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.

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.

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.

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.

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.

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.

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.

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.

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.

Genomic characterization and chromosomal mapping of 5 river buffalo skeletal muscle differentiation master genes.

River buffalo (Bubalus bubalis, 2n = 50, BBU) is a species of economic relevance in a number of countries. This species shows a very peculiar biology and a great capacity for environmental adaptation. There has been an increasing economic interest as well as a growing demand for a more detailed knowledge of molecular features in this species. From this perspective we report a genomic, transcriptional and cytogenetic analysis of 5 master genes involved in skeletal muscle development. Of these 5 genes, MYOD1, MYF5, MYF6 and MYOG belong to the basic helix-loop helix protein family while MSTN belongs to the TNF-B protein family. In mammals, these genes are involved in the early stages of skeletal muscle differentiation, development and regeneration. These pivotal biological functions are finely regulated in a tissue- and temporal-specific manner. We used a comparative genomic approach to obtain the buffalo specific sequences of MYOD1 and MYF6. The nucleotide sequence similarity and the protein domain conservation of the newly obtained sequences are analysed with respect to bovine and other mammalian species showing sequence similarity. The presence of a polymorphism in MYOD1 coding sequence is described and its possible effect discussed. Using a quantitative PCR approach, we compared the level of the 5 transcripts in adult and fetal muscle. These genes were physically localised on river buffalo R-banded chromosomes by FISH using bovine genomic BAC-clones. Here, we present a genomic and cytogenetic analysis which could offer a background to better characterise the buffalo genes involved in muscle function and which may be responsible for buffalo-specific meat features.

Ion, protein, phospholipid and energy substrate content of oviduct fluid during the oestrous cycle of buffalo (Bubalus bubalis).

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre-ovulatory, ovulatory, post-ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post-ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre-ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (µm/24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post-ovulatory phases (63.7 ± 11.2), with intermediate values in the pre-ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre-ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post-ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (µmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre-ovulatory and post-ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species-specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.

Cryotop vitrification of buffalo (Bubalus bubalis) in vitro matured oocytes: effects of cryoprotectant concentrations and warming procedures.

The aim of the work was to evaluate the in vitro developmental competence of in vitro-matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming-dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 M sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 M sucrose) in Groups B and D. Warming was performed in 1.25 M sucrose for 1 min, then in 0.62, 0.42 and 0.31 M sucrose, 30 s each (Groups A and B), or in 0.25 M sucrose for 1 min and in 0.15 M sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post-warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.

Corpus luteum function and pregnancy outcome in buffaloes during the transition period from breeding to non-breeding season.

The aim in this study was to investigate corpus luteum function and embryonic loss in buffaloes mated by artificial inseminations (AI) during the transitional period from breeding to non-breeding season. The study was carried out using 288 multiparous Italian Mediterranean Buffalo cows at 110 ± 4 days in milk. The buffaloes were mated by AI after synchronization of ovulation by the Ovsynch-TAI protocol 25 days after AI buffaloes underwent trans-rectal ultrasonography to assess embryonic development. Pregnancy diagnosis was confirmed on Days 45 and 70 after AI by rectal palpation. Buffaloes pregnant on Day 25 but not on Day 45 were considered to have undergone late embryonic mortality (LEM), whilst buffaloes pregnant on Day 45 but not on Day 70 were considered to have undergone foetal mortality (FM). Corpus luteum size and blood flow were determined by real-time B-mode/colour-Doppler on day 10 after AI in 122 buffaloes. The resistive index (RI) and pulsatility index (PI) were recorded at the time. Milk samples were collected on Days 10, 20 and 25 after AI in all inseminated buffaloes for the assay of whey P4 concentrations. Data were analysed by anova. Pregnancy rate on Day 25 after AI was 48.6% (140/288) and declined to 35.4% (102/288) and 30.6% (88/288) by Day 45 and Day 70 respectively. The incidences of LEM and FM were respectively 27.1% (38/140) and 13.7% (14/102). Pregnant buffaloes had greater (p < 0.01) whey concentrations of P4 from Day 20 onwards than buffaloes which showed LEM, whilst P4 in buffaloes that showed FM did not differ from the other two groups on Day 10 and Day 20. Corpus luteum blood flow on Day 10 after AI showed higher RI (p < 0.05) and PI (p = 0.07) values in buffaloes that subsequently were not pregnant on Day 25 compared with pregnant buffaloes. Buffaloes that were not pregnant on Day 45 also had a higher (p = 0.02) RI value on Day 10 than pregnant buffaloes, whilst PI values on Day 10 did not differ for the two groups of buffaloes. It was concluded that blood flow to the corpus luteum on Day 10 after AI influences corpus luteum function as judged by P4 secretion and also embryonic development and attachment in buffaloes.

Is a delayed treatment with GnRH, HCG or progesterone beneficial for reducing embryonic mortality in buffaloes?

The aims of this study were to verify the efficacy of delayed hormonal treatments performed on day 25 post-insemination on pregnancy rate at 45 and 70 days in buffalo. The trial was performed on 385 buffaloes synchronized by the Ovsynch/TAI protocol and submitted to artificial insemination (AI). Twenty-five days after AI, pregnant animals were assigned to four treatments: (1) GnRH agonist (n = 52), 12 microg of buserelin acetate; (2) hCG (n = 51), 1500 IU of human chorionic gonadotrophin; (3) Progesterone (n = 47), 341 mg of P4 intramuscular (im) every 4 days for three times; (4) Control (n = 54), treatment with physiological saline (0.9% NaCl). Milk samples were collected on days 10, 20 and 25 after AI in all buffaloes to determine progesterone concentration in whey by radioimmunoassay method. Statistical analysis was performed by anova. Pregnancy rate on day 25 after AI was 52.9%, but declined to 41.8% by day 45, indicating an embryonic mortality (EM) of 21%. If only control group is considered, the incidence of EM was 38.9%. Pregnant buffaloes had higher (p < 0.01) progesterone concentrations on day 20 and 25 after AI than both non-pregnant buffaloes and buffaloes that showed EM. The treatments on day 25 increased (p < 0.01) pregnancy rate, although in buffaloes with a low whey progesterone concentration on day 20 and 25 after AI (n = 22); all treatments were ineffective to reduce EM.

Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells.

Single cells from enzymatically dissociated chick embryo tibiae have been cloned and expanded in fresh or conditioned culture media. A cloning efficiency of approximately 13% was obtained using medium conditioned by dedifferentiated chondrocytes. A cloning efficiency of only 1.4% was obtained when conditioned medium from hypertrophic chondrocytes was used, and efficiencies of essentially 0 were found with fresh medium or medium conditioned by J2-3T3 mouse fibroblasts. Cell clones were selected by morphological criteria and clones showing a dedifferentiated phenotype (fibroblast-like) were further characterized. Out of 38 clones analyzed, 17 were able to differentiate to the hypertrophic chondrocyte stage and reconstitute hypertrophic cartilage when placed in the appropriate culture conditions. Cells from these clones expressed the typical markers of chondrocyte differentiation, i.e., type II and type X collagens. Clones not undergoing differentiation continued to express only type I collagen. Hypertrophic chondrocytes from differentiating clones were analyzed at the single cell level by immunofluorescence; all the cells were positive for type X collagen, while approximately 50% of them showed positivity for type II collagen.

Ovotransferrin and ovotransferrin receptor expression during chondrogenesis and endochondral bone formation in developing chick embryo.

Ovotransferrin expression during chick embryo tibia development has been investigated in vivo by immunocytochemistry and in situ hybridization. Ovotransferrin was first observed in the 7 day cartilaginous rudiment. At later stages, the factor was localized in the articular zone of the bone epiphysis and in the bone diaphysis where it was concentrated in hypertrophic cartilage, in zones of cartilage erosion and in the osteoid at the chondro-bone junction. When the localization of the ovotransferrin receptors was investigated, it was observed that chondrocytes at all stages of differentiation express a low level of the oviduct (tissue) specific receptor. Interestingly, high levels of the receptor were detectable in the 13-d old tibia in the diaphysis collar of stacked-osteoprogenitor cells and in the layer of derived osteoblasts. High levels of oviduct receptor were also observed in the primordia of the menisci. Metabolic labeling of proteins secreted by cultured chondrocytes and osteoblasts and Northern blot analysis of RNA extracted from the same cells confirmed and completed the above information. Ovotransferrin was expressed by in vitro differentiating chondrocytes in the early phase of the culture and, at least when culture conditions allowed extracellular matrix assembly, also by hypertrophic chondrocytes and derived osteoblast-like cells. Osteoblasts directly obtained from bone chips produced ovotransferrin only at the time of culture mineralization. By Western blot analysis, oviduct receptor proteins were detected at a very low level in extract from differentiating and hypertrophic chondrocytes and at a higher level in extract from hypertrophic chondrocytes undergoing differentiation to osteoblast-like cells and from mineralizing osteoblasts. Based on these results, the existence of autocrine and paracrine loops involving ovotransferrin and its receptor during chondrogenesis and endochondral bone formation is discussed.

Cell proliferation, extracellular matrix mineralization, and ovotransferrin transient expression during in vitro differentiation of chick hypertrophic chondrocytes into osteoblast-like cells.

Differentiation of hypertrophic chondrocytes toward an osteoblast-like phenotype occurs in vitro when cells are transferred to anchorage-dependent culture conditions in the presence of ascorbic acid (Descalzi Cancedda, F., C. Gentili, P. Manduca, and R. Cancedda. 1992. J. Cell Biol. 117:427-435). This process is enhanced by retinoic acid addition to the culture medium. Here we compare the growth of hypertrophic chondrocytes undergoing this differentiation process to the growth of hypertrophic chondrocytes maintained in suspension culture as such. The proliferation rate is significantly higher in the adherent hypertrophic chondrocytes differentiating to osteoblast-like cells. In cultures supplemented with retinoic acid the proliferation rate is further increased. In both cases cells stop proliferating when mineralization of the extracellular matrix begins. We also report on the ultrastructural organization of the osteoblast-like cell cultures and we show virtual identity with cultures of osteoblasts grown from bone chips. Cells are embedded in a dense meshwork of type I collagen fibers and mineral is observed in the extracellular matrix associated with collagen fibrils. Differentiating hypertrophic chondrocytes secrete large amounts of an 82-kD glycoprotein. The protein has been purified from conditioned medium and identified as ovotransferrin. It is transiently expressed during the in vitro differentiation of hypertrophic chondrocytes into osteoblast-like cells. In cultured hypertrophic chondrocytes treated with 500 nM retinoic acid, ovotransferrin is maximally expressed 3 d after retinoic acid addition, when the cartilage-bone-specific collagen shift occurs, and decays between the 5th and the 10th day, when cells have fully acquired the osteoblast-like phenotype. Similar results were obtained when retinoic acid was added to the culture at the 50 nM "physiological" concentration. Cells expressing ovotransferrin also coexpress ovotransferrin receptors. This suggests an autocrine mechanism in the control of chondrocyte differentiation to osteoblast-like cells.

Sex chromosome abnormalities and sterility in river buffalo.

Thirteen male river buffaloes, 119 females with reproductive problems (which had reached reproductive age but had failed to become pregnant in the presence of bulls) and two male co-twins underwent both clinical and cytogenetic investigation. Clinical analyses performed by veterinary practitioners revealed normal body conformation and external genitalia for most females. However, some subjects showed some slight male traits such as large base horn circumference, prominent withers and tight pelvis. Rectal palpation revealed damage to internal sex adducts varying between atrophy of Mullerian ducts to complete lack of internal sex adducts (with closed vagina). All bulls had normal karyotypes at high resolution banding, while 25 animals (23 females and 2 male co-twins) (20.7%) with reproductive problems were found to carry the following sex chromosome abnormalities: X monosomy (2 females); X trisomy (1 female); sex reversal syndrome (2 females); and free-martinism (18 females and 2 males). All female carriers were sterile.