NRF2 attenuation aggravates detrimental consequences of metabolic stress on cultured porcine parthenote embryos.

The nuclear factor erythroid 2-related factor 2 (NRF2) is a crucial transcription factor that plays a central role in regulating oxidative stress pathways by binding antioxidant response elements, but its involvement in early embryo development remains largely unexplored. In this study, we demonstrated that NRF2 mRNA is expressed in porcine embryos from day 2 to day 7 of development, showing a decrease in abundance from day 2 to day 3, followed by an increase on day 5 and day 7. Comparable levels of NRF2 mRNA were observed between early-cleaving and more developmental competent embryos and late-cleaving and less developmental competent embryos on day 4 and day 5 of culture. Attenuation of NRF2 mRNA significantly decreased development of parthenote embryos to the blastocyst stage. When NRF2-attenuated embryos were cultured in presence of 3.5 mM or 7 mM glucose, development to the blastocyst stage was dramatically decreased in comparison to the control group (15.9% vs. 27.8% for 3.5 mM glucose, and 5.4% vs. 25.3% for 7 mM glucose). Supplementation of melatonin moderately improved the development of NRF2-attenuated embryos cultured in presence of 0.6 mM glucose. These findings highlight the importance of NRF2 in early embryo development, particularly in embryos cultured under metabolically stressful conditions.

DCAF13 and RNF114 participate in the regulation of early porcine embryo development.

In brief: Ubiquitination plays a pivotal role in a multitude of cellular functions; however, the precise contributions of various ubiquitin ligases in governing early developmental processes remain largely unexplored. This study revealed that the E3 ubiquitin ligases DCAF13 and RNF114 are both necessary for the normal regulation of early porcine embryo development. Abstract: Ubiquitylation is required for normal regulation of many biological functions by modulating several protein facets such as structure, stability, interaction, localization, and degradation. In this study, we explored the roles of two E3 ubiquitin ligases (E3s), the DDB1- and CUL4-associated factor 13 (DCAF13) and the Ring finger protein 114 (RNF114), in the regulation of porcine embryo development. Attenuation of DCAF13 mRNA decreased embryo development at the blastocyst stage, while the development of RNF114-attenuated embryos was not significantly different than that of control embryos. The average number of cells per blastocyst was decreased in DCAF13-attenuated embryos and increased in RNF114-attenuated embryos compared to controls. The relative mRNA abundance of the histone methyltransferase SUV39H1, which regulates histone H3 lysine 9 trimethylation (H3K9me3), was increased in both DCAF13- and RNF114-attenuated embryos, but nuclear immunofluorescence signal for H3K9me3 on day 3 embryos was not significantly altered between attenuated and control embryos. Nuclear immunofluorescence signal for H3K4m3 was decreased in DCAF13-attenuated embryos, but it was increased in RNF114-attenuated embryos compared to controls. Attenuation of DCAF13 and RNF114 mRNAs increased transcript levels for the DNA recombinase RAD51 and decreased expression of phosphorylated histone H2A.X (γH2AX), which suggests an impact on DNA damage repair. In addition, lower mRNA expression of the lysine demethylases 5B (KDM5B) and 5C (KDM5C), both involved in embryo genome activation and DNA repair, was detected in DCAF13-attenuated embryos. These findings indicated that both DCAF13 and RNF114 have important roles in the regulation of the early development of porcine embryos.

Optimization of gonadotropin stimulation protocols for in vitro embryo production in prepubertal Mediterranean water buffalo.

Embryos can be produced from prepubertal donor animals using laparoscopic ovum pickup and in vitro embryo production technologies (LOPU-IVEP). Together, these tools can shorten the interval between generations, rapidly accelerating the rate of genetic gain. Here, we assessed the impact of different gonadotropin stimulation protocols in Mediterranean water buffalo heifer calves aged between 2 and 6 months old. Following gonadotropin stimulation, LOPU was performed at two-week intervals, with animals receiving different protocols on subsequent LOPUs. After collection, the cumulus-oocyte complexes (COCs) were matured and fertilized in vitro, and embryos were cultured to the blastocyst stage followed by transfer into synchronized adult recipients. The number and size of follicles aspirated during LOPU, the number and quality of COCs recovered, as well as cleavage, embryo development and pregnancy rates were assessed. First, we evaluated the impact of using FSH with and without eCG (administered 24-h prior to LOPU) and found that a combination of FSH and eCG was able to significantly improve embryo development rates (20.6 ± 2.0% vs. 9.0 ± 3.6%; P < 0.05). Second, we compared this protocol to a slow-release formulation of FSH reconstituted in hyaluronan. In addition to requiring less work to prepare the animals for LOPU, this slow-release formulation yielded numerically higher, but not statistically different, average number of recovered COCs (14.4 ± 2.1 vs. 10.3 ± 2.0; P > 0.05) and embryo development rates (22.9 ± 4.7% vs. 14.1 ± 5.2%; P > 0.05) compared to FSH given every 12 h. Next, we compared the length of gonadotropin treatment over 3-, 4- and 5-days prior to LOPU and found that as the length of gonadotropin treatment increased, although the number of COCs recovered steadily decreased (14.1 ± 2.4 vs. 8.7 ± 1.0 vs. 6.9 ± 0.7; P < 0.05), the embryo development rates steadily increased (14.4 ± 3.9 vs. 27.3 ± 4.4 vs. 35.9 ± 7.0; P < 0.05), presumably due to an increase in the proportion of large follicles at the time of LOPU. Numerically, the 4-day treatment yielded more transferrable embryos per donor per LOPU (2.70 ± 0.5) than 3-day (1.94 ± 0.6) and 5-day (2.25 ± 0.5) treatments. Finally, following embryo transfer, 26 of 90 recipient females became pregnant (28.9%). Pregnancies were established from all treatments, which suggests that post-implantation development was not affected among the gonadotropin treatments assessed.

Factors Affecting the Efficiency of In Vitro Embryo Production in Prepubertal Mediterranean Water Buffalo.

Embryos from prepubertal water buffalo can be produced using laparoscopic ovum pickup (LOPU) and in vitro embryo production (IVEP). However, to date, it is unclear what factors and environmental conditions can affect LOPU-IVEP efficiency in prepubertal animals, especially buffalo. In this study, we explored the impact of season, age and individual variation among female donor animals, as well as the effect of the sire used for in vitro fertilization. Donor animals between 2 and 6 months of age were stimulated using gonadotropins prior to LOPU, which was performed at two-week intervals. Following in vitro maturation and fertilization, the resulting embryos were then cultured to the blastocyst stage until they were either vitrified or transferred into recipient animals. The number of follicles available for aspiration and embryo development rates was stable throughout the year. As animals became older, there was a slight trend for fewer COCs recovered from LOPU and better embryo development. There was a large individual variation in both ovarian response and the developmental competence of oocytes among donors. The bull used for fertilization also had a significant impact on embryo development. Upon embryo transfer, pregnancy rates were not affected by the number of embryos transferred per recipient. The best pregnancy rates were achieved when transferring blastocysts, compared to compact morula or hatched blastocysts. Finally, vitrification had no effect on pregnancy rate compared to fresh embryos.

Simultaneous Inhibition of Histone Deacetylases and RNA Synthesis Enables Totipotency Reprogramming in Pig SCNT Embryos.

Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Optimizing swine in vitro embryo production with growth factor and antioxidant supplementation during oocyte maturation.

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.

DNA Damage Induction Alters the Expression of Ubiquitin and SUMO Regulators in Preimplantation Stage Pig Embryos.

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.

In Vitro Production of Embryos from Prepubertal Holstein Cattle and Mediterranean Water Buffalo: Problems, Progress and Potential.

Laparoscopic ovum pick-up (LOPU) coupled with in vitro embryo production (IVEP) in prepubertal cattle and buffalo accelerates genetic gain. This article reviews LOPU-IVEP technology in prepubertal Holstein Cattle and Mediterranean Water Buffalo. The recent expansion of genomic-assisted selection has renewed interest and demand for prepubertal LOPU-IVEP schemes; however, low blastocyst development rates has constrained its widespread implementation. Here, we present an overview of the current state of the technology, limitations that persist and suggest possible solutions to improve its efficiency, with a focus on gonadotropin stimulations strategies to prime oocytes prior to follicular aspiration, and IVEP procedures promoting growth factor metabolism and limiting oxidative and endoplasmic reticulum stress.

Tauroursodeoxycholic acid/TGR5 signaling promotes survival and early development of glucose-stressed porcine embryos†.

Conditions of impaired energy and nutrient homeostasis, such as diabetes and obesity, are associated with infertility. Hyperglycemia increases endoplasmic reticulum stress as well as oxidative stress and reduces embryo development and quality. Oxidative stress also causes deoxyribonucleic acid damage, which impairs embryo quality and development. The natural bile acid tauroursodeoxycholic acid reduces endoplasmic reticulum stress and rescues developmentally incompetent late-cleaving embryos, as well as embryos subjected to nuclear stress, suggesting the endoplasmic reticulum stress response, or unfolded protein response, and the genome damage response are linked. Tauroursodeoxycholic acid acts via the Takeda-G-protein-receptor-5 to alleviate nuclear stress in embryos. To evaluate the role of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling in embryo unfolded protein response, we used a model of glucose-induced endoplasmic reticulum stress. Embryo development was impaired by direct injection of tauroursodeoxycholic acid into parthenogenetically activated oocytes, whereas it was improved when tauroursodeoxycholic acid was added to the culture medium. Attenuation of the Takeda-G-protein-receptor-5 precluded the positive effect of tauroursodeoxycholic acid supplementation on development of parthenogenetically activated and fertilized embryos cultured under standard conditions and parthenogenetically activated embryos cultured with excess glucose. Moreover, attenuation of tauroursodeoxycholic acid/Takeda-G-protein-receptor-5 signaling induced endoplasmic reticulum stress, oxidative stress and cell survival genes, but decreased expression of pluripotency genes in parthenogenetically activated embryos cultured under excess glucose conditions. These data suggest that Takeda-G-protein-receptor-5 signaling pathways link the unfolded protein response and genome damage response. Furthermore, this study identifies Takeda-G-protein-receptor-5 signaling as a potential target for mitigating fertility issues caused by nutrient excess-associated blastomere stress and embryo death.

Histone Lysine Demethylases KDM5B and KDM5C Modulate Genome Activation and Stability in Porcine Embryos.

The lysine demethylases KDM5B and KDM5C are highly, but transiently, expressed in porcine embryos around the genome activation stage. Attenuation of KDM5B and KDM5C mRNA hampered embryo development to the blastocyst stage in fertilized, parthenogenetically activated and nuclear transfer embryos. While KDM5B attenuation increased H3K4me2-3 levels on D3 embryos and H3K4me1-2-3 on D5 embryos, KDM5C attenuation increased H3K9me1 on D3 embryos, and H3K9me1 and H3K4me1 on D5 embryos. The relative mRNA abundance of EIF1AX and EIF2A on D3 embryos, and the proportion of D4 embryos presenting a fluorescent signal for uridine incorporation were severely reduced in both KDM5B- and KDM5C-attenuated compared to control embryos, which indicate a delay in the initiation of the embryo transcriptional activity. Moreover, KDM5B and KDM5C attenuation affected DNA damage response and increased DNA double-strand breaks (DSBs), and decreased development of UV-irradiated embryos. Findings from this study revealed that both KDM5B and KDM5C are important regulators of early development in porcine embryos as their attenuation altered H3K4 and H3K9 methylation patterns, perturbed embryo genome activation, and decreased DNA damage repair capacity.

Tauroursodeoxycholic acid acts via TGR5 receptor to facilitate DNA damage repair and improve early porcine embryo development.

DNA damage associated with assisted reproductive technologies is an important factor affecting gamete fertility and embryo development. Activation of the TGR5 receptor by tauroursodeoxycholic acid (TUDCA) has been shown to reduce endoplasmic reticulum (ER) stress in embryos; however, its effect on genome damage responses (GDR) activation to facilitate DNA damage repair has not been examined. This study aimed to investigate the effect of TUDCA on DNA damage repair and embryo development. In a porcine model of ultraviolet light (UV)-induced nuclear stress, TUDCA reduced DNA damage and ER stress in developing embryos, as measured by γH2AX and glucose-regulated protein 78 immunofluorescence, respectively. TUDCA was equally able to rescue early embryo development. No difference in total cell number, DNA damage, or percentage of apoptotic cells, measured by cleaved caspase 3 immunofluorescence, was noted in embryos that reached the blastocyst stage. Interestingly, Dicer-substrate short interfering RNA-mediated disruption of TGR5 signaling abrogated the beneficial effects of TUDCA on UV-treated embryos. Quantitative PCR analysis revealed activation of the GDR, through increased messenger RNA abundance of DNAPK, 53BP1, and DNA ligase IV, as well as the ER stress response, through increased spliced XBP1 and X-linked inhibitor of apoptosis. Results from this study demonstrated that TUDCA activates TGR5-mediated signaling to reduce DNA damage and improve embryo development after UV exposure.

A fast and reliable protocol for activation of porcine oocytes.

Oocyte activation is physiologically triggered by the sperm during fertilization, however, production of porcine embryos by somatic cell nuclear transfer (SCNT), intracytoplasmic sperm injection (ICSI) or parthenogenetic activation (PA) requires artificial oocyte activation. Although effective protocols for artificial oocyte activation have been developed, current protocols require long exposures to non-specific inhibitors, which do not mimic the physiological process and may have detrimental consequences for embryo development. This study attempted to mimic the physiological activation events induced by fertilization, through the manipulation of Ca2+ and Zn2+ levels, and protein kinase C (PKC) as well as cyclin dependent kinase 1 (CDK1) activities, with the aim of developing an improved protocol for activation of porcine oocytes. In the first experiment, matured oocytes were exposed to ionomycin (Ion) for 5 min, and then treated with a specific CDK1 inhibitor (RO-3306) and/or PKC activator (OAG) for different time intervals. The highest rate of pronuclear (PN) formation (58.8%) was obtained when oocytes were treated with PKCa + CDK1i for 4 h. Second, PN formation and embryo development were evaluated in oocytes exposed for different times to a Zn2+ chelator (TPEN) following Ion treatment. This revealed that 15 min was the minimal exposure time to TPEN required to maximise oocyte activation and embryo development. Next, we observed that treatment with PKCa + CDK1i for 4 h after TPEN for 15 min decreased embryo development compared to TPEN alone. Lastly, we compared the efficiency of the Ion (5 min) plus TPEN (15 min) protocol (IT-20) with a control protocol used in our laboratory (CT-245) for production of PA, SCNT and ICSI embryos. In PA embryos, IT-20 resulted in higher cleavage (72% vs 49.2%) and blastocyst from cleaved embryos (65.5% vs 46.2%) compared to CT-245. In ICSI embryos, higher PN rates were obtained with the IT-20 protocol compared with CT-245 and the non-activated (N-A) group. Moreover, the two protocols were equally efficient for activation of SCNT embryos. Based on these findings, we propose that IT-20 is a fast and effective protocol for activation of porcine oocytes.

Granulosa cells of prepubertal cattle respond to gonadotropin signaling and upregulate genes that promote follicular growth and prevent cell apoptosis.

Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus-oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle-stimulating hormone [FSH] treated and three untreated) and eight FSH-treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real-time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.

Interval of gonadotropin administration for in vitro embryo production from oocytes collected from Holstein calves between 2 and 6 months of age by repeated laparoscopy.

Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6 months at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72 h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12 h (FSH12h), or every 8 h (FSH8h) or every 8 h until -36 h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7 ±â€¯16 vs. 38.5 ±â€¯25 vs. 31.1 ±â€¯22), mean oocytes recovered (26.5 ±â€¯14 vs. 21.6 ±â€¯10 vs. 19.4 ±â€¯14) and cleavage rate (66.0 ±â€¯14 vs. 61.1 ±â€¯11 vs. 72.2 ±â€¯8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5 ±â€¯8%) compared with FSH12h (8.9 ±â€¯5%, P < 0.05). Oocytes from follicles of ≥5 mm in diameter yielded a higher rate (P < 0.05) of development to the blastocyst stage (13.8%) than those collected from <5 mm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 and > 130 days of age, did not affect the mean number of follicles (34.2 ±â€¯15 vs. 39.3 ±â€¯26 vs. 31.6 ±â€¯25), the mean number of oocytes recovered (21.2 ±â€¯10 vs. 24.5 ±â€¯15 vs. 22.6 ±â€¯17), and the cleavage rate (68.6 ±â€¯11 vs. 61.7 ±â€¯12 vs. 70.7 ±â€¯10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9 ±â€¯6 vs. 9.5 ±â€¯8%, P < 0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1 ±â€¯47 vs. 91.5 ±â€¯25, P < 0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50 µM tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1 ±â€¯8 vs. 18.6 ±â€¯4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.

The effect of age and length of gonadotropin stimulation on the in vitro embryo development of Holstein calf oocytes.

The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.