The effect of maternal HMB supplementation on bone mechanical and geometrical properties, as well as histomorphometry and immunolocalization of VEGF, TIMP2, MMP13, BMP2 in the bone and cartilage tissue of the humerus of their newborn piglets.

The presented experiment focuses on assessing the impact of HMB (hydroxy-ß-methobutyrate) supplementation of mothers during pregnancy on the development of the skeletal system of their offspring. For this purpose, an experiment was carried out on 12 clinically healthy sows of the Great White Poland breed, which were divided randomly into two groups the control and the HMB group. All animals were kept under standard conditions and received the same feed for pregnant females. In contrast, females from the HMB group between 70 and 90 days were supplemented with 3-hydroxy-3-methylbutyle in the amount of 0.2g/kg b.w/day. Immediately after birth, the piglets were also divided into groups based on: sex, and presence or lack HMB supplementation, and subsequently were euthanized and humerus bones from all piglets were collected. Mother's HMB supplementation during pregnancy affected the multiple index of their offspring. The higher humerus mass and length was observed with the greater effect in males. Maternal supplementation also influenced on the geometrical and mechanical properties of the humerus as in the case of mass, this effect was higher in males. Also, the collagen structure of the compacted and trabecular bone changed under the HMB addition. Maternal supplementation also affected the expression of selected proteins in growth cartilage and trabecular bone. The obtained results show that the administration to the mother during pregnancy by the HMB significantly affects the development of the humerus in many ways. The obtained results also confirm the utility of such experiments in understanding of the importance of the pregnancy diet as an develop and adaptable factor of offspring organisms and are the base for further research in that area as well as in the protein markers expression area.

Maternal supplementation of organic selenium during gestation improves sows and offspring antioxidant capacity and inflammatory status and promotes embryo survival.

Selenium (Se) is an essential trace element in humans and sows, having a biological function mediated in part by its incorporation into selenoproteins. This study was conducted to investigate the effects of maternal 2-hydroxy-4-methylselenobutanoic acid (HMSeBA), an organic Se source, on reproductive performance, antioxidant capacity and inflammatory status of sows and their offspring. Forty-three Landrace × Yorkshire sows were randomly allocated to receive one of the following three diets during gestation: control diet (control, basal diet, n = 15), sodium selenite (Na2SeO3) supplemented diet (Na2SeO3, basal diet + Na2SeO3 at 0.3 mg Se per kg, n = 13), and HMSeBA supplemented diet (HMSeBA, basal diet + HMSeBA at 0.3 mg Se per kg, n = 15). Blood samples of sows and piglets, placentas and piglet liver samples were analyzed for selenium status, antioxidant capacity and inflammatory cytokines. Results showed that, as compared to the control group, HMSeBA supplementation increased the number of born alive piglets and plasma concentrations of total selenium and selenoprotein P in both sows and piglets. Besides, the activities of antioxidant enzymes in the blood of sows, umbilical cord and piglets, placentas and piglets' liver were increased by dietary HMSeBA supplementation as compared to the control group, while malondialdehyde concentration (p < 0.05) was decreased in the blood of sows, umbilical cord and newborn piglets. In addition, maternal HMSeBA intake during gestation up-regulated antioxidant-related selenoprotein gene expression in the placenta (GPx2, GPx3, p < 0.05) and in the liver of newborn piglets (GPx1, GPx2, GPx3, TXNRD2, p < 0.05). Moreover, as compared to the control group, sows and newborn piglets in the Na2SeO3 and HMSeBA groups had a lower serum interleukin-6 (p < 0.05) concentration, and placentas in the HMSeBA group had lower IL-1ß, IL-6 and IL-8 gene expression (p < 0.05). In conclusion, maternal supplementation of HMSeBA during pregnancy improved antioxidant capacities and reduced the inflammation level in mater, placenta, and fetus. This finding may highlight the important role of selenoproteins (especially GPXs) in preventing negative consequences of over-production of free radicals and inflammatory cytokines during gestation and at births.

Fatty acid induced lipolysis influences embryo development, gene expression and lipid droplet formation in the porcine cumulus cells†.

The pig oocyte maturation protocol differs from other mammalian species due to dependence on follicular fluid (FF) supplementation. One of the most abundant components of the porcine follicular fluid are fatty acids (FAs). Although evidence from other mammalian models revealed a negative impact of saturated fatty acids (SFA) on developmental competence of oocytes, pig has not yet been widely analyzed. Therefore, we aimed to investigate whether supplementation of IVM medium with 150 µM of stearic acid (SA) and oleic acid (OA) affects lipid content and expression of genes related to fatty acid metabolism in porcine cumulus-oocyte complexes and parthenogenetic embryo development. We found significant influence of fatty acids on lipid metabolism in cumulus cells without affecting the oocyte proper. The expression of ACACA, SCD, PLIN2, FADS1, and FADS2 genes was upregulated (P < 0.01) in cumulus cells, while their expression in oocytes did not change. The increase in gene expression was more pronounced in the case of OA (e.g., up to 30-fold increase in PLIN2 transcript level compared to the control). The number of lipid droplets and occupied area increased significantly in the cumulus cells and did not change in oocytes after SA treatment. Oleic acid improved the blastocyst rate (48 vs 32% in control), whereas stearic acid did not affect this parameter (27%). Additionally, we have discovered a phenotypic diversity of LD in cumulus cells in response to FA supplementation, suggesting extensive lipolysis in response to SA. Stearic acid excess in maturation media led to the formation of multiple micro lipid droplets in cumulus cells.

Inhibitory Activity of Scutellaria baicalensis Flavonoids against Tick-Borne Encephalitis Virus.

We studied virus-inhibiting activity of Baikal skullcap (Scutellaria baicalensis) flavonoids against tick-borne encephalitis virus using various model schemes. The half-maximum cytotoxic concentration (CC50) for the plant extract was found (363.9±58.6 µg/ml). Based on the CC50 and IC50, selective index (SI) was calculated for viricidal (53.4), preventive (50.5), and direct antiviral actions (39.1) and for-intracellular replication of the virus (40.4). Suppression of virus reproduction ≥2.0 lg TCID50 was observed at extract concentration ≥5 µg/ml (viricidal effect), ≥11.2 µg/ml (preventive and direct antiviral effects), and ≥9 µg/ml (intracellular replication). Flavonoids of Baikal skullcap extract produced an in vitro inhibitory effect on tick-borne encephalitis virus due to their direct viricidal activity and direct inhibition of adsorption and intracellular replication of tick-borne encephalitis virus, which determines their value as highly effective antiviral drugs.

Melatonin alleviates defects induced by zearalenone during porcine embryo development.

Zearalenone (ZEA), which is produced by several fusarium mycotoxins, is found in animal feed and food products, and can exert estrogen-like activity. Melatonin (MT) is emerging as a supplement that can fight the toxic effects of mycotoxins. With a variety of physiological functions that play crucial roles in the development of animal germ cells and embryos, melatonin regulates circadian rhythms and has an anti-inflammatory and anti-oxidative role. This study investigated the protective effects of melatonin against ZEA in porcine early embryonic development. Our results showed that ZEA adversely affected this development, while melatonin supplementation ameliorated the toxic effects. ZEA exposure increased oxidative stress and impaired mitochondrial function, which may affect blastocyst formation. Moreover, we found that ZEA exposure promotes apoptosis, DNA damage, and autophagy in porcine blastocysts. The toxic effects of ZEA on early embryos may be the result of oxidative stress-mediated early apoptosis, while melatonin treatment significantly improved these phenotypes in ZEA-exposed porcine early embryos. Taken together, our results indicate that melatonin has a protective effect on defects caused by ZEA during early porcine embryonic development.

Asiatic acid supplementation during the in vitro culture period improves early embryonic development of porcine embryos produced by parthenogenetic activation, somatic cell nuclear transfer and in vitro fertilization.

Asiatic acid is a pentacyclic triterpene enriched in the medicinal herb Centella asiatica, and it has been suggested to possess free radical scavenging and anti-apoptotic properties. The purpose of the current study was to explore the effects of asiatic acid on porcine early-stage embryonic development and the potential mechanisms for any observed effects. The results showed that 10 µM asiatic acid supplementation during the in vitro culture period dramatically improved developmental competence in porcine embryos derived from parthenogenetic activation (PA), somatic cell nuclear transfer (SCNT) and in vitro fertilization (IVF). Further analysis revealed that asiatic acid attenuated H2O2-induced intracellular reactive oxygen species (ROS) generation. Notably, asiatic acid not only enhanced intracellular GSH levels but also attenuated mitochondrial dysfunction. Gene expression analysis revealed that asiatic acid upregulated expression of the antioxidant-related gene Sod-1 and the blastocyst formation related gene Cox-2, while downregulating expression of the apoptosis-related gene Caspase-9 in SCNT blastocysts. These results suggest that asiatic acid exerts beneficial effects on early embryonic development in porcine embryos and that asiatic acid may be useful for improving the in vitro production of porcine embryos.

Review: Mammary gland development in swine: embryo to early lactation.

Milk production by the sow is a major factor limiting the growth and survival of her litter. Understanding the process of morphogenesis of the sow's mammary gland and the factors that regulate mammary development are important for designing successful management tools that may enhance milk production. Primordia of the mammary glands are first observable in the porcine embryo at approximately 23 days of gestation. The glands then progress through a series of morphologically distinct developmental stages such that, at birth, each mammary gland is composed of the teat, an organized fat pad and two separate lactiferous ducts each with a few ducts branching into the fat pad. The glands continue to grow slowly until about 90 days of age when the rate of growth increases significantly. The increased rate of mammary gland growth coincides with the appearance of large ovarian follicles and an increase in circulating estrogen. After puberty, the continued growth of the gland and elongation and branching of the duct system into the fat pad takes place in response to the elevated levels of estrogen occurring as part of the estrous cycles. After conception, parenchymal mass of each gland increases slowly during early pregnancy and then grows increasingly rapidly during the final trimester. This growth is in response to estrogen, progesterone, prolactin and relaxin. Lobuloalveolar development occurs primarily during late pregnancy. By parturition, the fat pad of the mammary gland has been replaced by colostrum-secreting epithelial cells that line the lumen of the alveoli, lobules and small ducts. All mammary glands develop during pregnancy, however, the extent of development is dependent on the location of the mammary gland on the sow's underline. The mammary glands undergo significant functional differentiation immediately before and after farrowing with the formation of colostrum and the transition through the stages of lactogenesis. Further growth of the glands during lactation is stimulated by milk removal. Individual glands may grow or transiently regress in response to the intensity of suckling during the initial days postpartum. Attempts to enhance milk production by manipulation of mammary development at stages before lactation generally have met with limited success. A more in depth understanding of the processes regulating porcine mammary gland morphogenesis at all stages of development is needed to make further progress.

Effects of polyunsaturated fatty acids on the development of pig oocytes in vitro following parthenogenetic activation and on the lipid content of oocytes and embryos.

As oocytes and embryos of pigs have greater lipid content in the cytoplasm than those of other species, supplementation of the medium for in vitro maturation (IVM) of oocytes with omega-3 polyunsaturated fatty acids (PUFA) may help to improve embryo development. This study was conducted to evaluate effects of the inclusion of the docosaexaenoic (DHA) and of the eicosapentaenoic acids (EPA) in the IVM medium on the development of pig oocytes and on the lipid content of oocytes and embryos. In all experiments, control media consisted of porcine follicular fluid and oocytes were activated through parthenogenesis. In Experiment 1, there were four treatments for each PUFA: one control; and three treatments including EPA or DHA in the IVM medium at 12.5 µM, 25.0 µM and 50.0 µM). In Experiment 2, inclusion of 50 µM DHA was compared against the control. Cleavage rates in the IVM medium including 12.5 µM EPA and blastocyst development rates in media at any EPA concentration were less than for the control in Experiment 1 (P < 0.05). Compared to the control, inclusion of 50 µM DHA in the IVM medium was related to greater cleavage rates and greater number of embryo cells, in Experiment 1, and lesser lipid content in oocytes after 22 and 44 h and in embryos after 7 days, in Experiment 2 (both P < 0.05). Addition of DHA in the IVM medium may benefit the development of pig oocytes, but EPA appears to be cytotoxic.

A polyphenol-rich extract from an oenological oak-derived tannin influences in vitro maturation of porcine oocytes.

Tannins have been demonstrated to have antioxidant and various health benefit properties. The aim of this study was to determine the effect of an ethanol extract (TRE) of a commercial oenological tannin (Quercus robur toasted oak wood, Tan'Activ R®) on female gamete using an in vitro model of pig oocyte maturation (IVM) and examining nuclear maturation, cytoplasmic maturation, intracellular GSH and ROS levels and cumulus cell steroidogenesis. To this aim, during IVM performed in medium either supplemented (IVM A) or not supplemented (IVM B) with cysteine and ß-mercaptoethanol, TRE was added at different concentrations (0, 1, 5, 10, 20 µg/ml). The addition of TRE at all the concentration tested to either IVM A or IVM B, did not influence oocyte nuclear maturation. When IVM was performed in IVM A, no effect was induced on cytoplasmic maturation by TRE at the concentration of 1, 5 and 10 µg/ml, while TRE 20 µg/ml significantly reduced the penetration rate after IVF (p < 0.05) and the blastocyst rate after parthenogenetic activation (p < 0.01). Oocyte maturation in IVM B, compared to IVM A group, decreased GSH (p < 0.001) and increased ROS (p < 0.01) intracellular levels and in turn impaired oocyte cytoplasmic maturation reducing the ability to sustain male pronuclear formation after IVM (p < 0.001) and the developmental competence after parthenogenetic activation (p < 0.001). TRE supplementation to IVM B significantly reduced ROS production (5, 10, 20 µg/ml TRE) to levels similar to IVM A group, and increased GSH levels (10, 20 µg/ml TRE) compared to IVM B (p < 0.05) without reaching those of IVM A group. TRE supplementation to IVM B at the concentrations of 1, 5 and 10 µg/ml significantly improved (p < 0.001) oocyte cytoplasmic maturation enhancing the ability to sustain male pronuclear formation without reaching, however, IVM A group levels. TRE addition at all the concentration tested to both IVM A and IVM B, did not induce any effect on E2 and P4 secretion by cumulus cells suggesting that the biological effect of the ethanol extract is not exerted thought a modulation of cumulus cell steroidogenesis. In conclusion, TRE, thanks to its antioxidant activity, was partially able to reduce the negative effect of the absence of cysteine and ß-mercaptoethanol in IVM B, while TRE at high concentration in IVM A was detrimental for oocyte cytoplasmic maturation underlying the importance of maintaining a balanced redox environment during oocyte maturation.

Leucine Promotes the Growth of Fetal Pigs by Increasing Protein Synthesis through the mTOR Signaling Pathway in Longissimus Dorsi Muscle at Late Gestation.

Leucine (Leu) plays an important role in protein synthesis and metabolism. The present study tested whether Leu supplementation in the diet for sows during late pregnancy could improve piglet birth weight, and it also investigated the possible underlying mechanism. Two hundred sows at day 70 of pregnancy were selected and assigned to four groups fed with following four diets until farrowing, respectively: corn and soybean meal-based diet group (CON), CON + 0.40% Leu, CON + 0.80% Leu, and CON + 1.20% Leu. We found that supplementing with 0.80% Leu significantly increased mean piglet birth weight ( P < 0.05). Supplementation with 0.40, 0.80, and 1.20% Leu increased the plasma concentration of Leu, while decreasing the plasma concentrations of valine (Val) and isoleucine (Ile) in both farrowing sows and newborn piglets ( P < 0.05). The protein expressions of amino acid transporters (including LAT1, SNAT1, SNAT2, 4F2hc, and rBAT) in duodenum, jejunum, ileum, longissimus dorsi muscle of newborn piglets, and placenta of sows showed a difference among the CON group and Leu supplemented groups. Expressions of p-mTOR, p-4E-BP1, and p-S6K1 in longissimus dorsi muscle were also enhanced in each of the supplemental Leu groups compared to CON ( P < 0.05). Collectively, these results indicated that 0.40-0.80% Leu supplementation during late gestation enhanced birth weight of fetal pigs by increasing protein synthesis through modulation of the plasma amino acids profile, amino acid transporters expression, and mTOR signaling pathway.

Exogenous ascorbic acid enhances vitrification survival of porcine in vitro-developed blastocysts but fails to improve the in vitro embryo production outcomes.

In this study, the effects of addition of the antioxidant ascorbic acid (AsA) were evaluated during porcine in vitro embryo production (IVP) and vitrification. In experiment 1, the effects of AsA supplementation during IVM, IVF and IVC were evaluated, using a total of 2744 oocytes in six replicates. The IVM, IVF and embryo IVC media were supplemented or not (control) with 50 µg/mL AsA in all possible combinations. No significant effects of AsA were detected in any of the maturation, fertilization or embryo development parameters assessed. In experiment 2, we evaluated the effects of adding AsA to vitrification-warming media on the post-warming survival and quality of blastocysts. Day-6 in vitro-produced blastocysts (N = 588) from six replicates were randomly divided in two groups, with vitrification and warming media either supplemented with 50 µg/mL AsA (VW + group) or un-supplemented (VW- control). Addition of AsA increased (P < 0.05) blastocyst survival rate after vitrification compared with that of VW- control embryos. Vitrification and warming increased (P < 0.05) the production of oxygen species (ROS) and reduced (P < 0.05) the glutathione levels in blastocysts. Although VW + blastocysts displayed higher (P < 0.05) ROS levels than those of fresh control blastocysts, the levels were lower (P < 0.05) than those found in VW- control blastocysts. In conclusion, under the experimental conditions, supplementation of IVM/IVF/IVC media with AsA did not improve the embryo production in vitro. By contrast, the addition of AsA to chemically defined vitrification and warming media increased the survival of in vitro-produced porcine blastocysts by decreasing ROS production.

Successful vitrification of pronuclear-stage pig embryos with a novel cryoprotective agent, carboxylated ε-poly-L-lysine.

Vitrification is a powerful tool for the efficient production of offspring derived from cryopreserved oocytes or embryos in mammalian species including domestic animals. Genome editing technologies such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas)9 are now available even for domestic species, suggesting that the vitrification of embryos at the pronuclear stage (PN) will be more important because they could provide genomic host cells to be targeted by TALENs or CRISPR/Cas9. Although we reported the successful production of piglets derived from vitrified PN embryos by a solid-surface vitrification method with glutathione supplementation, further improvements are required. The cryoprotective agent (CPA) carboxylated ε-poly-L-lysine (COOH-PLL) was introduced in 2009. COOH-PLL reduces the physical and physiological damage caused by cryopreservation in mammalian stem cells and the vitrification of mouse oocytes and embryos. Those results suggested that vitrification of COOH-PLL may help improve the developmental ability of pig embryos vitrified at the PN stage. However, it remains unclear whether COOH-PLL is available as a CPA for the vitrification of embryos in domestic species. In this study, we evaluated COOH-PLL as a CPA with ethylene glycol (EG) and Cryotop as a device for the vitrification of PN pig embryos. Exposure to vitrification solution supplemented with COOH-PLL up to 30% did not decrease developmental ability to the 2-cell stage and the blastocyst stage. After warming, most of the vitrified embryos survived regardless of the concentration of COOH-PLL (76.0 ± 11.8% to 91.8 ± 4.6%). However, the vitrified embryos without COOH-PLL showed a lower development rate up to the blastocyst stage (1.3 ± 1.0%) compared to the fresh embryos (28.4 ± 5.0%) (p<0.05). In contrast, supplementation of 20% (w/v) COOH-PLL in the vitrification solution dramatically improved the developmental ability to blastocysts of the vitrified embryos (19.4 ± 4.6%) compared to those without COOH-PLL (p<0.05). After the transfer of embryos vitrified with 30% (v/v) EG and 20% (w/v) COOH-PLL, we successfully obtained 15 piglets from 8 recipients. Taken together, our present findings demonstrate for the first time that COOH-PLL is an effective CPA for embryo vitrification in the pig. COOH-PLL is a promising CPA for further improvements in the vitrification of oocytes and embryos in mammalian species.

Pyridoxine (Vitamin B6) and the Glutathione Peroxidase System; a Link between One-Carbon Metabolism and Antioxidation.

Vitamin B6 (B6) has a central role in the metabolism of amino acids, which includes important interactions with endogenous redox reactions through its effects on the glutathione peroxidase (GPX) system. In fact, B6-dependent enzymes catalyse most reactions of the transsulfuration pathway, driving homocysteine to cysteine and further into GPX proteins. Considering that mammals metabolize sulfur- and seleno-amino acids similarly, B6 plays an important role in the fate of sulfur-homocysteine and its seleno counterpart between transsulfuration and one-carbon metabolism, especially under oxidative stress conditions. This is particularly important in reproduction because ovarian metabolism may generate an excess of reactive oxygen species (ROS) during the peri-estrus period, which may impair ovulatory functions and early embryo development. Later in gestation, placentation raises embryo oxygen tension and may induce a higher expression of ROS markers and eventually embryo losses. Interestingly, the metabolic accumulation of ROS up-regulates the flow of one-carbon units to transsulfuration and down-regulates remethylation. However, in embryos, the transsulfuration pathway is not functional, making the understanding of the interplay between these two pathways particularly crucial. In this review, the importance of the maternal metabolic status of B6 for the flow of one-carbon units towards both maternal and embryonic GPX systems is discussed. Additionally, B6 effects on GPX activity and gene expression in dams, as well as embryo development, are presented in a pig model under different oxidative stress conditions.

The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture.

The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0% and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos.

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA-fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7-5.4%) of DNA-fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.

Effect of zinc on in vitro development of porcine embryos.

This study aimed to investigate the effect of zinc on in vitro development of porcine embryos. We evaluated the effects of zinc on blastocysts formation and investigated gene expression at zinc-deficient and supplemented conditions. Zinc-deficient in vitro condition was induced by 10-µM N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylendiamine (TPEN) (zinc chelator) treatment during IVC. On parthenogenetic activated embryos, this treatment significantly decreased cleavage rate and blastocyst formation compared with the control (0.0% and 0.0% vs. 69.0% and 36.0%, respectively). And time effect of the zinc deficiency exposure is observed. Blastocyst formation rate was significantly decreased as zinc-deficient time increases (54.1%, 31.0%, 9.0%, and 1.2% for zinc deficiency during 0, 3, 5, and 7 hours). However, zinc supplementation during IVC supported in vitro embryonic development. On parthenogenetic activated embryos, supplementation of 0.8 µg/mL of zinc during IVC significantly increased blastocyst formation compared with other groups (43.9%, 57.8%, 67.1%, 51.4%, and 52.6% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 µg/mL). In vitro-fertilized (IVF) embryos showed similar results. The blastocyst formation rate was significantly higher in the 0.8 µg/mL of zinc-supplemented group than in the other groups (21.3%, 24.1%, 36.1%, 25.9%, and 25.2% for zinc supplementation of 0, 0.4, 0.8, 1.2, and 1.6 µg/mL). PCNA, POU5F1, and Bcl2 messenger RNA expressions were unregulated in IVF-derived blastocysts in the 0.8 µg/mL of zinc-supplemented group compared with the control. These results suggest that zinc is required for embryonic development, and supplementation with adequate zinc concentrations during IVC improved the viability of porcine embryos, possibly by increasing PCNA, POU5F1, and Bcl2 gene expression of embryos.

Supplementing culture and vitrification-warming media with l-ascorbic acid enhances survival rates and redox status of IVP porcine blastocysts via induction of GPX1 and SOD1 expression.

The present study sought to determine the effect of adding l-ascorbic acid (AC) to (1) in vitro culture medium and (2) vitrification and warming solutions on redox status and developmental ability and quality of IVP porcine embryos. In both experiments, embryo quality was analysed in terms of total cell number (TCN), DNA fragmentation, intracellular peroxide levels and expression of three oxidative stress-related genes: glutathione peroxidase 1 (GPX1), superoxide dismutase 1 (SOD1) and 2 (SOD2). In the first experiment, fresh blastocysts were found to upregulate SOD1 expression when cultured with medium supplemented 100 µM AC. No differences were found between culture groups in the other analysed parameters. In the second experiment, blastocysts cultured with or without AC were divided into two groups: vitrified and warmed with solutions containing 0 or 100 µM AC. Addition of AC during culture and vitrification-warming upregulated the expression of GPX1 and SOD1 genes, enhanced survival rates and decreased peroxide levels at 24h post-warming. In addition, peroxide levels were negatively correlated with relative GPX1- and SOD1-transcript abundances, whereas GPX1 was positively correlated with embryo survival at 24h post-warming. No effects of AC-supplementation were seen for TCN, DNA fragmentation or relative SOD2-transcript abundance in vitrified blastocysts. In conclusion, the addition of AC to culture and vitrification-warming media increases gene expression of antioxidant enzymes SOD1 and GPX1. This appears to improve redox balance and is suggested to ultimately enhance embryo cryosurvival.

Glutathione and cysteine enhance porcine preimplantation embryo development in vitro after intracytoplasmic sperm injection.

Because intracytoplasmic sperm injection (ICSI) had been introduced to animal science, not only reproductive biology of domestic animals, but also medicine to treat infertility has been developed. This assisted reproductive technology is beneficial for generating transgenic animals, especially pigs, because polyspermy is the greatest hurdle in porcine IVF when researchers make highly qualified preimplantation embryos. However, ICSI-derived embryos expressed high level of reactive oxygen species (ROS), which are known to cause serious dysfunction during preimplantation development. The objective of this study was to investigate the developmental competence, ROS level, and apoptosis index when glutathione (GSH) or cysteine was supplemented into the in vitro culture medium for ICSI-derived porcine embryos. First, we evaluated the effect of different concentrations of GSH or cysteine on developmental ability of porcine ICSI-derived embryos. The cleavage rate (79.6%) and the blastocyst formation rate (20.9%) were significantly improved in culture medium supplemented with 1 mmol/L GSH compared with other concentrations or no supplementation. Also, 1.71 mmol/L cysteine showed a significantly higher proportion of cleavage (80.7%) and blastocyst formation (22.5%) than other cysteine-supplemented groups. Next, we confirmed that intracellular ROS level was significantly reduced in the group of blastocysts cultured with GSH or cysteine after ICSI compared with the no supplementation group. Finally, we found that terminal uridine nick-end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased in blastocysts when ICSI-derived embryos were cultured with supplementation of 1.71 mmol/L cysteine or 1 mmol/L GSH. Taken together, these results strongly indicate that GSH or cysteine can improve the developmental competence of porcine ICSI-derived embryos by reducing intracellular ROS level and the apoptosis index.

Improved developmental ability of porcine oocytes grown in nude mice after fusion with cytoplasmic fragments prepared by centrifugation: a model for utilization of primordial oocytes.

Primordial oocytes are a potential resource for medical and zoological application, but those of large animals have not yet been reported to show efficient embryonic development. In the present study, we established a pig model for production of blastocysts from primordial oocytes that had been grafted into nude mice and matured in vitro, in combination with fusion of cytoplasmic fragments. Neonatal porcine ovaries in which most follicles are at the primordial stage were minced and grafted into nude mice (Crlj:CD1-Foxn1(nu)). About 60 days after detection of vaginal opening, the mice were given 62.5 U/mL porcine FSH for 2 weeks by infusion to enhance follicular development. Developmentally competent oocytes collected from porcine ovaries (conventional oocytes) were matured in vitro and subjected to serial centrifugation to prepare cytoplasmic fragments without a metaphase plate (cytoplasts). Three cytoplasts were fused by electrostimulation to an oocyte retrieved from a host mouse (xenogeneic oocyte) and matured in vitro. Then these fused oocytes were fertilized and subsequently cultured in vitro. No blastocysts were generated from xenogeneic oocytes without fusion of cytoplasm. When xenogeneic oocytes had been fused with three cytoplasts, the blastocyst rate increased significantly to 14.3%, comparable to that for untreated conventional oocytes (20.0%). The numbers of cells in blastocysts for these fused oocytes (37.2 cells/blastocyst) were not significantly different from those for conventional oocytes (25.4 cells/blastocyst). Our findings show that it is possible to use primordial oocytes of large mammals in combination with xenografting of ovarian tissue and also ooplasmic fusion.

Leptin and nonessential amino acids enhance porcine preimplantation embryo development in vitro by intracytoplasmic sperm injection.

Intracytoplasmic sperm injection (ICSI) has been considered one of the strong assisted reproductive technologies for producing transgenic animals as well as treating infertility in animals and humans. However, in porcine ICSI, embryos produced by in vitro methods show low pregnancy rates with high abnormal offspring and blastocyst formation rate as well as quality are poor compared with those in other species. For these reasons, developing a protocol for porcine ICSI is essential to efficiently generate transgenic pigs. Since amino acids were introduced to embryo development because of their beneficial effects, many embryologists have been using nonessential amino acid (NEAA) in culture medium for embryonic development in pig and other species. Leptin also has been shown to be beneficial in embryonic development for increasing rate of cleavage and blastocyst development. However, the effects of NEAA and leptin were not fully understood in the development of porcine ICSI-derived embryos. Here we investigated the optimization of NEAA and leptin supplementation in culture medium to improve developmental competence and quality of preimplantation embryos after ICSI in pig. The proportion of embryos that developed to the blastocyst stage was significantly greater when 1% vol/vol NEAA (24.6%) or 100 ng/mL leptin (27.1%) was supplemented in the culture medium compared with other concentrations or no supplement. When NEAA and leptin (24.8%) were supplemented together, blastocyst formation was significantly higher than other single supplementation groups. We also evaluated the effects of different supplementation periods of NEAA or leptin on the preimplantation embryonic development after ICSI. Both NEAA and leptin showed that supplementation for the entire 7 days significantly increased the blastocyst formation rate compared with the other groups of supplementation for the first 4 days and for the subsequent 3 days. A second goal of our research was to evaluate the quality of developed blastocysts after ICSI. The supplementation of 100 ng/mL leptin in culture medium made blastocysts express less of the proapoptosis genes BAX and BAK and more of the antiapoptosis genes BCL-XL and BCL-2 after the ICSI procedure. Furthermore, terminal deoxynucleotidyl transferase dUTP nick end labeling index, fragmentation, and total apoptosis were significantly decreased and the total cell number was significantly increased when the ICSI-derived embryos were cultured to blastocyst stage in the presence of the combination of NEAA and leptin. These results suggest that NEAA and leptin could improve not only the quantity but also quality of ICSI-derived porcine embryos during in vitro culture with the optimal concentration of each reagent.