Studio delle dinamiche di mercato e analisi del contesto competitivo, riguardante il settore dell'artigianato orafo, attraverso l'elaborazione del Business Plan, per un'impresa artigiana in fase di start-up

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Analysis of factors affecting embryo recovery in superovulated buffaloes

The main objective of this thesis was to investigate the different factors affecting the embryo recovery in superovulated buffaloes. Several hypotheses have been formulated over the years to explain the low embryo recovery recorded in this species after superovulatory treatment, among which the most likely is a failure of ovum capture. Before addressing the major issue, other experiments were carried out in order to select both the donors and the most suitable superstimulation treatment. Indeed, another critical factor limiting the application of this technology in buffalo is the high between-animal variability in the number of embryos produced. Therefore, the aim of Experiment 1 was to evaluate whether the Anti-Mullerian hormone (AMH) can be used as a reliable marker to select good donors in addition to the screening of the antral follicular count (AFC), before enrolling buffaloes in embryo production programs. In order to do so, 54 multiparous Italian Mediterranean Buffalo cows were synchronized by double prostaglandin administration spaced 11 days apart and were slaughtered at a local authorized abattoir 6 days after the second administration. On all animals AFC, recovery rate and oocyte quality were recorded, and both plasma and follicular fluid (FF) were collected for AMH determination. In a representative number (n=22) the mRNA expression of six genes (AMHR2, CYP19A1, FSHR, LHR, TP53INP1, and CASP3) was analysed in granulosa cells (GCs). Finally, buffaloes were classified according to the AFC as good (≥ 12 follicles) and bad (< 12 follicles) donors, in order to evaluate the differences in reproductive parameters. Differences in AMH concentration in FF and gene expression in GCs among classes of follicles were analysed by ANOVA. Pearson correlation analysis was carried out to evaluate the relationship between AMH and the AFC, as well as among other reproductive parameters. The differences in AMH plasma levels and reproductive parameters between good and bad donors were analysed by Student’s t test. The results of Experiment 1 showed a positive correlation (r = 0.31; P<0.05) between intrafollicular AMH concentration and the AFC in buffalo. Interestingly, good donors had a higher (P < 0.05) concentration of AMH in FF and AMHR2 levels in small follicles and higher (P < 0.05) LHR levels in large follicles than bad donors. However, no correlation was found with the plasmatic AMH levels, likely due to the poor sensitivity of the available kits. Thus, one of the future objectives could be to assess a more reliable specific assay in order to use this as a marker to screen donors. The aim of Experiment 2 was to evaluate the efficacy of replacing the last four FSH injections with a single administration of 1000 IU of eCG compared to the conventional FSH decreasing doses superovulation protocol. The study was carried out on 20 multiparous Italian Mediterranean buffaloes synchronized with a common double prostaglandin protocol. Then, animals were divided in two groups: one treated with a superovulatory protocol with FSH decreasing doses and one with the eCG replacing the last four FSH administrations. All cows were artificially inseminated at oestrus and ultrasonographic investigations in the two groups were carried out to assess follicular dynamics and to establish the number and size of corpora lutea on the ovaries. Differences between treatments were analysed by Student’s t test. It was demonstrated that the replacement of the last four injections of FSH with a single administration of eCG within a superovulation protocol in buffalo is not recommended, due to the presence of a high number of anovulatory follicles at the moment of flushing, and a reduced number of ova compared to the conventional FSH treatment (3.0 ± 0.6 vs 6.6 ± 0.9 and 2.0 ± 1.2 vs 0.8 ± 0.6 respectively). However, a poor recovery rate in relation to the number of corpus luteum (CL) recorded in both protocols remains. In order to select the most reliable control group for the superovulation study, the aim of Experiment 3 was to evaluate the relationship between the ovarian follicular response at the start of an Ovsynch program and the pregnancy outcome. The study involved 116 pluriparous buffaloes that were synchronized by Ovsynch–TAI program, involving the administration of GnRH on Day 0 (GnRH1), PGF2a on Day 7, and GnRH on Day 9 (GnRH2), with timed artificial insemination (TAI) carried out on Day 10. Ovarian ultrasound examinations were undertaken on days 0, 2, 7, 9, 10, and 11 to record total follicles, the dimensions of follicles greater than 0.75 cm, dimensions of the CL, and the occurrence of ovulation. Blood concentrations of progesterone (P4) were measured on days 0 and 10 and 10 days after TAI. Pregnancy status was determined on days 27 and 45 after TAI to calculate late embryonic mortality. The data were analysed by ANOVA and multiple logistic regression. The results of Experiment 3 showed that only 60% of the buffaloes ovulated after the first GnRH on day 0 of the Ovsynch-TAI program. A higher proportion (P <0.01) of buffaloes that ovulated after GnRH1 had a vascularized corpus luteum (CL) on day 7. Furthermore, a greater proportion (P<0.05) of buffaloes that ovulated after GnRH1 was in oestrus and both a higher ovulation rate and pregnancy to AI were observed compared to buffaloes that did not ovulate. The results of the experiment 3 showed how important is the ovulation after the 1st GnRH to the pregnancy outcome, during Ovsynch-TAI program. This is due to both an optimized response of the CL to PGF2α on day 7 and a better response of the preovulatory follicle to second GnRH on day 9, increasing the likelihood of pregnancy to TAI. The results of this experiment allowed us to better plan the experiment 4, by selecting the animals that ovulated after the 1st GnRH as a control group to compare with superovulated animals. Therefore, the aim of Experiment 4 was to compare the morphological parameters of the follicles and the steroid profile (progesterone and oestradiol) both in plasma and follicular fluid in the peri-ovulatory period in superovulated vs synchronized buffaloes. Thirty-five multiparous Italian Mediterranean Buffalo cows were randomLy divided into two groups: a group (n = 25) was synchronized by Ovsynch and another group (n=10) was superovulated with conventional FSH protocol and all of them were sacrificed 18 h after GnRH. On all animals AFC, recovery rate and oocyte quality were recorded, and both plasma and FF were collected for steroid profile determination. In addition, out of 10 animals (5/group), GCs were collected to analyse the mRNA expression of gonadotropin receptors LHR and FSHR while oviducts were collected to evaluate the mRNA expression of steroid receptors ER1 and PGR, VEGF and the VEGF receptor FLK1. Differences in morphological follicular parameters, as well as in the concentration of progesterone and oestradiol both in plasma and in follicular fluid were analysed by ANOVA, while differences in the recovery rate and in the percentage of oocytes showing cumulus expansion were analysed by Chi Square Test. Experiment 4 demonstrated that in superovulated animals the E2 and P4 concentrations in FF were lower (P<0.05) than in synchronized animals. Interestingly, both the recovery rate and the percentage of oocytes exhibiting proper cumulus expansion decreased (P<0.05) in the superovulated animals. Finally, in superovulated buffaloes a decreased expression of both ER1 and the VEGF receptor FLK1 in the infundibulum and an increased expression of FSHR in granulosa cells were observed (P<0.05). The results of the latter experiment suggest that the exogenous FSH treatment has probably increased the expression of FSHR in granulosa cells that was not followed by a parallel increase of oestradiol synthesis, eliciting an alteration of the granulosa cell function. The consequent altered steroid profile could affect in turn both the cumulus cells expansion during maturation, and the contraction-relaxation of the infundibulum, confirming the hypothesis of a failure in ovum capture in superovulated buffaloes. In conclusion, the results of this thesis taken together highlight the limitations of MOET in buffalo, suggesting that at present the only way to improve the intensity of genetic selection, through the maternal lineage, in this species is the OPU-IVEP technology. Indeed, buffaloes do respond to the gonadotropin treatment with multiple ovulations but the ova capture is severely impaired, resulting in poor embryo recovery. Nevertheless, the outputs of this thesis led to an improved knowledge and understanding of the limiting factors of the superovulation technique in this species

Enhanced Glutathione Levels and Oxidoresistance Mediated by Increased Glucose-6-phosphate Dehydrogenase Expression *

Glucose-6-phosphate dehydrogenase (G6PD) is the key enzyme of the pentose phosphate pathway that is responsible for the generation of NADPH, which is required in many detoxifying reactions. We have recently demonstrated that G6PD expression is induced by a variety of chemical agents acting at different steps in the biochemical pathway controlling the intracellular redox status. Although we obtained evidence that the oxidative stress-mediated enhancement of G6PD expression is a general phenomenon, the functional significance of such G6PD induction after oxidant insult is still poorly understood. In this report, we used a GSH-depleting drug that determines a marked decrease in the intracellular pool of reduced glutathione and a gradual but notable increase in G6PD expression. Both effects are seen soon after drug addition. Once G6PD activity has reached the maximum, the GSH pool is restored. We suggest and also provide the first direct evidence that G6PD induction serves to maintain and regenerate the intracellular GSH pool. We used HeLa cell clones stably transfected with the human G6PD gene that display higher G6PD activity than the parent HeLa cells. Although the activities of glutathione peroxidase, glutathione reductase, and catalase were comparable in all strains, the concentrations of GSH were significantly higher in G6PD-overexpressing clones. A direct consequence of GSH increase in these cells is a decreased reactive oxygen species production, which makes these cells less sensitive to the oxidative burst produced by external stimuli. Indeed, all clones that constitutively overexpress G6PD exhibited strong protection against oxidants-mediated cell killing. We also observe that NF-kappa B activation, in response to tumor necrosis factor-alpha treatment, is strongly reduced in human HeLa cells overexpressing G6PD

Early Prediction of Corpus Luteum Functionality Using an Imaging Software

The present study aimed to assess the applicability of luteal blood flow data acquired through the use of color Doppler ultrasonography and a post-processing analysis tool (ImageJ) for predicting pregnancy in buffaloes (Bubalus bubalis). The experiment was carried out on 59 multiparous Italian Mediterranean buffaloes that underwent synchronization of estrus and fixed-time artificial insemination (TAI). Corpus luteum features (size: CLS and blood flow: BFA) were taken from Day 5 to 10 after TAI and retrospectively measured with ImageJ. In the same period, blood samples were taken to assess progesterone (P4) concentrations. Pregnancy diagnosis was carried out on Day 45 by ultrasound and confirmed on Day 70 post-TAI. Differences in CLS, BFA, and P4 concentrations from Day 5 to 10 after TAI measured between groups were analyzed by ANOVA repeated measures as were differences within each day of measuring. Buffaloes that established a pregnancy (n = 29; 55%) had larger CLS (2.2 ± 0.1 vs. 1.9 ± 0.1 cm2; P < 0.01), higher BFA (0.6 ± 0.0 vs. 0.4 ± 0.0 cm2; P < 0.01), and higher P4 blood level (1.8 ± 0.1 vs. 1.4 ± 0.1; P < 0.01) during Day 5–10 as compared to not-pregnant buffaloes (n = 22). Throughout the entire period, the first feature that changed between groups was P4 blood concentration at Day 7 (1.7 ± 0.1 vs. 1.2 ± 0.1; P < 0.05) followed by BFA at Day 8 (0.6 ± 0.0 vs. 0.5 ± 0.0; P < 0.05), respectively, in pregnant and not-pregnant animals. The ROC analyses indicated that P4 was able to predict pregnancy since Day 5 (P < 0.05) although a more reliable result could be obtained from Day 8 (P < 0.01). At Day 10, it was possible to set a cutoff value for every parameter taken into account. The logistic regression analysis showed that pregnancy was positively influenced by P4 concentration (odds ratio 534.127; P < 0.01) and BFA (odds ratio 744.893; P < 0.01). In conclusion, the use of color Doppler ultrasonography, together with ImageJ, identified different patterns of BFA between pregnant and not-pregnant buffaloes starting from Day 8 post-TAI

Pregnancies following Protocols for Repetitive Synchronization of Ovulation in Primiparous Buffaloes in Different Seasons

Primiparous buffaloes were tested in two periods of the year characterized, by either low or high reproductive efficiency. They were subjected to two protocols for synchronization of ovulation: (i) Ovsynch (OV) and (ii) progesterone based (P4) treatment. After calving, the animals underwent a series of four cycles of re-synchronization protocols. The season did not affect pregnancy rates when the results of the two treatments were pooled together with regard to the first synchronization protocol, followed by AI. Pregnancy rates were similar during the low breeding season (50.3% vs. 57.4% in OV and P4, respectively), but different during the high breeding season (50.4% vs. 67.7% in OV and P4, respectively; p = 0.000). Logistic regression confirmed a significant effect of treatment and season interaction on pregnancy (p = 0.003). Following re-synchronization, a treatment by season interaction was detected during the low breeding season (odds ratio = 2.233), in favor of P4. Finally, a survival analysis showed a better response of animals subjected to P4 treatment from the second AI onward. In conclusion, the pooled data of pregnancy rates from both treatments between seasons are not different following AIs. Better results, though, were obtained from the implementation of P4 treatment, and are recorded in a season-fashioned mode when the comparison is made following first or cumulative AIs

Relationship among Milk Conductivity, Production Traits, and Somatic Cell Score in the Italian Mediterranean Buffalo

The measurement of milk electrical conductivity (EC) is a relatively simple and inexpensive technique that has been evaluated as a routine method for the diagnosis of mastitis in dairy farms. The aim of this study was to obtain further knowledge on relationships between EC, production traits and somatic cell count (SCC) in Italian Mediterranean Buffalo. The original dataset included 5411 records collected from 808 buffalo cows. Two mixed models were used to evaluate both the effect of EC on MY, PP and FP and EC at test-day, and the effect of EC on somatic cell score (SCS) by using five different parameters (EC_param), namely: EC collected at the official milk recording test day (EC_day0), EC collected 3 days before official milk recording (EC_day3), and three statistics calculated from EC collected 1, 3 and 5 days before each test-day, respectively. All effects included in the model were significant for all traits, with the only exception of the effect of EC nested within parity for FP. The relationship between EC and SCS was always positive, but of different magnitude according to the parity. The regression of EC on SCS at test-day using different EC parameters was always significant except when the regression parameter was the slope obtained from a linear regression of EC collected over the 5-day period. Moreover, in order to evaluate how well the different models fit the data, three parameters were used: the Average Information Criteria (AIC), the marginal R2 and the conditional R2. According to AIC and to both the Marginal and Conditional R2, the best results were obtained when the regression parameter was the mean EC estimated over the 5-day period

Effect of photoperiod on follicular IGF-1 and oocyte quality independently of metabolic status in buffalo heifers

The aim of this study was to determine whether buffalo heifers maintained at a constant live weight (LW) and body condition score (BCS) by a restricted diet show changes in reproductive function in response to changes in day length. Heifers were fed a low energy diet (86% daily intake recommended for the species) throughout the study, which involved an 8-week period of decreasing day length and a 7-week period of increasing day length. Weekly, LW, BCS and ovarian follicular/oocyte population were assessed. Blood and follicular fluid samples were taken at the time of ovum pick-up for hormone analyses and at 30-day intervals to measure metabolic substrates. Buffalo heifers maintained LW and BCS throughout the study and blood concentrations of the main metabolic hormones, such as GH, insulin, IGF-1 and thyroid hormones were not influenced by photoperiod. Likewise, there were no differences in metabolic substrates such as glucose, NEFA, triglycerides and HDL cholesterol, suggesting that the metabolic condition remained essentially the same. During increasing day length periods, the number of total follicles (8.4 ± 0.5 vs. 11.9 ± 0.6; p < .01), cumulus-oocyte complexes (2.2 ± 0.3 vs 4.3 ± 0.5; p < .01) and superior quality Grade A + B COCs (0.2 ± 0.1 vs 1.1 ± 0.2; p < .01) decreased. These features were associated to reduced oestradiol levels both in plasma (3.9 ± 0.4 vs 9.2 ± 0.5 pg/mL; p < .01) and follicular fluid (49.7 ± 12.9 vs. 109.1 ± 25.8 ng/mL; p = .05), as well as to reduced intrafollicular IGF-1 levels (45.5 ± 4.0 vs. 58.5 ± 3.9 ng/mL; p < .05). These findings suggest that the ovarian function in buffalo heifers is influenced by photoperiod independent of nutritional and metabolic status.Highlights Metabolic profile, LW and BCS do not change in buffalo heifers on a restricted diet Photoperiod affects ovarian function in heifers regardless of metabolic status Follicular and oocyte population decrease in increasing day length periods Oestradiol and IGF-1 levels in follicular fluid are affected by photoperio

Productive Performance and Meat Characteristics of Kids Fed a Red Orange and Lemon Extract

This study evaluates the animal performance and meat characteristics of 60 Saanen suckling kids daily fed a red orange and lemon extract (RLE), rich in anthocyanins. In our methodology, after colostrum administration, animals are randomly assigned to two treatments: Treatment group (Group RLE; n = 30) that received RLE (90 mg/kg live body weight) as oral food additive, and a control group (Group CON; n = 30) that received a standard diet. Animals are slaughtered after 40 days. The RLE administration did not influence daily weight gain, carcass measurements, or incidences (expressed as a percentage) of different anatomical regions on the whole carcass weight. On the contrary, RLE supplementation significantly improved the oxidative profile of the meat seven days after slaughtering, as demonstrated by the reduced levels of thiobarbituric acid reactive substances (TBARS; p &lt; 0.01) and hydroperoxides (p &lt; 0.01) in Group RLE compared to Group CON. A significant influence of RLE administration is observed on day 7 for yellowness (p &lt; 0.01). There are also lower saturated and higher monounsaturated and polyunsaturated fatty acids concentration in Group RLE meat (p &lt; 0.01), which also shows lower atherogenic and thrombogenic indexes (p &lt; 0.01) compared to Group CON. The study demonstrates that the supplementation of a diet with RLE rich in anthocyanins is effective to improve the meat quality