of these gametes.

Evaluation of the post-implantation development of mouse embryos derived from round spermatid injection.

Round spermatid injection (ROSI), one of the assistant reproductive technologies, was used to treat partial infertility patients suffering from non-obstructive azoospermia. However, the development efficiency and birth rate of ROSI embryos are extremely low, and it is urgent to investigate the underlying mechanisms of low efficiency to improve the clinical application of ROSI technology. Here, we analyzed and compared the genome stability of the mouse blastocyst and the post-implantation development between ROSI and ICSI embryos. We first sequenced the genome of blastocysts from mouse ROSI embryos that can correctly form male and female pronuclei (2 PN) and found that the genomes of 7 blastocysts were normal. Furthermore, the implantation rate of ROSI 2 PN embryos on embryonic day 7.5 is similar to that of ICSI embryos, and at this time, 37.50% (9/24) of deciduas have no normal gestational sac. The proportion of embryos that survived to embryonic day 11.5 in the ROSI 2 PN group, ROSI non-2 PN group, parthenogenesis group, and ICSI 2 PN group was 51.61%, 7.14%, 0.00%, and 55.00%, respectively. And two smaller fetuses were found in the ROSI 2 PN group, which is not found in the other three groups. In addition, the physiological indexes, including fetus and placenta weight, sex ratio, growth rate, and the natural breeding ability for the offspring obtained from mouse ROSI, were evaluated; ROSI mice exhibited no obvious defect or abnormality and implied that the progeny were safe. Our results provided new evidence to promote the clinical application of ROSI technology.

Time-lapse imaging and developmental competence of donkey eggs after ICSI: Effect of preovulatory follicular fluid during oocyte in vitro maturation.

Equus members exhibit very divergent karyotype, genetic plasticity, and significant differences in their reproductive physiology. Despite the fact that somatic cell nuclear transfer and intracytoplasmic sperm injection (ICSI) has gained relevance in the last few years in horses, few reports have been published exploring ovum pick up (OPU) and in vitro maturation (IVM) of cumulus-oocyte complexes (COCs) in donkeys. Yet, some donkey species and breeds are considered endangered, and these assisted-reproductive technologies could help to preserve the genetic of valuable individuals. In this study, we tested the hypothesis that supplementation with jenny preovulatory follicular fluid (PFF) during IVM could improve oocyte developmental competence in the donkey. For this, in vitro nuclear maturation rates, cumulus cell expansion, and embryo development after ICSI of donkey COCs matured in culture media supplemented with fetal bovine serum (FBS) or donkey PFF, with a known metabolomic profile, were assessed. Time-lapse imagining was performed after ICSI of horse and donkey oocytes. Eight OPU sessions were done in five jennies with an average recovery rate of 69.2% (n = 45 COCs). Although lower cumulus cells expansion was observed in oocytes of PFF group (P = 0.0010), no significant differences were described in nuclear maturation rates and preimplantation embryo development between groups. Donkey ICSI embryos showed similar morphokinetics to horse ICSI embryos. Our study shows that supplementing IVM media with FBS or donkey PFF supports nuclear maturation and early preimplantation embryo development after ICSI in donkeys. To our knowledge, the present study is the first report of ICSI, time-lapse imaging and in vitro blastocyst production in donkey.

Sperm factors associated with the production of equine blastocysts by intracytoplasmic sperm injection (ICSI) using frozen/thawed semen.

Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro-matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro-matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses.

Bovine ICSI: limiting factors, strategies to improve its efficiency and alternative approaches.

Intracytoplasmic sperm injection (ICSI) is an assisted reproductive technique mainly used to overcome severe infertility problems associated with the male factor, but in cattle its efficiency is far from optimal. Artificial activation treatments combining ionomycin (Io) with 6-dimethylaminopurine after piezo-ICSI or anisomycin after conventional ICSI have recently increased the blastocyst rate obtained. Compounds to capacitate bovine spermatozoa, such as heparin and methyl-ß-cyclodextrin and compounds to destabilize sperm membranes such as NaOH, lysolecithin and Triton X-100, have been assessed, although they have failed to substantially improve post-ICSI embryonic development. Disulfide bond reducing agents, such as dithiothreitol (DTT), dithiobutylamine and reduced glutathione, have been assessed to decondense the hypercondensed head of bovine spermatozoa, the two latter being more efficient than DTT and less harmful. Although piezo-directed ICSI without external activation has generated high fertilization rates and modest rates of early embryo development, other studies have required exogenous activation to improve the results. This manuscript thoroughly reviews the different strategies used in bovine ICSI to improve its efficiency and proposes some alternative approaches, such as the use of extracellular vesicles (EVs) as 'biological methods of oocyte activation' or the incorporation of EVs in the in vitro maturation and/or culture medium as antioxidant defence agents to improve the competence of the ooplasm, as well as a preincubation of the spermatozoa in estrous oviductal fluid to induce physiological capacitation and acrosome reaction before ICSI, and the use of hyaluronate in the sperm immobilization medium.

Overnight holding aids in selection of developmentally competent equine oocytes.

The demand for equine in vitro produced embryos has increased over the last decade. The aim of this study was to compare the effects of an extended IVM or a prolonged period before fertilization, including holding time, on equine immature oocyte developmental competence. Oocytes, collected from abattoir-derived ovaries, were divided into 4 groups: H0/24 (n = 165) 0 h holding + standard 24-26 h IVM; H8/36 (n = 160) 8 h holding + 36 h IVM; H20/24 (n = 187) 20 h holding + 24 h IVM; H0/44 (n = 164) 0 h holding + 44 h IVM. Oocytes matured to MII were fertilized by intracytoplasmic sperm injection (ICSI) and cultured for 10 days. The oocyte degeneration rate was higher (P < 0.05) for H20/24 than the other groups (H0/24 38.2 %, H8/36 43.1 %, H20/24 54.5 %, H0/44 32.9 %). Cleavage was higher (P < 0.05) in H20/24 (70 %) compared to H0/24 (45 %) and H8/36 (54 %) but not to H0/44 (63 %). No differences among groups were observed in the number of blastocysts per oocyte. Injected oocytes that reached the blastocysts stage were higher (P < 0.05) for H20/24 (20 %) than H0/24 (7 %) and H0/44 (7 %) but not H8/36 (12 %). For cleaved oocytes, a higher blastocyst rate (P < 0.05) was observed for H20/24 (28 %) than H0/44 (11 %), while H0/24 (15 %) and H8/36 (21 %) were not different from any group (P > 0.05). Timing of blastocyst development was not different among groups. Overnight holding of equine immature oocytes followed by a standard IVM interval may induce a pre-selection of the most competent oocytes thereby improving cleavage and embryo development rates after ICSI.

High survival of bovine mature oocytes after nylon mesh vitrification, as assessed by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) is an alternative technique to in vitro fertilization (IVF) for producing transferable blastocysts, especially in combination with cryopreserved oocytes, when the IVF system does not work sufficiently. The present study was conducted to directly compare the efficacy of producing bovine blastocysts by ICSI and IVF from vitrified-warmed and fresh oocytes. Denuded oocytes with a detectable first polar body were vitrified-warmed using a nylon mesh device. In the non-vitrified control group, blastocyst yields 8 days after IVF and ICSI were 32.0 and 26.8%, respectively. Oocyte vitrification and subsequent IVF resulted in an impaired blastocyst yield (15.0%); however, such a loss of efficacy due to vitrification was not observed in the ICSI group (blastocyst yield, 25.2%). The alignment of cortical granules beneath the oolemma was comparable between the fresh control and vitrified-warmed oocytes. Here, we report the high survival of vitrified-warmed bovine oocytes, as assessed by ICSI.

Effect of day of estrus cycle at time of transvaginal follicle aspiration for oocyte recovery on rates of in vitro maturation and blastocyst production after intracytoplasmic sperm injection.

OBJECTIVE: To determine the effect of stage of estrus cycle (day after ovulation) at the time of transvaginal ultrasound-guided follicle aspiration (TVA) on parameters related to the success of in vitro equine embryo production. ANIMALS: 14 healthy mares were used; 11 completed the study and were included for analysis. PROCEDURES: Mares underwent TVA of all follicles ≥ 5 mm diameter at each of 3 timepoints: 7 days after ovulation, 14 days after ovulation, and S-DSF (subordinate to a dominant stimulated follicle), during estrus at 24 hours after gonadotropin administration. The 3 treatments were assigned to each mare in random order; mares underwent follicle growth and ovulation between treatments. Recovered oocytes were matured in vitro, subjected to intracytoplasmic sperm injection (ICSI), and cultured to the blastocyst stage in vitro. RESULTS: Total follicle numbers differed significantly between individual mares but did not differ between treatments. The number of follicles of different sizes significantly (P < 0.05) differed between treatments, with mares in the Day 7 treatment having more follicles 5 to 9 mm in diameter and fewer follicles 20 to 24 mm in diameter than mares in the other 2 treatments. After in vitro maturation culture, there were significantly more mature oocytes in the S-DSF treatment than in the other 2 treatments. There were no differences in blastocyst rate after ICSI among treatment groups. CLINICAL RELEVANCE: Timing of TVA for aspiration of S-DSFs may increase the number of mature oocytes available for ICSI. Understanding of the effects of timing of TVA will help veterinarians to maximize the efficiency of this procedure.

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.

Cattle production by intracytoplasmic sperm injection into oocytes vitrified after ovum pick-up.

Intracytoplasmic sperm injection (ICSI), oocyte vitrification after ovum pick-up (OPU), and in vitro maturation are reproductive technologies with incredible potential for efficient cattle production. However, the developmental competence of embryos produced by ICSI using vitrified OPU oocytes remains unknown. Here, we aimed to evaluate the developmental competence of these embryos from the early embryo period to full term. The cleavage rate in the ICSI embryos using vitrified OPU oocytes during in vitro culture was significantly lower than those in control in vitro fertilized (IVF) embryos using fresh OPU oocytes (30.9 ± 4.5% v.s. 65.9 ± 7.0%) (P < 0.05), but the proportion of blastocysts to cleaved embryos was significantly higher than those of IVF embryos using vitrified OPU oocytes (55.9 ± 10.8% v.s. 23.2 ± 9.3%) (P < 0.05). To further investigate the transcription levels of genes related to cell differentiation in ICSI embryos using vitrified OPU oocytes, the relative abundance of mRNAs (OCT4, NANOG, SOX2, CDX2, GATA3, and IFNT) was analyzed by quantitative reverse-transcription PCR. There were no significant differences in the expression levels between ICSI embryos using vitrified OPU oocytes and control IVF embryos. Finally, developmental competence to term in ICSI embryos using vitrified OPU oocytes was examined by embryo transfer, and two healthy calves were born. These findings confirmed that ICSI and vitrification decrease developmental rates in vitro, but both procedures can lead to full-term development of bovine embryos. These results demonstrate that ICSI embryos using vitrification OPU oocytes are viable for cattle production.

Past, present and future of ICSI in livestock species.

During the past 2 decades, intracytoplasmic sperm injection (ICSI) has become a routine technique for clinical applications in humans. The widespread use among domestic species, however, has been limited to horses. In horses, ICSI is used to reproduce elite individuals and, as well as in humans, to mitigate or even circumvent reproductive barriers. Failures in superovulation and conventional in vitro fertilization (IVF) have been the main reason for the use of this technology in horses. In pigs, ICSI has been successfully used to produce transgenic animals. A series of factors have resulted in implementation of ICSI in pigs: need to use zygotes for numerous technologies, complexity of collecting zygotes surgically, and problems of polyspermy when there is utilization of IVF procedures. Nevertheless, there have been very few additional reports confirming positive results with the use of ICSI in pigs. The ICSI procedure could be important for use in cattle of high genetic value by maximizing semen utilization, as well as for utilization of spermatozoa from prepubertal bulls, by providing the opportunity to shorten the generation interval. When attempting to utilize ICSI in ruminants, there are some biological limitations that need to be overcome if this procedure is going to be efficacious for making genetic improvements in livestock in the future. In this review article, there is an overview and projection of the methodologies and applications that are envisioned for ICSI utilization in these species in the future.

Genome activation in equine in vitro-produced embryos.

Embryonic genome activation is a critical event in embryo development, in which the transcriptional program of the embryo is initiated. The timing and regulation of this process are species-specific. In vitro embryo production is becoming an important clinical and research tool in the horse; however, very little is known about genome activation in this species. The objective of this work was to identify the timing of genome activation, and the transcriptional networks involved, in in vitro-produced horse embryos. RNA-Seq was performed on oocytes and embryos at eight stages of development (MII, zygote, 2-cell, 4-cell, 8-cell, 16-cell, morula, blastocyst; n = 6 per stage, 2 from each of 3 mares). Transcription of seven genes was initiated at the 2-cell stage. The first substantial increase in gene expression occurred at the 4-cell stage (minor activation), followed by massive gene upregulation and downregulation at the 8-cell stage (major activation). An increase in intronic nucleotides, indicative of transcription initiation, was also observed at the 4-cell stage. Co-expression network analyses identified groups of genes that appeared to be regulated by common mechanisms. Investigation of hub genes and binding motifs enriched in the promoters of co-expressed genes implicated several transcription factors. This work represents, to the best of our knowledge, the first genomic evaluation of embryonic genome activation in horse embryos.

Effects of intracytoplasmic sperm injection timing and fertilization methods on the development of bovine spindle transferred embryos.

Cytoplasmic replacement by spindle transfer (ST) technique can be applied to improve the developmental competence of poor qualitied or aged oocytes. In cattle, ST technology has not been well established for producing embryos and the calves successfully using intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF). The objective of this study was to develop a novel procedure for producing bovine ST embryos, which could be fundamental to applying ST technology in other mammals. In the present study, the efficacies of performing ICSI before (ICSI-ST) or after (ST-ICSI) and IVF on the development of ST bovine embryos were investigated. Results indicated that the blastocyst rate of ST embryos produced by ICSI-ST (24.7%) was higher (P < 0.05) than that produced by ST-ICSI (5.9%). On the other hand, ST-IVF had the highest fertilization rate (97.3%), polyspermy rate (24.7%), and lowest blastocyst rate (22.7%) when compared to denuded oocytes (DO), zona cut oocytes (ZC), and cumulus-oocyte complexes (COCs)-IVF groups. Finally, the in vitro development rates of ICSI-ST (24.5%) and ST-IVF (25.2%) were not significantly different (P > 0.05). However, the pregnancy rate (46.7%) and birth rate (33.3%) of ST-IVF were higher (P < 0.05) than those of ICSI-ST (6.3% and 0%, respectively). The percentage of mitochondrial DNA (mtDNA) heteroplasmy derived from donor karyoplasts of the 5 claves was between 2% and 18%. Taken together, performing ICSI prior to ST can improve the embryonic development of ST bovine embryos. Moreover, using IVF, instead of ICSI, for ST oocyte fertilization dramatically increased the pregnancy rate and birth rate of ST calves, in which mtDNA heteroplasmy derived from donor karyoplasts exists.

Successful blastocyst production by intracytoplasmic injection of sperm after in vitro maturation of follicular oocytes obtained from immature female squirrel monkeys (Saimiri boliviensis).

Advanced reproductive technologies are being applied for the propagation of squirrel monkeys, to ensure their preservation as a genetic resource and the effective use of their gametes in the future. In the present study, oocytes and spermatozoa were collected from live squirrel monkeys, following which piezo intracytoplasmic sperm injection (ICSI) was performed using these gametes. Follicular development was induced by administering equine chorionic gonadotropin (eCG) containing inhibin antiserum to an immature squirrel monkey female. The unilateral ovary was excised after the administration of human chorionic gonadotropin (hCG), to induce ovulation, following which the larger developed follicular oocytes were collected. Follicular oocytes were prepared for ICSI using sperm from the epididymal tail of a unilateral testis extracted from a mature male. The embryos were continuously incubated in CMRL 1066 medium supplemented with 10% (v/v) fetal bovine serum. Embryo culture was performed with cumulus cells. Two experiments of ICSI carried out with three females resulted in 14 mature oocytes from the 49 cumulus-oocyte complexes collected and five embryos, three of which developed into blastocysts. These blastocysts were vitrified, thawed, and transferred to recipient monkeys, but no pregnancies resulted. In conclusion, the present study is the first to successfully produce ICSI-derived blastocysts from MII oocytes obtained by means of hormone administration (a combination of eCG+inhibin antiserum and hCG) and in vitro maturation in immature squirrel monkeys.

In vitro maturation of equine oocytes followed by two vitrification protocols and subjected to either intracytoplasmic sperm injection (ICSI) or parthenogenic activation.

The aim of this study was determine the viability and developmental competence of equine oocytes after IVM and vitrification using the Rapid-I method, as part of an effort to develop an effective equine oocyte vitrification protocol. Equine oocytes were collected by scraping ovarian follicles of slaughtered mares. A total of 1052 ovaries were used in this study, from which 3135 oocytes were obtained. Of the 2853 oocytes retrieved, 2557 underwent in vitro maturation for approximately 36 h. After in vitro culture, 1202 oocytes (47%) had a first polar body. To evaluate the toxicity of the solutions (Experiment I), oocytes were exposed to vitrification media without cryopreservation. Of all the experimental groups evaluated, the best results were obtained for IVM oocytes exposed to EquiproVitKit media (IVM + TOX EquiVitKit), with a viability rate of 69.5%. In the Experiment II, oocytes, either freshly collected from the ovary or after in vitro maturation (IVM), were vitrified using either the EquiPro VitKit or an in-house medium containing 18% Ficoll, 40% ethylene glycol and 0.3 M sucrose. Oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate viability. In vitro matured oocytes vitrified using EquiproVitKit media (IVM + VIT EquiVitKit) had a cryosurvival rate of 63%. In the last part of the study (Experiment III), vitrified IVM oocytes were activated by 7.5 µM ionomycin in TCM-199 for 5 min TCM 199 (5 min) combined with 2 mM 6-DMAP in TCM-99 with 10% FBS (4.5 h) or in vitro fertilized using ICSI. Development of potential embryos after activation in TCM-199 medium, showed a cleavage rate was 10.2%, compared to 22.5% of oocytes cultured in G1/G2 medium. ICSI of vitrified IVM oocytes resulted in 20% embryo development to the 16-cell stage, compared to 33.3% in the control. The vitrification of oocytes after IVM by Rapid-I method is a good way to preserve genetic material in horses.

A high glucose concentration during early stages of in vitro equine embryo development alters expression of genes involved in glucose metabolism.

BACKGROUND: Equine embryos exhibit an unusual pattern of glucose tolerance in vitro and are currently cultured in hyperglycaemic conditions. OBJECTIVE: Our main objective was to analyse the effect of different glucose concentrations on in vitro-produced equine embryo development and quality. STUDY DESIGN: Experiments comparing in vitro and in vivo produced embryos. METHODS: Oocytes (n = 641) were collected from post-mortem ovaries, matured in vitro and fertilised by intracytoplasmic sperm injection (ICSI). Embryo culture was divided from Day 0 to Day 4 and from Day 4 to Day 9 in three groups: 5-10 (5 and 10 mmol/L glucose respectively; n = 87); 5-17 (5 and 17.5 mmol/L; n = 66); and 10-17 (10 and 17.5 mmol/L; n = 117). A control group of 20 in vivo produced blastocysts was included. Cleavage and blastocyst rates were evaluated and embryos were snap-frozen for analysis of the relative mRNA expression of genes related to mitochondrial function, DNA methylation, apoptosis, glucose transport and metabolism. RESULTS: No differences were observed in the cleavage or blastocyst rates among in vitro groups. Under high glucose conditions in vitro (10-17 group), BAX/BCL2 was higher, and PFKP, LDHA and COX2 were overexpressed compared to all other groups. The two groups with 5 mmol/L glucose concentration during the first culture stage (5-10 and 5-17) displayed similar patterns which differed to the 10-17 group. MAIN LIMITATIONS: Conclusions related to embryo quality are based on gene expression patterns. Transfer of in vitro-produced embryos would reveal whether the observed differences improve embryo developmental competence. CONCLUSIONS: Five mM glucose during the first days of culture seems to be preferable to avoid over-activation of embryonic glycolytic pathways. Further studies are necessary to determine whether this improves embryo developmental competence.