Aim: In vitro and in vivo challenge studies were undertaken to develop an in-feed additive of microencapsulated propionic, sorbic acids and pure botanicals to control Campylobacter jejuni in broilers at slaughter age. Methods and Results: Organic acids (OA) and pure botanicals were tested in vitro against Camp. jejuni, whereas in vivo, chickens were fed either a control diet, or increasing doses of the additive for 42 days (experiment 1); in the second experiment, chickens received the additive at 0Á1 or 0Á3% from day 0 to 21 or from day 22 to 42. The additive consistently reduced Camp. jejuni caecal counts at any given dose (exp. 1) or inclusion plan (exp. 2). Moreover, it was able to reduce the number of goblet cells and modify mucin glycoconjugates biosynthesis pattern. Conclusions: We developed an additive that was effective in reducing Camp. jejuni in slaughter-age chickens even at low doses (0Á1%). That efficacy was the result of the synergistic action between OA and botanicals. Significance and Impact of the Study: This study provides a strategy to reduce Camp. jejuni in broilers and, as a consequence, to improve the safety of the food chain. Moreover, data suggest that a treatment limited to the last weeks before slaughter would allow to save on inclusion of the additive throughout the whole production cycle.

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.

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The reduction of Salmonella prevalence in broilers is a priority in European Union agricultural policies because treatment with antibiotics is forbidden by Regulation (EC) 2160/2003. Two trials were conducted to evaluate the efficacy of a microencapsulated blend of sorbic acid and nature-identical compounds (i.e., chemically synthesized botanicals; SAB) on the reduction of the cecal prevalence and contents of Salmonella enterica serovars Hadar and Enteritidis in experimentally infected chickens. In the first trial, 125 one-day-old Lohmann specific-pathogen-free chickens were assigned to one of the following treatments: negative control (not challenged and not treated), positive control (challenged and not treated), SAB0.3, SAB1, or SAB5 (challenged and treated with the microencapsulated blend included in the feed at 0.03, 0.1, or 0.5%, respectively). At 30 d of age, birds were infected with 10(6) cfu of Salmonella Hadar, and after 5, 10, or 20 d postinfection, 5, 10, and 10 birds per treatment, respectively, were killed and the cecal contents and liver and spleen samples were analyzed for Salmonella Hadar. In the second trial, 100 one-day-old Ross 708 chickens were assigned to 1 of 5 treatments: control (not treated), SAB0.3, SAB1, SAB2, or SAB5 (treated with the blend included in the feed at 0.03, 0.1, 0.2, or 0.5%, respectively). At 7 d of age, the birds were challenged with 10(5) cfu of Salmonella Enteritidis, and after 7, 14, or 24 d after challenge, 5, 5, and 10 birds per treatment, respectively, were killed and cecal contents were analyzed for Salmonella Enteritidis. Results showed that in the early stage of infection Salmonella prevalence was high in both studies, whereas at the end of the observation periods, the blends at 0.03, 0.1, and 0.5 in the challenge with Salmonella Hadar and at 0.2 and 0.5% in the challenge with Salmonella Enteritidis significantly reduced (by 2 log(10) cfu) the cecal content of Salmonella. This study showed that intestinal delivery of microencapsulated sorbic acid and nature-identical compounds can result in a 100-fold reduction of Salmonella at the intestinal level in broilers at slaughter age.

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.Keywords: boar, spermatozoa quality, extender, semen storage IntroductionArtificial insemination (AI) is used routinely because it is simple, economical and successful (Vishwanath, 2003); it remains the main vehicle, together with embryo transfer, for the rapid dispersal of valuable genes and reduces the sexual transmission of diseases (Thacker et al., 1984). The use of fresh diluted semen, preserved at 15 to 208C, is predominant (99% AI carried out worldwide/year) while AI with frozen semen, due to the low sperm survival after thawing, accounts for only 1% (Gerrits et al., 2005). For this reason, there has been increased interest 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 swine spermatozoa manipulation and biotechnological applications as the production of transgenic pigs utilising the sperm-mediated gene transfer technique (Lavitrano et al., 2002;Lavitrano et al., 2003), with good fertility results as well as in AI or surgical or laparoscopic inseminations Manzini et al., 2006). Nevertheless, this SFM has never been evaluated methodically in comparison to other well-defined commercial diluents.Even if extenders, in association with low temperature, can prolong the spermatozoa lifespan, the physiological senescence of sperm cells still cannot be completely avoided. Such a phenomenon can be composed of non-regulated capacitation-like modifications (Bailey et al., 2000), structural and functional changes (Johnson et al., 2000), loss of DNA integrity (Fraser and Strzezek, 2004) and membrane fatty acid peroxidation (Cerolini et al., 2000); these modifications can be postponed by using various extenders (Huo et al., 2002). Long-term extenders have some advantages; they allow better organisation at semen collection centres, longdistance transport and the possibility of conducting tests on the semen before use (Gadea, 2003); h...