The aim of this study was to investigate 96 single-nucleotide polymorphisms (SNPs) from 54 candidate genes, and test the associations of the polymorphic SNPs with milk yield, composition, milk urea nitrogen (MUN) content and somatic cell score (SCS) in individual milk samples from Italian Brown Swiss cows. Milk and blood samples were collected from 1271 cows sampled once from 85 herds. Milk production, quality traits (i.e. protein, casein, fat and lactose percentages), MUN and SCS were measured for each milk sample. Genotyping was performed using a custom Illumina VeraCode GoldenGate approach. A Bayesian linear animal model that considered the effects of herd, days in milk, parity, SNP genotype and additive polygenic effect was used for the association analysis. Our results showed that 14 of the 51 polymorphic SNPs had relevant additive effects on at least one of the aforementioned traits. Polymorphisms in the glucocorticoid receptor DNA-binding factor 1 (GRLF1), prolactin receptor (PRLR) and chemokine ligand 2 (CCL2) were associated with milk yield; an SNP in the stearoyl-CoA desaturase (SCD-1) was related to fat content; SNPs in the caspase recruitment domain 15 protein (CARD15) and lipin 1 (LPIN1) affected the protein and casein contents; SNPs in growth hormone 1 (GH1), lactotransferrin (LTF) and SCD-1 were relevant for casein number; variants in beta casein (CSN2), GH1, GRLF1 and LTF affected lactose content; SNPs in beta-2 adrenergic receptor (ADRB2), serpin peptidase inhibitor (PI) and SCD-1 were associated with MUN; and SNPs in acetyl-CoA carboxylase alpha (ACACA) and signal transducer and activator of transcription 5A (STAT5A) were relevant in explaining the variation of SCS. Although further research is needed to validate these SNPs in other populations and breeds, the association between these markers and milk yield, composition, MUN and SCS could be exploited in gene-assisted selection programs for genetic improvement purposes.

Subclinical mastitis is a costly disease and its diagnosis is difficult. Besides the somatic cell count (SCC) and bacteriology, the differential inflammatory cell count (DICC) is a meaningful tool for mastitis detection. As microscopy is very subjective because of the low number of events to be counted, flow cytometry has often been proposed for the differentiation of milk cells. The objective of this study was to determine whether it is possible to identify subclinical mastitis in cattle at an early stage by a simple and fast flow cytometric method. The aim was to identify the main leucocyte populations in flow cytometric dotplots (polymorphonuclear neutrophils (PMN), lymphocytes and macrophages) and, with these, to elaborate a method of mastitis prognostics. Milk from 15 German Holstein cows was sampled in cross-sectional studies and SCC determined. After preparation, the milk cells were incubated with different specific antibodies that bind to different cell types and also to propidium iodide (PI), which differs between viable and non-viable cells. This procedure made it possible to localize cell types in a flow cytometric dot plot and to differentiate between viable and non-viable PMN. Percentages of viable PMN can be determined by a procedure consisting of a simple centrifugation, incubation with PI, and flow cytometric measurement. So it is possible to quickly determine the stage of the inflammation even in quarters with a low SCC.

Pooled milk samples from 115 Bergamasca meat sheep were collected aseptically five times from lambing to weaning to determine the prevalence of intramammary infection, somatic cell counts and milk quality parameters (protein, fat and lactose), and effects of infection on lamb weight gain. The global prevalence of subclinical intramammary infection was 51·2%. The Staphylococcus genus was responsible for the greatest prevalence (53·3% among infected udders). Staphylococcus aureus was isolated in 8·4% of infected milk samples. Infection status had significant effects on fat and protein percentage and on somatic cell count. Lamb growth was greatest for lambs of ewes with no infection and decreased as the number of infected samples increased. No significant differences were detected in the growth of lambs with dams infected by different bacterial species.

As an important step in the nuclear transfer (NT) procedure, we evaluated the effect of three different treatments for oocyte activation on the in vitro and in vivo developmental capacity of bovine reconstructed embryos: (1) strontium, which has been successfully used in mice but not yet tested in cattle; (2) ionomycin and 6-dimethylaminopurine (6-DMAP), a standard treatment used in cattle; (3) ionomycin and strontium, in place of 6-DMAP. As regards NT blastocyst development, no difference was observed when strontium (20.1%) or ionomycin/6-DMAP (14.4%) were used. However, when 6-DMAP was substituted by strontium (3), the blastocyst rate (34.8%) was superior to that in the other activation groups (p <0.05). Results of in vivo development showed the possibility of pregnancies when NT embryos activated in strontium were transferred to recipient cows (16.6%). A live female calf was obtained when ionomycin/strontium were used, but it died 30 days after birth. Our findings show that strontium can be used as an activation agent in bovine cloning procedures and that activation with a combination of strontium and ionomycin increased the in vitro developmental capacity of reconstructed embryos. This is the first report of a calf produced by adult somatic cell NT in Latin America.

G2/M somatic nuclei were introduced into enucleated meiotically competent oocytes and subsequently cultured in TCM199 plus 10% fetal calf serum (FCS). Pseudo-first polar bodies could be extruded, but the chromosomes failed to arrange normally. Kinetochores were traced with immunofluorescent microscopy using autoimmune sera from patients with CREST (Calcinosis, Raynaud's phenomenon, Esophageal dysmotility, Sclerodactyly, Telangiectasia) scleroderma. In vitro matured oocytes arrested at second meiotic metaphase and kinetochores were detectable as paired structures aligned at the spindle equator. At meiotic anaphase, present or past the kinetochores separated and remained aligned at the distal sides of the chromosomes until telophase, when their alignment perpendicular to the spindle axis was lost. Kinetochores failed to arrange normally after transferring somatic nuclei into oocytes. Our results suggest that somatic cell nuclei are unable to proceed normally through meiosis when introduced into oocyte meiotic cytoplasm.