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Abstract: Oocyte and embryo cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig oocytes, because of their greater susceptibility to cryoinjuries, have shown a reduced ability to be fertilized in vitro and a lower developmental competence. The aim of this study was to evaluate the apoptotic status of porcine oocytes vitrified by Cryotop method. We assessed three parameters linked to the activation of the apoptotic cascade: the exteriorization of phosphatidylserine using Annexin V, the caspase activation using FITC-VAD-FMK and the alteration of plasma membrane permeability employing YO-PRO-1. These assays were performed on control oocytes, oocytes exposed to vitrification solutions (toxicity control) and vitrified oocytes either immediately after warming or after incubation for 2h into maturation medium. The exposition to vitrification solutions triggered an increase of the percentage of oocytes both faintly (VAD+ PI−) and strongly (VAD++ PI−) labeled by FITC-VAD-FMK but not a significant modification of the number of oocytes Annexin V (A+ PI−, early apoptotic) and YO-PRO-1(YP+ PI−, apoptotic) positive in comparison with control oocytes. Oocytes submitted to the whole vitrification procedure showed a rise of the percentage of early apoptotic oocytes (A+ PI−) and FITC-VAD-FMK positive oocytes (VAD+/VAD++ PI−) and a contemporaneous increase of the number of dead oocytes (PI+). On the contrary, vitrified oocytes analyzed immediately after warming did not show a significant increase in the percentage of apoptotic oocytes (YO-PRO-1+/PI−) as compared with the control. Post warming incubation for 2h into maturation medium, in comparison with oocytes analyzed immediately after warming, did not induce any increase in the percentage of early apoptotic (A+ P−) oocytes while a decrease of the percentage of VAD+/PI− oocytes and a contemporaneous increase of VAD−/PI− oocytes were observed. Moreover, the post-warming incubation induced a rise of the percentage of apoptotic oocytes (YO-PRO-1+/PI−). All these data confirm the involvement of apoptotic mechanisms on the injuries induced by vitrification procedure in pig oocytes; explanation of this phenomenon could be useful to improve oocytes’ cryopreservation protocols. [Copyright &y& Elsevier]

Abstract: Sorting procedures submit sperm cells to a set of stressful steps that can trigger an increase of ROS production and consequently reduce sorted semen quality. This study evaluated the effect of supplementation with different antioxidants (EGCG, Na pyruvate+catalase, SOD) on acrosome and plasma membrane integrity, activation of caspases (as assayed by FITC-VAD/PI staining) and immunolocalization of Hsp70 of boar spermatozoa after sperm preparation (Hoechst 33342 staining) and sorting procedure. The effect of antioxidants, with or without seminal plasma, on sorted spermatozoa stored for 24h at 15°C was also evaluated. Antioxidants did not exert any preventive action on sperm modification induced by staining and sorting. After 24h of storage at 15°C, sorted samples supplemented with either EGCG or SOD plus seminal plasma showed a significant (p <0.05) increase of the percentage of viable spermatozoa, while no positive effect on the other tested parameters was observed; EGCG seems to exert an inhibition on caspase activation in that a decrease of the number of dead cells FITC-VAD+/PI+ was recorded. In conclusion, our results indicate that EGCG and SOD in association with seminal plasma are effective in exerting some compensatory protection against the detrimental effects of storage of sorted semen while their action is not evident during steps of the sorting procedure. [ABSTRACT FROM AUTHOR]

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Abstract: Three experiments were designed to evaluate the effects of vitrification using Cryotop method on MII porcine oocyte viability, chromosomes configuration, meiotic spindle morphology and in vitro fertilization; to do this, in vitro matured oocytes were subjected to the cryoprotectant treatment excluding the plunging into liquid nitrogen, the whole vitrification/warming/rehydration procedure or no treatment (control). In experiment 1 viable oocytes were not reduced by either cryoprotectants or vitrification when they were evaluated immediately after warming and cryoprotectant dilution. However, after a 2h incubation, the survival rate significantly decreased (P <0.05). In experiment 2 cryoprotectant exposure significantly (P <0.05) influenced spindle morphology even if chromosome organization did not vary, while vitrification significantly (P <0.05) increased oocytes with damaged spindles and chromosomes displaced from the metaphase plate. No significant improvements in these parameters were observed after 2h of incubation but, on the contrary, the rate of oocytes with normal chromosome configuration was reduced. In experiment 3 significant differences among the three groups in the fertilization rate but not in the percentages of monospermy fertilization were recorded; in addition, exposure to cryoprotectants and vitrification significantly (P <0.05) increased degenerated oocyte rate. Overall, these findings confirm that porcine oocytes at MII stage are very sensitive to vitrification, which reduces the rate of viable oocytes and alters microtubule organization, thus impairing fertilization; in addition, incubation of oocytes for 2h after devitrification seems to be detrimental rather than ameliorative. Further improvements of the current protocol will be necessary in order to optimize the Cryotop method for vitrifying pig matured oocytes. [Copyright &y& Elsevier]

Abstract: Single layer centrifugation (SLC) is a useful technique to select porcine spermatozoa for further artificial insemination practices. The aim of this study was to determine possible side-effects related to capacitation due to the process. Semen viability, acrosome integrity and capacitation status were determined through fluorescent probes (SYBR14-PI, FITC-PSA, CTC staining) and Hsp70 immunolocalization and protein tyrosine phosphorylation (by western blotting and immunolocalization) in different groups: control, after SLC with Androcoll (AND), after SLC and washing (AND-Wash) and after SLC, washing and storage for 2h at 17°C with 2.5% of seminal plasma (AND-Wash-SP). Neither viability nor acrosome integrity were impaired by the different treatments; as far as CTC staining, we observed a significant increase (p <0.05) in the capacitation related pattern in AND and AND-Wash, while after exposure for 2h to seminal plasma (AND-Wash-SP group), the increase became less evident; the same trend was observed in Hsp70 immunolocalization for the EL pattern. Neither immunolocalization nor western blotting for tyrosine phosphorylated proteins had an increase in capacitated pattern or in phosphorylation status, except for a 25kDa band that increased in AND and AND-Wash groups and decreased in AND-Wash-SP group. SLC using Androcoll-P induces some capacitation-related changes in boar sperm membrane, as demonstrated by CTC staining and Hsp70 immunolocalization. For protein tyrosine phosphorylation, only a 25kDa protein showed some changes that should be investigated further. [Copyright &y& Elsevier]

Background: The expression of taste receptors (TASRs) and their signalling molecules in the gastrointestinal (GI) epithelial cells, including enteroendocrine cells (EECs), suggests they participate in chemosensing mechanisms influencing GI physiology via the release of endocrine messengers. TASRs mediate gustatory signalling by interacting with different transducers, including α-gustducin (Gαgust) and α-transducin (Gαtran) G protein subunits. This study tested whether Gαtran and Gαgust immunoreactive (-IR) cells are affected by a short-term (3 days) and long-term (30 days) high protein (Hp) diet in the pig GI tract., Result: In the stomach, Gαgust and Gαtran-IR cells contained serotonin (5-HT) and ghrelin (GHR), while in the small and large intestine, Gαgust and Gαtran-IR colocalized with 5-HT-, cholecystokinin (CCK)- and peptide YY (PYY)-IR. There was a significant increase in the density of Gαtran-IR cells in the pyloric mucosa in both short- and long-term Hp diet groups (Hp3 and Hp30) vs. the control group (Ctr) (P<0.05), while the increase of Gαgust-IR cells in the pyloric mucosa was significant in Hp30 group vs. Ctr and vs. Hp3 (P<0.05); these cells included Gαtran / 5HT-IR and Gαtran / GHR-IR cells (P<0.05 and P<0.001 vs. Ctr, respectively) as well as Gαgust /5-HT-IR or Gαgust / GHR-IR cells (P<0.05 and P<0.01 vs. Ctr, respectively). In the small intestine, we recorded a significant increase in Gαtran-IR cells in the duodenal crypts and a significant increase of Gαgust-IR cells in the jejunal crypts in Hp3 group compared to HP30 (P<0.05). With regard to the number of Gαtran-Gαgust IR cells colocalized with CCK or 5-HT, there was only a significant increase of Gαtran / CCK-IR cells in Hp3 group compared to Ctr (P = 0.01)., Conclusion: This study showed an upregulation of selected subpopulations of Gαgust / Gαtran-IR cells in distinct regions of the pig GI tract by short- and long-term Hp diet lending support to TASR-mediated effects in metabolic homeostasis and satiety mechanisms.

Vertebrates perceive a variety of exogenous substances using two main chemosensory systems, taste and olfaction. The taste perception occurs through the interaction of taste receptors associated with specific G protein subunits such as α-transducin (Gαtran) and α-gustducin (Gαgust). Aquatic vertebrates are also provided with a chemosensory system consisting of solitary chemosensory cells distributed to the oropharynx and skin. In this study, we identified Gαtran and Gαgust-immunoreactive cells intermingled with non-labeled epithelial cells in the gastric mucosa of the common sole. A long-term diet with increasing concentrations of mussel meal in the protein component of a conventional fish meal-based diet induced a dose-dependent increase in the gastric epithelial area and density of Gαtran and Gαgust immunoreactive cells. These findings suggest that taste-related molecules are regulated by changes in diet formulation in common sole aquaculture.

In vertebrates, chemosensitivity of nutrients occurs through the activation of taste receptors coupled with G-protein subunits, including α-transducin (G(αtran)) and α-gustducin (G(αgust)). This study was aimed at characterising the cells expressing G(αtran) immunoreactivity throughout the mucosa of the sea bass gastrointestinal tract. G(αtran) immunoreactive cells were mainly found in the stomach, and a lower number of immunopositive cells were detected in the intestine. Some G(αtran) immunoreactive cells in the stomach contained G(αgust) immunoreactivity. Gastric G(αtran) immunoreactive cells co-expressed ghrelin, obestatin and 5-hydroxytryptamine immunoreactivity. In contrast, G(αtran) immunopositive cells did not contain somatostatin, gastrin/cholecystokinin, glucagon-like peptide-1, substance P or calcitonin gene-related peptide immunoreactivity in any investigated segments of the sea bass gastrointestinal tract. Specificity of G(αtran) and G(αgust) antisera was determined by Western blot analysis, which identified two bands at the theoretical molecular weight of ~45 and ~40 kDa, respectively, in sea bass gut tissue as well as in positive tissue, and by immunoblocking with the respective peptide, which prevented immunostaining. The results of the present study provide a molecular and morphological basis for a role of taste-related molecules in chemosensing in the sea bass gastrointestinal tract.

Taste signalling molecules are found in the gastrointestinal (GI) tract suggesting that they participate to chemosensing. We tested whether fasting and refeeding affect the expression of the taste signalling molecule, α-transducin (Gαtran ), throughout the pig GI tract and the peptide content of Gαtran cells. The highest density of Gαtran -immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most Gαtran -IR cells contained chromogranin A. In the stomach, many Gαtran -IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin-IR and a few somatostatin-IR. Gαtran -IR and Gαgust -IR colocalized in some cells. Fasting (24 h) resulted in a significant decrease in Gαtran -IR cells in the cardiac mucosa (29.3 ± 0.8 versus 64.8 ± 1.3, P < 0.05), pylorus (98.8 ± 1.7 versus 190.8 ± 1.9, P < 0.0 l), caecum (8 ± 0.01 versus 15.5 ± 0.5, P < 0.01), descending colon (17.8 ± 0.3 versus 23 ± 0.6, P < 0.05) and rectum (15.3 ± 0.3 versus 27.5 ± 0.7, P < 0.05). Refeeding restored the control level of Gαtran -IR cells in the cardiac mucosa. In contrast, in the duodenum and jejunum, Gαtran -IR cells were significantly reduced after refeeding, whereas Gαtran -IR cells density in the ileum was not changed by fasting/refeeding. These findings provide further support to the concept that taste receptors contribute to luminal chemosensing in the GI tract and suggest they are involved in modulation of food intake and GI function induced by feeding and fasting., (© 2013 The Authors. Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Mammalian cells use glucides as a substrate that can be catabolized through glycolitic pathways or oxidative phosphorylation, used as a source of reducing potential, or used for anabolic aims. An important role in supplying cells with energy is played by different membrane proteins that can actively (sodium-dependent glucose transporters) or passively (glucose transporters; GLUT) transport hexoses through the lipidic bilayer. In particular, GLUTs are a family of 13 proteins that facilitate the transport of sugars and have a peculiar distribution in different tissues as well as a particular affinity for substrates. These proteins are also present in mature sperm cells, which, in fact, need carriers for uptake energetic sources that are important for maintaining cell basic activity as well as specific functions, such as motility and fertilization ability. Likewise, several GLUTs have been studied in various mammalian species (man, bull, rat, mouse, boar, dog, stallion, and donkey) to point out both their actual presence or absence and their localization on plasma membrane. The aim of this work is to give an overall picture of the studies available on GLUTs in mammalian spermatozoa at this moment, pointing out the species peculiarity, the possible role of these proteins, and the potential future research on this item.

Daidzein, an isoflavone abundant in soybeans and other legumes, displays estrogen like properties. This study was aimed at evaluating the effect of daidzein (1 and 10 microM) on nuclear and cytoplasmic maturation of pig oocytes and on steroidogenic activity of cumulus cells. Daidzein supplementation during IVM had no effect on nuclear maturation and on fertilization traits. By contrast, both concentrations significantly (P < 0.05) inhibited progesterone production by cumulus cells after 24 and 48 h of culture while they did not induce any effect on estradiol production. Furthermore, daidzein did not exert any effect on the percentage of embryos that developed to blastocyst stage, on the number of blastomeres per blastocyst, or on the level of Hsp-70 and -90 gene transcript. Overall, our data demonstrate that daidzein added during oocyte maturation does not affect pig embryo development even if it markedly inhibits progesterone production by cumulus cells. Further studies are needed to evaluate the possible effect of daidzein during embryonic development., (Copyright 2010 Elsevier Inc. All rights reserved.)