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

©2019. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Accepted version of a Published Work that appeared in final form in Theriogenology. To access the final edited and published work see https://doi.org/10.1016/j.theriogenology.2019.05.045The 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

The impact of in vitro embryo production on placental and umbilical cord vascularization is minimized by the addition of reproductive fluids

© 2023 The Authors. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Published version of a Published Work that appeared in final form in Theriogenology. To access the final edited and published work see https://doi.org/10.1016/j.theriogenology.2023.05.029Animals born from in-vitro-produced (IVP) embryos show changes in the placenta and umbilical cord vascularization. This study compares the placental and umbilical vascular morphometry in pigs (n = 19) born through artificial insemination (AI group) or after transfer of IVP embryos cultured with (RF-IVP group) or without (C-IVP group) reproductive fluids. The relationship between vascular parameters and animal growth during the first year of life was also analyzed. Samples were collected at birth, fixed, paraffin-embedded, cut in sections, stained, and photographed for vascular and morphometric analysis with ImageJ® and Slide Viewer®. The average daily weight gain was individually scored from birth to the first year of life. No differences were found in placental vascular morphometry among groups, except for the vascular area of small vessels (arterioles, venules, and small vessels) that was higher in the C-IVP group. Regarding the umbilical cord, the values for perimeter (AI: 26.40 ± 3.93 mm; IVP: 30.51 ± 4.74 mm), diameter (AI: 8.35 ± 1.01 mm; IVP: 10.26 ± 1.85 mm), area (AI: 43.18 ± 12.87; IVP: 56.61 ± 14.89 mm2), and Wharton's jelly area (AI: 36.86 ± 12.04 mm2; IVP 48.88 ± 12.80 mm2) were higher in IVP-derived than AI-derived animals, whereas arterial and venous morphometric data were similar between groups. A correlation study showed that placental and umbilical cord vascular phenotypes affect the further growth of pigs. In conclusion, assisted reproductive technologies impact small caliber vessels in the placenta and morphometric parameters in the umbilical cord. The addition of reproductive fluids in IVP-embryo contributes to reduce the differences with in vivo-derived animals

Growth analysis and blood profile in piglets born by embryo transfer

©2022. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Published, version of a Published Work that appeared in final form in Research in Veterinary Science. To access the final edited and published work see https://doi.org/10.1016/j.rvsc.2021.11.010Assisted 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

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

Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM

Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits. Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group. Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.MINECO, FEDER, Fundación Senec

Uso del simulador informático PhysioEX en la asignatura de Fisiología Veterinaria: valoración del alumnado

[SPA]El empleo de animales, material biológico y otros materiales/reactivos de laboratorio conlleva, en muchos casos, ciertas dificultades y un coste elevado, por lo que cada día se hace más extenso el uso de diferentes métodos como alternativas docentes. Entre ellas se encuentran los simuladores informáticos, que permiten de una forma sencilla y económica extrapolar la metodología del laboratorio a un ordenador, con las ventajas que ello supone en cuanto a tiempo de uso, posibilidad de repetición de los ejercicios o acceso ilimitado. El objetivo de nuestro estudio fue conocer la opinión de los alumnos (planes de estudio de Licenciatura y Grado en Veterinaria ) sobre el uso del simulador informático PhysioEx en la asignatura de Fisiología Veterinariacomo complemento a los contenidos teóricos y prácticos. Para ello, los alumnos contestaron tras realizar la práctica una breve encuesta de 6 preguntas. En total se analizaron 249 encuestas. Los resultados obtenidos fueron muy positivos ya que aproximadamente el 90% de los alumnos consideraron satisfactorio o muy satisfactorio el uso de estos simuladores en la impartición de la asignatura de Fisiología. [ENG]The use of animals, biological material and other laboratory materials/reagents causes, in many cases, certain difficulties and a high cost. Nowadays the widespread use of different teaching methods as alternatives is increasing. These methods include computer simulators, which allow a simple and affordable method for computer usage as an alternative to laboratory methodologies. The benefits of this usage are the possibility of repeating the exercises as often as students like and unlimited access and use of the program. The aim of our study was to determine students´ point of view on the use of computer simulation “PhysioEx” on Physiology teaching as a complement to the theoretical and laboratory sessions. To this end, students were asked to complete a survey made up of 6 questions. In total 249 surveys were analyzed. The results were very positive since approximately 90% of students considered good or very good the use of these simulators in the teaching of Physiology.Campus Mare Nostrum, Universidad Politécnica de Cartagena, Universidad de Murcia, Región de Murci

DNA methylation and gene expression changes derived from assisted reproductive technologies can be decreased by reproductive fluids

[EN]The number of children born since the origin of Assisted Reproductive Technologies (ART) exceeds 5 million. The majority seem healthy, but a higher frequency of defects has been reported among ART-conceived infants, suggesting an epigenetic cost. We report the first wholegenome DNA methylation datasets from single pig blastocysts showing differences between in vivo and in vitro produced embryos. Blastocysts were produced in vitro either without (C-IVF) or in the presence of natural reproductive fluids (Natur-IVF). Natur-IVF embryos were of higher quality than C-IVF in terms of cell number and hatching ability. RNA-Seq and DNA methylation analyses showed that Natur-IVF embryos have expression and methylation patterns closer to in vivo blastocysts. Genes involved in reprogramming, imprinting and development were affected by culture, with fewer aberrations in Natur-IVF embryos. Methylation analysis detected methylated changes in C-IVF, but not in Natur-IVF, at genes whose methylation could be critical, such as IGF2R and NNAT.SIThe authors thank CEFU, SA and El Pozo, SA for providing the biological material; Juan Antonio Carvajal and Soledad Rodriguez for collecting the oviducts, uteri and ovaries at the slaughterhouse; Carmen Mata´ s for technical support with IVF and Kristina Tabbada for sequencing RNA-Seq and BS-seq libraries. Funding: Work in GK’s laboratory was supported by the UK Biotechnology and Biological Sciences Research Council and Medical Research Council. Work in PC’s laboratory was supported by grants AGL2012–40180 C03-01 and AGL2015–66341-R from the Ministry of Economy and Competitiveness (Spain), and 20040/GERM/16 from Fundacio´ n Se´ neca. PC stay at The Babraham Institute was funded by a mobility grant of the Spanish Ministry of Education, Culture and Sports (PRX14/00348

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

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

Mammalian spermatozoa and cumulus cells bind to a 3D model generated by recombinant zona pellucida protein-coated beads

The egg is a spherical cell encapsulated by the zona pellucida (ZP) which forms a filamentous matrix composed of several glycoproteins that mediate gamete recognition at fertilization. Studies on molecular mechanisms of sperm-egg binding are limited in many mammalian species by the scarcity of eggs, by ethical concerns in harvesting eggs, and by the high cost of producing genetically modified animals. To address these limitations, we have reproduced a three-dimensional (3D) model mimicking the oocyte’s shape, by means of magnetic sepharose beads coated with recombinant ZP glycoproteins (BZP) and cumulus cells. Three preparations composed of either ZP2 (C and N-termini; BZP2), ZP3 (BZP3) or ZP4 (BZP4) were obtained and characterized by protein SDS-PAGE, immunoblot and imaging with confocal and electron microscopy. The functionality of the model was validated by adhesion of cumulus cells, the ability of the glycoprotein-beads to support spermatozoa binding and induce acrosome exocytosis. Thus, our findings document that ZP-beads provide a novel 3D tool to investigate the role of specific proteins on egg-sperm interactions becoming a relevant tool as a diagnostic predictor of mammalian sperm function once transferred to the industry.Supported by Fundación Seneca-Agencia de Ciencia y Tecnología de la Región de Murcia “Ayudas a la realización de proyectos para el desarrollo de investigación científica y técnica por grupos competitivos 2018” (20887/PI/18), MINECO and FEDER (AGL2015 70159 P), Groups and Units of Scientific Excellence of the Region of Murcia (20040/GERM/16) and the AGL2015–66341‐R from the Ministry of Economy and Competitiveness (Spain)