Expression of Perilipin 2 (PLIN2) in Porcine Oocytes During Maturation

Perilipins have been reported to limit the interaction of lipases with neutral lipids within the droplets, thereby regulating neutral lipid accumulation and utilization. This study aimed to identify the location and expression of PLIN1 and PLIN2 in porcine oocytes during maturation. Quantitative real‐time polymerase chain reaction (qRT‐PCR), immunostaining and Western blot methods were used to characterize the expression and distribution patterns of PLIN1 and PLIN2 in porcine oocytes. The results showed that PLIN1 was not detectable in porcine oocytes. PLIN2 and BODIPY 493/503‐detected neutral lipid droplets appeared identical distribution patterns and extensive colocalization in both GV and MII porcine oocytes. PLIN2 protein expression was higher in GV oocytes than that in MII oocytes (p < 0.05), although PLIN2 mRNA expression was similar in both groups. These findings suggested that PLIN2 was a major lipid droplet‐associated protein in porcine oocytes.     

Fertilization and blastocyst development in oocytes obtained from prepubertal and adult pigs

Polyspermic fertilization and embryo quality are important issues for the in vitro production of pig embryos. We hypothesized that oocyte donor (prepubertal gilt vs. sow) affects polyspermy and blastocyst development in vitro and that the sexual maturity of the oocyte donor affects the response to sperm concentration in the fertilization medium. In Exp. 1, oocytes of sows and gilts were mounted and stained 12 h after insemination to provide fertilization data. In Exp. 2, putative embryos were developed in vitro to 144 h post-insemination before mounting. In both experiments, cumulus-oocyte complexes (COC) were collected from ovaries of prepubertal gilts and adult sows. Sperm were added after maturation of COC for 40 to 44 h. Sperm from two boars at 0.5 to 5.0 x 10(6) sperm/mL was used for insemination. More (P < 0.01) monospermic fertilizations were observed in oocytes derived from gilts than for oocytes from sows. There were fewer (P < 0.02) penetrated sperm per fertilized oocyte in oocytes from gilts compared with sows. There were effects of semen donor (boar) on the percentage of monospermic (P < 0.01) and polyspermic (P < 0.002) fertilizations, and on the number of penetrated sperm/fertilized oocyte (P < 0.02). In Exp. 2, cleavage and blastocyst formation was evaluated at 2 and 6 d postinsemination, respectively. More (P < 0.001) blastocysts developed from sow-derived oocytes than from gilt-derived oocytes. More (P < 0.05) total cells per blastocyst were observed in embryos from sow-derived oocytes than from gilt-derived oocytes. Semen donor affected the percentage of oocytes cleaving (P < 0.02), and a boar x sperm concentration interaction affected (P < 0.05) the incidence of blastocyt formation. Results indicate that sexual maturity of the donor is not responsible for the high incidence of polyspermy in porcine in vitro fertilization. However, blastocyst development is improved by the use of oocytes from sows rather than from prepubertal gilts.     

Effects of melatonin on oocyte developmental competence and the role of melatonin receptor 1 in juvenile goats

Melatonin enhances in vitro embryo development in several species by improving the oocyte developmental competence during in vitro maturation (IVM). Melatonin has a wide range of actions, from scavenging reactive oxygen species (ROS) to regulating gene expression, and it can also act by way of melatonin receptors. The aim of this study was to determine the mechanism of action of melatonin during the IVM of juvenile goat oocytes and the role of the membrane receptors. Melatonin receptor 1 was immunolocalized in cumulus cells and oocytes before and after 24 hr of IVM. The effect of melatonin on oocyte developmental competence was tested in three experimental IVM groups: (a) control, (b) 10^?7 M melatonin, and (c) 10^?7 M melatonin +10^?7 M luzindole (an inhibitor of both melatonin receptors). After IVM oocytes were assessed for ROS levels, mitochondrial activity, adenosine 5′‐triphosphate (ATP) concentration and relative gene expression (ACTB, SLC1A1, SOD1, GPx1, BAX, DNMT1, GCLC and GDF9). IVM‐oocytes were in vitro fertilized and cultured under conventional conditions. Blastocyst rate and quality (differential cell count) were assessed at 8 days post‐fertilization. Melatonin decreased ROS levels, increased mitochondrial activity and ATP content and increased blastocyst quality compared to control group (55.8 vs. 30.4 inner cell mass ICM, p < 0.05). There was no effect on the relative gene expression due to treatment with melatonin. In conclusion, we have showed that melatonin improves oocyte developmental competence in juvenile goats by reducing ROS levels and improving mitochondrial activity.     

Evidence that oxidative stress is higher in replacement gilts than in multiparous sows

The recent success obtained in term of increasing the litter size of sows has not correlated with a reduction of replacement rate. There is thus an increased economic demand for gilts with optimal reproductive potential and longevity. Unfortunately, replacement gilts are known to be more susceptible to diseases and less productive than multiparous sows. Interestingly, reproductive performance, resistance to diseases and longevity could all be largely affected by oxidative stress. To investigate whether oxidative stress conditions could account for the poor longevity of gilts, three distinct groups of conventional Yorkshire × Landrace sows were formed based on their similar age and parity (gilts, second parity sows as well as fourth to fifth parity sows). All animals were slaughtered during the post‐ovulatory period, and blood as well as tissue samples were collected. Biomarkers of oxidative damage to proteins (carbonyls) and DNA (8‐OHdG) were analysed in samples. Specific mRNA expression of major antioxidants such as glutathione peroxidases 1, 3 and 4 (GPx1, GPx3, GPx4) as well as superoxide dismutases 1 and 2 (Sod1, Sod2) were monitored in liver and kidney samples by quantitative RT‐PCR. Specific enzymatic activities of both GPx and SOD were measured by spectrophotometric assays. The plasma concentration of protein carbonyls was significantly different between the three groups with the highest concentration being observed in gilts (p ≤ 0.001). The mRNA expression levels of GPx1 and GPx4 were also significantly increased in the liver of gilts when compared to multiparous sows (p ≤ 0.05). SOD2 enzymatic activity was found to be higher in the liver of gilts than multiparous sows (p ≤ 0.05). Taken together, these results indicate that replacement gilts sustain significantly higher oxidative conditions than multiparous sows. Current findings may contribute to the design of nutritional regimens that will increase the productivity of gilts by counteracting oxidative stress.     

Post-mortem recovery,in vitro maturation and fertilization of fallow deer (Dama dama,Linnaeus 1758) oocytes collected during reproductive and no reproductive season

Habitat degradation leads to small and fragmented populations, lower genetic variability and fertility overtime. Assisted reproductive techniques represent important tools to cope with the dramatic loss of biodiversity. Fallow deer (Dama dama), beyond its high commercial value and wide distribution, may represent the most suitable model to study endangered cervids. In this study, oocytes were recovered post-mortem from fallow deer during the breeding and no breeding seasons and were in vitro matured (IVM). The ability of cryopreserved thawed sperm samples recovered by electroejaculation from four adult males was tested by in vitro fertilization of IVM oocytes. The number of oocytes collected per ovary did significantly vary across seasons from 6.2 ± 0.92 during breeding season to 10.4 ± 1.26 during no breeding season (p = .006). Oocytes collected during the breeding season showed higher in vitro fertilization rate compared to the no breeding season (p = .045). However, no embryos reached the blastocyst stage. Semen samples obtained by electroejaculation were successfully cryopreserved, although the cryopreservation process negatively affected most kinetic parameters, mainly at 2 hr post-thawing. Moreover, the percentage of rapid spermatozoa significantly decreased between fresh samples and at 2 hr post-thawing, whereas the percentage of slow spermatozoa increased across the same period (p < .05). Our study provides the logistic steps for the application of assisted reproductive techniques in fallow deer and might be of great interest for genetic resource bank planning.     

Presence of chlorogenic acid during in vitro maturation protects porcine oocytes from the negative effects of heat stress

Chlorogenic acid (CGA) is known to protect oocytes from oxidative stress. Here we investigated the effects of CGA on porcine oocyte maturation under heat stress and subsequent embryonic development after parthenogenetic activation. For in vitro maturation (IVM) at 41.0°C (hyperthermic condition), supplementation of the maturation medium with 50 μM CGA significantly improved the percentage of matured oocytes and reduced the rate of apoptosis relative to oocytes matured without CGA (p < .05). CGA treatment of oocytes during IVM under hyperthermia tended to increase (p < .1) percentage of blastocyst formation after parthenogenesis and significantly increased (p < .05) the total cell number per blastocyst relative to oocytes matured without CGA. For IVM at 38.5°C (isothermic condition), CGA significantly improved the rate of blastocyst development compared with oocytes matured without CGA (p < .05), but did not affect oocyte maturation, apoptosis rate or the number of cells per embryo. Omission of all antioxidants from the IVM medium significantly reduced the rate of oocyte maturation, but the rate was restored upon addition of CGA. These results demonstrate that CGA is a potent antioxidant that protects porcine oocytes from the negative effects of heat stress, thus reducing the frequency of apoptosis and improving the quality of embryos.     

Peri‐conceptional under‐nutrition alters the expression of TRIM28 and ZFP57 in the endometrium and embryos during peri‐implantation period in domestic pigs

DNA methylation is maintained by the main elements of methylation complex—tripartite motif containing 28 (TRIM28) and zinc finger protein 57 (ZFP57). Previously, it was found that the activity of TRIM28 and ZFP57 determines the process of DNA methylation and preserves over‐expression of genes. We hypothesized that restricted diet applied during peri‐conceptional period may induce changes in the expression of methylation complex in porcine endometrium and embryos during the peri‐implantation period. The aim of this study was to detect and determine the expression of TRIM28 and ZFP57 in the endometrium and embryos harvested from gilts during the peri‐implantation period (days 15–16 of pregnancy) fed restricted (n = 5) or normal (n = 5) diet during peri‐conceptional period. In restricted‐diet‐fed gilts, endometrial expression of TRIM28 and ZFP57 mRNAs was decreased in comparison with normal‐diet‐fed gilts (p ≤ .01), while the embryonic expression of TRIM28 and ZFP57 mRNAs was increased in restricted‐diet‐fed gilts (p ≤ .05). The immunofluorescence showed the presence of TRIM28 and ZFP57 in luminal epithelial (LE), glandular epithelial (GE) and stromal cells (ST) of the endometrium as well as in the embryos. Total endometrial and embryonic abundance of TRIM28 and ZFP57 proteins was significantly higher (p ≤ .05) in restricted‐diet‐fed gilts than in normal‐diet‐fed gilts. Female under‐nutrition during peri‐conceptional period affects the expression of two main elements of methylation complex in the endometrium and in embryos during the peri‐implantation period and may have the impact on DNA methylation in these tissues.     

Association between methylation potential and nutrient metabolism throughout the reproductive cycle of sows

DNA methylation is an important epigenetic strategy for embryo development and survival. The one‐carbon metabolism can be disturbed by inadequate provision of dietary methyl donors. Because of the continuous selection for larger litters, it is relevant to explore if highly prolific sows might encounter periods of methyl donor deficiency throughout their reproductive cycles. This study, therefore, assesses the fluctuation(s) in methylation potential (MP) and aims to link possible methyl donor deficiencies to nutrient metabolism. In total, 15 hybrid sows were followed from weaning of the previous reproductive cycle (d‐5) to weaning of the present cycle. Blood samples were taken at d‐5, 0, 21, 42, 63, 84 and d108 of gestation, the day of parturition (d115), two weeks of lactation (d129) and at weaning (d143). Blood plasma samples were analysed for S‐adenosylmethionine (SAM), S‐adenosylhomocysteine (SAH), free methionine, free glycine, acetylcarnitine and 3‐hydroxybutyrylcarnitine. Serum samples were analysed for urea and creatinine. Generally, MP (i.e. ratio SAM:SAH) increased throughout gestation (p = 0.009), but strongly fluctuated in the period around parturition and weaning. From d108 to parturition, absolute plasma levels of SAM (p < 0.001), SAH (p = 0.031) and methionine (p = 0.001) increased. The first two weeks of lactation were characterised by an increase in MP (p = 0.039) due to a remaining high value of SAM and a distinct decrease in SAH (p = 0.008). During the last two weeks of lactation, MP decreased (p = 0.038) due to a decrease in SAM (p < 0.001) and a stable value for SAH. The methylation reactions seem to continue after weaning, a period crucial for the follicular and embryonic development of the subsequent litter. This study thus demonstrates that the methylation status fluctuates substantially throughout a sow's reproductive cycle, and further research is needed to identify the factors affecting methylation status.     

Buffalo SCNT embryos exhibit abnormal gene expression of ERK/MAPK pathway and DNA methylation

Inhibition of ERK/MAPK pathway has been shown to decrease DNA methylation via down‐regulation of DNA methyltransferases (DNMTs) in several studies suggesting that this pathway plays an important role in regulation of DNA methylation. We examined the relative expression level of seven important genes related to ERK/MAPK pathway and DNMTs (DNMT1, DNMT3a and DNMT3b) by quantitative real‐time PCR in buffalo blastocysts produced by Hand‐made cloning and compared it with that in blastocyst‐stage embryos produced by in vitro fertilization (IVF). The expression level of six of seven genes related to ERK/MAPK pathway examined i.e., p21RAS, RAF1, AKT1, ERK2, PIK3R2 and c‐Myc was significantly higher (p < 0.05) in cloned than in IVF embryos. However, the expression level of FOS was lower (p < 0.005) in cloned than in IVF embryos. The relative expression level of DNMT3a and DNMT3b but not that of DNMT1 was significantly higher (p < 0.05) in cloned than in IVF embryos. These results indicate that the cloned embryos exhibit an abnormal expression of several important genes related to ERK/MAPK pathway and DNMTs. Although a direct link between ERK/MAPK pathway and DNMTs was not examined in the present study, it can be speculated that ERK/MAPK pathway may have a role in regulating the expression of DNMTs in embryos, as also observed in other tissues.     

Transporting bovine oocytes in a medium supplemented with different macromolecules and antioxidants: Effects on nuclear and cytoplasmic maturation and embryonic development in vitro

We investigated whether supplementing the medium used to transport bovine oocytes with different macromolecules [foetal calf serum (FCS) or bovine serum albumin (BSA)] or a mixture of antioxidants (cysteine, cysteamine and catalase) affects their nuclear and cytoplasmic maturation and thereby affects their subsequent embryonic development and cryotolerance. Oocytes were transported for 6 hr in a portable incubator and then subjected to standard in vitro maturation (IVM) for 18 hr. The oocytes in the control groups were cultured (standard IVM) for 24 hr in medium containing 10% FCS (Control FCS) or 10% FCS and the antioxidant mixture (Control FCS+Antiox). The intracellular concentrations of reactive oxygen species (ROS) at the end of IVM period were lower in the oocytes subjected to simulated transport in the presence of a macromolecular supplement or the antioxidant mixture than that of the control group (FCS: 0.62 and BSA: 0.66 vs. Control FCS: 1.00, p < .05; and Transp: 0.58 and Transp Antiox: 0.70 vs. Control FCS: 1.00, p < .05). After IVM, the mitochondrial membrane potentials of the transported oocytes were lower than those of the non‐transported oocytes (FCS: 0.41 and BSA: 0.57 vs. Control FCS: 1.00, p < .05; and Transp: 0.48 and Transp Antiox: 0.51 vs. Control FCS: 1.00 and Control Antiox: 0.84, p < .05). The blastocyst formation rates (36.9% average) and the re‐expansion rates of vitrified‐warmed blastocysts (53%, average) were unaffected (p > .05) by the treatments. In conclusion, supplementing the medium in which bovine oocytes are transported with antioxidants or different macromolecules did not affect their in vitro production of embryos or their cryotolerance.     

Theca cell-conditioned medium added to in vitro maturation enhances embryo developmental competence of buffalo (Bubalus bubalis) oocytes after parthenogenic activation

Theca cells (TCs) play a key role in follicular growth and atresia. TCs synthesize androgens that act as substrate for granulosa cells (GCs) aromatization to estrogens needed for oocyte maturation. However, the effects of TCs in the form of conditioned medium on in vitro maturation (IVM) and developmental competence of buffalo oocytes remain unclear. In the present study, we examined the impacts of TC-conditioned medium (TCCM) on maturation efficiency and embryo development of buffalo oocytes after parthenogenic activation (PA). Our results showed that TCCM that was collected on day 2 and added to IVM medium at a 20% proportional level (2 days & 20%) exerted no significant effect on IVM rate (43.06% vs. 44.71%), but significantly (p  < .05) enhanced embryo development (oocyte cleavage, 80.93% vs. 69.66%; blastocyst formation, 39.85% vs. 32.84%) of buffalo oocytes after PA compared with the control group. However, monolayer TC significantly (p < .05) promoted both maturation efficiency (48.84% vs. 44.53%) and embryo development (oocyte cleavage, 80.39% vs. 69.32%; blastocyst formation, 35.38% vs. 29.25%) of buffalo oocytes after PA compared to that in the control group. Furthermore, TCs secreted some testosterone into the conditioned medium, which significantly (p < .05) promoted the expression levels of oestrogen synthesis-related genes (CYP11A1, CYP19A1 and 17β-HSD) in buffalo cumulus–oocyte complexes (COCs). Our study indicated that TCCM (2 days & 20%) did not significantly affect IVM efficiency, but enhanced embryo developmental competence of oocytes after PA principally by stimulating the secretion of testosterone and facilitating estradiol synthesis of buffalo COCs.     

Estimates of genetic parameters associated with lactation feed intake and growth and composition traits measured during a performance test

Days to reach 113.4 kg, adjusted backfat depth and adjusted loin muscle area to 113.4 kg were evaluated on pure‐bred Landrace (n = 15 660) and Yorkshire (n = 14 808) boars and gilts. Daily lactation feed intake (LFI) was recorded within parity records from pure‐bred Yorkshire (n = 1587) and Landrace (n = 2197) females during day 1–22 of lactation, and LFI curves were predicted using a mixed model. Evaluation of feed intake differences between 2 consecutive days of lactation resulted in the following periods: day 1–6 (PB1), day 7–10 (PB2) and day 11–18 (PB3). Average rate of change in intake (ARC), average daily intake (ADI) and variation from predicted daily LFI values (VAR) metrics were computed for each period of lactation. Gibbs sampling was used to estimate the genetic covariance between LFI metrics and grow‐finish traits. Genetic correlations were strongest between grow‐finish traits, and LFI metrics in first parity sows and were generally favourable, but correlations with LFI metrics during parity 2 or greater were low and not different from 0 (p > 0.05). Genetic correlations between LFI metrics in parity 1 sows with growth and composition traits varied greatly in strength and direction. Selection for leaner, heavier muscled gilts had a limited effect on LFI metrics. However, selection for increased growth rate was associated with higher ARC and ADI and smaller VAR values.     

Influence of mastitis metritis agalactia (MMA) on bone and fat metabolism

Mastitis metritis agalactia (MMA) is a common disease in post-partum sows and has a negative effect on sows’ longevity as well as on sows’ and piglets’ health. MMA leads to an inflammatory state. The aim was to investigate the impact of MMA on bone and fat metabolism. The hypothesis was that it is possible to predict MMA by measuring ketone bodies and bone markers. Blood samples from 175 sows were taken within 72 hr after farrowing. Serum was analysed for 25-hydroxyvitamin D (25-(OH)-D), serum crosslaps (CTX-I), osteocalcin (OC), alkaline phosphatase (ALP), calcium (Ca), magnesium (Mg), phosphorus (P), parathyroid hormone (PTH), triglycerides (TG), beta-hydroxybutyric acid (BHB), tumour necrosis factor-alpha (TNF-α) and haptoglobin. Spontaneous urine was collected, and pH value was measured in addition to Ca and P. A proximate analysis of the sows’ diets was performed. Age, litter size, body condition score (BCS) and clinical signs of MMA were recorded for each sow. A multivariable logistic regression was undertaken with disease status (MMA or healthy) as the dependent variable. Significance was accepted at p < .05. MMA sows had a poorer BCS (p < .001) in relation to healthy sows. Age and number of piglets did not differ. MMA sows showed increased serum CTX-I (p = .004) and decreased serum OC (p < .001). Concentrations of P (p = .007), activity of ALP (p = .002) and BHB (p = .019) as well as TNF-α (p < .001) and haptoglobin (p = .048) concentrations were increased in MMA sows. No difference in urinary pH value between MMA and healthy sows was found. Our results are in accordance with the known fact that sows are in an extreme catabolic state peripartum. Bone metabolism in MMA sows is much more negatively affected than in healthy sows post-partum, due to inflammatory processes shown by higher concentrations of pro-inflammatory cytokines.     

Effect of high‐density lipoprotein on oocyte maturation and bovine embryo development in vitro

High‐density lipoprotein (HDL) is the main lipoprotein in the follicular fluid, and it has anti‐inflammatory, antioxidant and cryoprotectant properties. The anti‐inflammatory potential and antioxidant potential are derived from its lipid composition, especially the apolipoprotein AI (ApoAI) and paraoxonase 1 (PON1). The aim of this study was to evaluate the effect of HDL during in vitro maturation (IVM) on oocyte maturation and early bovine embryo development. For this, cumulus–oocyte complexes (COCs) were obtained from bovine ovaries collected at a local slaughterhouse. COCs (n = 2,250) were allocated into three groups (n = 50 COCs/group) according to the addition of HDL protein (HDL‐P) during IVM for 22 hr: 0 (control), 50 and 150 mg/dl. After IVM, COCs were inseminated (in vitro fertilization) and cultivated for 7 days. Total cholesterol concentration, total protein, triglycerides and ApoAI concentrations on IVM medium increased proportionally to HDL‐P addition. However, PON1 activity was not detected in any treatment. The addition of HDL‐P did not affect nuclear maturation rate, endogenous reactive oxygen species and glutathione levels in COCs (p > 0.05). The highest HDL‐P concentration (150 mg/dl) decreased cleavage and blastocyst rate (p < 0.05). Moreover, the HDL‐P 150 mg/dl group had lower cellular count/blastocyst than the 50 mg/dl group (p < 0.05). However, the addition of HDL‐P did not affect relative gene expression of evaluated genes. In conclusion, the complex HDL/ApoAI obtained from human plasma, in the absence of PON1 activity during in vitro oocyte maturation, decreased initial embryo development.     

Granulosa cells in three-dimensional culture: A follicle-like structure for domestic cat vitrified oocytes

Follicle-like structures are three-dimensional matrices joint with living cells that allow the in vitro development of female gametes in more physiological conditions. They have been shown to be beneficial to fresh oocytes in different species, and in this study, domestic cat (Felis silvestris catus) granulosa cells were used to create a functional follicle-like structure aimed at supporting the in vitro maturation of conspecific vitrified oocytes, key players of fertility preservation programmes that usually struggle to acquire their full developmental competence after warming. Cat granulosa cells were cultured for up to 6 days in three-dimensional barium alginate microcapsules (i.e. follicle-like structures) or in two-dimensional monolayers, and their steroidogenic ability (estradiol and progesterone secretion) was assessed to confirm their functionality. The same systems were used (on day 2 or 6 of granulosa cells culture) for the in vitro maturation (IVM) of Cryotop^® vitrified immature cat oocytes and compared with microdrops of IVM medium without cells (control). Granulosa cells were able to maintain their functionality in vitro in both the conditions, even if with a different extent of hormonal secretion along culture (p = .02). Vitrified oocytes resumed meiosis at higher rates when cultured with 2 days old granulosa cells (p = .03), but full maturation rates slightly raised when granulosa cells were cultured longer, albeit without differences with the control group. This study paved the road for the creation of enriched culture systems in the domestic cat, but innovations are strongly needed for vitrified oocytes that deserve better chances to develop in vitro.     

Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.     

Effects of the E1 activating enzyme UBA2 on porcine oocyte maturation,apoptosis, and embryonic development in vitro

The purpose of this study was to investigate the effect of the E1 activating enzyme UBA2 on the expression of the SUMO-1 protein during in vitro maturation (IVM) of pig oocytes and embryonic development. In the 5 μg/ml UBA2 treatment group, the expression of the anti-apoptotic gene Bcl-2 and the embryo cleavage rate was significantly increased, while the proapoptotic gene Bax was significantly reduced. When 10 μg/ml UBA2 was added, the in vitro maturation rate, blastocyst rate, and SUMO-1 protein content of oocytes increased significantly (p < .05), and the expression of proapoptotic gene Caspase3 was significantly decreased (p < .05), while the viability of cumulus cells was extremely significantly reduced (p < .01). In summary, UBA2 can regulate the content of the SUMO-1 protein in mature pig oocytes in vitro, which in turn affects the maturation rate of oocytes, expression of apoptosis genes, cumulus cell viability, and the development of embryos after fertilization.     

Effects of deficiency and surplus dietary threonine on reproductive performance of primiparous pregnant gilts

Dietary threonine imbalance is known to impair reproductive performances of gestating sows, but the underlying mechanisms are largely unknown. In this study, effects of deficiency and surplus dietary threonine during gestation on reproductive performance, serum metabolites and hormones concentration, and colostral nutrient and immunoglobulin contents of primiparous sows were investigated. Ninety primiparous pregnant gilts were assigned to one of the three dietary treatments with different standardized ileal digestible threonine/lysine ratios at 0.59, 0.72 and 0.85, which represented deficient (DT), adequate (AT) and surplus (ST) dietary threonine concentration respectively. Maternal body weight gain from day 80–110 of gestation was highest (p < .05) for gilts fed AT than for gilts fed DT or ST. On days 30 and 110, serum threonine concentration increases in a dose‐dependent manner with the increasing of dietary threonine concentration in (p < .01), serum urea nitrogen concentration was lower (p < .01) in gilts fed AT than DT or ST, and serum insulin‐like growth factor‐I (IGF‐I) was lowest (p < .05) for gilts fed DT. On day 110, gilts fed AT had lower serum progesterone concentration but higher concentrations of serum prolactin (p < .05) compared to DT and ST. Concentration of colostral immunoglobulin A and G from gilts fed DT was lower (p < .05) compared with gilts fed AT or ST. In conclusion, gilts with the adequate threonine intake were more able to conserve dietary amino acids to support foetal and maternal tissue gain. Deficient or ST threonine intake may induce a delay in changes in progesterone and prolactin concentrations during the prepartum period impeding the transition from pregnancy to lactation.