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We evaluated the effects of a Bacillus-based direct-fed microbial (DFM) on total in vitro gas production, dry matter (DM), neutral detergent fiber (NDF), and starch disappearance of different feedstuffs and total mixed rations (TMR) in three different experiments. In experiment 1, six single fiber-based feedstuffs were evaluated: alfalfa hay, buffalo grass, beet pulp, eragrostis hay, oat hay, and smutsvinger grass. Experimental treatments were control (with no probiotic inoculation; CON) or incubation of a probiotic mixture containing Bacillus licheniformis and B. subtilis (3.2 × 109 CFU/g; DFM). The calculation of DFM dose under in vitro conditions was based on the assumption of a rumen capacity of 70 liter and the dose of 3 g of the DFM mixture/head/d (9.6 × 109 CFU). Total in vitro gas production, DM, and NDF disappearance were evaluated at 24- and 48 h posttreatment incubation. Mean treatment effects were observed at 24- and 48 h gas production (P < 0.0001), as DFM incubation increased in vitro gas production by 5.0% and 6.5%, respectively. For nutrient digestibility, mean DM digestibility was increased at 48 h (P = 0.05), whereas mean NDF digestibility increased at both timepoints by incubating DFM in vitro (P ≤ 0.02). In experiment 2, nine commercial dairy TMR were collected and evaluated for the same variables and treatments described in experiment 1, with the additional analysis of starch digestibility at 7 h post in vitro incubation. The only difference was the concentration of the DFM included, being representative for a dosage of 8.8 × 109 CFU/head/d. In vitro gas production was increased only at 48 h due to DFM incubation (P = 0.05), whereas DM and NDF digestibility were improved at 24 and 48 h (P ≤ 0.02). No treatment effects were observed on in vitro starch digestibility (P = 0.31). In experiment 3, a combined analysis of DM and NDF digestibility was performed by using quality values (NDF and crude protein or CP) of 16 substrates. Regardless of CP and NDF levels of the substrates, DFM improved in vitro 24 and 48 h DM and NDF digestibility (P ≤ 0.03). In summary, incubating a Bacillus-based DFM (B. licheniformis and B. subtilis; BOVACILLUS) improved mean in vitro gas production, DM, and NDF digestibility of single feedstuffs and commercial dairy TMR, highlighting the potential of this combination of Bacillus spp. to improve nutrient utilization, mainly fiber.

The evaluation of sperm functionality and morphology allows discerning between high and low quality ejaculates, but does not give detailed predictive information regarding in vivo fertility. The current developments in statistical modeling have helped in carrying out reproductive studies, but their biggest limitation is in the size of the dataset to be used. The aim of the present observational study was to evaluate whether advanced statistical approaches, such as mixed effects regression models and bootstrap resampling, can help in assessing the predictive ability of semen parameters in terms of in vivo fertility (farrowing rate and litter size), on a small/medium farm with a limited number of animals. Data regarding 33 ejaculates, including viability, subjective motility and acrosome reaction, were collected. Two hundred and thirty-five sows were inseminated with an outcome of 167 deliveries and 1734 newborn piglets. In order to evaluate the relationships among the parameters measured and fertility, mixed effects regression statistical models were used. Once the covariates to be included in the final models were identified, non-parametric bootstrapping was used. The results showed that the farrowing rate was highly associated with the total number of spermatozoa and subjective motility, while litter size was associated with percentage of acrosome reaction. In conclusion, the proposed statistical approach seemed to be suitable for studies regarding reproduction and fertility, even for relatively small sample sizes. Nonetheless, larger data sets are still preferable and required in order to achieve higher reliability.

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 16.58C 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.

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

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.

Lipopolysaccharide (LPS) is a highly proactive molecule that causes in vivo a systemic inflammatory response syndrome and activates in vitro the inflammatory pathway in different cellular types, including endothelial cells (EC). Because the proinflammatory status could lead to EC injury and apoptosis, the expression of proinflammatory genes must be finely regulated through the induction of protective genes. This study aimed at determining whether an LPS exposure is effective in inducing apoptosis in primary cultures of porcine aortic endothelial cells and in stimulating heat shock protein (Hsp)70 and Hsp32 production as well as vascular endothelial growth factor (VEGF) secretion. Cells between third and eighth passage were exposed to 10 microg/mL LPS for 1, 7, 15, and 24 hours (time-course experiments) or to 1, 10, and 100 microg/mL LPS for 7 and 15 hours (dose-response experiments). Apoptosis was not affected by 1 microg/mL LPS but significantly increased in a dose-dependent manner with the highest LPS doses. Furthermore, apoptosis rate increased only till 15 hours of LPS exposure. LPS stimulated VEGF secretion in a dose-dependent manner; its effect became significant after 7 hours and reached a plateau after 15 hours. Both Hsp70 and Hsp32 expressions were induced by LPS in a dose-dependent manner after 7 hours. Subsequent studies were addressed to evaluate the protective role of Hsp32, Hsp70, and VEGF. Hemin, an Hsp32 inducer (5, 20, 50 microM), and recombinant VEGF (100 and 200 ng/mL), were added to the culture 2 hours before LPS (10 microg/mL for 24 hours); to induce Hsp70 expression, cells were heat shocked (42 degrees C for 1 hour) 15 hours before LPS (10 microg/mL for 24 hours). Hemin exposure upregulated Hsp32 expression in a dose-dependent manner and protected cells against LPS-induced apoptosis. Heat shock (HS) stimulated Hsp70 expression but failed to reduce LPS-induced apoptosis; VEGF addition did not protect cells against LPS-induced apoptosis at any dose tested. Nevertheless, when treatments were associated, a reduction of LPS-induced apoptosis was always observed; the reduction was maximal when all the treatments (HS + Hemin + VEGF) were associated. In conclusion, this study demonstrates that LPS is effective in evoking "the heat shock response" with an increase of nonspecific protective molecules (namely Hsp70 and Hsp32) and of VEGF, a specific EC growth factor. The protective role of Hsp32 was also demonstrated. Further investigations are required to clarify the synergic effect of Hsp32, Hsp70, and VEGF, thus elucidating the possible interaction between these molecules.

Semen quality assessment represents a fundamental step for obtaining successful artificial insemination (AI). In commercial settings, the semen employed for AI should be of high quality but traditional semen quality estimates are not sufficiently sensitive to discriminate between differences among samples in terms of fertilising ability. Therefore, more discriminative sperm characteristics need to be identified in order to better predict fertility outcome. In the present study, a series of molecular aspects of semen, represented by heat shock proteins, oxidative stress status, antioxidant potential and tumour necrosis factor (TNF)-alpha were evaluated and analysed. Several relationships between traditional and investigated molecular semen quality estimates were found by using a multivariate analysis approach. Tumour necrosis factor (TNF)-alpha was identified in boar seminal plasma resulting in positive correlations with several sperm quality aspects and particularly with motility. The protective roles of antioxidant molecules and heat shock proteins have been demonstrated confirming the data previously published in the literature.

Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins. (c) 2005 Wiley-Liss, Inc.

Stress proteins are induced in response to a wide range of biological and physicochemical stresses; HSC70 (constitutive) and HSP70 (inducible) belong to the HSP70 stress protein family. The present study aimed at investigating whether environmental stress, particularly thermal and oxidative stress, is involved in modulating HSP70/HSC70 expression in in vitro porcine embryos from two/four cell stage to blastocyst. For oxidative stress, embryos were cultured at 38.5 degrees C under a 5% O2-5% CO2 atmosphere or 5% CO2 in air (approximately 20% O2); for thermal stress, embryos were cultured at 38.5 degrees C under 5% O2, 5% CO2, and exposed to heat shock (1 hr at 42 degrees C). At the end of culture, embryos were analysed by Western blotting, using specific antibodies discriminating HSP70 from HSC70. Embryos cultured under 20% O2 showed HSC70 levels significantly higher (P < 0.005) than embryos cultured under 5% O2, while heat shocked embryos presented HSP70 levels higher (P < 0.01) than control group. In addition, the developmental rate of embryos was negatively affected by the higher oxygen tension (P < 0.05). Our data indicate that porcine embryos express both HSP70 and HSC70 and could differentially respond to both oxidative and heat stress by up-regulating HSC70 and HSP70, respectively.

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 (P0.05). 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.