Expression and regulation of α-transducin in the pig 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, a-transducin (Gatran), throughout the pig GI tract and the peptide content of Gatran cells. The highest density of Gatran-immunoreactive (IR) cells was in the pylorus, followed by the cardiac mucosa, duodenum, rectum, descending colon, jejunum, caecum, ascending colon and ileum. Most Gatran-IR cells contained chromogranin A. In the stomach, many Gatran-IR cells contained ghrelin, whereas in the upper small intestine many were gastrin/cholecystokinin-IR and a few somatostatin-IR. Gatran-IR and Gagust-IR colocalized in some cells. Fasting (24 h) resulted in a significant decrease in Gatran-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 Gatran-IR cells in the cardiac mucosa. In contrast, in the duodenum and jejunum, Gatran-IR cells were significantly reduced after refeeding, whereas Gatran-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

Storage of boar sexed semen

Gli spermatozoi di suino sottoposti alla procedura di sessaggio mediante citofluorimetria presentano una serie di modificazioni morfo-funzionali che compromettono nel tempo la loro sopravvivenza e la capacità fecondante. Questi spermatozoi, inoltre, a causa della sensibilità ai danni indotti dalla crioconservazione, vengono solitamente conservati allo stato liquido a 15-17°C, con conseguente ulteriore peggioramento nel tempo della qualità delle cellule spermatiche sessate. Lo scopo della ricerca è stato quello di valutare le modificazioni di alcune caratteristiche morfo-funzionali degli spermatozoi in seguito a sex-sorting e conseguente conservazione. Successivamente si è cercato di migliorare i parametri qualitativi del seme sessato mediante l’aggiunta di sostanze antiossidanti e la messa a punto di una nuova metodica di conservazione. I risultati ottenuti hanno evidenziato che la procedura di sessaggio e la conseguente conservazione per 24-26 ore a 15°C hanno indotto un peggioramento significativo delle caratteristiche morfo-funzionali (vitalità, integrità acrosomiale, quantità e distribuzione dell’Hsp70, capacità fecondante). Mentre l’azione degli antiossidanti non si è rivelata efficace nel miglioramento della qualità degli spermatozoi durante le fasi di colorazione e passaggio attraverso il citofluorimetro, l’azione congiunta del plasma seminale e degli antiossidanti superossido-dismutasi ed epigallocatechina-3-gallato ha indotto un miglioramento significativo della vitalità degli spermatozoi. Per la conservazione del seme di suino è stata testata la tecnica di incapsulazione in membrane di alginato di bario che permette, durante l’inseminazione artificiale, un rilascio graduale degli spermatozoi e l’utilizzo di un quantitativo inferiore di materiale seminale. L’applicazione di tale tecnica per la conservazione degli spermatozoi di suino sessati non sembra provocare un calo significativo della vitalità, dell’integrità acrosomiale e dell’efficienza totale di fecondazione rispetto al seme sortato e conservato diluito suggerendo futuri studi in vivo. Una migliore conoscenza dei danni indotti da queste tecnologie e la loro minimizzazione potrà stimolare in futuro l’utilizzo su vasta scala del seme sessato nel suino.Boar spermatozoa submitted to the sorting procedure show several morpho-functional modifications effective in compromising their survival and fertilization ability. Moreover ,boar spermatozoa, because of their susceptibility to damages induced by cryopreservation, are usually stored at 15-17°C after the sorting procedure; however, also the conservation at liquid state implies the worsening of semen quality. The aims of this research were: 1) to evaluate morpho-functional characteristics of sperm cells submitted to sex-sorting and consequent storage; 2) to try to improve the quality of sorted semen by the addition of antioxidants; 2) to set up a new storage method. Our results evidence a decreased quality of boar sorted-stored spermatozoa in term of: viability, acrosome integrity, amount and localization of Hsp70, fertilizing ability. During the staining step and the passage through the cytofluorimeter, antioxidants were not effective in improving sperm cells morpho-functional characteristics, while the addition of superoxide dismutase or epigallocatechin-3-gallate associated with seminal plasma induced an increase of viability of sorted boar spermatozoa stored 24 h at 15°C. Some researchers have utilized encapsulation in barium alginate membrane to store boar sperm cells. This technique allows a constant release of spermatozoa in sow reproductive system, avoiding the double/triple intervention of insemination and reducing the number of spermatozoa/insemination. The application of this technique in order to store boar sperm cells after sorting did not induce any impairment of sperm morpho-functional characteristics (viability, acrosome integrity, total efficiency of insemination) compared to sorted spermatozoa stored at liquid state, thus demonstrating the possibility to use this method to improve the reproductive performance of boar sorted semen

Effects of single layer centrifugation with Androcoll-P on boar sperm

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, FITCPSA, 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 2 h at 17 \u25e6C 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 2 h 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 25 kDa 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 25 kDa protein showed some changes that should be investigated further

a-Transducin and a-gustducin immunoreactive cells in the stomach of common sole (Solea solea) fed with mussel meal

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 a-transducin (Gatran) and a-gustducin (Gagust). 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 Gatran and Gagust-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 Gatran and Gagust immunoreactive cells. These findings suggest that taste-related molecules are regulated by changes in diet formulation in common sole aquaculture

Capacitation-like changes in sex-sorted boar spermatozoa

Sex-sorting process induces several changes in sperm cell functionality which are defined as capacitation-like modifications. The aim of the present study was to evaluate protein tyrosine phosphorylation (TP) status and Chlortetracycline (CTC) staining pattern in boar spermatozoa after sex-sorting procedure and to compare these parameters with those of freshly ejaculated, capacitated and acrosome reacted cells. TP status was evaluated by immunofluorescence and three different patterns were recognized; each of them was considered to be typical of fresh (F), capacitated (C) and acrosome reacted(AR) cells. In sorted spermatozoa the most expressed pattern was F (80.2 ± 6.6% Mean ± SD), while C and AR patterns were 8.5 ± 5.9% and 11.3 ± 5.6%, respectively. These pattern distribution is similar to that observed in freshly ejaculated sperm cells (F 87.5 ± 7.3%; C 9.9 ± 6.1%; AR 2.6 ± 2%). CTC positivity was assayed in that it is considered a sensitive capacitation index. Sex sorted cells presented the following pattern distribution: F 68.4 ± 5.2%; C 26.3 ± 4.3%; AR 5.3 ± 0.9%, which are very similar to the those observed in capacitated spermatozoa (F 67.8 ± 6.1%; C 25.2 ± 2.5%; AR 7 ± 4.3%). These results suggest that sex sorting procedure induces a capacitation-like switch in sperm subpopulations of boar ejaculates, as registered with CTC technique. As for protein TP immunoreactivity, it evidences a fresh-like subpopulation trend, with an increase of AR pattern, probably due to mechanical damage. Further studies would be necessary to better define the pathways involved in sex sortinginduced modifications

Actin distribution and tyrosine phosphorylation in sex-sorted bull spermatozoa

Sexing semen has reached a high commercial level in bovine, even if fertility after sorting is still variable because of stresses due to the process. This study was aimed at evaluating actin rearrangement and protein tyrosine phosphorylation (TP) in sexed spermatozoa, as compared to freshly ejaculated, capacitated and acrosome reacted sperm, in order to determine possible capacitation-like changes. As for TP, sexed spermatozoa showed two main patterns: cells positive in both acrosome and equatorial subsegment (EQSS) (49.3 ± 10.3%, mean ± SEM, five replicates) and cells with acrosome immunoreactivity (43.6 ± 11%). The remaining population was equally divided into EQSS positive and negative spermatozoa. This condition is in-between the fresh (77.2 ± 12.6% acrosome positive) and capacitated (84.9 ± 7.4% acrosome and EQSS positive) spermatozoa pattern distribution. As for actin, three different patterns (F, C and R, typical of fresh, capacitated and acrosome reacted cells, respectively) were observed. In fresh cells, F 92.7 ± 0.4%, C 5 ± 0.4%, R 2.3 ± 0.8%; in capacitated cells, F 47.7 ± 2.4%; C 44.4 ± 2.1%; R 7.9 ± 0.6%; in acrosome reacted cells, F 5.2 ± 0.5, C 55.8 ± 5.8%, R 39 ± 5.5%. Sex-sorting determined a capacitation-like distribution, with an increase of C pattern: F 28 ± 9%, C 67 ± 11%, R 5 ± 2%. In conclusion, sex sorting in bull sperm cells seems to induce capacitation-like changes that could be responsible for reducing semen quality; other studies on possible functional modifications could be useful to improve sexed semen performance

Regulation of \u3b1-Transducin and \u3b1-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract

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 \u3b1-gustducin (G\u3b1gust) and \u3b1-transducin (G\u3b1tran) G protein subunits. This study tested whether G\u3b1tran and G\u3b1gust 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

Correction: Regulation of α-Transducin and α-Gustducin Expression by a High Protein Diet in the Pig Gastrointestinal Tract.

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

Encapsulation of sex sorted boar semen: Sperm membrane status and oocyte penetration parameters

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 \ub1 14.38% vs. 44.32 \ub1 11.72%, respectively) and acrosome integrity (74.32 \ub1 12.17% vs. 66.07 \ub1 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, 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