Effect of season and cryopreservation on oxidative status of stallion spermatozoa

The aim of this study was to investigate 1) the effect of different ROS and lipid peroxidation on sperm quality, and 2) differences in ROS between non-breeding and breeding seasons. Eighteen ejaculates from six stallions were collected in January and July (N = 36), processed for freezing. After 90’ of cooling, some straws were not frozen (unfrozen), some were frozen (frozen). Rapid sperm (RAP, CASA), membrane-acrosome integrity (MAI), high mitochondrial membrane potential (Mpos), intracellular Ca2+ (Fneg), lipid peroxidation (BODIPY), ROS (DCFH, MitoSOX) and chromatin fragmentation (DFI%) were evaluated by flow cytometry during incubation at +37°C at T0 (after 90 min at +4°C and after thawing), 3, 6, 12 and 24h. In winter, ROS and BODIPY were higher and faster (P < 0.0001) in frozen than unfrozen; DFI% was similar at 0h (P > 0.05) but higher in frozen after 3h of incubation (P < 0.0001). RAP, PMAI, Mpos and Fneg were lower in frozen compared to unfrozen (P < 0.0001). Summer and winter data were compared. Overall, ROS concentrations and BODIPY were higher and faster (P < 0.001) in winter, DFI% was lower in winter (P < 0.001), but similar between the two groups within seasons after thawing. Differences were found at 3h and 12h for DFI%, and for DCFH and MitoSOX at 0h and 12h of incubation in winter and summer respectively. A moderate positive correlations was found between DFI% and MitoSOX, DCFH, BODIPY, whereas a negative correlation, stronger in winter, between RAP, PMAI, Mpos, Fneg and BODIPY, DCFH, MitoSOX. DFI was not different in unfrozen and frozen, despite a significant higher ROS level in winter, and incubation allowed to asses differences in DFI, suggesting that incubation should be included when evaluating stallion frozen semen. Higher level of ROS and BODIPY in winter was less detrimental than freezing-thawing.

Embryo transfer as a tool to disseminate k-casein BB genotype and improve milk clotting properties

Dairy industries are asked to be increasingly competitive and efficient. Despite the increasing trend in milk yield and protein content during the last decade genetic selection, milk coagulation ability has diminished and even if the absolute amount of cheese produced has increased, the relative cheese yield from a set amount of milk, has decreased. As casein content and variants, along with milk clotting properties (MCP) are determined to a large extent at DNA level, genetic selection and embryo transfer can provide efficacious tools to reverse this trend and achieve improvements. The aim of the proposed research was to determine how rapidly and to what extent milk coagulation properties could be improved by using embryo transfer (ET) as a tool to increase the frequency of k-casein BB genotype cattle and reducing A and E variants in an Italian Holstein herd with a low prevalence of the favourable genotype. In the effort to optimize superovulation protocols and results, synchronization of wave emergence was performed through manual transrectal ablation of the largest (dominant) ovarian follicle on days 7 or 8 of the cycle (estrus = day 0); different drugs and dosage for the superstimulation protocol were experimented trying to overcome the negative effects of stress and the perturbance of LH secretion in superovulated highly producing lactating cows and the use of SexedULTRA™ sex-sorted semen, for artificial insemination of superovulated cows was reported for the first time. The selection program carried out in this research, gave evidence and gathered empirical data of feasible genetic improvements in cheesemaking ability of milk by means of k-casein BB selection. In conclusion, in this project, selection of k-casein BB genotype markedly enhanced cheese-making properties of milk, providing an impetus to include milk coagulation traits in genetic selection and breeding programs for dairy cattle.