Human social buffer in goats and dogs.

The primary goal of this study was to explore the social buffering effect that humans offer to goats and dogs with limited exposure to human socialization, particularly in situations involving interactions with unfamiliar humans. A total of 13 dogs and 14 goats were selected for the study, all of which had limited prior socialization with humans. Each animal was placed in a testing room with unfamiliar humans for 15 min. Three experimenters aimed to establish a comfortable environment, encouraging social interaction by offering food to the animals and assessing the animals' willingness to accept food and their response to being approached and petted. If both conditions were satisfied, the animals were classified as "social". If one or none of the conditions were met, the animals were classified as "not social". Cortisol levels were measured by collecting blood samples before and after the test. Non-parametric tests together with a GzLM showed that the effect of human social buffering in goats was different in comparison to dogs: goats exhibited higher cortisol levels after the test, while dogs did not show a significant change. Further analysis demonstrated that "social" goats had a lower likelihood of experiencing significant changes in cortisol levels than dogs. Thus, once human interactions are accepted, both species could benefit from social buffering. In summary, this study enhances our understanding of how dogs and goats respond to social interactions with humans in the social buffering effect.

The Italian Cilentana goat breed: productive performances and economic perspectives of goat farming in marginal areas.

In the internal areas of Cilento, province of Salerno (Campania), the Cilentana, a small native goat breed population, is reared quite widespread, due to its dual-purpose attitude and to its typical productions. The extensive livestock system adopted for this breed allows the use of otherwise abandoned territories and a sustainable farming capable of ensuring high-quality levels. In addition, Cilentana goat farming represents an important source of income for the local communities and also preserves the territory itself guaranteeing the protection of biodiversity and the conservation of local activities that have a historical tradition. The aim of this study is twofold: give an overview of Cilentana breed morphological and productive traits linked to the historical and gastronomic tradition of the area and emphasize the economic role of this breed in the perspective of the ongoing new Common Agricultural Policy (CAP) rules.

Effects of ecotrofin™ on milk yield, milk quality and serum biochemistry in lactating goats.

A nutritional supplement (Ecotrofin™, by Vetoquinol Italia S.r.l) recommended in ruminants feeding to strengthen the physiological condition and improve digestive performance was tested in 20 pluriparae grazing goats divided in two groups (control and treated) to assess its possible effects on milk yield and quality and to assess eventual adverse effects. Animals from both groups also received 400 g/day of corn meal, and the treated group was supplemented with 20 g/head/day of the nutritional supplement. At the doses suggested by the manufacturer, despite a transient increase after 30 days of supplementation, Ecotrofin™ did not show significant effects on milk yield and, although some changes were found in the fatty acids profile, no significant improvement of MUFA and PUFA, as well as of omega-6:omega-3 ratio and CLA content were seen. Therefore, in our experimental conditions the supplementation of diet with Ecotrofin™ did not appear useful to improve goat's performance. A significant effect on kidney health markers (27 vs. 22.5 for urea and 0.83 vs. 0.76 for creatinine, p < 0.05) suggested a beneficial effect on renal function but, since levels fell in the normal ranges in both groups, such hypothesis would need further studies to be addressed.

Milk from cows fed a diet with a high forage:concentrate ratio improves inflammatory state, oxidative stress, and mitochondrial function in rats.

Excessive energy intake may evoke complex biochemical processes characterized by inflammation, oxidative stress, and impairment of mitochondrial function that represent the main factors underlying noncommunicable diseases. Because cow milk is widely used for human nutrition and in food industry processing, the nutritional quality of milk is of special interest with respect to human health. In our study, we analyzed milk produced by dairy cows fed a diet characterized by a high forage:concentrate ratio (high forage milk, HFM). In view of the low n-6:n-3 ratio and high content of conjugated linoleic acid of HFM, we studied the effects of this milk on lipid metabolism, inflammation, mitochondrial function, and oxidative stress in a rat model. To this end, we supplemented for 4 wk the diet of male Wistar rats with HFM and with an isocaloric amount (82 kJ, 22 mL/d) of milk obtained from cows fed a diet with low forage:concentrate ratio, and analyzed the metabolic parameters of the animals. Our results indicate that HFM may positively affect lipid metabolism, leptin:adiponectin ratio, inflammation, mitochondrial function, and oxidative stress, providing the first evidence of the beneficial effects of HFM on rat metabolism.

Nutritional Characteristics of Forage Grown in South of Benin.

In order to provide recommendations on the most useful forage species to smallholder farmers, eleven grass and eleven legume forages grown in Abomey-Calavi in Republic of Benin were investigated for nutritive value (i.e. chemical composition and energy content) and fermentation characteristics (i.e. gas and volatile fatty acid production, organic matter degradability). The in vitro gas production technique was used, incubating the forages for 120 h under anaerobic condition with buffalo rumen fluid. Compared to legume, tropical grass forages showed lower energy (8.07 vs 10.57 MJ/kg dry matter [DM]) and crude protein level (16.10% vs 19.91% DM) and higher cell wall content (neutral detergent fiber: 63.8% vs 40.45% DM), respectively. In grass forages, the chemical composition showed a quite high crude protein content; the in vitro degradability was slightly lower than the range of tropical pasture. The woody legumes were richer in protein and energy and lower in structural carbohydrates than herbaceous plants, however, their in vitro results are influenced by the presence of complex compounds (i.e. tannins). Significant correlations were found between chemical composition and in vitro fermentation characteristics. The in vitro gas production method appears to be a suitable technique for the evaluation of the nutritive value of forages in developing countries.

Characterization and effect of year of harvest on the nutritional properties of three varieties of white lupine (Lupinus albus L.).

BACKGROUND: Three cultivars of Lupinus albus L. (Lutteur, Lublanca and Multitalia) were assessed for proximate composition, fatty acids, alkaloids and in vitro fermentation characteristics over three harvest years. RESULTS: The chemical composition varied greatly during the three harvest years. Crude protein content ranged from 353 to 456 g kg(-1) dry matter (DM), neutral detergent fiber content from 209 to 321 g kg(-1) DM and lignin content from 3.0 to 63.9 g kg(-1) DM. Lublanc showed the highest crude protein (417 g kg(-1) DM) and lignin (35 g kg(-1) DM) contents. High levels of lipids (89.9 g kg(-1) DM) and starch (93.3 g kg(-1) DM) were found in all samples. Alkaloid content ranged from 3.63 to 165 mg per 100 g. Lutteur and Lublanc showed more favorable n-3/n-6 polyunsaturated fatty acid ratios (from 0.44 to 0.73) and lower values of the anti-quality factor 'erucic acid' (from 5.8 to 20.9 g kg(-1) ) than Multitalia. Lutteur showed higher degradability (897 g kg(-1) ), gas production (330 mL g(-1) organic matter (OM)) and volatile fatty acid production (117 mmol g(-1) OM) than the other varieties. CONCLUSION: The present data suggest L. albus L. cv. Lutteur to be a promising crop as food thanks to its high nutritive traits and most constant yield over time.

Growth Performance of Buffalo Calves in Response to Different Diets with and without Saccharomyces cerevisiae Supplementation.

The aim of the present trial was to evaluate the growth performance of buffalo calves fed on diets characterized by different forage/concentrate ratios, with or without Saccharomyces cerevisiae supplementation (CBS 493.94, Yea-Sacc®). Twenty-four male buffalo calves (mean age of 145.1 ± 16.1 days; mean weight of 108.0 ± 18.7 kg) were assigned randomly to 4 groups, homogeneous in age, that were fed four different diets: diet 1, F:C ratio 50:50; diet 2, F:C ratio 30:70; diet 3, F:C ratio 50:50 + Yea-Sacc®; and diet 4, F:C ratio 30:70 + Yea-Sacc®. Buffalo calves were individually weighted before the start of the experiment and the data were used as a covariate, being taken monthly until the end of the trial. Dry matter intake (DMI), daily weight gain (DWG) and feed conversion ratio (FCR) were calculated. The differences in diets composition significantly (p < 0.01) affected all these parameters. In particular, the animals fed diet 1 and diet 3 showed higher values of DWG (0.91 and 0.88 g/d vs. 0.68 and 0.66 for group 2 and 4) and DMI (5.8 and 5.3 kg/d, respectively) compared to the other groups (4.3 and 4.4 kg/d for group 2 and 4), as well as a higher final body weight (370.5 and 334.1 kg for group 1 and 3 vs. 272.8 and 273.1 kg of group 2 and 4, respectively). Indeed, the supplementation with Yea-Sacc® at the dosage of 1 × 10E8 did not affect buffaloes' growth performance.

The Effects of Hemp Hay (Canapa sativa L.) in the Diets of Grazing Goats on Milk Production and Fatty Acid Profile.

Hemp (Cannabis sativa L.) is a cosmopolitan annual herbaceous plant used in the past as a source of textile fiber. Currently, hemp is receiving great interest as animal feed due to its chemical and nutritional properties. The aim of this study was to explore the effects of supplementing goats' diets with hemp hay on the milk yield, chemical composition, and fatty acid profile. Twenty multiparous goats, immediately after kidding, were divided into two homogenous groups (C: control vs. H: hemp); the goats had free access to the pasture, and both groups received a supplement of 500 g/head/day of a barley and corn meal mixture (50/50). In addition, group H was given 250 g/head/day of hemp hay while group C received the same amount of alfalfa hay. The milk yield was measured daily, and milk samples were collected monthly 4 times to evaluate the milk composition and fatty acid profile. The milk yield was significantly (p < 0.05) higher in the experimental group, while no differences were found in the milk chemical composition. Concerning the fatty acid profile, the milk from group H was characterized by significantly lower concentrations of C11:0, C12:0, C13:0, C14:0, C15:0, and C17:0 and higher C16:0 and C18:0. Among the polyunsaturated FA, C18:2 n6 and C20:4 were significantly (p < 0.001) lower, and C20:5 n3 was significantly (p < 0.05) higher in the milk from group H than that from group C. The n6/n3, LA/ALA and AA/EPA ratios were significantly (p < 0.001) lower in the milk from group H, while the CLAs were unaffected by the treatment.

Diet supplementation with hemp (Cannabis sativa L.) inflorescences: effects on quanti-qualitative milk yield and fatty acid profile on grazing dairy goats.

Hemp (Cannabis sativa L.) is an annual plant belonging to the family of Cannabaceae with several varieties characterized by different fatty acid profile, content in flavonoids, polyphenols, and cannabinoid compounds. Hemp is mostly used in livestock nutrition as oil or as protein cake, but not as inflorescences. The aim of this study was to evaluate the effect of dietary hemp inflorescences on milk yield and composition in grazing dairy goats. Twenty Camosciata delle Alpi goats at their 3rd parity and with a mean body weight of 45.2 ± 2.0 kg, immediately after kidding, were equally allocated into two groups (G: Grazing and GH: grazing and hemp). For three months, all goats were fed on a permanent pasture and received 700/head/day of concentrate; diet of group GH was supplemented with 20 g/head/day of hemp inflorescences. Goats' body weight did not change during the trial. Individual milk yield was daily recorded and samples collected every 20 days for chemical composition and fatty acid profile analysis. No significant differences were found for milk yield and chemical composition. Caproic (C6:0) (1.80 vs. 1.74%; p < 0.01) and lauric acids (C12:0) were significantly higher in milk of group GH (4.83 vs. 4.32%; p < 0.01) as well as linoleic (C18:2) (2.04 vs. 1.93%; p < 0.05), adrenic acid (C22:4) (0.046 vs. 0.031%, p < 0.05), omega-6/omega-3 ratio (3.17 vs. 2.93, p < 0.05) and total conjugated linoleic acids (CLAs) (0.435 vs. 0.417%; p < 0.01). The results of this study suggest that the supplementation of grazing goats' diet with hemp inflorescences may enhance the milk nutritional characteristics by increasing its content of CLAs and other beneficial fatty acids.

A biochemical and cellular approach to explore the antiproliferative and prodifferentiative activity of Aloe arborescens leaf extract.

Aloe arborescens Miller, belonging to the Aloe genus (Liliaceae family), is one of the main varieties of Aloe used worldwide. Although less characterized than the commonest Aloe vera, Aloe arborescens is known to be richer in beneficial phytotherapeutic, anticancer, and radio-protective properties. It is commonly used as a pharmaceutical ingredient for its effect in burn treatment and ability to increase skin wound healing properties. However, very few studies have addressed the biological effects of Aloe at molecular level. The aim of the research is to provide evidences for the antiproliferative properties of Aloe arborescens crude leaf extract using an integrated proteomic and cellular biological approach. We analysed the composition of an Aloe arborescens leaf extract by gas chromatography-mass spectrometry analysis. We found it rich in Aloe-emodin, a hydroxylanthraquinone with known antitumoral activity and in several compounds with anti-oxidant properties. Accordingly, we show that the Aloe extract has antiproliferative effects on several human transformed cell lines and exhibits prodifferentiative effects on both primary and immortalized human keratinocyte. Proteomic analysis of whole cell extracts revealed the presence of proteins with a strong antiproliferative and antimicrobial activity specifically induced in human keratinocytes by Aloe treatment supporting its application as a therapeutical agent.

Role of Milk Micronutrients in Human Health.

The aim of this review is to provide an overview of different compositions, in terms of main minerals and vitamins, of milk from animal species that represent the most common source of this food for humans, highlighting the uniqueness of nutritional qualities linked to animal species. It is known that milk is an important and valuable food for human nutrition, representing an excellent source of nutrients. Indeed, it contains both macronutrients (proteins, carbohydrates, and fat) that contribute to its nutritive and biological value and micronutrients represented by minerals and vitamins, which play a relevant role in the body's various vital functions. Although their supply is represented by small quantities, vitamins and minerals are important components for a healthy diet. Milk composition in terms of minerals and vitamins differs between various animal species. Micronutrients are important components for human health as their deficiency is causes of malnutrition. Furthermore, we report on the most significant metabolic and beneficial effects of certain micronutrients in the milk, emphasizing the importance of this food for human health and the need for some milk enrichment procedures with the most relevant micronutrients to human health.

Forage:Concentrate Ratio Effects on In Vivo Digestibility and In Vitro Degradability of Horse's Diet.

Determination of digestibility represents the first step for the evaluation of the net energy content of feed for livestock animals. The aim of this study was to evaluate the in vivo digestibility and in vitro degradability of five diets characterized by different forage/concentrate ratios (F:C) in horses. The in vitro degradability was determined by the Gas Production Technique (GPT), using as an inoculum source the feces of the same subjects used for the in vivo test. Five diets consisting of poliphyte hay, straw and grains of barley and oats with a different F:C ratio [90/10 (Diet 1); 78/22 (Diet 2); 68/32 (Diet 3); 60/40 (Diet 4); 50/50 (Diet 5) were formulated and administered in succession, starting with Diet 1. In the in vivo results, no significant differences emerged, despite the different F:C content. In in vitro fermentation, four diets out of the five (2, 3, 4, 5) presented a similar trend of the curve of gas production, showing good activity of the fecal micro population during the first hours of incubation. An important correlation between gas and Volatile Fatty Acid (VFA) were found, suggesting that the processes linked to the micro population deriving from the horse's caecum follow metabolic pathways whose products can be modeled in the same way as for the rumen. The GPT could represent the correct method for studying the nutritional characteristics of feed for horses, using feces as the source of inoculum, even if further investigations must be performed to improve the technique.

Colostrum Composition, Characteristics and Management for Buffalo Calves: A Review.

In this review, the composition, characteristics, and management of dairy buffalo calves were examined and compared with bovines. The neonatal period is critical for buffalo calves and is characterized by a high mortality rate (more than 40%). The early intake of high-quality colostrum (IgG > 50 mg/mL) is the one way to improve the immune system of calves (serum IgG > 10 mg/mL after 12 h), thus increasing their chances of survival. Mainly in intensive farms, the availability of high-quality colostrum is necessary; thus, good quality colostrum is often stored to provide newborn calves which cannot be fed by their mothers. Also, the manipulation of the immunological status of animals through vaccination has been depicted since the quality of colostrum tended to be influenced by vaccination against pathogens. Buffalo breeding is constantly expanding in Italy, mainly thanks to the Mozzarella cheese production that represents the excellence of the "Made in Italy" and is exported worldwide. Indeed, high calf mortality rates directly affect the profitability of the business. For these reasons, the aim of this review was to examine specific research on buffalo colostrum that, compared with other species, are scarce. Improving the knowledge of buffalo colostrum, in terms of characteristics and management, is critical to guarantee buffalo newborns' health in order to reduce their mortality rate. Importantly, considering the knowledge on cattle valid also for buffalo is a widespread, and often erroneous, habit in several fields, including colostrum feeding. Therefore, the two species were compared in this review.

Serum Oxytocin, Cortisol and Social Behavior in Calves: A Study in the Impossible Task Paradigm.

In this study, we explored the correlations between circulating levels of oxytocin, cortisol, and different social behaviors toward humans in 26 Italian Red Pied calves (all females, with an average age of 174 ± 24 days) using the impossible task paradigm. This paradigm has proved fruitful in highlighting the effect of socialization on the willingness to interact with humans in several domesticated species. The test consists of the violation of an expectation (recovering food from an experimental apparatus) while a caregiver and a stranger are present. Immediately after the end of the test (less than one minute), blood was collected from the coccygeal vein. Statistics were performed by the Spearman's rank correlation; significant differences were adjusted according to Bonferroni's correction. Cortisol correlates positively (ρ = 0.565; p < 0.05) with the latency of behaviors directed at the caregiver, and the duration of behaviors directed at the apparatus correlates negatively with both the caregiver (ρ = -0.654; p < 0.05) and a stranger (ρ = -0.644; p < 0.05). Contrary to what is reported in the literature on cows, no correlations were found between oxytocin levels and direct behaviors toward the caregiver. This highlights a different behavioral strategy between calves and cows when placed in front of an impossible task.

Nutritional Characterization of Hay Produced in Campania Region: Analysis by the near Infrared Spectroscopy (NIRS) Technology.

Since the dietary characteristics of hays can be very variable, it is of great importance for nutritionists to know their chemical composition in order to formulate adequate rations for the animals. Laboratory analyses are time-consuming and expensive while the Near Infrared Spectroscopy offers several advantages, including obtaining information on feeds nutritional characteristics very quickly and in situ at the farm, thanks to portable NIRS. In this trial, over 400 hay samples collected in the Campania region (Italy) were analyzed with portable NIRS device. The final aim was to analyze the differences in hay quality in different production areas, highlighting the possible factors involved and suggesting possible corrective measures. All the analyzed hays (polyphite, Gramineae and alfalfa) were significantly (p < 0.05) influenced by the area of cultivation/preservation. In particular, the polyphite and Gramineae hays produced in most of the areas of Campania region showed poor nutritional value due to the low protein content and high structural carbohydrate that significantly reduced its digestibility. The use of high-quality forages in the ration represents a pivotal factor to allow the production of high-quality products of animal origin. The use of NIRS seems to be a valuable strategy to select potential treatments that can increase feed digestibility and to avoid long chemical analysis.

Serum Oxytocin in Cows Is Positively Correlated with Caregiver Interactions in the Impossible Task Paradigm.

This study explored a possible relationship between the circulating oxytocin, cortisol, and the willingness of dairy cows to engage in social behaviors with humans in an experimental context. The behaviors of twenty-nine cows were recorded during the impossible task paradigm, a procedure aimed at creating a violation of expectancy, in the presence of the caregiver and a stranger. The results showed that serum oxytocin levels were positively correlated with duration and negatively correlated with the latency of the cows' social interactions with the caregiver. This research provides a clear correlation between circulating oxytocin and a willingness to engage in social contact with the caregiver, excluding the possible effect of different cortisol levels on such behavior.

Effects of Linseed Supplementation on Milk Production, Composition, Odd- and Branched-Chain Fatty Acids, and on Serum Biochemistry in Cilentana Grazing Goats.

The purpose of this study was to investigate the effects of linseed supplementation on milk yield and quality, serum biochemistry and, in particular, to evaluate its possible effects on the production of odd- and branched-chain fatty acids (OBCFA) in the milk of Cilentana grazing goats. Twelve pregnant Cilentana dairy goats were divided into two groups (CTR, control, and LIN, linseed supplementation group). After kidding, the goats had free access to the pasture and both groups received a supplement of 400 g/head of concentrate, but the one administered to the LIN group was characterized by the addition of linseed (in a ratio of 20% as fed) to the ingredients. During the trial, milk samples were taken from April to August in order to evaluate milk production, composition, and fatty acid profile. In addition, blood samples were taken for evaluating the effects of linseed supplementation on goats' health status. The health status of the goats was not influenced by the linseed supplementation, as confirmed by blood analyses. Concerning the effects on milk, the supplementation positively affected (p < 0.001) milk production and fat percentage and the fatty acid profile was markedly influenced by the lipid supplementation. In particular, milk from the LIN group was characterized by significantly lower concentrations of saturated fatty acids (FA; p < 0.001) and higher proportions of monounsaturated FA, polyunsaturated FA, and conjugated linoleic acids (CLAs) than milk from the CTR group (p < 0.001). In contrast, the OBCFA were negatively influenced by the linseed supplementation (p < 0.0001). Further studies are needed to test the effects of different fat sources and other nutrients on the diets.

Effects of a Nutritional Supplement (DìRelaxTM) on Anxiety in Dogs in a Randomized Control Trial Design.

This study aimed to investigate the efficacy of DìRelaxTM, a nutraceutical formulated to reduce anxiety in dogs, using a randomized controlled trial (RCT) design. The C-BARQ questionnaire, some clinical investigations, and the impossible task test were performed in dogs before and after treatment. The C-BARQ questionnaire is particularly useful for assessing the frequency and severity of problematic behaviors. The impossible task paradigm provides insight into the decision-making processes in the realm of expectancy frustration. Results showed an ameliorative effect on the performances of treated dogs during the solvable phases, with a significant decrease in the time needed to solve the task. No behavioral difference was found between treated and untreated anxious dogs during the unsolvable phase. According to the results from the C-BARQ questionnaire, some of the behaviors appeared to improve. Clinical investigations, including a complete blood cell count and blood chemistry, showed no difference between groups, thus suggesting the safety of the product. In general, this study suggests that DìRelaxTM can be safely administered with no adverse effects and can exercise a beneficial effect on anxious dogs by enhancing their cognitive abilities, but further studies should investigate the best method of administration.

Management of struvite uroliths in dogs.

Urolithiasis is a common clinical problem in dogs. Struvite and calcium oxalate are the predominant mineral types in dog urolithiasis. The aim of the present study was to compare the effect of two commercial dry foods formulated for the management of struvite urolithiasis with different anion-cation balance on urinary pH. For the trial, twelve privately owned adult dogs showing struvite urolithiasis were studied. The dogs were randomly divided into two groups (A and B) and fed two dissolving diets for 3 months. The analyses of urine were repeated six times. In both diets, the anion-cation balance was negative ( - 203 and - 192 for diets A and B, respectively). At the first urine analysis, pH values of all the dogs were close to 8.0, and bacteria were present in about 70 % of the samples and thus an antimicrobial was administered for 1 week. Both groups showed a progressive decrease in pH values, and after 2 months, in both cases, the recommended pH values for stone dissolution were achieved. From the sampling at 30 d, group A showed pH values significantly (P < 0.05) lower than group B, probably due to the lower anion-cation balance of diet A. The combination of antimicrobial and dietary therapy allowed the dissolution of struvite uroliths in both groups, even if the utilisation of the diet characterised by the lower anion-cation balance seems to decrease the urinary pH more rapidly. In this case, it seems necessary to interrupt the dietary treatment in order to avoid the risk of other diseases.

In vitro fermentation characteristics of diets with different forage/concentrate ratios: comparison of rumen and faecal inocula.

BACKGROUND: The aim of this trial was to evaluate the replacement of rumen fluid with faeces as inoculum in studying the in vitro fermentation characteristics of diets for ruminants using the in vitro gas production technique. Six iso-protein diets with different forage/concentrate ratios were incubated with rumen fluid (RI) or faeces (FI) collected from sheep. RESULTS: Most of the fermentation parameters were influenced by diet and inoculum (P < 0.01). With both inocula, organic matter degradability (dOM), cumulative gas production (OMCV) and maximum fermentation rate (R(max) ) increased as the amount of concentrate in the diet increased. R(max) was lower with FI vs RI (P < 0.01); dOM was higher with FI vs RI and the diet × inoculum interaction was significant. As expected, with both inocula, R(max) increased as the neutral detergent fibre content of the diet decreased. Significant correlations were obtained using both inocula between OMCV/dOM and gas/volatile fatty acid (VFA), while the correlation VFA/dOM was significant only with FI. The microbial biomass yield calculated by stoichiometric analysis for all diets was higher with FI vs RI. With FI the organic matter used for microbial growth showed an overall decreasing trend as the amount of concentrate in the diet increased. CONCLUSION: The results indicate that both faeces and rumen fluid from sheep have the potential to be used as inoculum for the in vitro gas production technique.