Beyond the surface: Understanding obsessive symptoms and body perceptions, from shape concerns to fear of blushing.

INTRODUCTION: Recent reclassifications have expanded the understanding of Obsessive-Compulsive Disorders (OCDs), now incorporated into a broader category known as Obsessive-Compulsive Disorder and Related Disorders (OCRDs). This study sought to assess obsessive-compulsive symptoms and body uneasiness among outpatients seeking treatment for Eating Disorders (ED). Additionally, we aimed to explore associations and potential mediation effects between obsessive-compulsive symptoms and body uneasiness. This investigation extended beyond concerns related solely to body shape and weight, encompassing fears associated with specific body components (such as facial features, abdominal region, and limbs) or functions (including sweating, blushing, emitting noises, and releasing odors). METHODS: Psychometric assessments included the Obsessive-Compulsive Inventory-Revised (OCI-R) and the Body Uneasiness Test (BUT). Statistical analyses involved bivariate correlations, linear regression, and mediation analysis to explore the associations and potential mediation effects between obsessive-compulsive symptoms and different manifestations of body uneasiness. RESULTS: The sample (N = 210) demonstrated substantial obsessive-compulsive symptoms and notable body discomfort. OCI-R scores positively correlated with various dimensions of body dissatisfaction, including shape, weight, and specific body components or functions. Linear regression revealed significant associations between OCI-R scores and overall body uneasiness (BUT-A) as well as concerns about body components or functions (BUTB). Mediation analysis indicated that BUT-A mediated the relationship between obsessive-compulsive symptoms and BUTB. CONCLUSION: This study offers new insights into the comprehensive landscape of OCRDs. It specifically emphasizes the association between obsessive-compulsive symptoms and body uneasiness, embracing not only concerns about body shape and weight but also extending to body components and functions.

Dietary supplementation with green tea extract improves the antioxidant status and oocyte developmental competence in Italian Mediterranean buffaloes.

The aim of this work was to assess the antioxidant status and the developmental competence of oocytes recovered by ovum pick-up (OPU) in Italian Mediterranean buffaloes supplemented with green tea extracts (GTE) for 90 days. Buffalo cows (n = 16) were randomly assigned to a control group receiving no supplement and a treatment group, receiving GTE starting 90 days before OPU, carried out for five consecutive sessions. Blood samples were collected before the start of supplementation with GTE (T0) and at day 45 (T1) and day 90 (T2) of supplementation, to measure ferric reducing activity (FRAP), total antioxidant capacity (TAC), superoxide dismutase (SOD) and catalase (CAT). The antioxidant status of follicles was measured as TAC on the follicular fluid collected from the dominant follicle just prior OPU, coinciding with T2, and at the end of five repeated OPU sessions (T3). Another objective was to assess in vitro the protective effects of green tea extracts on hepatic cells exposed to methanol insult. Different concentrations of GTE (0.5 µM and 1 µM) were tested on cultured hepatic cells and viability, morphology and SOD activity were assessed at 24, 48 and 72 h. Supplementation with GTE increased (P < 0.05) the number of total follicles (8.7 ± 0.5 vs 6.9 ± 0.5), the number and the percentage of Grade A + B cumulus-oocyte complexes (COCs) compared with the control (3.7 ± 0.4 vs 2.3 ± 0.3 and 57.5 ± 4.2 vs 40.4 ± 4.9 %, respectively). Oocyte developmental competence was improved in the GTE group as indicated by the higher (P < 0.05) percentages of Grade 1,2 blastocysts (44.8 vs 29.1 %). In the GTE group, plasma TAC was higher both at T1 and T2, while FRAP increased only at T2, with no differences in SOD and CAT. The TAC of follicular fluid was higher (P < 0.05) in the GTE compared to the control both at T2 and at T3 The in vitro experiment showed that co-treatment with methanol and 1 µM GTE increased (p < 0.01) cell viability at 24 h (P < 0.01), 48 h (P < 0.05) and 72 h (P < 0.01) compared with the methanol treatment co-treatment with 1 µM GTE prevented the decrease in SOD activity observed with methanol at 24 and 48 h of culture. In conclusion, the results of in vivo and in vitro experiments suggest that supplementation with GTE increases buffalo oocyte developmental competence, by improving oxidative status and liver function.

Genetic parameters for reproductive traits in the Italian Mediterranean buffalo using milk yield as a correlated trait.

Until now, the genetic evaluation of the Italian Mediterranean Buffalo has been mainly focused on production traits. However, female fertility affects the efficiency of the dairy industry as it is essential to maintain the profitability of dairy farms. Indeed, the estimation of its genetic component is crucial for its improvement. In this study, 3 measures of buffalo's fertility were analyzed: the age at first calving (AFC), the interval between first and second calving (CIV1), and the interval between second and successive calvings (CIV2_12). Milk yield at 270 d (MY270) was used as a correlated trait. First, genetic parameters were estimated using 7,915 buffalo cows with first calving from 1991 to 2018, then breeding values were calculated from 236,087 buffalo cows. Genetic parameters were estimated by Bayesian inference fitting a multiple-trait animal model using the GIBBS1F90 program, and BLUPF90 was used for estimation of breeding value. The heritability and repeatability estimates of fertility traits were low. The genetic correlations among fertility traits ranged from 0.10 (AFC-CIV1) to 0.92 (CIV1-CIV2_12). Genetic correlation between MY270 and fertility traits was unfavorable, ranging from 0.23 to 0.48. The results from this study can be used as a basis for the future genetic improvement of fertility traits in the Italian Mediterranean Buffaloes.

Buffalo milk and rumen fluid metabolome are significantly affected by green feed.

The use of green feed for livestock breeding is an important strategy to encounter both the increasing demand for animal derived products and the perceptions of the consumers regarding animal welfare and sustainability. The aim of this study was to compare different feeding strategies in lactating water buffaloes by using a metabolomic approach. The study was carried out on 32 milking buffaloes that were randomly divided into two groups for a total period of 90 days (3 sampling times). DD Group (dry diet) received a standard total mixed ratio (TMR) characterized by dry forages and concentrates; ZG Group (zero grazing) fed an isoenergetic and isoproteic diet obtained using 30% of sorghum as green forage. Samples of milk and rumen fluid were analyzed by liquid chromatography-mass spectrometry (LC-MS) techniques. Data analyses revealed the presence of several differentially accumulated metabolites and among these, ten compounds were putatively identified in milk samples (i.e. L-carnitine, acetylcarnitine, propionylcarnitine, butyrylcarnitine, 2-methylbutyroylcarnitine, 2-hexenoylcarnitine, hexanoylcarnitine, glycerophosphocholine, δ-valerobetaine and γ-butyrobetaine) and four in rumen fluid (3-(2-hydroxyphenyl) propanoate, Indole-3-acrylic acid, oleamide (cis-9,10-octadecenoamide) and 20-carboxy-leukotriene B4). The modulation of these molecules in buffalo milk is significantly related to the green/dry based feeding and some the natural compound detected could be considered as health-promoting nutrients.

Milk somatic cell count and its relationship with milk yield and quality traits in Italian water buffaloes.

In Southern Italy, buffalo (Bubalus bubalis) milk is mostly intended for the manufacture of Mozzarella di Bufala Campana Protected Denomination of Origin (PDO) cheese. Despite the economic boost of the last 2 decades, the buffalo farming system should be improved to maximize the efficiency of the dairy industry, improve yield and quality of milk and cheese, and work toward better animal welfare. Milk somatic cell count (SCC) is used worldwide as an indicator of udder health in individual milk and is useful for monitoring farm hygiene in bulk milk. Mastitis data are currently not available on a large scale in Italy; thus, SCC is essential for identifying animals with suspected udder infection and inflammation. Moreover, high milk SCC is associated with altered composition and acidity, and poor technological properties of milk. However, payment systems of the PDO area are based simply on the delivered volume of milk rather than on quality characteristics. Hence, currently there are no penalties for elevated SCC in bulk milk in the Italian buffalo dairy industry. In addition, SCC for buffalo milk is not mentioned by either the European Community regulations or the PDO protocol, evidencing a lack of rules for the maximum SCC limit. To provide a phenotypic characterization of SCC at the population level and to improve knowledge on buffalo milk quality, 876,299 test-day records of 70,156 buffaloes reared in the PDO area were analyzed. Data revealed that around 11% of herd-test-dates (≥5 animals sampled each) showed average milk SCC ≥400,000 cells/mL (i.e., above the threshold fixed by the European Community for bovine milk). This suggests that there is room to improve SCC at both the farm and individual level. Within first parity, more than 28 and 15% of lactations had average SCC ≥200,000 and ≥300,000 cells/mL, respectively. Both percentages increased with parity and were 39 and 25% in sixth parity, respectively. Supporting this, the proportion of lactations with average SCC ≥500,000 cells/mL increased from 6% in first parity to 12% in sixth parity. Milk yield and SCC were negatively correlated with each other, especially when SCC level was high. An ANOVA was carried out on test-day record milk yield and composition traits, with fixed effects of parity, lactation stage, class of somatic cell score (n = 6), month of calving, and their interactions; buffalo, herd-test-date, and residual were considered random effects. Significantly lower milk yield and lactose percentage were estimated in progressively higher classes of somatic cell score, whereas no significant differences were observed for fat and protein percentages. This is the first attempt to investigate milk SCC in a large data set of Italian dairy buffaloes. These findings may be helpful for defining reliable and effective SCC thresholds to be adopted whenever specific penalties for high SCC are included in milk payment systems. Finally, these results could be used in mastitis monitoring plans aiming to reduce SCC and udder issues at both the individual and farm levels in the Italian buffalo population.

Integrating RNA-seq and GWAS reveals novel genetic mutations for buffalo reproductive traits.

Genome-wide association study (GWAS) has been applied in buffalo breeding programs and been used to identify a number of candidate genes associated with buffalo reproductive traits. The genetic code of specific genes underlying buffalo reproductive traits remains unclear. Association study that measures both genetic and transcriptional variation has been applied for the investigation of complex traits. To investigate genes involved in buffalo reproductive traits, integrated RNA-seq results were investigated of buffalo granulosa cells and candidate genes which were reported to be associated with buffalo reproductive traits in a previous GWAS. A large number of variants were detected by RNA-seq, and 214 variants were located within the buffalo reproductive candidate genes identified by GWAS. A further association study in 462 Italian Mediterranean buffalo indicated that 25 SNPs distributed in 13 genes were associated with reproductive traits. Of the 13 genes, 11 were expressed in granulosa cells of all antral follicle development stages, and significant difference was found in the expression of NDUFS2 between follicles of diameter <8 mm and > 8 mm. These findings extend the results of GWAS by expanding the knowledge about new and potentially functional single-nucleotide polymorphisms and provide useful information about regulatory genes affecting buffalo reproductive traits.

Genome-wide association studies to identify quantitative trait loci affecting milk production traits in water buffalo.

Water buffalo is the second largest resource of milk supply around the world, and it is well known for its distinctive milk quality in terms of fat, protein, lactose, vitamin, and mineral contents. Understanding the genetic architecture of milk production traits is important for future improvement by the buffalo breeding industry. The advance of genome-wide association studies (GWAS) provides an opportunity to identify potential genetic variants affecting important economical traits. In the present study, GWAS was performed for 489 buffaloes with 1,424 lactation records using the 90K Affymetrix Buffalo SNP Array (Affymetrix/Thermo Fisher Scientific, Santa Clara, CA). Collectively, 4 candidate single nucleotide polymorphisms (SNP) in 2 genomic regions were found to associate with buffalo milk production traits. One region affecting milk fat and protein percentage was located on the equivalent of Bos taurus autosome (BTA)3, spanning 43.3 to 43.8 Mb, which harbored the most likely candidate genes MFSD14A, SLC35A3, and PALMD. The other region on the equivalent of BTA14 at 66.5 to 67.0 Mb contained candidate genes RGS22 and VPS13B and influenced buffalo total milk yield, fat yield, and protein yield. Interestingly, both of the regions were reported to have quantitative trait loci affecting milk performance in dairy cattle. Furthermore, we suggest that buffaloes with the C allele at AX-85148558 and AX-85073877 loci and the G allele at AX-85106096 locus can be selected to improve milk fat yield in this buffalo-breeding program. Meanwhile, the G allele at AX-85063131 locus can be used as the favorable allele for improving milk protein percentage. Genomic prediction showed that the reliability of genomic estimated breeding values (GEBV) of 6 milk production traits ranged from 0.06 to 0.22, and the correlation between estimated breeding values and GEBV ranged from 0.23 to 0.35. These findings provide useful information to understand the genetic basis of buffalo milk properties and may play a role in accelerating buffalo breeding programs using genomic approaches.

Crocetin improves the quality of in vitro-produced bovine embryos: Implications for blastocyst development, cryotolerance, and apoptosis.

The aim of this work was to assess the effect of supplementation of bovine culture medium with the natural antioxidant crocetin on in vitro blastocyst development and quality. This was evaluated as cryotolerance, apoptosis index, and total cells number and allocation. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid medium, supplemented with 0, 1, 2.5, and 5 µM crocetin (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On Day 7, embryo yields were assessed and the blastocysts were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO, and 0.5 M sucrose. Finally, blastocysts produced on Day 8 in the absence (control) and presence of 1 µM crocetin were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and differential staining to evaluate, respectively, the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm (TE) lineages (experiment 2). Embryo development was higher in the 1 µM crocetin group compared to the control, both in terms of total embryo output (37.7 ± 4.2%, 52.9 ± 6.3%, 40.9 ± 7.6%, and 42.4 ± 8.7%, respectively, with 0, 1, 2.5, and 5 µM; P < 0.01) and grade 1 and 2 blastocysts (33.6 ± 4.9%, 46.1 ± 7.3%, 37.8 ± 7.9%, and 39.4 ± 7.9%, respectively, with 0, 1, 2.5, and 5 µM; P < 0.05). Moreover, the percentage of fast-developing embryos increased in 1 µM crocetin group compared to the control (23.4 ± 4.7%, 32.7 ± 6.6%, 27.2 ± 6.6%, and 30.1 ± 7.2%, respectively, with 0, 1, 2.5, and 5 µM; P < 0.05). In addition, the enrichment of culture medium with 1 µM crocetin improved embryo cryotolerance compared to the control, as indicated by higher hatching rates recorded after 48 hours postwarming culture (46.5% vs. 60.4%; P < 0.05). Furthermore, 1 µM crocetin decreased both the average number (9.9 ± 0.4 vs. 7.1 ± 0.3) and the percentage of apoptotic cells (7.1 ± 0.4 vs. 4.2 ± 0.2) in blastocysts compared to the control (P < 0.01). However, no differences were recorded in the average number of ICM, TE, and total cells between 1 µM crocetin and control groups. In conclusion, the enrichment of bovine culture medium with 1 µM crocetin increased both blastocyst yield and quality, as indicated by the improved chronology of embryo development, increased resistance to cryopreservation, and reduced incidence of apoptosis.

L-ergothioneine supplementation during culture improves quality of bovine in vitro-produced embryos.

The aim of this study was to evaluate whether supplementation of bovine culture medium with the natural antioxidant L-ergothioneine (LE), improves in vitro blastocyst development and quality, assessed as resistance to cryopreservation, total cells number, cellular differentiation, and apoptosis index. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, presumptive zygotes were cultured in synthetic oviduct fluid with 0, 0.05 mM, 0.1 mM, 0.5 mM, and 1 mM of LE (experiment 1) at 39 °C under humidified air with 5% CO2, 7% O2, and 88% N2. On the basis of the results of this dose-response trial, the range of concentrations to test was reduced in experiment 2, in which presumptive zygotes were cultured with 0, 0.05 mM, and 0.1 mM of LE. On Day 7, embryo yields were assessed, and the blastocysts (BL) were vitrified by Cryotop method in 16.5% ethylene glycol, 16.5% DMSO and 0.5 M sucrose. Finally, BL produced on Day 8 in the absence (control) and presence of 0.1 mM LE were used for transferase-mediated dUTP nick end labeling and differential staining to evaluate, respectively the apoptotic rate and the allocation of cells into inner cell mass (ICM) and trophectoderm lineages (experiment 3). Despite similar blastocyst yields, supplementation of culture medium with 0.1 mM LE improved the cryotolerance of in vitro-produced (IVP) embryos compared to the control group, as indicated by higher (P < 0.05) hatching rates recorded after 48-hour post-warming culture (48.5%, 50.0%, and 63.8%, respectively with 0, 0.05, and 0.1 mM LE). Interestingly, when embryos were cultured in the presence of 0.1 mM LE, the percentage of BL with the most physiological ICM:total cells ratio (20%-40%) increased (85.1 vs. 66.0%, P < 0.05), confirming a beneficial effect on embryo quality. Furthermore, 0.1 mM LE decreased (P < 0.01) both the average number (4.3 ± 0.2 vs. 9.1 ± 0.3) and the proportion (3.6 ± 0.3 vs. 8.1 ± 0.5) of apoptotic cells in BL compared to the control. In conclusion, the enrichment of bovine culture medium with 0.1 mM LE improves embryo quality, as indicated by the improved cryotolerance, the lower apoptotic rate, and the higher percentage of BL with the most physiological ICM:total cells ratio.

Effect of Relaxin on Fertility Parameters of Frozen-Thawed Buffalo (Bubalus bubalis) Sperm.

The aim of this work was to evaluate the effect of relaxin on fertility parameters of buffalo frozen/thawed sperm. Sperm were incubated in the absence of capacitating agents (negative control), with a known capacitating agent such as heparin (positive control) and with 50 and 100 ng/ml relaxin for 2 and 4 h. Sperm viability, motility, capacitation and the effect of relaxin on the fertilizing ability after heterologous IVF were evaluated. Although viability was not affected, relaxin increased (p < 0.05) sperm motility compared to the negative and positive controls both after 2 h (60.0 ± 2.0, 60.0 ± 3.1, 68.3 ± 1.7 and 69.4 ± 2.7, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin) and 4 h (55.0 ± 2.5, 53.3 ± 3.0, 62.2 ± 3.0 and 65.0 ± 3.2, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin) incubation. When sperm were incubated with both 100 ng/ml relaxin and heparin, a decrease (p < 0.01) of pattern A, that is low capacitation level, was observed compared to the negative control both after 2 h (54.4, 34.3 and 36.4%, respectively, in negative control, positive control and 100 ng/ml relaxin) and 4 h (51.9, 35.0 and 34.3%, respectively, in negative control, positive control and 100 ng/ml relaxin). Moreover, an increase (p < 0.01) of pattern EA, that is high capacitation level, was recorded with 100 ng/ml relaxin and heparin compared to the negative control both after 2 h (44.1, 59.3 and 57.7%, respectively, in negative control, positive control and 100 ng/ml relaxin) and after 4 h (43.0, 54.4 and 56.0%, respectively, in negative control, positive control and 100 ng/ml relaxin). Finally, relaxin increased (p < 0.01) cleavage rate compared to the negative control (57.1 ± 4.4, 72.5 ± 6.0, 71.4 ± 5.5 and 73.6 ± 2.9, respectively, in negative control, positive control, 50 and 100 ng/ml relaxin). In conclusion, relaxin has a beneficial effect on motility, capacitation and fertilizing ability of frozen-thawed buffalo sperm.

Effect of pour-on alphacypermethrin on feed intake, body condition score, milk yield, pregnancy rates, and calving-to-conception interval in buffaloes.

The aims of this study were to assess the efficacy of alphacypermethrin (ACYP) on pediculosis due to Haematopinus tuberculatus and to evaluate the influence of the treatment on productive and reproductive performance in buffaloes (Bubalus bubalis) reared in an intensive system. The trial was performed on 56 pluriparous buffaloes at 86.8 ± 8.1 d in milk. The animals underwent individual louse count and were divided into 2 homogenous groups according to louse count, age, number of lactations, days in milk, live BW, BCS, pregnancy status, and milk yield. Group A (n = 28) was treated by a pour-on formulation of ACYP, and Group S (n = 28) was treated by pour-on saline solution. Individual louse counts were performed weekly on 10 buffaloes in each group. Feed intake was recorded daily and the total mixed ration, individual ingredients, and orts were analyzed to calculate DM ingestion. Individual milk yield was recorded daily and milk samples were analyzed at the beginning of the trial, after 4 wk, and at the end of the trial to assess milk composition. Individual BCS was also evaluated simultaneously. Finally, the animals underwent synchronization of ovulation starting 4 wk after treatment and the pregnancy rate and the calving-conception interval were evaluated. Data were analyzed by the Mann-Whitney test and ANOVA for repeated measures. The infestation was constant in Group S, whereas no lice were present in Group A throughout the study. Daily DMI was similar in the 2 groups (16.7 ± 0.4 vs. 16.3 ± 0.3 kg/d in Group A vs. Group S, respectively), although buffaloes in Group A showed higher (P < 0.05) BCS score at the end of the trial (7.39 ± 0.1 vs. 7.14 ± 0.1 in Group A vs. Group S, respectively). The average milk yield/buffalo was higher (P < 0.05) in Group A compared to Group S (10.58 ± 0.1 vs. 10.39 ± 0.1 kg in Group A vs. Group S, respectively) and this was mainly due to the higher milk production recorded in buffaloes at less than 75 d in milk (11.81 ± 0.1 vs. 11.45 ± 0.1 kg in Group A vs. Group S, respectively). Despite of a similar fertility rate (90.5 vs. 80.9% in Group A vs. Group S, respectively), a lower (P < 0.05) calving-conception interval was recorded in Group A compared to Group S (118 ± 16 vs. 177 ± 16 d in Group A vs. Group S, respectively). In addition to the pour-on treatment against pediculosis, productive and reproductive performance were also improved. This represents a significant improvement in dairy buffalo herd management.

Influence of oocyte donor on in vitro embryo production in buffalo.

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n=40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A+B COC recorded in each 4-PS period had great repeatability (r=0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A+B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r=0.60; P<0.001), SFL (r=0.68; P<0.001), COC (r=0.48; P<0.01) and Grade A+B COC (r=0.40; P<0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P<0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r=0.80; P<0.05) and COC (r=0.76; P<0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.

Immunological detection of m- and µ-calpains in the skeletal muscle of Marchigiana cattle.

Calpains are Ca(2+)-dependent proteases able to cleave a large number of proteins involved in many biological functions. Particularly, in skeletal muscle they are involved in meat tenderizing during post mortem storage. In this report we analyzed the presence and expression of µ- and m-calpains in two skeletal muscles of the Marchigiana cattle soon after slaughter, using immunocytochemical and immunohistochemical techniques, Western blotting analysis and Casein Zymography. Therefore, the presence and the activity of these proteases was investigated until 15th day post-mortem during normal process of meat tenderizing. The results showed m- and µ-calpain immunosignals in the cytoplasm both along the Z disk/I band regions and in the form of intracellular stores. Moreover, the expression level of µ-calpain but not m-calpain decreased after 10 days of storage. Such a decrease in µ-calpain was accompanied by a gradual reduction of activity. On the contrary, m-calpain activity persisted up to 15 days of post-mortem storage. Such data indicate that expression and activity of both µ-calpain and m-calpain analyzed in the Marchigiana cattle persist longer than reported in literature for other bovines and may be related to both the type of muscle and breed examined.

Short communication: hair cortisol concentrations in Holstein-Friesian and crossbreed F1 heifers.

The aim of this study was to evaluate the hypothalamic-pituitary-adrenal (HPA) axis activity of Holstein-Friesian and crossbreed F1 heifers by analysis of the cortisol concentrations in hair samples. Cortisol, the primary hormone of the HPA axis, is the biological endpoint for the investigation of the HPA response. The study was conducted on 290 prepubertal heifers; 142 heifers were pure Holstein-Friesian and 148 were crossbreed F1 heifers obtained from the 3-way rotational system with Swedish Red and Montbéliarde breeds. Extraction was performed on the hair using methanol, and cortisol concentrations were determined by a radioimmunoassay method. Cortisol concentrations measured in regrown hair of crossbreed F1 heifers were significantly lower than those in hair of Holstein-Friesian heifers. This result helps us to better understand the differences in HPA activity and allostatic load between Holstein-Friesian and crossbreed F1 heifers and allows us to better assess the adaptability of these animals to the environment and the importance of crossbreed traits for profitability in dairy farming.

Influence of season on corpus luteum structure and function and AI outcome in the Italian Mediterranean buffalo (Bubalus bubalis).

The aim was to ascertain whether relationships between corpus luteum (CL) vascularization, CL function, and pregnancy outcome in AI in buffaloes were consistent across the breeding season and transition period to the nonbreeding season in a Mediterranean environment. Stage of the estrous cycle in Italian Mediterranean buffaloes was synchronized using the Ovsynch with timed AI program and buffaloes were mated by AI in both the breeding season (N = 131) and transition period (N = 125). Detailed investigation of CL structure and function was undertaken in 39 buffaloes at each of the respective times using realtime B-mode/color-Doppler ultrasonography on Days 10 and 20 after AI. Progesterone (P4) concentrations were determined by RIA in all buffaloes. Pregnancy rate on Day 45 after AI was greater (P < 0.05) during the breeding season (58.0%) than the transitional period (45.6%) and this was primarily the result of a lower (P < 0.05) late embryonic mortality during the breeding season (7.3%) compared with the transition period (23%). Circulating concentrations of P4 on Days 10 and 20 after AI were greater (P < 0.01) during the breeding season (4.6 ± 0.3 and 3.4 ± 0.2, respectively) than during the transition period (1.6 ± 0.12 and 1.8 ± 0.2, respectively), and this was independent of reproductive status as there was no interaction between pregnancy and season. Corpus luteum time average medium velocity at Day 10 after AI was greater (P < 0.01) during the breeding season (19.3 ± 1.5) than in the transitional period (8.3 ± 0.7). There were positive correlations in pregnant buffaloes between CL time average medium velocity and P4 concentrations on Day 10 (r = 0.722; P < 0.01) and Day 20 (r = 0.446; P < 0.01) after AI. The findings were interpreted to indicate that relationships between CL vascularization, CL function, and pregnancy outcome in AI in buffaloes are consistent across the breeding season and transition period to the nonbreeding season. The distinction between the breeding season and the transition period is the relatively low proportion of buffaloes that have CL function and P4 concentrations required to establish a pregnancy during the transition period, which is manifested in a greater incidence of embryonic mortality.

Corpus luteum development and function and relationship to pregnancy during the breeding season in the Mediterranean buffalo.

The aim of this study was to ascertain corpus luteum (CL) development and function in buffaloes synchronized and mated by artificial insemination (AI) during the breeding season. Italian Mediterranean buffalo cows (n = 43) at 86.5 ± 2.7 days postpartum were synchronized by the Ovsynch-TAI Program and inseminated using frozen thawed semen at 20 and 44 h after the second injection of GnRH. The CL dimensions (diameter and area) and blood flow were examined on Days 5, 10, 15, 20, and 25 after AI by realtime B-mode/colour-Doppler ultrasonography. The resistive index (RI), pulsatility index (PI) and time average medium velocity (TAMV) were recorded at each time, together with CL dimensions. Blood samples were taken on the days of ultrasonography for progesterone (P4) assay by RIA. Data were grouped into pregnant or non-pregnant and retrospectively analyzed by repeated measure ANOVA and correlation analyses. Dimensions of the CL on Days 10, 20, and 25 after AI were greater (P < 0.01) in buffaloes pregnant on Day 45 (n = 18) compared with non-pregnant buffaloes (n = 25). The former buffaloes also showed a greater (P < 0.01) rate of CL growth between Days 5 and 10 after AI. Blood flow to the CL on Day 10 after AI showed a higher TAMV (P < 0.01) and lower RI (P < 0.05) in pregnant buffaloes compared with non-pregnant buffaloes. Negative correlations were observed on Day 10 after AI between CL diameter and RI (r = -0.61; P < 0.01) and PI (r = -0.60; P < 0.01); P4 concentrations and RI (r = -0.46; P < 0.02); and RI and pregnancy (r = 0.45; P < 0.02). Positive correlations were observed between pregnancy and CL size (r = 0.54; P < 0.01), ΔCL diameter between Days 5 and 10 (r = 0.52; P < 0.01), ΔCL area between Days 5 and 10 (r = 0.48; P < 0.015), and ΔP4 between Days 5 and 10 (r = 0.50; P < 0.01). Based on these findings it is concluded that the period between Day 5 and 10 is very important for CL growth and crucial in evaluating pregnancy. Accordingly, the assessment of CL parameters during the period from Day 5 to Day 10 after AI might be used to predict the likelihood of an ongoing pregnancy.

Efficacy of PGF(2α) on pre-ovulatory follicle and corpus luteum blood flow.

The aim of this study was to evaluate the effect of cloprostenol administration on the blood flow of pre-ovulatory follicle (PF) and corpus luteum (CL), progesterone secretion and pregnancy outcome in buffaloes subjected to AI. The trial was performed on 75 Italian buffaloes at 182 ± 8 days in milk. Synchronized animals were randomly divided into two groups on the day of oestrus: Group T (n = 37) received a 0.524 mg intramuscular injection of cloprostenol and Group C (n = 38) received saline. Ultrasound examinations of the ovaries were performed 5 h after AI on the PF and 10 and 20 days after AI on the CL. Resistive (RI) and pulsatily index (PI) were calculated by colour-Doppler mode in each examination. Blood samples were collected on days 10, 20 and 25 after AI for progesterone assay and 25 days after AI, ultrasonography was performed to assess pregnancy, which was confirmed on day 45. Subjects pregnant on day 25 but not on day 45 were considered to have undergone late embryonic mortality (LEM). Statistical analysis was performed by anova. No differences were found in PF dimensions, CL size and blood flow on day 10 and 20 after AI between treated and control groups. Pre-ovulatory follicle area was higher in buffaloes that resulted pregnant on day 25 after AI compared to those that were non-pregnant (2.13 vs 1.66 cm in pregnant and non-pregnant buffaloes, respectively), while non-pregnant buffaloes showed higher values of RI (0.49 vs 0.30; p < 0.05) and PI (1.0 vs 0.37; p = 0.07) compared to pregnant subjects. Treatment by cloprostenol did not influence pregnancy rate both on day 25 (31/75; 41.3%) and 45 (27/75; 36.0%), progesterone levels and incidence of LEM (4/31; 12.9%). In conclusion, cloprostenol administration at the time of AI does not seem to affect PF and CL blood flow.

The influence of gamete co-incubation length on the in vitro fertility and sex ratio of bovine bulls with different penetration speed.

The objectives of this work were to evaluate whether the sperm penetration speed is correlated to the in vitro fertility and whether adapting the gamete co-incubation length to the kinetics of the bull improves in vitro fertility and affects the sex ratio. In vitro matured oocytes were co-incubated with spermatozoa from four different bulls (A-D). At various post-insemination (p.i.) times (4, 8, 12, 16 and 20 h), samples of oocytes were fixed and stained with DAPI for nuclei examination, while the remaining ones were transferred into culture to evaluate embryo development. The blastocysts produced were sexed by PCR. Two bulls (A and B) had faster kinetics than the others (C and D), as shown by the higher penetration rates recorded at 4 h p.i. (43%, 30%, 11% and 6%, respectively for bulls A, B, C and D; p<0.01). The differences in the kinetics among bulls did not reflect their in vitro fertility. The incidence of polyspermy was higher for faster penetrating bulls (36%, 24%, 16% and 4%, respectively for bulls A, B, C and D; p<0.01) and at longer co-incubation times (0%, 16%, 19%, 30% and 34%, respectively at 4, 8, 12, 16 and 20 h p.i.; p<0.01). The fertilizing ability of individual bulls may be improved by adapting the co-incubation length to their penetration speed. A sperm-oocyte co-incubation length of 8 h ensured the greatest blastocyst yields for the two faster penetrating bulls. On the contrary, 16 h co-incubation was required to increase (p<0.01) cleavage rate of the two slower bulls. Bulls with a faster kinetics did not alter the embryo sex ratio towards males. The female/male (F/M) ratios recorded were 2.1, 1.4, 1.2, 1.3 and 1.6, respectively at 4, 8, 12, 16 and 20 h p.i.

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis).

The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.

Growth, metabolic status and ovarian function in buffalo (Bubalus bubalis) heifers fed a low energy or high energy diet.

The aim was to establish the capacity of buffalo heifers to adapt their metabolic requirements to a low energy diet. Murrah buffalo (Bubalus bubalis) heifers undergoing regular estrous cycles were randomly assigned by age, live weight (LW) and body condition score (BCS) to a high energy group (HE, 5.8 milk forage units (MFU)/day, n=6) or low energy group (LE, 3.6 MFU/day, n=6). Circulating concentrations of metabolic substrates, metabolic hormones and reproductive hormones were determined weekly for 19 weeks. Ovarian follicular characteristics and oocyte parameters were also ascertained weekly. Heifers fed the LE diet had a better dry matter conversion than heifers fed the HE diet and the calculated daily energy provision was negative for heifers fed the LE diet (-0.248 MFU) and positive for heifers fed the HE diet (5.4 MFU). Heifers fed the HE diet had an increase in 50 kg LW over the duration of the study whereas LW remained constant for heifers fed the LE diet. The BCS of heifers fed the HE diet (4.2) was greater (P<0.05) than the BCS for heifers fed the LE diet (3.4). Heifers fed the HE diet had greater (P<0.05) circulating concentrations of metabolic substrates (glucose, total cholesterol and HDL cholesterol) and metabolic hormones (insulin, glucagon, leptin and T3) compared with heifers fed the LE diet. There were no significant differences in circulating reproductive hormones between the two groups of heifers. Ovarian follicular characteristics were similar for the two groups of heifers while heifers fed the LE diet tended to have oocytes of reduced quality compared with heifers fed the HE diet. The most notable finding was that heifers fed the LE diet had a negative calculated daily energy provision but were able to maintain LW and reproductive activity. It was concluded that buffalo heifers may potentially have the capacity to undergo metabolic adjustment and reduce their energy requirements when dietary energy is limiting. This adaptive capacity would explain why buffaloes remain productive in environments that are limiting to other ruminants.