Elisabetta Salimei

Measurement of thyroid hormones in donkey (Equus asinus) blood and milk: validation of ELISA kits and evaluation of sample collection, handling and storage

Donkeys milk is well tolerated by human infants with cows milk allergy and is useful in the treatment of human immune-related diseases and in the prevention of atherosclerosis. Thyroid hormones (TH) stimulate lactation and active triiodothyronine (T3) in colostrum and milk could take paracrine action supporting lactogenesis in the mother, and play physiological roles for the suckling offspring (systemic or within the gastrointestinal tract). The aims were to measure TH concentrations in donkey blood and milk, validate ELISA methods, evaluate the effects of sample collection and post-collection handling and the stability of TH in milk and blood serum and plasma samples. In milk and blood samples obtained from lactating jennies total concentrations of TH were assayed using competitive-type ELISA kits. Good validation results were obtained for both TH concentrations in blood serum and plasma and T3 in milk samples extracted with cold (-20°C) ethanol alkalinized (pH 9·0) with NH4OH. In most of the milk extract samples, thyroxine (T4) concentrations resulted below the sensitivity threshold. Intra- and inter-assay coefficients of variations of TH concentrations in different blood and milk samples were below 10%. Parallelism tests gave displacement lines parallel to those of the calibrators for both TH in blood serum and plasma and for T3 in milk extracts. Mean recovery rates were between 95% and 123%, but the concentration values approaching the highest calibrators were overestimated. Therefore, serum and plasma samples for T3 assay must be previously diluted with buffer. Both TH concentrations in blood serum and plasma and T3 in milk did not change during storage for up to 6 months at -20°C. In conclusion, the ELISA methods tested in the present study are suitable for determination of both TH concentrations in donkey blood samples, and for T3 measurement in milk, after extraction with cold alkaline ethanol.

Ca, Mg, Zn, Fe, Cu and Mn content of ass’s milk

Abstract The aim of this study was to determine Ca, Mg, Zn, Fe, Cu and Mn content of ass’s milk. Twenty four individual milk samples were collected from 4 lactating asses. During the experimental period milk samples were collected every 3 weeks interval, using a milking machine; asses were housed with the foals that were separated from the jennets 3 hours before milking. Milk was analysed for Ca, Mg, Zn, Fe, Cu and Mn content by atomic absorption spectrometry. The concentration mean (±SD) of Ca Mg, Zn, Fe, and Cu were respectively 334.61±39.80, 58.46±8.43, 1.99±0.51, 1.15±0.52, 0.16±0.06 mg/kg. Mn was found only at trace level. Iron content of ass’s milk was the most variable ranging from 0.43 to 1.88 mg/kg. Correlation coefficients were positive and significant between Ca and Mg (r=0.63), Zn and Mg (r=0.45), Zn and Fe (r=0.49) and Zn and Cu (r=0.50). In this study, except for Fe, mean concentration of Ca, Mg, Zn, and Cu in ass’s milk was similar to those reported in literature for human milk.

Isoelectric Focusing and ELISA for Detecting Adulteration of Donkey Milk with Cow Milk

Donkey milk has been recently revalued intensely due to its nutritional properties. Moreover, donkey milk has been proposed as an effective alternative food for some infants with cow milk allergy. Two fast analytical methods were proposed to detect the fraudulent practice of blending cow milk to donkey milk. Detection of cow αs1-casein bands along the profiles of experimental donkey-cow milk mixtures analyzed by isoelectric focusing was adequate to estimate cow milk used as adulterant of donkey milk starting from 5% (v/v). An ELISA-based method using the antipeptide antibodies raised against the 1-28 sequence stretch of cow β-casein was also developed for an accurate definition of composition of donkey-cow milk mixtures. The presence of cow milk at levels as low as 0.5% (v/v) was detected in donkey-cow milk mixtures prepared at laboratory scale and assayed by ELISA.

Microbiological characteristics of raw ass’s milk: manual vs. machine milking

Bearing in mind the objectives of this study to investigate the Barbary sheep fertility under our climatic conditions and to evaluate the efficiency of the prostaglandin f2α(pgf2α)injections in induction of fertile estrus in different seasons. For these objectives, some experiments were performed during the period from July,2008 to March, 2009. A total number of 300 Libyan Barbary ewes(3-6 years old, weighing 40-60 kg) was used in this experiment. Ewes were kept in privet farms, fed and managed similarly. Ewes were divided to four season groups(summer, autumn, winter and spring groups). Each season group was divided into treated and control. Animals in the treated groups were injected with double injections of 125 μg of prostaglandin f2α intramuscularly(i/m), 11 days apart. While those in control groups were injected with two injections of 1.0 ml of 0.9% NACL saline solution simultaneously with the treated ewes. At the same day (day 11) rams wearing painted sponges on their briskets regions were introduced for natural mating. Treated groups showed shorter estrus response time than control groups in all seasons(P<0.05). Estrus duration was longer in winter and spring than in summer and autumn (P<0.001),but no difference was found between treated and control groups inside seasons. Treated group showed higher pregnancy rate(P<0.001) in winter season than control group. Percentage of ewes lambed in winter was significantly high (P<0.001) among treated ewes than control (80 vs 38%). Lambing rate differed significantly (P<0.001) among treated groups in all seasons. Data were collected and calculated statistically using SPSS system for percentages, means, standard deviation(mean ± sd) analysis of variance (anova), Chi square and Dunacan’s test were used accordingly. Other values were measured, calculated and analyzed similarly. Adoption of technology in sheep farms of La Mancha, Spain J. Rivas1, C. De Pablos2, J. Perea1, C. Barba1, R. Dios-Palomares1, M. Morantes1 and A. García1 1Universidad de Córdoba, Campus Rabanales, 14014, Córdoba, Spain, 2Universidad Rey Juan Carlos, Paseo de los Artilleros, 28034, Madrid, Spain; pa1gamaa@uco.es Recently in Spain the milk production coming from sheep farms shows higher degrees of specialization based on the adoption of technologies. The aim of this research is to examine the pattern of adoption of technologies in sheep farms of La Mancha. Based on previous researches, from 77 questions, only 38 questions were selected by using qualitative and participatory methods; the chosen variables were grouped into six technology packages (TP): management, feeding, animal health and milk quality, pasture and land use, equipment and facilities, and reproduction and breeding program. The survey was applied to a sample of 157 farms. Using descriptive statistics each TP was characterized and the pattern of adoption was determined. The average of technologies adopted was of 18.4±6.0 (48.3%). TP showing higher degrees of implementation are animal health and milk quality (67.8%), feeding (56.0%) and management (55.7%), but their adoption is not sequential or responds to independent events. This research facilitates the identification of a number of technologies that must be implemented from an organizational strategy point of view. Moreover, all technologies are seeking a dynamic balance system that allows firms migrate to more efficient processes without losing their main attributes. As a technological challenge, an andrologic evaluation of ram, gynecologic evaluation of ewes prior to mating, early detection of non-productive animals are proposed; and a better use of productive records for the decision-making; aspects are recommended. The results of this analysis will have an impact on future research that attempts to improve the use of subproducts, forage reserves and improved rangeland management and hygiene control system, taking the quality milk as an strategic asset, so further research is necessary to assess the impact of each technology on the operating of the mixed system in the Mancha region. Session 40 Poster 20 Session 40 Poster 19In Italy, as in other European countries, since 2005 a program of selection for scrapie resistance in sheep, based on the PRNP polymorphism, has been implemented with the aim of increasing ARR ‘resistant’ allele and eliminating VRQ ‘susceptible’ allele. In a small breed, the ARR-carriers may be more related to each other than ranndomly chosen animals; as a consequence, for an equal number of reproducers the effective size may be smaller than expected in a pure genetic drift condition (1). In the Sambucana sheep breed (reared in Piemonte region, north-west Italy) the ARR allele frequency was higher than in other breeds, like Biellese for example, before the selection plan started (2). On the other hand, due to the reduced number of animals (3500), this breed is considered at risk of extinction (Piemonte Regional Rural Development Plan for 2007-2013 period). The aim of the present investigation was to evalute the impact of scrapie resistance selection on genetic variation of the Sambucana. INTRODUCTIONCONCLUSIONS During the last decade there has been growing scientific interest in the use of genomic information as an additional tool in conventional dairy cows’ breeding schemes. It is now well established that dairy cows’ fertility has declined during the last decades and this may not be remedied only by improved management. Given that there is a substantial genetic background in this decline, emphasis should be placed in efforts to improve dairy cows’ fertility through genetic selection. Although there are substantial genetic effects that contribute to this infertility, little progress has been made on the identification of major genes affecting reproduction traits. There is an urgent need to identify the genetic factors responsible for the decline in fertility in cattle. The objective of this study was to investigate the association of two specific polymorphisms, the SNP12195 and SNP11646 polymorphisms of the Signal Transducer and Activator of Transcription 5A (STAT5A) and Fibroblast Growth Factor 2 (FGF2) genes respectively with fertility in cattle. These genes have been previously reported to be associated with in vitro fertilization and embryonic survival rate in cattle.In European grasslands, grazing and mowing regimes are major drivers of the maintenance of many wild species, either through direct or indirect effects. To date, both types of effects were studied separately, mainly at the field scale and with no quantification of performance. Objective of this study was to model the trade-offs between ecological and productive performance of a grassland agro-ecosystem at two nested scales i.e. field and farm scales. We developed a dynamic model linking grass dynamics controlled by grazing or mowing to stochastic population dynamics of two grassland bird species. Bird dynamics were driven by both direct and indirect effects of management. Viable control framework was used to predict productive and ecological performance. At field scale, results showed that the best ecological performance was obtained at intermediate levels of productive performance (60 to 108 grazing days /ha /year). Above 108 grazing days, no grazing strategy was viable for either species due to the negative direct effects of management. Between 108 and 240 grazing days, the ecological-productive relationship showed a concave Pareto like frontier. Any improvement in productive performance entailed a strong decrease in ecological performance. As expected, at farm scale the best ecological performance was obtained with extensive farms. However, the ecological-productive relationship showed a convex Pareto like frontier indicating the occurrence of compensations among management regimes. In intensive farms, it was necessary to allocate 40% of farm area in low-intensity grazing in order to compensate for the negative effects of mowing and high-intensity grazing on birds. However this land allocation involved a 25% reduction in productive performance. Finally, we explore several forms of complementarities among farm types to improve overall performance.

Technological use of donkey milk in cheesemaking

The efficacy of donkey milk as an alternative to hens egg white lysozyme in preventing cheese blowing during ripening of Italian Grana cheeses was tested. Indices of proteolysis and lipolysis did not differ between the different cheesemaking processes. All tested products showed good microbiological and sensorial quality.

Hygiene and health parameters of donkey’s milk

Bearing in mind the objectives of this study to investigate the Barbary sheep fertility under our climatic conditions and to evaluate the efficiency of the prostaglandin f2α(pgf2α)injections in induction of fertile estrus in different seasons. For these objectives, some experiments were performed during the period from July,2008 to March, 2009. A total number of 300 Libyan Barbary ewes(3-6 years old, weighing 40-60 kg) was used in this experiment. Ewes were kept in privet farms, fed and managed similarly. Ewes were divided to four season groups(summer, autumn, winter and spring groups). Each season group was divided into treated and control. Animals in the treated groups were injected with double injections of 125 μg of prostaglandin f2α intramuscularly(i/m), 11 days apart. While those in control groups were injected with two injections of 1.0 ml of 0.9% NACL saline solution simultaneously with the treated ewes. At the same day (day 11) rams wearing painted sponges on their briskets regions were introduced for natural mating. Treated groups showed shorter estrus response time than control groups in all seasons(P<0.05). Estrus duration was longer in winter and spring than in summer and autumn (P<0.001),but no difference was found between treated and control groups inside seasons. Treated group showed higher pregnancy rate(P<0.001) in winter season than control group. Percentage of ewes lambed in winter was significantly high (P<0.001) among treated ewes than control (80 vs 38%). Lambing rate differed significantly (P<0.001) among treated groups in all seasons. Data were collected and calculated statistically using SPSS system for percentages, means, standard deviation(mean ± sd) analysis of variance (anova), Chi square and Dunacan’s test were used accordingly. Other values were measured, calculated and analyzed similarly. Adoption of technology in sheep farms of La Mancha, Spain J. Rivas1, C. De Pablos2, J. Perea1, C. Barba1, R. Dios-Palomares1, M. Morantes1 and A. García1 1Universidad de Córdoba, Campus Rabanales, 14014, Córdoba, Spain, 2Universidad Rey Juan Carlos, Paseo de los Artilleros, 28034, Madrid, Spain; pa1gamaa@uco.es Recently in Spain the milk production coming from sheep farms shows higher degrees of specialization based on the adoption of technologies. The aim of this research is to examine the pattern of adoption of technologies in sheep farms of La Mancha. Based on previous researches, from 77 questions, only 38 questions were selected by using qualitative and participatory methods; the chosen variables were grouped into six technology packages (TP): management, feeding, animal health and milk quality, pasture and land use, equipment and facilities, and reproduction and breeding program. The survey was applied to a sample of 157 farms. Using descriptive statistics each TP was characterized and the pattern of adoption was determined. The average of technologies adopted was of 18.4±6.0 (48.3%). TP showing higher degrees of implementation are animal health and milk quality (67.8%), feeding (56.0%) and management (55.7%), but their adoption is not sequential or responds to independent events. This research facilitates the identification of a number of technologies that must be implemented from an organizational strategy point of view. Moreover, all technologies are seeking a dynamic balance system that allows firms migrate to more efficient processes without losing their main attributes. As a technological challenge, an andrologic evaluation of ram, gynecologic evaluation of ewes prior to mating, early detection of non-productive animals are proposed; and a better use of productive records for the decision-making; aspects are recommended. The results of this analysis will have an impact on future research that attempts to improve the use of subproducts, forage reserves and improved rangeland management and hygiene control system, taking the quality milk as an strategic asset, so further research is necessary to assess the impact of each technology on the operating of the mixed system in the Mancha region. Session 40 Poster 20 Session 40 Poster 19In Italy, as in other European countries, since 2005 a program of selection for scrapie resistance in sheep, based on the PRNP polymorphism, has been implemented with the aim of increasing ARR ‘resistant’ allele and eliminating VRQ ‘susceptible’ allele. In a small breed, the ARR-carriers may be more related to each other than ranndomly chosen animals; as a consequence, for an equal number of reproducers the effective size may be smaller than expected in a pure genetic drift condition (1). In the Sambucana sheep breed (reared in Piemonte region, north-west Italy) the ARR allele frequency was higher than in other breeds, like Biellese for example, before the selection plan started (2). On the other hand, due to the reduced number of animals (3500), this breed is considered at risk of extinction (Piemonte Regional Rural Development Plan for 2007-2013 period). The aim of the present investigation was to evalute the impact of scrapie resistance selection on genetic variation of the Sambucana. INTRODUCTIONCONCLUSIONS During the last decade there has been growing scientific interest in the use of genomic information as an additional tool in conventional dairy cows’ breeding schemes. It is now well established that dairy cows’ fertility has declined during the last decades and this may not be remedied only by improved management. Given that there is a substantial genetic background in this decline, emphasis should be placed in efforts to improve dairy cows’ fertility through genetic selection. Although there are substantial genetic effects that contribute to this infertility, little progress has been made on the identification of major genes affecting reproduction traits. There is an urgent need to identify the genetic factors responsible for the decline in fertility in cattle. The objective of this study was to investigate the association of two specific polymorphisms, the SNP12195 and SNP11646 polymorphisms of the Signal Transducer and Activator of Transcription 5A (STAT5A) and Fibroblast Growth Factor 2 (FGF2) genes respectively with fertility in cattle. These genes have been previously reported to be associated with in vitro fertilization and embryonic survival rate in cattle.In European grasslands, grazing and mowing regimes are major drivers of the maintenance of many wild species, either through direct or indirect effects. To date, both types of effects were studied separately, mainly at the field scale and with no quantification of performance. Objective of this study was to model the trade-offs between ecological and productive performance of a grassland agro-ecosystem at two nested scales i.e. field and farm scales. We developed a dynamic model linking grass dynamics controlled by grazing or mowing to stochastic population dynamics of two grassland bird species. Bird dynamics were driven by both direct and indirect effects of management. Viable control framework was used to predict productive and ecological performance. At field scale, results showed that the best ecological performance was obtained at intermediate levels of productive performance (60 to 108 grazing days /ha /year). Above 108 grazing days, no grazing strategy was viable for either species due to the negative direct effects of management. Between 108 and 240 grazing days, the ecological-productive relationship showed a concave Pareto like frontier. Any improvement in productive performance entailed a strong decrease in ecological performance. As expected, at farm scale the best ecological performance was obtained with extensive farms. However, the ecological-productive relationship showed a convex Pareto like frontier indicating the occurrence of compensations among management regimes. In intensive farms, it was necessary to allocate 40% of farm area in low-intensity grazing in order to compensate for the negative effects of mowing and high-intensity grazing on birds. However this land allocation involved a 25% reduction in productive performance. Finally, we explore several forms of complementarities among farm types to improve overall performance.

Purification and identification of alpha(s-1) and beta-caseins from asses milk

Recent clinical studies have demonstrated that feeding with asses milk is a safe and valid treatment for infants affected by protein intolerance to dairy cows milk (Businco et al., 2000; Iacono et al., 1992). In fact, the composition of asses milk is similar to humans milk, especially concerning the lipid and protein fraction contents (Fantuz et al., 2001; Salimei et al., 2004). Furthermore, the protein fraction in asses milk is characterized by low casein and β-lactoglobulin content, probably responsible for the hypoallergenic characteristics of this milk, and by a quite high lysozime content (Fantuz et al., 2001; Salimei et al., 2004). In any case, there is not much information regarding the caseinic fractions of asses milk in the literature. Therefore, in the present study αS1and β-caseins were purified from asses milk by gel filtration chromatography followed by anion exchange chromatography on HPLC. The obtained caseins were characterized by SDS-PAGE and, after blotting on a PVDF membrane, were identified by N-terminal sequencing.