Can Microfiltered Seminal Plasma Preserve the Morphofunctional Characteristics of Porcine Spermatozoa in the Absence of Antibiotics? A Preliminary Study.

Artificial insemination is extensively performed in pig farms in Europe, the United States and Canada. Antibiotics are typically added to the inseminating dose to limit bacterial growth during liquid phase storage at 16°C, as bacterial contamination is unavoidable. The World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) take action to control and reduce antibiotic use in animals as more bacteria are becoming resistant to antimicrobials. To avoid the use of antibiotics, we prepared inseminating doses using microfiltered seminal plasma (SP). Microfiltration is a common technology used to reduce bacterial contamination but may retain seminal substances, influencing sperm quality during storage. Therefore, the aim of this study was to evaluate the morphofunctional parameters of spermatozoa during storage at 16°C in doses prepared with or without microfiltered SP, with or without the addition of antibiotics, in a Latin square design. Artificial insemination doses with microfiltered SP and without antibiotic addition preserved spermatozoa viability, mitochondrial membrane potential, acrosome integrity and objective motility, with absolute values equal or even better than those observed in conventional doses. In conclusion, although the results could be considered preliminary due to the small sample size, this study suggests that microfiltration of SP can be a simple method, feasible on farms, to replace antibiotic use in extended doses stored in the liquid phase at 16°C for up to 7 days.

Initial ontogeny of digestive enzymes in the early life stages of captive-bred European eels during fasting: A partial characterization.

The European eel has recently been included on the Red List of the International Union for Conservation of Nature (IUCN) as a critically endangered species. The rearing of Anguilla larvae is seen as a key bottleneck to the mass production of glass eels since very little ecological information is available regarding their natural nutrition. Studies of digestive physiology and ontogenetic development in eel larvae could provide useful information for solving some of the puzzles regarding larval fish culture. The aim of this study was to characterize the ontogeny of pancreatic enzymes (trypsin, lipase and amylase) and a peptide hormone regulator of pancreatic secretion (cholecystokinin) in terms of gene expression in European eel larvae from day 0 (P0) of hatching to 5, 10, 15 and 20 days post hatching during fasting. The results in the present study showed that all the genes selected were present, with different levels of expression and increasing trends, during larval development. At P0, the increase in the gene expression of lipase and amylase was higher than that of trypsin and cholecystokinin, confirming that enzymatic activity began before mouth opening and that larvae, provided with a complete enzymatic set, might have the capacity of digesting and absorbing various nutrients.

Relative abundance of heat shock proteins and clusterin transcripts in spermatozoa collected from boar routinely utilised in an artificial insemination centre: preliminary results.

It is widely accepted that mature sperm contains RNA. The first hypothesis was that sperm RNAs have no functions of their own but are simply residues of spermatogenesis reflecting the events that occurred during their formation in the testes. More recently new discoveries have essentially expanded these views, showing that sperm mRNAs constitute a population of stable full-length transcripts, many of which are selectively retained during spermatogenesis and delivered to oocytes contributing to early embryo development. It is well known that semen quality can be influenced by occasional physical stress, infection, and variation in temperature and the definition of new markers for evaluation of semen could offer knowledge about the fertility potential of a semen sample. The aim of the present study was to evaluate the presence and the relative quantity of transcripts and protein of heat shock protein 70 (HSP70), 90 (HSP90) and clusterin (CLU) in Percoll-selected spermatozoa collected from seven adult boars of proven fertility routinely employed for artificial insemination. Our results showed the presence of HSP70, HSP90 and CLU transcripts with different level of expression: high for HSPs and low for CLU transcripts. The transcript level of both HSPs are similar among selected spermatozoa derived from high quality sperm with the exception of one boar that showed a reduced content of HSP70 and HSP90 mRNA together with a lower semen quality. At protein level, both HSPs were detected with similar amount among all seven boars whilst no band was evidenced for CLU protein.

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs.

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi-organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well-characterized model of LPS-induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS-induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL-1beta in response to LPS, while augmenting the anti-inflammatory cytokine IL-10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS-induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Embelin supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of pig oocytes.

Oxidative stress caused from in vitro culture contributes to inadequate oocyte maturation which leads to a poor embryo development. Therefore, it is important to protect oocytes and embryos against oxidative stress. This study was aimed at evaluating the effect of Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone), an antioxidant with various pharmacologic activities, on nuclear and cytoplasmic maturation of pig oocytes as well as on steroidogenesis of cumulus cells (CCs). Another objective was to determine the influence of Embelin on developmental competence of pig oocytes as well as the expression levels of three key genes (Nanog, Sox2 and Oct4) involved in the control of pluripotency in parthenogenetically activated embryos. Embelin (0, 10, 20 and 40 µM) was added during in vitro maturation of cumulus oocyte complexes; media of both the first and the second day of culture were collected and assayed for progesterone and estradiol-17ß. At the end of the maturation period, the oocytes were fixed (to determine nuclear maturation) or partenogenically activated to evaluate cytoplasmic maturation and genes expression. Embelin did not exert any effect on the proportion of MII oocytes, steroidogenesis of CCs, percentage of embryos that developed to blastocyst stage and the number of blastomeres/blastocyst. Moreover, no significant differences of Oct4, Nanog and Sox2 transcripts were detected in blastocyst stage embryos. In conclusion, Embelin did not influence the reproductive parameters assessed, confirming that it is not possible to predict whether the beneficial effect exerted by an antioxidant in a particular tissue could be present also in another one.

Laparoscopic insemination technique with low numbers of spermatozoa in superovulated prepuberal gilts for biotechnological application.

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36 h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 x 10(9)spermatozoa/5 mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 x 10(8), 1.5 x 10(7), 1 x 10(7), 5 x 10(6) or 1 x 10(6)) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P < 0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 +/- 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 +/- 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.

Effect of tributyltin on mammalian endothelial cell integrity.

Tributyltin (TBT), is a man-made pollutants, known to accumulate along the food chain, acting as an endocrine disruptor in marine organisms, with toxic and adverse effects in many tissues including vascular system. Based on the absence of specific studies of TBT effects on endothelial cells, we aimed to evaluate the toxicity of TBT on primary culture of porcine aortic endothelial cells (pAECs), pig being an excellent model to study human cardiovascular disease. pAECs were exposed for 24h to TBT (100, 250, 500, 750 and 1000nM) showing a dose dependent decrease in cell viability through both apoptosis and necrosis. Moreover the ability of TBT (100 and 500nM) to influence endothelial gene expression was investigated at 1, 7 and 15h of treatment. Gene expression of tight junction molecules, occludin (OCLN) and tight junction protein-1 (ZO-1) was reduced while monocyte adhesion and adhesion molecules ICAM-1 and VCAM-1 (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) levels increased significantly at 1h. IL-6 and estrogen receptors 1 and 2 (ESR-1 and ESR-2) mRNAs, after a transient decrease, reached the maximum levels after 15h of exposure. Finally, we demonstrated that TBT altered endothelial functionality greatly increasing monocyte adhesion. These findings indicate that TBT deeply alters endothelial profile, disrupting their structure and interfering with their ability to interact with molecules and other cells.

Effect of photoperiod on endocrine profiles and vitellogenin expression in European eels Anguilla anguilla during artificially induced ovarian development.

The aim of this work was to determine the effects of dark and light conditions on the E2, testosterone and thyroid hormones levels and on the gene expression levels (vitellogenin 1, vitellogenin 2, and estradiol receptor one) in European eels (Anguilla anguilla) during ovarian development induced by increasing doses of carp pituitary extracts (CPEs). The subjects were divided into 2 groups: 14-hour light:10-hour dark (Light Group) and 24-hour darkness (Dark Group). All the eels received intramuscular injections with CPE at a dosage of 10 mg/kg body weight (BW) once a week for the first 3 weeks, 20 mg/kg BW fourth-sixth week, 30 mg/kg BW seventh-ninth week, and 40 mg/kg up to the end of the experiment (13th week). Vitellogenin and estradiol receptor expression levels did not show significant differences between the two housing conditions whereas in both groups vitellogenin mRNA increased starting from first CPE injection. Testosterone and 17-beta estradiol plasma levels were significantly greater in the Dark Group compared with the Light Group starting from the ninth and the 13th week, respectively. These results suggest that darkness could be a useful variable for standardizing gonadal maturation in eels kept in captivity.

A study on some welfare-related parameters of hDAF transgenic pigs when compared with their conventional close relatives.

Pigs are increasingly used in medical research as transgenic laboratory animals; however, little knowledge is presently available concerning their welfare assessment. The aim of the present study was to investigate some welfare-related parameters of transgenic pigs intended for xenotrasplantation (human decay-accelerating factor (hDAF)) when compared with their conventional (i.e. not transgenic) close relatives (full sibs and half sibs). A total of 14 Large White female transgenic pigs and 10 female non-transgenic (conventional) pigs from four litters were used. All pigs were from the same conventional boar, donor of the semen treated for sperm-mediated gene transfer. During the experiment, BW ranged from 50 to about 80 kg and pigs were weighed at the beginning and at the end of the experiment. Animals were subjected to a set of behavioural tests: a human approach test (HAT), a novel object test (NOT) and an open-door test (ODT). Food preferences were tested through the offer of different foods (banana, apple, carrot, cracker and lemon). During a 4-day period, pigs were diurnally videotaped to study the prevalence of the different behaviours and social interactions (aggressive and non-aggressive interactions). At the end of the trial, cortisol level had been assessed on bristles. No significant differences (P>0.05) were observed between hDAF transgenic and conventional pigs with respect to growth traits, reactivity towards unexpected situations (HAT, NOT, ODT), food preferences, main behavioural traits, social interactions and hair cortisol.

Orexin system expression in the gastrointestinal tract of pigs.

The aim of the present study was to characterize the expression of both proteins and gene transcripts for orexins (OXA and OXB) and their cognate receptors (OX1R and OX2R) in the different gastrointestinal sections of pigs. Using immunohistochemistry, OXA and OXB were found to be co-expressed in the same endocrine cells localized in the basal third of the glands of the body portion of the stomach. Using double immunostaining technique, these orexin-immunoreactive (IR) cells co-stored ghrelin and gastrin. Apparently, OX1R was also expressed within the same cells, forming the tubular gastric gland which displayed positive immunostaining for orexins and the other peptides. Neurons of the enteric nervous system of the stomach were not immunolabeled. We did not find any definite OXA- or OXB-IR cells as well as any immunosignal for orexin receptors in sections of the duodenum, ileum, cecum and rectum. PPOX, OX1R, OX2R mRNA were similarly expressed in all the gastrointestinal tracts. Gastrin and ghrelin showed the highest levels of expression in the gastric mucosa, but their abundance decreased along the subsequent tracts. Thus, in pigs, orexins do not play any role in the local control of intestinal motility and secretion but may rather be involved as endocrine modulators for the regulation of feeding and metabolic homeostasis. However, the co-localization of ghrelin and gastrin with both orexins in the same endocrine cells of the gastric glands suggests that these gut peptides may collaborate in the regulation of gastric secretion, energy homeostasis, body weight and food intake.

Pulmonary kinetic expression of the endothelin system in a swine model of endotoxic shock.

Endothelin (ET)-1 is a potent vasoconstrictor peptide involved in the derangement of respiratory mechanics during endotoxic shock. We measured the kinetics of pulmonary mRNA expression of the key components of the ET system [i.e., ET-1, ET-converting enzyme (ECE), and ETA and ETB receptors] by quantitative real-time reverse transcriptase-polymerase chain reaction in a swine model of endotoxic shock (0, 1, 2, 3, and 4 h of continuous LPS infusion at 40 microg/kg/hour; sham group, 4 hour saline infusion). A significant increase in mRNA expression levels was observed for ET-1 in LPS-treated piglets; the increase began as early as 1 hour. In contrast, no significant variations were observed for the ECE, ETA, or ETB genes. Small gene expression differences observed with respect to our previous results suggest a possible effect of the anesthesia or surgical protocol on ET system regulation.

Evaluation of swine fertilisation medium (SFM) efficiency in preserving spermatozoa quality during long-term storage in comparison to four commercial swine extenders.

In pig production, artificial insemination is widely carried out and the use of fresh diluted semen is predominant. For this reason, there are increasing interests in developing new extenders and in establishing the optimal storage conditions for diluted spermatozoa. In the last few decades, we utilised a homemade diluent (swine fertilisation medium (SFM)) for spermatozoa manipulation and biotechnological application as the production of transgenic pigs utilising the sperm-mediated gene transfer technique. The purpose of the present study is therefore to analyse the ability of SFM, in comparison to four commercial extenders, in preserving the quality of diluted boar semen stored at 16.5°C till 15 days. We utilised some of the main predictive tests as objectively measured motility, acrosome and sperm membrane integrity, high mitochondrial membrane potential and pH. Based on our in vitro study, SFM could be declared as a good long-term extender, able to preserve spermatozoa quality as well as Androhep Enduraguard for up to 6 to 9 days and more.

Sperm-mediated gene transfer-treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine.

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100 microg/mL) of DNA than usual (5 microg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.

Stimulatory effects of fasting on vascular endothelial growth factor (VEGF) production by growing pig ovarian follicles.

The aim of this study was to investigate the effect of fasting on both vascular endothelial growth factor (VEGF) production and VEGF mRNA expression in growing ovarian follicles (>5 mm in diameter) from gilts at 48 h after equine chorionic gonadotrophin (eCG) treatment. The concentrations of VEGF and albumin were measured in the follicular fluid of single follicles, and VEGF mRNA was determined in the follicle wall. Fasting resulted in a significant increase in VEGF concentrations in follicular fluid (20.64+/-0.72 versus 10.79+/-0.86 ng ml(-1), P<0.001), but it did not affect the total amount of VEGF mRNA in the follicle wall compared with that of fed animals. However, VEGF mRNA in the theca and granulosa compartments increased and decreased, respectively, compared with that of fed animals. The concentrations of albumin measured in follicular fluid as an index of vessel permeability were higher in fasted than in animals fed normally, most likely as a result of the increased VEGF production. Follicular steroidogenesis was impaired in fasted animals. Progesterone was the most abundant steroid in the follicular fluid and oestradiol was present in lower concentrations, thus indicating an alteration in the steroidogenic enzymatic cascade. In conclusion, fasting induces an increase in both VEGF production and vessel permeability. Such a reaction is unable under severe food deprivation to preserve follicle function, but may represent a mechanism that regulates blood vessel extension and distribution in relation to tissue requirements and availability of systemic nutrient.

Follicle activation involves vascular endothelial growth factor production and increased blood vessel extension.

The authors evaluated the relationship between vascular endothelial growth factor (VEGF) production, blood vessel extension, and steroidogenesis in small (<4 mm), medium (4-5 mm), and large (>5 mm) follicles isolated from gilts treated with eCG. VEGF and estradiol levels were measured in follicular fluid by an enzyme immunoassay and radioimmunoassay, respectively, and then each follicle wall was used to evaluate VEGF mRNA content and for the immunohistochemical analysis of blood vessels. VEGF production was low in small follicles (<3 ng/ml), high in large follicles (>10 ng/ml), and markedly differentiated in medium follicles; 44% exhibited values up to 15 ng/ml, whereas the levels never exceeded 3 ng/ml in the remaining aliquot. Medium follicles were then used as a model to investigate angiogenesis. Reverse transcription-polymerase chain reaction for VEGF mRNA demonstrated that granulosa cells represent the main component involved in the production of VEGF. The follicle wall, which presents two distinct concentric vessel networks, showed a vascular area (positive stained area/percent of field area) that was significantly wider in high VEGF follicles than in low VEGF follicles (2.54% +/- 0.58% vs. 1.29% +/- 0.58%, respectively). Medium follicles with high VEGF levels and extensive vascularization accumulated high estradiol levels (150-300 ng/ml), whereas follicles with low VEGF levels had basal estradiol levels that never exceeded 30 ng/ml. Early atretic medium-size follicles had undetectable levels of VEGF and estradiol paralleled by a marked reduction in blood vessel. The data presented propose an improved model for follicle dynamics in which the production of VEGF, stimulated by gonadotropin, creates the vascular conditions required for follicle growth and activity.