Swine in vitro embryo production: Potential, challenges, and advances.

Pig production, a vital sector of the meat industry, faces demands for improved quality, efficiency, and sustainability. Advancements in breeding, disease control, and artificial insemination have enhanced production, while biotechnologies such as in vitro embryo production (IVP) and genetic engineering offer further progress. In vitro embryo production could facilitate the global exchange of valuable genetic material, accelerate breeding programs, and improve productivity, and it is essential for generating genetically modified (GM) pigs. These GM pigs have two main applications: first, they allow for targeted modifications aimed at improving production traits relevant to pig production in agriculture, such as meat quality and disease resistance. Second, they serve as valuable biomedical models for human disease research, regenerative medicine, and organ transplantation. Yet, despite notable advancements in recent decades, the efficiency of the current IVP systems for porcine embryos remains a challenge. Compared to the in vivo environment, suboptimal culture conditions lead to issues such as elevated polyspermy, poor embryo development, and the production of low-quality blastocysts. This review provides an overview of the key steps and recent advancements in porcine IVP technology. We will emphasize the promising utilization of oocytes from live females of high genetic value through ovum pick-up and the incorporation of extracellular vesicles and cytokines into IVP media. These innovative strategies hold immense potential to significantly enhance embryo development and overall success rates in porcine IVP, and could open the door for significant progress in both agriculture and biomedicine applications.

Vitrification of pig embryos dysregulates the microRNA transcriptome profile.

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4-6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139-3p, miR-214 and miR-885-3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-ß signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-ß (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

Cytokine profile in peripheral blood mononuclear cells differs between embryo donor and potential recipient sows.

Introduction: Pregnancy success relies on the establishment of a delicate immune balance that requires the early activation of a series of local and systemic immune mechanisms. The changes in the immunological profile that are normally occurring in the pregnant uterus does not take place in cyclic (non-pregnant) uterus, a fact that has been widely explored in pigs at the tissue local level. Such differences would be especially important in the context of embryo transfer (ET), where a growing body of literature indicates that immunological differences at the uterine level between donors and recipients may significantly impact embryonic mortality. However, whether components of peripheral immunity also play a role in this context remains unknown. Accordingly, our hypothesis is that the immune status of donor sows differs from potential recipients, not only at the tissue local level but also at the systemic level. These differences could contribute to the high embryonic mortality rates occurring in ET programs. Methods: In this study differences in systemic immunity, based on cytokine gene expression profile in peripheral blood mononuclear cells (PBMCs), between embryo-bearing donor (DO group; N = 10) and potential recipient sows (RE group; N = 10) at Day 6 after the onset of the estrus were explored. Gene expression analysis was conducted for 6 proinflammatory (IL-1α, IL-1ß, IL-2, GM-CSF, IFN-γ, and TNF-α) and 6 anti-inflammatory (IL-4, IL-6, IL-10, IL-13, TGF-ß1, and LIF) cytokines. Results and discussion: All cytokines were overexpressed in the DO group except for IL-4, suggesting that stimuli derived from the insemination and/or the resultant embryos modify the systemic immune profile in DO sows compared to RE (lacking these stimuli). Our results also suggest that certain cytokines (e.g., IL-1α and IL-1ß) might have a predictive value for the pregnancy status.

In-depth proteome characterization of endometrium and extraembryonic membranes during implantation in pig.

BACKGROUND: Proteome characterization of the porcine endometrium and extraembryonic membranes is important to understand mother-embryo cross-communication. In this study, the proteome of the endometrium and chorioallantoic membrane was characterized in pregnant sows (PS) during early gestation (d 18 and 24 of gestation) and in the endometrium of non-pregnant sows (NPS) during the same days using LC-MS/MS analysis. The UniProtKB database and ClueGO were used to obtain functional Gene Ontology annotations and biological and functional networks, respectively. RESULTS: Our analysis yielded 3,254 and 3,457 proteins identified in the endometrium of PS and NPS, respectively; of these, 1,753 being common while 1,501 and 1,704 were exclusive to PS and NPS, respectively. In addition, we identified 3,968 proteins in the extraembryonic membranes of PS. Further analyses of function revealed some proteins had relevance for the immune system process and biological adhesion in endometrium while the embryonic chorion displayed abundance of proteins related to cell adhesion and cytoskeletal organization, suggesting they dominated the moment of endometrial remodeling, implantation and adhesion of the lining epithelia. Data are available via ProteomeXchange with identifier PXD042565. CONCLUSION: This is the first in-depth proteomic characterization of the endometrium and extraembryonic membranes during weeks 3 to 4 of gestation; data that contribute to the molecular understanding of the dynamic environment during this critical period, associated with the majority of pregnancy losses.

Cryopreservation of highly extended pig spermatozoa remodels its proteome and counteracts polyspermic fertilization in vitro.

BACKGROUND: Currently, high polyspermy remains a significant obstacle to achieving optimal efficiency in in vitro fertilization (IVF) and in vitro embryo production (IVP) systems in pigs. Developing strategies that would prevent polyspermy is essential in overcoming this challenge and maximizing the potential of this reproductive biotechnology. Previous results have demonstrated that using boar spermatozoa subjected to a high-extension and reconcentration procedure and then cryopreserved resulted in significant improvements in IVF/IVP systems with high rates of monospermy and penetration. OBJECTIVE: The aim of the present study was to unveil the molecular mechanisms that may underlie the changes in fertilization patterns exhibited by highly extended and cryopreserved boar spermatozoa. MATERIALS AND METHODS: To achieve this goal, we used quantitative proteomic analysis (LC‒ESI‒MS/MS SWATH) to identify differentially abundant proteins (DAPs) between highly extended (HE) and conventionally (control; CT) cryopreserved boar spermatozoa. Prior to the analysis, we evaluated the in vitro post-thawing fertilizing ability of the sperm samples. The results demonstrated a remarkable improvement in monospermy and IVF efficiency when using HE spermatozoa in IVF compared with CT spermatozoa. RESULTS: At the proteomic level, the combination of high-extension and cryopreservation had a significant impact on the frozen-thawed sperm proteome. A total of 45 proteins (24 downregulated and 21 upregulated) were identified as DAPs (FC > 1 or ≤1; p < 0.05) when compared with CT spermatozoa. Some of these proteins were primarily linked to metabolic processes and the structural composition of sperm cells. The dysregulation of these proteins may have a direct or indirect effect on essential sperm functions and significantly affect spermatozoa-oocyte interaction and, therefore, the sperm fertilization profile under in vitro conditions. While these findings are promising, further research is necessary to comprehend how the disturbance of specific proteins affects sperm fertilization ability.

Cryotop vitrification of large batches of pig embryos simultaneously provides excellent postwarming survival rates and minimal interference with gene expression.

The most commonly used technique to vitrify pig embryos is the super open pulled straw (SOPS), where a maximum of 6 embryos can be vitrified simultaneously per device without compromising the minimum volume necessary for optimal preservation. Since optimal embryo transfer (ET) demands a transfer of 20-40 embryos per recipient, the customary use of SOPS complicates embryo warming and ET in field conditions. Such complications could be avoided when using the Cryotop® (OC) system, which has been proven to be an effective option for vitrifying at least 20 porcine embryos simultaneously. This study aimed to investigate the changes in the transcriptome of blastocysts caused by vitrification using both systems. In vivo-derived blastocysts were OC- (n = 60; 20 embryos/device) and SOPS- (n = 60; 4-6 embryos/device) vitrified and cultured for 24 h after warming. Nonvitrified blastocysts (n = 60) cultured for 24 h postcollection acted as controls. At the end of culture, 48 viable embryos from each group (6 pools of 8 embryos) were selected for microarray (GeneChip® Porcine Genome Array, P/N 900624, Affymetrix) analysis of differentially expressed genes (DEGs). The survival rate of embryos vitrified with the OC and SOPS systems (>97%) was similar to that of the control embryos (100%). Microarray analysis of each vitrification system compared to the control group showed 245 DEGs (89 downregulated and 156 upregulated) for the OC system and 210 (44 downregulated and 166 upregulated) for the SOPS system. Two pathways were enriched for the DEGs specifically altered in each vitrification system compared to the control (glycolysis/gluconeogenesis and carbon metabolism pathways for the OC system and amino sugar and nucleotide sugar metabolism and lysosome pathways in the SOPS group). The OC group showed 31 downregulated and 24 upregulated genes and two enriched pathways (mineral absorption and amino sugar and nucleotide sugar metabolism pathways) when compared to the SOPS group. In summary, vitrification with the OC system altered fewer genes related to apoptosis and activated genes related to cell proliferation. We conclude that vitrification with either the OC or SOPS system has a moderate to low effect on the transcriptome of in vivo-derived porcine blastocysts. Further investigation is needed to elucidate how the differences in the transcriptome of embryos vitrified with these systems affect their subsequent developmental ability after ET.

The Use of a Brief Synchronization Treatment after Weaning, Combined with Superovulation, Has Moderate Effects on the Gene Expression of Surviving Pig Blastocysts.

The combination of estrus synchronization and superovulation (SS) treatments causes alterations in ovarian and endometrial gene expression patterns, resulting in abnormal follicle and oocyte growth, fertilization, and embryo development. However, the impact of combined SS treatments on the transcriptome of the surviving embryos remains unidentified. In this study, we examined gene expression changes in day 6 blastocysts that survived a brief regimen of synchronization treatment combined with superovulation. The sows were included in one of three groups: SS7 group (n = 6), sows were administered Altrenogest (ALT) 7 days from the day of weaning and superovulated with eCG 24 h after the end of ALT treatment and hCG at the onset of estrus; SO group (n = 6), ALT nontreated sows were superovulated with eCG 24 h postweaning and hCG at the onset of estrus; control group (n = 6), weaned sows displaying natural estrus. Six days after insemination, the sows underwent a surgical intervention for embryo collection. Transcriptome analysis was performed on blastocyst-stage embryos with good morphology. Differentially expressed genes (DEGs) between groups were detected using one-way ANOVA with an un-adjusted p-value < 0.05 and a fold change 1.5. The effect of SO treatment on the number of altered pathways and DEGs within each pathway was minimal. Only four pathways were disrupted comprising only a total of four altered transcripts, which were not related to reproductive functions or embryonic development. On the other hand, the surviving blastocysts subjected to SS7 treatments exhibited moderate gene expression changes in terms of DEGs and fold changes, with seven pathways disrupted containing a total of 10 transcripts affected. In this case, the up-regulation of certain pathways, such as the metabolic pathway, with two up-regulated genes associated with reproductive functions, namely RDH10 and SPTLC2, may suggest suboptimal embryo quality, while the down-regulation of others, such as the glutathione metabolism pathway, with down-regulated genes related to cellular detoxification of reactive oxygen species, namely GSTK1 and GSTO1, could depress the embryos' response to oxidative stress, thereby impairing subsequent embryo development. The gene expression changes observed in the present study in SS7 embryos, along with previous reports indicating SS7 can negatively affect fertilization, embryo production, and reproductive tract gene expression, make its use in embryo transfer programs unrecommendable.

Extracellular vesicles would be involved in the release and delivery of seminal TGF-ß isoforms in pigs.

Introduction: Pig seminal plasma (SP) is rich in active forms of all three isoforms (1-3) of transforming growth factor ß (TGF-ß), a chemokine modulatory of the immune environment in the female genital tract once semen is delivered during mating or artificial insemination (AI). The present study aimed to examine how TGF-ßs are secreted by the epithelium of the male reproductive tract and how they are transported in semen, emphasizing the interplay with seminal extracellular vesicles (sEVs). Methods: Source of TGF-ßs was examined by immunohistochemistry in testis, epididymis, and accessory sex glands, by immunocytochemistry in ejaculated spermatozoa, and by Luminex xMAP® technology in SP and sEVs retrieved from healthy, fertile male pigs used as breeders in AI programs. Results: All three TGF-ß isoforms were expressed in all reproductive tissues explored and would be released into ductal lumen either in soluble form or associated with sEVs. Ejaculated spermatozoa expressed all three TGF-ß isoforms, both inside and outside, probably the outer one associated with membrane-bound sEVs. The results confirmed that pig SP contains all three TGF-ß isoforms and demonstrated that a substantial portion of them is associated with sEVs. Discussion: Seminal EVs would be involved in the cellular secretion of the active forms of seminal TGF-ß isoforms and in their safe transport from the male to the female reproductive tract.

Combined synchronization and superovulation treatments negatively impact embryo viability possibly by the downregulation of WNT/ß-catenin and Notch signaling genes in the porcine endometrium.

The combination of estrus synchronization and superovulation treatments introduces molecular modifications whose effects are yet to be disclosed. Here, reproductive parameters and gene expression changes in ovaries and endometrium were explored on day 6 after artificial insemination (AI), when synthetic progestin altrenogest (ALT) was combined with gonadotropins. Sows were administered ALT for 7 d beginning on the day of weaning and superovulated with equine chorionic gonadotropin (eCG) 24 h later and human chorionic gonadotropins (hCG) at the onset of estrus (SS-7 group; n = 6). The controls were either superovulated sows with eCG 24 h postweaning and hCG at the onset of estrus (SC group; n = 6) or sows with postweaning spontaneous estrus (NC group; n = 6). Ovary examination and embryo and tissue collection were performed in all sows via laparotomy on day 6 post-AI. RNA-Seq was conducted to analyze differentially expressed genes (DEGs) between groups. Statistical analysis of the reproductive parameters was conducted with ANOVA and Tukey post hoc tests. DEGs were analyzed with an ANOVA (fold changes ≥2 or ≤2, P value <0.05). Hormonal treatments almost doubled (P < 0.03) the number of corpora lutea (39.8 ± 10.2 and 38.3 ± 11.1 in SS-7 and SC sows, respectively) compared with that in the NC group (23.1 ± 3.8). In contrast, embryo viability significantly decreased (P < 0.003) in response to SS-7 treatment (75.1% ± 15.2%) compared to SC and NC groups (93.8 ± 7.6% and 91.8 ± 6.9%, respectively). RNA-Seq analyses revealed 675 and 1,583 DEGs in the SS-7 group compared to both SC and NC groups in endometrial and ovarian samples, respectively. Interestingly, many genes with key roles in the Wnt/ß-catenin and Notch signaling pathways were differentially expressed in SS-7 sows relative to SC and NC groups (e.g., Ctnnb1, Myc, Gli3, Scyl2, Ccny, Daam1, Ppm1n, Rbpj, and Usp8). A key finding in this study was the downregulation of ß-catenin (Ctnnb1) gene expression in the SS-7 endometrium, suggesting that this treatment influences embryo-uterine dialogue by triggering a cascade of events leading to embryo maldevelopment. These data explain the proliferative defects in SS-7 embryos and suggest a novel mechanism of a porcine embryo-maternal crosstalk.

Methods for porcine superovulation (increasing the number of ovulated oocytes per cycle) and estrus synchronization (grouping estrus sows on the same day) are available for assisted reproductive technologies, using hormonal treatments. The main goal of the present study was to understand how hormones used for these purposes influence gene expression patterns in the female reproductive tract (ovaries and endometrium). We observed that hormonal treatments (synchronization combined with superovulation) have the potential to alter ovarian and endometrial gene expression patterns, triggering improper follicle development and oocyte growth, and leading to abnormal embryonic development before implantation. Genes involved in two key metabolic pathways for embryo development (Wnt/ß-catenin and Notch signaling pathways) were dysregulated in reproductive tissues.

The Open Cryotop System Is Effective for the Simultaneous Vitrification of a Large Number of Porcine Embryos at Different Developmental Stages.

The Superfine Open Pulled Straw (SOPS) system is the most commonly used method for vitrification of pig embryos. However, this system only allows the vitrification of four to seven embryos per straw. In this study, we investigated the effectiveness of the open (OC) and closed (CC) Cryotop® systems to simultaneously vitrify a larger number of porcine embryos. Morulae, early blastocysts and full blastocysts were vitrified with the open Cryotop® (n = 250; 20 embryos per device) system, the closed Cryotop® (n = 158; 20 embryos per device) system and the traditional superfine open pulled straw (SOPS; n = 241; 4-7 embryos per straw) method. Fresh embryos from each developmental stage constituted the control group (n = 132). Data expressed as percentages were compared with the Fisher's exact test. The Kruskal-Wallis test was used to analyze the effect of the different vitrification systems on the embryo quality parameters and two-by-two comparisons were accomplished with the Mann-Whitney U test. Differences were considered statistically significant when p < 0.05. Vitrified and control embryos were incubated for 24 h and examined for viability and quality. At the warming step, the embryo recovery rate for the CC system was 51%, while all embryos were recovered when using OC and SOPS. There were no differences between the vitrification and control groups in the postwarming viability of full blastocysts. In contrast, morulae and early blastocysts that were vitrified-warmed with the SOPS system had lower viability (p < 0.01) compared to those from the OC, CC and control groups. The embryonic viability was similar between the OC and control groups, regardless of the developmental stage considered. Moreover, the embryos from the OC group had comparable total cell number and cells from the inner cell mass and apoptotic index than the controls. In conclusion, the OC system is suitable for the simultaneous vitrification of 20 porcine embryos at different developmental stages and provides comparable viability and quality results to fresh embryos subjected to 24 h of in vitro culture.

Exogenous Melatonin in the Culture Medium Does Not Affect the Development of In Vivo-Derived Pig Embryos but Substantially Improves the Quality of In Vitro-Produced Embryos.

Cloned and transgenic pigs are relevant human disease models and serve as potential donors for regenerative medicine and xenotransplantation. These technologies demand oocytes and embryos of good quality. However, the current protocols for in vitro production (IVP) of pig embryos give reduced blastocyst efficiency and embryo quality compared to in vivo controls. This is likely due to culture conditions jeopardizing embryonic homeostasis including the effect of reactive oxygen species (ROS) influence. In this study, the antioxidant melatonin (1 nM) in the maturation medium, fertilization medium, or both media was ineffective in enhancing fertilization or embryonic development parameters of in vitro fertilized oocytes. Supplementation of melatonin in the fertilization medium also had no effect on sperm function. In contrast, the addition of melatonin to the embryo culture medium accelerated the timing of embryonic development and increased the percentages of cleaved embryos and presumed zygotes that developed to the blastocyst stage. Furthermore, it increased the number of inner mass cells and the inner mass cell/total cell number ratio per blastocyst while increasing intracellular glutathione and reducing ROS and DNA damage levels in embryos. Contrarily, the addition of melatonin to the embryo culture medium had no evident effect on in vivo-derived embryos, including the developmental capacity and the quality of in vivo-derived 4-cell embryos or the percentage of genome-edited in vivo-derived zygotes achieving the blastocyst stage. In conclusion, exogenous melatonin in the embryo culture medium enhances the development and quality of in vitro-derived embryos but not in in vivo-derived embryos. Exogenous melatonin is thus recommended during embryo culture of oocytes matured and fertilized in vitro for improving porcine IVP efficiency.

Immunological uterine response to pig embryos before and during implantation.

The establishment of a successful pregnancy can only occur through a concerted functioning of the entire female reproductive system, allowing for fertilization, subsequent embryo development and implantation of the conceptus. In this context, the uterine immunological responses responsible for rejection or tolerance of the conceptus are of critical importance. The aim of the present review is to summarize our current knowledge about those cellular and molecular immunological events occurring at the uterine level during pre-implantation and implantation stages of pregnancy in the pig. Advancing our understanding of the immune mechanisms involved in the success or failure of pregnancy will provide cues to develop novel strategies augmenting endometrial receptivity, finally increasing the efficiency of assisted reproductive technologies in pigs.

Equilibration time with cryoprotectants, but not melatonin supplementation during in vitro maturation, affects viability and metaphase plate morphology of vitrified porcine mature oocytes.

The aims of this study were to investigate the effects of different equilibration times with cryoprotectants on viability and metaphase plate morphology of vitrified-warmed porcine mature oocytes (Experiment 1) and to evaluate the effects of supplementation with 10-9 M melatonin during in vitro maturation on these parameters (Experiment 2). In Experiment 1, 2,392 mature oocytes were vitrified using different equilibration times of oocytes with cryoprotectants (3, 10, 15, 20, 30, 40, 60 and 80 min). Fresh oocytes matured in vitro for 44 hr (n = 509) were used as controls. In Experiment 2, a total of 573 COCs were used. COCs were matured with 10-9 M melatonin supplementation or without melatonin (control). Some oocytes from each group were vitrified with a 60-min equilibration time with cryoprotectants according to the results of Experiment 1. The remaining oocytes from each maturation group were used as fresh control groups. In both experiments, oocytes were stained with 2',7'-dichlorodihydrofuorescein diacetate and Hoechst 33342 to assess viability and metaphase plate morphology, respectively. Vitrification and warming affected (p < .01) oocyte viability compared with controls, which were all viable after 44 hr of IVM. In Experiment 1, the longer the equilibration time with cryoprotectants, the higher the viability. Oocytes equilibrated for 60 and 80 min had the highest (p < .05) viability and similar metaphase plate characteristics to the fresh control oocytes. In Experiment 2, supplementation with melatonin during in vitro maturation had no effect on oocyte viability or metaphase plate morphology of vitrified-warmed oocytes. In conclusion, under our experimental conditions, vitrified porcine mature oocytes equilibrated with cryoprotectants for 60 or 80 min exhibited the highest viability and similar metaphase plate characteristics to fresh controls. Furthermore, supplementation with 10-9 M melatonin during in vitro maturation had no effect on these parameters.

Neither frozen-thawed seminal plasma nor commercial transforming growth factor-ß1 infused intra-utero before insemination improved fertility and prolificacy in sows.

Seminal plasma (SP) affects reproduction, inducing cell and molecular changes in the female genital tract. A main active component in SP is the modulatory transforming growth factor-ß (TGF-ß), particularly its TGF-ß1 isoform, which affects the synthesis of other cytokines as granulocyte-macrophage colony-stimulating factor, relevant for embryo development and pregnancy. This study evaluated the effect of pooled frozen-thawed SP and commercial TGF-ß1 infused during oestrus in sows post-cervically inseminated with liquid extended semen, containing ~4 ml of residual SP, on their fertility and prolificacy. For this, 250 sows in their post-weaning oestrus were used. Sows were randomly assigned to one of the following groups to be post-cervically treated 30 min before insemination: (i) SP group: infused with 40 ml of SP (N = 57); ii) Group TFGß1 : infused with 40 ml of BTS extender containing 3 ng/ml of porcine TGF-ß1 (N = 64); iii) BTS group: infused with 40 ml of BTS extender (N = 60); and iv) Control Group: sows catheterized but not infused prior to AI (N = 69). Farrowing rates (range: 86.7% to 91.3%) and numbers of live-born piglets (range: range: 12.8 ± 2.9 to 13.4 ± 3.1) were not affected by any treatment compared with Controls, indicating that neither pre-infusions of SP nor TGF-ß1 30 min before AI influenced subsequent fertility and prolificacy.

A Short-Term Altrenogest Treatment Post-weaning Followed by Superovulation Reduces Pregnancy Rates and Embryo Production Efficiency in Multiparous Sows.

Although embryo transfer (ET) is a biotechnology ready for the swine industry, there are factors to be solved, the availability of embryo donors as one. Multiparous sows as donors ought to be considered since weaning is a natural and efficient method for estrus synchronization. In addition, superovulation treatments at weaning are effective in increasing the efficiency of donor embryo production. However, ET programs typically require more donors than those available from a single weaning, imposing grouping several weanings to establish a batch for ET. Since short-term administration of Altrenogest is effective in delaying estrus after weaning without effects on ovulation and embryo development, we investigated how Altrenogest combined with superovulation would affect reproductive parameters and embryo quality and quantity of weaned multiparous donor sows. The sows were administered Altrenogest from the day of weaning for 14 (SS-14 group; N = 26), 7 (SS-7 group; N = 31) and 4 (SS-4 group; N = 32) days. The sows were superovulated with eCG 24 h after the last administration of Altrenogest and with hCG at the onset of estrus. Sows not treated with Altrenogest that were superovulated with eCG 24 h post-weaning and hCG at the onset of estrus (SC group; N = 37) and sows with natural estrus after weaning (C group; N = 34) were used as control groups. The percentage of sows showing estrus within 10 days was not affected by the treatment, but the interval from Altrenogest withdrawal to estrus was longer (P < 0.05) in the SS groups than the interval from weaning to estrus in the controls. SS treatments increased (P < 0.05) the percentage of sows with ovarian cysts and the development of polycystic ovaries. The pregnancy and the fertilization rates, and the overall embryo production efficiency were also negatively affected by the SS treatments (P < 0.05). Interestingly, almost 70% of the structures classified as unfertilized oocytes or degenerated embryos in sows from the SS groups were immature oocytes. In conclusion, although superovulation of weaned sows was highly efficient, short-term administration of Altrenogest in combination with superovulation had negative effects on most of the reproductive parameters assessed, particularly affecting the overall efficiency of pregnancy and embryo production.

Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae.

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70-75%), the pregnancy loss is 5-15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

Post-Thaw Sperm Quality and Functionality in the Autochthonous Pig Breed Gochu Asturcelta.

Genetic resource banks (GRB) preserve the genetic material of endangered, valuable individuals or genetically relevant breeds. Semen cryopreservation is a crucial technique to reach these goals. Thus, we aimed to assess the sperm parameters of semen doses from the native pig breed Gochu Asturcelta stored at the GRB of Principado de Asturias (GRB-PA, Gijón, Spain), focusing on intrinsic and extrinsic (boar, season) factors. Two straws per boar (n = 18, 8-71 months of age) were thawed, pooled, and assessed after 30 and 150 min at 37 °C by CASA (computer-assisted sperm analysis system; motility and kinematic parameters) and flow cytometry (viability, acrosomal status, mitochondrial activity, apoptosis, reactive oxygen species, and chromatin status). The effects of age, incubation, and season on post-thawing quality were determined using linear mixed-effects models. Parameters were on the range for commercial boar breeds, with chromatin status (SCSA: fragmentation and immaturity) being excellent. Incubation decreased sperm quality and functionality. The boar age did not have a significant effect (p > 0.05), but the between-boar variability was significant (p < 0.001). The season significantly affected many parameters (motility, kinematics, viability, acrosomal status, mitochondrial activity), especially after 150 min of incubation. In general, samples collected in spring and summer showed higher quality post-thawing, the lowest in winter. In conclusion, the sperm doses from the Gochu Asturcelta breed stored at the GRB-PA showed excellent chromatin status and acceptable characteristics after thawing. Therefore, boar and seasonal variability in this autochthonous breed could be relevant for cryobank management.

Intrauterine Infusion of TGF-ß1 Prior to Insemination, Alike Seminal Plasma, Influences Endometrial Cytokine Responses but Does Not Impact the Timing of the Progression of Pre-Implantation Pig Embryo Development.

Seminal plasma (SP) in the female genital tract induces changes that affect multiple reproductive processes. One of the active components in SP is the transforming growth factor ß1 (TGF-ß1), which has major roles in embryo development and pregnancy. Embryo transfer (ET) technology is welcomed by the pig industry provided that embryo quality at embryo collection as well as the fertility and prolificacy of the recipients after the ET is increased. This study evaluated different intrauterine infusion treatments at estrus (40 mL of SP, TGF-ß1 cytokine in the extender, or the extender alone (control)) by mimicking an ET scenario in so-called "donor" (inseminated) and "recipient" (uninseminated) sows. On day 6 (day 0-onset of estrus), all "donors" were laparotomized to determine their pregnancy status (presence and developmental stage of the embryos). In addition, endometrial explants were collected from pregnant "donors" and cyclic "recipients," incubated for 24 h, and analyzed for cytokine production. SP infusions (unlike TGF-ß1 infusions) positively influenced the developmental stage of day 6 embryos. Infusion treatments differentially influenced the endometrial cytokine production, mainly in donors. We concluded that SP infusions prior to AI not only impacted the porcine preimplantation embryo development but also influenced the endometrial cytokine production six days after treatment, both in donors and recipients.

Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model.

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGß, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

Allogeneic Embryos Disregulate Leukemia Inhibitory Factor (LIF) and Its Receptor in the Porcine Endometrium During Implantation.

Despite its advantages for pig breeding, embryo transfer (ET) has a major handicap: high embryo mortality during the pre- and implantation period, probably caused by divergent phenomena of tolerance between the immunologically unrelated (i.e., allogeneic) embryos and the recipient sow. Thus, to reach a similar maternal tolerance as in conventional breeding by artificial insemination (AI) would be the key to ET-success. For this reason, we studied the expression of the leukemia inhibitory factor (LIF) cytokine and its receptor in the pig endometrium during the implantation period (days 18 and 24) in sows subjected to ET (AL group) vs. post-cervical-AI controls (Hemi-AL group). Quantification of expression was performed at both mRNA (rt-qPCR) and protein (WB) levels. The expression of endometrial LIF on day 24 was considerably lower in ET than in AI pregnancies. Correlations between endometrial mRNA levels of LIF and LIF-R showed that, contrary to early AI-pregnancies, ET-pregnancies lack an inverse relation between cytokine and receptor levels. In conclusion, ET-pregnancies lack sufficient endometrial levels of LIF to develop adequate immunotolerance mechanisms to prevent the rejection of allogeneic ET-embryos.