Oocyte electroporation prior to in vitro fertilization is an efficient method to generate single, double, and multiple knockout porcine embryos of interest in biomedicine and animal production.

Genetically modified pigs play a critical role in mimicking human diseases, xenotransplantation, and the development of pigs resistant to viral diseases. The use of programmable endonucleases, including the CRISPR/Cas9 system, has revolutionized the generation of genetically modified pigs. This study evaluates the efficiency of electroporation of oocytes prior to fertilization in generating edited gene embryos for different models. For single gene editing, phospholipase C zeta (PLC ζ) and fused in sarcoma (FUS) genes were used, and the concentration of sgRNA and Cas9 complexes was optimized. The results showed that increasing the concentration resulted in higher mutation rates without affecting the blastocyst rate. Electroporation produced double knockouts for the TPC1/TPC2 genes with high efficiency (79 %). In addition, resistance to viral diseases such as PRRS and swine influenza was achieved by electroporation, allowing the generation of double knockout embryo pigs (63 %). The study also demonstrated the potential for multiple gene editing in a single step using electroporation, which is relevant for xenotransplantation. The technique resulted in the simultaneous mutation of 5 genes (GGTA1, B4GALNT2, pseudo B4GALNT2, CMAH and GHR). Overall, electroporation proved to be an efficient and versatile method to generate genetically modified embryonic pigs, offering significant advances in biomedical and agricultural research, xenotransplantation, and disease resistance. Electroporation led to the processing of numerous oocytes in a single session using less expensive equipment. We confirmed the generation of gene-edited porcine embryos for single, double, or quintuple genes simultaneously without altering embryo development to the blastocyst stage. The results provide valuable insights into the optimization of gene editing protocols for different models, opening new avenues for research and applications in this field.

Optimizing oocyte electroporation for genetic modification of porcine embryos: Evaluation of the parthenogenetic activation.

In reproductive biology, understanding the effects of novel techniques on early embryo development is of paramount importance. To date, the effects of electrical activation on oocytes prior to in vitro fertilization (IVF) are not well understood. The aim of this study was to investigate the effects of oocyte electroporation prior to IVF on embryo development and to differentiate between true embryos and parthenotes by using a TPCN2 knock-out (KO) male to evaluate the presence of the KO allele in the resulting blastocysts. The study consisted of three experiments. The first one examined oocyte electroporation with and without subsequent IVF and found that electroporated oocytes had higher activation rates, increased occurrence of a single pronucleus, and no effect on sperm penetration. Cleavage rates improved in electroporated oocytes, but blastocyst rates remained constant. Genotype analysis revealed a significant increase in the proportion of parthenotes in the electroporated groups compared to the IVF control (30.2 % vs. 6.8 %). The second experiment compared two electroporation media, Opti-MEM and Nuclease-Free Duplex Buffer (DB). DB induced higher oocyte degeneration rates, and lower cleavage and blastocyst rates than Opti-MEM, while parthenogenetic formation remained consistent (60.0 and 48.5 %). In the third experiment, the timing of electroporation relative to IVF was evaluated (1 h before IVF, immediately before IVF and 7 h after IVF). Electroporation immediately before IVF resulted in higher activation rates and different pronuclear proportions compared to the other timing groups. The penetration rate was higher in the immediate electroporation group, and cleavage rate improved in all electroporated groups compared to the control. Blastocyst rates remained constant. Genotyping revealed no significant differences in parthenote proportions among the timing groups, but these were higher than the control (56.25 %, 63.89 %, 51.61 %, 2.44 %, respectively), and showed higher mutation rates when electroporation was performed 7 h after IVF. Overall, this comprehensive study sheds light on the potential of electroporation for creating genetically modified embryos and the importance of media selection and timing in the process, the best media being the Opti-MEM and the more efficient timing regarding mutation rate, 7 h post-IVF, even when the parthenote formation did not differ among electroporated groups. Further studies are needed to reduce the parthenogenetic activation while maintaining high mutation rates to optimize the use of this procedure for the generation of gene-edited pig embryos by oocyte/zygote electroporation.

Information Flow between Parents and Offspring Is Essential for Successful Development.

Over the last several decades, the sciences of developmental biology and physiology have expanded and intertwined their scope enormously [...].

Production of Genetically Modified Porcine Embryos via Lipofection of Zona-Pellucida-Intact Oocytes Using the CRISPR/Cas9 System.

The generation of genetically modified pigs has an important impact thanks its applications in basic research, biomedicine, and meat production. Cloning was the first technique used for this production, although easier and cheaper methods were developed, such as the microinjection, electroporation, or lipofection of oocytes and zygotes. In this study, we analyzed the production of genetically modified embryos via lipofection of zona-pellucida-intact oocytes using LipofectamineTM CRISPRMAXTM Cas9 in comparison with the electroporation method. Two factors were evaluated: (i) the increment in the concentration of the lipofectamine-ribonucleoprotein complexes (LRNPC) (5% vs. 10%) and (ii) the concentration of ribonucleoprotein within the complexes (1xRNP vs. 2xRNP). We found that the increment in the concentration of the LRNPC had a detrimental effect on embryo development and a subsequent effect on the number of mutant embryos. The 5% group had a similar mutant blastocyst rate to the electroporation method (5.52% and 6.38%, respectively, p > 0.05). The increment in the concentration of the ribonucleoprotein inside the complexes had no effect on the blastocyst rate and mutation rate, with the mutant blastocyst rate being similar in both the 1xRNP and 2xRNP lipofection groups and the electroporation group (1.75%, 3.60%, and 3.57%, respectively, p > 0.05). Here, we showed that it is possible to produce knock-out embryos via lipofection of zona-pellucida-intact porcine oocytes with similar efficiencies as with electroporation, although more optimization is needed, mainly in terms of the use of more efficient vesicles for encapsulation with different compositions.

Generation of Calpain-3 knock-out porcine embryos by CRISPR-Cas9 electroporation and intracytoplasmic microinjection of oocytes before insemination.

Limb girdle muscular dystrophy type R1 (LGMDR1) is an autosomal recessive myopathy described in humans resulting from a deficiency of calpain-3 protein (CAPN3). This disease lacks effective treatment and an appropriate model, so the generation of KO pigs by CRISPR-Cas9 offers a way to better understand disease ethology and to develop novel therapies. Microinjection is the main method described for gene editing by CRISPR-Cas9 in porcine embryo, but electroporation, which allows handling more embryos faster and easier, has also recently been reported. The objective of the current study was to optimize porcine oocyte electroporation to maximize embryo quality and mutation rate in order to efficiently generate LGMDR1 porcine models. We found that the efficiency of generating CAPN3 KO embryos was highest with 4 electroporation pulses and double sgRNA concentration than microinjection. Direct comparison between microinjection and electroporation demonstrated similar rates of embryo development and mutation parameters. The results of our study demonstrate that oocyte electroporation, an easier and faster method than microinjection, is comparable to standard approaches, paving the way for democratization of transgenesis in pigs.

Broadening the educational pipeline: the global landscape of master of science programs in reproductive science and medicine.

Reproductive health underpins overall health, and thus, research in reproductive science and medicine is essential. This requires a pipeline of trained scientists and clinicians, which is challenging given the relatively small size of the field. Educational programs outside the traditional doctorate or medical degrees are needed to generate and maintain a well-trained reproductive science and medicine workforce. Master's programs have gained traction as a viable way for students to receive educational value relative to their career goals. Our hypothesis is master's degree programs in the fundamental, applied, and allied health reproductive fields broadens the workforce and increases impact. An internet web search identified 73 programs that conferred an MS degree in the areas of animal science, clinical/embryology, and reproductive health/biology. These programs are spread across the globe in Europe (47%), North America (23%), Asia (14%), South America (7%), Oceania (5%), and Africa (4%). To evaluate global exemplars, we profiled the mission and structure, curriculum, and program impact of two established master's degree programs: the Master of Science in Reproductive Science and Medicine at Northwestern University in the United States and the Biology and Technology of Reproduction in Mammals at the University of Murcia in Spain. Elements of infrastructure and support, program connectivity, and alumni networks were analyzed for their role in the success of the programs. These two programs have formally trained >375 individuals, demonstrating master's degree programs in reproductive science are an important educational mechanism. The educational best practices shared here serve as templates for expanding training programs worldwide.

Effect of Aphidicolin, a Reversible Inhibitor of Eukaryotic Nuclear DNA Replication, on the Production of Genetically Modified Porcine Embryos by CRISPR/Cas9.

Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 µM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.

Reproductive fluids, added to the culture media, contribute to minimizing phenotypical differences between in vitro-derived and artificial insemination-derived piglets.

The addition of reproductive fluids (RF) to the culture media has shown benefits in different embryonic traits but its long-term effects on the offspring phenotype are still unknown. We aimed to describe such effects in pigs. Blood samples and growth parameters were collected from piglets derived from in vitro-produced embryos (IVP) with or without RF added in the culture media versus those artificially inseminated (AI), from day 0 to month 6 of life. An oral glucose tolerance test was performed on day 45 of life. We show here the first comparative data of the growth of animals produced through different assisted reproductive techniques, demonstrating differences between groups. Overall, there was a tendency to have a larger size at birth and faster growth in animals derived from in vitro fertilization and embryo culture versus AI, although this trend was diminished by the addition of RFs to the culture media. Similarly, small differences in hematological indices and glucose tolerance between animals derived from AI and those derived from IVP, with a sex-dependent effect, tended to fade in the presence of RF. The addition of RF to the culture media could contribute to minimizing the phenotypical differences between the in vitro-derived and AI offspring, particularly in males.

Growth analysis and blood profile in piglets born by embryo transfer.

Assisted reproductive technologies (ART), besides solving several reproductive problems, it has also been used as a tool to improve the animal productivity that is required for feeding the human population. One of these techniques, the embryo transfer (ET), has presented limitations in the porcine species, which could constrain its use in the porcine industry. To clarify the potential of this technique, we aimed to compare the impact of using ET or artificial insemination (AI) on the phenotype of the offspring during its first days of age, in terms of growth and blood parameters. At birth, the body weight was higher for ET-females than AI-females, but this difference was no longer observed at day 15. On day 3, it was observed a higher concentration of red blood cells, haemoglobin, and haematocrit in females-ET and a higher concentration of white blood cells in both ET-derived piglets (males and females) when compared to AI groups. On day 3, the biochemical analysis showed a higher level of albumin for ET-derived males, and a lower level of bilirubin for ET-females than AI controls. However, all values were within the normal ranges. Our results indicate that piglets derived from ET seem to be phenotypically similar to those born by AI, which provides preliminary evidence that the ET procedure is a safe technique, but additional studies beyond 15 days of life are requested to conclude its global impact. Furthermore, the presented reference values of blood parameters in this species are interesting data for the pig industry.

Photo Stimulation of Seminal Doses with Red LED Light from Duroc Boars and Resultant Fertility in Iberian Sows.

In pigs, it has been reported that increased farrowing rates and litter size have been induced by photostimulating the seminal doses for artificial insemination with red LED light. As the reproductive characteristics, production system, and outcome parameters of Iberian breed pigs are different from other commercial breeds, the aim of this study was to evaluate the possible effect of illuminating seminal doses from Duroc boars with red LED light and the fertility outcomes of Iberian females. Semen samples were obtained from 38 fertile Duroc boars. Photostimulation of the artificial insemination (AI) seminal doses was carried out by illuminating the samples with a red LED for 10 min, followed by 10 min of darkness, and finally 10 additional minutes of red light. The fertility study was conducted on two commercial farms using multiparous Iberian sows (farm A, n = 824; farm B, n = 2131), that were randomly assigned to LED (L) or control (C) groups. No differences were found between L and C groups in both farms (p > 0.05) for parity, pregnancy rate, duration of pregnancy, farrowing rate, and litter size (total, alive, and stillborn piglets). Farrowing rates in farm A were 88.8% (n = 383) for control and 89.6% (n = 441, p = 0.67) for the LED group. In farm B, farrowing rates were C:90.5% (n = 1030) and L: 90.1% (n = 1101, p = 0.48). In farm A, total born piglets were 8.69 ± 0.11 for C and 8.71 ± 0.11 for L (p = 0.87). In farm B, the results were 8.72 ± 0.7 for C and 8.70 ± 0.06 (p = 0.82) for L. Under the production conditions for the Iberian breed, the photostimulation with red LED light using Duroc pig seminal doses was not effective in improving the fertility of Iberian sows.

Replacement of Albumin by Preovulatory Oviductal Fluid in Swim-Up Sperm Preparation Method Modifies Boar Sperm Parameters and Improves In Vitro Penetration of Oocytes.

More suitable and efficient methods to protect gametes from external harmful effects during in vitro handling can be achieved by adding preovulatory porcine oviductal fluid (pOF) to in vitro culture media. The objective of this study was to assess the swim-up procedure's suitability as a sperm selection method using a medium supplemented with 1mg/mL BSA, 1% preovulatory pOF (v/v), 1% v/v pOF plus 1mg/mL BSA, and 5mg/mL BSA. After selection, various sperm parameters were studied, such as sperm recovery rate, sperm morphology, motility (by CASA), vitality, acrosome status and intracellular calcium (by flow cytometry) and ability to penetrate oocytes in vitro. Around 2% of sperm were recovered after swim-up, and the replacement of BSA by pOF showed a beneficial reduction of motility parameters calcium concentration, resulting in an increased penetration rate. The combination of albumin and oviductal fluid in the medium did not improve the sperm parameters results, whereas a high concentration of BSA increased sperm morphological abnormalities, motility, and acrosome damage, with a reduction of calcium concentration and penetration rate. In conclusion, the replacement of albumin by preovulatory oviductal fluid in the swim-up sperm preparation method modifies boar sperm parameters and improves the in vitro penetration of oocytes.

Generation of Nonmosaic, Two-Pore Channel 2 Biallelic Knockout Pigs in One Generation by CRISPR-Cas9 Microinjection Before Oocyte Insemination.

Studies of knockout (KO) mice with defects in the endolysosomal two-pore channels (TPCs) have shown TPCs to be involved in pathophysiological processes, including heart and muscle function, metabolism, immunity, cancer, and viral infection. With the objective of studying TPC2's pathophysiological roles for the first time in a large, more humanlike animal model, TPC2 KO pigs were produced using CRISPR-Cas9. A major problem using CRISPR-Cas9 to edit embryos is mosaicism; thus, we studied for the first time the effect of microinjection timing on mosaicism. Mosaicism was greatly reduced when in vitro produced embryos were microinjected before insemination, and surgical embryo transfer (ET) was performed using such embryos. All TPC2 KO fetuses and piglets born following ET (i.e., F0 generation) were nonmosaic biallelic KOs. The generation of nonmosaic animals greatly facilitates germ line transmission of the mutation, thereby aiding the rapid and efficient generation of KO animal lines for medical research and agriculture.

Seminal plasma components from fertile stallions involved in the epididymal sperm freezability.

BACKGROUND: Seminal plasma (SP) plays a crucial role in sperm protection and functionality. However, the effect of SP on the sperm cryopreservation is dependent on the stallion and SP composition. The use of epididymal spermatozoa incubated in the presence of SP could help the identification of the components of SP that are able to confer protection upon the spermatozoa during freezing. OBJECTIVE: The aims of this study were (i) to identify SP components involved in the potential protection of epididymal spermatozoa during the freeze-thawing process and (ii) to identify and evaluate the proteins likely related to sperm freezability, using two-dimensional difference gel electrophoresis (2D-DIGE). MATERIALS AND METHODS: Epididymal spermatozoa from 4 stallions were incubated with SP (80%, v/v) or without SP (control) before freezing. Sperm parameters were evaluated after thawing (viability, chromatin condensation, acrosomal integrity, reactive oxygen species [ROS]) and SP composition: total antioxidant capacity (TAC), fatty acid composition, total protein concentration, and protein components by 2D-DIGE. RESULTS: After thawing, the proportions of viable and acrosome-intact spermatozoa were higher than control when SP from two stallions was used (F and O). The SP of all stallions reduced ROS production in comparison with the control. After analyzing the SP components, it was found that total protein concentration, TAC, polyunsaturated fatty acids (PUFA), and eight specific proteins identified by 2D-DIGE were different between stallions. DISCUSSION: These studies allow the identification of SP components that could be involved in sperm protection or cryotolerance. Use of this information could help in the selection of stallions according to their semen freezing capacity. CONCLUSION: The composition of the SP probably contributes to semen cryotolerance capacity. Total protein, TAC, PUFA, and some proteins such as cysteine-rich secreted protein 3 could be used as biomarkers for the selection for sperm cryotolerance.

Effect of oviductal fluid on bull sperm functionality and fertility under non-capacitating and capacitating incubation conditions.

This study investigated the effect of bovine oviductal fluid from late follicular (LF) and early luteal (EL) phases on bull sperm functionality under non-capacitating (NCAP) and capacitating (CAP) conditions. Frozen-thawed semen samples from five bulls were thawed and incubated (0, 1 or 2 h) in NCAP and CAP media supplemented with 1% bovine oviductal fluid (LF and EL groups) and in absence of fluid (C group). Motion parameters were assessed by CASA; sperm viability, acrosomal integrity and membrane lipid disorder parameters were evaluated by flow cytometry; and sperm DNA fragmentation was evaluated by the Comet assay. Finally, in vitro fertilization with sperm treated under CAP conditions was performed and further embryo culture results evaluated. In NCAP medium, addition of LF and EL fluid increased the total and progressive motility, and LF fluid improved the stability of sperm DNA. However, under CAP conditions addition of LF and EL fluid decreased some sperm motion parameters and some parameters of sperm DNA stability. Proportion of viable sperm cells with low lipid disorder was higher in NCAP than CAP medium and addition of LF fluid markedly increased the proportion of viable spermatozoa with high lipid disorder and acrosome alteration (spontaneous acrosome reaction). Under current conditions, incubation of bull sperm with oviductal fluid before insemination did not affect detrimentally the IVF results nor embryo development, being blastocyst rate similar between CAP-LF, CAP-EL and control groups. In conclusion, oviductal fluid positively influences sperm functionality and modulate in vitro capacitation.

Addition of exogenous proteins detected in oviductal secretions to in vitro culture medium does not improve the efficiency of in vitro fertilization in pigs.

This work was designed to study whether HSP70-1A, HSP90α, ezrin or PDI4, proteins previously identified in porcine oviductal secretions, have a role in zona pellucida (ZP) resistance to enzymatic digestion, in vitro fertilization (IVF) and sperm viability. In vitro matured porcine cumulus oocyte complexes were denuded and i) incubated for 1 h in TALP medium supplemented or not with each exogenous oviductal protein and in presence or absence of heparin to assess ZP digestion time by pronase; and ii) inseminated with fresh ejaculated boar spermatozoa in medium supplemented or not with each exogenous oviductal protein to assess their effect on fertilization results. Finally, spermatozoa were incubated in Tyrode's medium (0, 1 and 20 h) supplemented or not with HSP-701A, HSP-90α or ezrin, to assess simultaneously sperm viability and acrosome status by means of flow cytometry. Although all proteins increased the ZP digestion time, this increase was lower than 1 min, being ezrin the protein with a stronger effect. Presence of heparin in the medium reinforced the ZP hardening effect of ezrin and HSP-701A up to one more min, but not HSP-90α nor PDI4. Sperm penetration, but not IVF efficiency, increased when gametes were cocultured in medium containing PDIA4 whereas sperm penetration and polyspermy rates decreased in presence of ezrin and HSP proteins. This reduction was not the result of a detrimental effect of proteins on sperm viability or acrosome reaction. In conclusion, addition of exogenous proteins detected in oviductal secretions to artificial media does not reproduce the effect of adding such secretions nor improve the final efficiency of the porcine IVF system.

Livestock Gene Editing by One-step Embryo Manipulation.

The breakthrough and rapid advance of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has enabled the efficient generation of gene-edited animals by one-step embryo manipulation. Clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 delivery to the livestock embryos has been typically achieved by intracytoplasmic microinjection; however, recent studies show that electroporation may be a reliable, efficient, and practical method for CRISPR/Cas9 delivery. The source of embryos used to generate gene-edited animals varies from in vivo to in vitro produced, depending mostly on the species of interest. In addition, different Cas9 and gRNA reagents can be used for embryo editing, ranging from Cas9-coding plasmid or messenger RNA to Cas9 recombinant protein, which can be combined with in vitro transcribed or synthetic guide RNAs. Mosaicism is reported as one of the main problems with generation of animals by embryo editing. On the other hand, off-target mutations are rarely found in livestock derived from one-step editing. In this review, we discussed these and other aspects of generating gene-edited animals by single-step embryo manipulation.

The Addition of Lactobacillus spp., Enrofloxacin or Doxycycline Negatively Affects the Viability of Mycoplasma bovis in Diluted Bovine Semen.

Mycoplasma bovis is an important etiologic agent of bovine mycoplasmosis in cattle. Different transmission routes have been described, including those related to reproduction. The presence of mycoplasma in semen has led to its appearance in infection-free areas through artificial insemination (AI). Semen was recently reported to be the initial source of two M. bovis mastitis outbreaks in two closed dairy herds in Finland. This questions the effectiveness of the antimicrobials currently used in semen extenders to control the pathogens in contaminated semen. They should be re-evaluated, or alternative measures to antimicrobials should be tested to obtain M. bovis-free semen. This in vitro study aimed to assess different strategies to reduce the risk of transmission of M. bovis through AI technologies. The viability of M. bovis (PG45, NCTC 10131) in bull semen diluted (DS) in a Tris-citrate-fructose solution was tested, after the addition of enrofloxacin, doxycycline or a Lactobacillus spp.-based probiotic. The data show the susceptibility of the pathogen to the addition of 0.125 µg/mL of enrofloxacin or 0.0625 µg/mL of doxycycline and to the addition of the probiotic at a concentration of 3.24 × 106 colony forming units (CFU)/mL or 3.24 × 108 CFU/mL in DS. The Tris-citrate-fructose medium negatively affected the viability of M. bovis, although this effect was lower than that observed after the addition of the probiotic and antimicrobials (p < 0.05). Our results may support new strategies for reducing the risk of M. bovis transmission through AI.

Assessment and preservation of liquid and frozen-thawed Black crested mangabey (Lophocebus aterrimus) spermatozoa obtained by transrectal ultrasonic-guided massage of the accessory sex glands and electroejaculation.

The Black Crested Mangabey (Lophocebus aterrimus) is an African monkey listed as Near Threatened by the IUCN and in captivity the population is limited to 34 males. The aim of this study was to evaluate two Black Crested Mangabey males, maintained in captivity in a zoological garden and suspected of infertility, with a complete examination of their genital tract using ultrasonography, followed by recovery of semen using transrectal ultrasonic massage of the accessory sexual glands (TUMASG) and electroejaculation. One male had small testicular and accessory sex gland sizes indicative of senile hypoplasia. The other male was suspected of infertility. Four semen samples were obtained. Fresh semen was initially evaluated, diluted in Refrigeration Medium Test Yolk buffer, cooled at 15 °C and cryopreserved. Endocrine profiles (testosterone, oestradiol, FSH, LH, cortisol), prostatic specific antigen and semen variables (volume, concentration, motility by CASA, viability and acrosome status using flow cytometry, morphology, morphometry utilising TEM) were evaluated in raw, cooled and cryopreserved samples. There was no detrimental effect of cooling or cryopreservation on sperm viability and acrosomal integrity. Similar percentages of viable and acrosome-intact spermatozoa were present in cooled (for 6 h) and frozen-thawed semen samples (75.1% compared with 69.0%, P > 0.05), while progressive motility was greater in cooled, compared with frozen-thawed samples (81.5% compared with 67.3%). This study was the first in which there was evaluation of sperm variables in this species and, although this study is limited by the number of animals it provides background information for further studies using assisted reproductive technologies.

Pig in vitro fertilization: Where are we and where do we go?

The pig is an important livestock animal. Biotechnological interest in this species has increased due to its use, among others, in the generation of transgenic animals for use in biomedicine based on its greater physiological proximity to the human species than other large domestic animals. This development has paralleled an improvement in Assisted Reproduction Techniques (ART) used for this species. However, the ability to generate animals from embryos produced entirely in vitro is still limited and a wide margin for improvement remains. Here we review the procedures, additives, and devices used during pig in vitro fertilization (IVF), focusing on the main points of each step that have offered the best results in terms of increased efficiency of the system. The lack of standardized protocols and consensus on the parameters to be assessed makes it difficult to compare results across different studies, but some conclusions are drawn from the literature. We anticipate that new physiological protocols will advance the field of swine IVF, including induction of prefertilization ZP hardening with oviductal fluid, sperm preparation by swim-up method, increased viscosity through the addition of inert molecules or reproductive biofluids, and the incorporation of 3D devices. Here we also reflect on the need to expand the variables on which the efficiency of pig IVF is based, providing new parameters that should be considered to supply more objective and quantitative assessment of IVF additives and protocols.

Generation and characterization of a novel knockin minipig model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients.