This study aims to compare the efficacy of computer-assisted sperm analysis (CASA) and smartphone-applied sperm analysis (SASA) in assessing the quality of frozen-thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA-37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA-25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA-37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen-thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p > .05). However, significant differences among the groups were observed for total motile spermatozoa values (p < .001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p < .001). The immotile spermatozoa values were significant between the groups (p < .001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen-thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis.

A seasonal effect on sperm quality parameters was observed previously. Although identification of the bull semen microbiota by 16S rRNA sequencing was performed previously, it has not been carried out in commercial semen samples from different seasons, and its connection with sperm quality parameters has not been evaluated yet. The objectives in this study were; (i) to evaluate diversity of bull semen microbiota and sperm quality parameters in different seasons, and (ii) to find if specific bacteria were associated with seasonal differences in specific sperm quality parameters. Bull semen microbiota was identified in 54 commercial bull semen samples from 3 seasons (winter, spring, summer). Sperm quality was analysed by Computer Assisted Sperm Analyses (CASA) and Flow Cytometry (FC). From 28 phyla in all samples, six phyla were identified in samples from all seasons, with observed seasonal differences in their distribution. At genus level, 388 genera were identified, of which 22 genera had a relative abundance over 1 % and showed seasonal differences in bacterial diversity, and 9 bacteria genera were present in all seasons. Differences between spring and summer (P < 0.05) were observed for live hydrogen peroxide positive sperm cells. A trend towards significance (0.10 > P > 0.05) was observed for some CASA kinematics (VCL and LIN) and FC parameters (High respiratory activity, and live hydrogen peroxide positive sperm cells) between seasons. Nevertheless, associations between sperm quality parameters and specific bacteria were observed in spring.

The purpose of this study was to improve the quality of frozen-thawed Piedmontese bull semen by incorporating MitoTEMPO (MT) in extended semen before cryopreservation. Semen was collected from 4 fertile bulls, using an artificial vagina, once weekly for 6 consecutive weeks. Semen samples were pooled, diluted with Bullxcell® extender, and supplemented with different concentrations of MT (0 as control, 5, 10, 20, 40, and 80 μM) before cooling, equilibration, and freezing procedures. The frozen-thawed semen was assessed for motility, vitality, acrosome intactness, plasma membrane integrity, DNA integrity, apoptosis, mitochondrial membrane potential, intracellular ROS level and in vitro fertilizing capability. The results showed that MT at concentrations of 10, 20, and 40 μM improved the total, progressive, and rapid motility directly after thawing while, at the highest tested concentration (80 μM), it decreased the progressive and rapid motility after 1, 2, and 3 h of incubation. The sperm kinetics including STR and LIN were noticeably increased at concentrations of 10, 20, and 40 μM directly after thawing (0 h), whereas the MT effect was variable on the other sperm kinetics during the different incubation periods. MitoTEMPO improved the sperm vitality at all tested concentrations, while the acrosomal and DNA integrity were improved at 20 μM and the mitochondrial membrane potentials was increased at 80 μM. The cleavage and blastocyst formation rates were significantly increased by using semen treated with 20 μM MT compared with controls. These findings suggest a potential use of MT mainly at a concentration of 20 μM as an additive in the cryopreservation media of bull semen to improve sperm quality.

Up to now, the definitive conclusion of the positive effects of rapid transient thawing at higher temperatures for shorter durations has not been obtained yet and is still under discussion due to some contradictory findings and limited assessment of post-thawed parameters. The purpose of the current study was to evaluate the effectiveness of rapid thawing in water at 70 °C by using various post-thawed parameters of frozen bull spermatozoa. Experiment 1, monitoring the change of temperature inside frozen bull straw thawed in water at different temperatures. Experiment 2, evaluation of various post-thawed characteristics of frozen bull spermatozoa thawed in water at different temperatures by using a computer-assisted sperm analysis, flow cytometry and immunocytochemistry. The time it took for the temperature inside the straw to warm up to 15 °C was nearly twice as faster when the straw was thawed in 70 °C water compared with 39 °C. Although there were differences among bulls, viability, motility, and mitochondrial membrane potential of spermatozoa thawed at 70 °C for 8 seconds and stabilized at 39 °C for 52 seconds were significantly higher than those of controls (thawed at 39 °C for 60 seconds) at 0 and 3 h after thawing. Just after thawing, however, there were no differences in acrosome integrity and distribution of phospholipase C zeta1, whereas mitochondrial reactive oxygen species production was significantly lower in spermatozoa thawed at 70 °C. From these results, we conclude that rapid thawing at 70 °C and then stabilization at 39 °C significantly improves viability, motility and mitochondrial health of bull spermatozoa rather than conventional thawing at 39 °C. The beneficial effect of rapid transient thawing could be due to shorter exposure to temperatures outside the physiological range, consequently maintaining mitochondrial health.

Oxidative stress leads to a decrease in semen quality during semen cryopreservation and fresh semen production. Grape seed proanthocyanidins (GSPs) are endowed with well-recognized antioxidant, anti-inflammatory, anti-cancer, and anti-aging activities. Therefore, the objective of this experiment was to explore the effects of GSPs on the quality of fresh and cryopreserved semen to provide a basis for GSPs as a new dietary additive and semen diluent additive for males\' reproduction. Fresh semen from three healthy bulls aged 3 to 5 years old were gathered and mixed with semen diluents dissolved with 0 µg/mL, 30 µg/mL, 40 µg/mL, 50 µg/mL, and 60 µg/mL GSPs respectively. The motility, physiological structures (acrosome integrity, membrane integrity, mitochondrial activity), and antioxidant capacity of frozen-thawed sperm were measured after storage in liquid nitrogen for 7 days (d). Bulls were fed with 20 mg/kg body weight (BW) GSPs in their diet for 60 days; the weight of the bull is about 600 kg. Then, the reproductive performance and antioxidant indexes of bulls were measured before and after feeding. The results demonstrated that GSPs supplementation significantly increased sperm motility, physiological structures, GSH-Px, and CAT enzyme activities and significantly decreased MDA content in sperm during semen cryopreservation. The optimal concentration of GSPs was 40 µg/mL (p < 0.05). After 20 mg/kg (body weight) GSP supplementation, sperm motility was significantly heightened (p < 0.05), the sperm deformity rate was significantly reduced (p < 0.05), and antioxidant enzyme activities (such as SOD, CAT, and GSH-Px) were significantly enhanced (p < 0.05), and the production of MDA was significantly suppressed (p < 0.05) in serum compared with that before feeding. In conclusion, these results reveal that a certain concentration of GSPs has a good protective effect on sperm damage caused by semen cryopreservation and the reproductive performance reduction caused by stress in bulls, which may be attributed to the antioxidant function of GSPs. In summary, GSPs are a useful cryoprotective adjuvant and dietary additive for bull sperm quality.

Sperm survives for a very short time in fresh semen, and slow cooling to 5.00 ˚C kills a large number of sperms. This study was aimed to compare the semen quality parameters and anti-oxidant levels in four extenders (manual, Triladyl, Steridyl and AndroMed). Semen samples were obtained from a total number of 12 dual-purpose Simmental bulls kept in the Simmental Cattle Breeding Center for a period of 3 months using an artificial vagina. Sperm viability, motility, abnormal morphology, plasma membrane integrity, DNA damage, chromatin quality, total antioxidant capacity (TAC) and lipid peroxidation were evaluated. The highest progressive motility, viability, plasma membrane integrity, and TAC and the lowest levels of malondialdehyde in the frozen-thawed semen belonged to the semen group frozen with Triladyl. Parameters of motility were higher in the frozen-thawed semen with Triladyl than in other groups, indicating a significant difference from the manual extender. Among the extenders studied, Triladyl was the most suitable for semen freezing in Simmental bulls.

Semen samples contain bacteria originating from the animal urogenital tract, environment, and/or contamination during semen processing, negatively affecting sperm quality by producing toxins and/or competing for nutrients in extenders. The aims of this study were to evaluate two methods of Single-layer centrifuges (SLC), high and low density colloid, as a method for bacterial removal from bull semen, and to evaluate sperm quality after treatment. In total, semen samples from 20 bulls (3 ejaculates per bull) were used in this study. Bacterial reduction was evaluated by bacterial quantification (colony forming unit - CFU/mL) while bacterial identification was performed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after culturing bacteria on blood agar. Sperm motility parameters were evaluated by Computer Assisted Sperm Analyses (CASA), and sperm chromatin structure assay (SCSA) by Flow cytometry. Both, High and Low density SLC reduced number of bacteria significantly (p < 0.001) compared with control. The difference in bacterial count between High and Low SLC was also significant (p < 0.001). Furthermore, High density SLC was successful in removing almost all Bacillus and Proteus spp. Most CASA parameters were significantly improved after both treatments (p < 0.001, p < 0.01, p < 0.05). The Deoxyribonucleic acid (DNA) fragmentation index evaluated by SCSA in High (p < 0.01) and Low (p < 0.05) SLC group differed significantly compared with control. Single-layer centrifugation (SLC) with either a high or a low density colloid is a suitable method for bacterial removal in bull semen.

Flaxseed oil (FO) and vitamin E (VE) both have antioxidant effects on sperm. The present study investigated the effects of dietary supplementation with FO and/or VE on semen quality.
16 fertile Simmental bulls were selected and randomly divided into 4 groups (n = 4): the control group (control diet), FO group (control diet containing 24 g/kg FO), VE group (control diet containing 150 mg/kg VE) and FOVE group (control diet containing 150 mg/kg VE and 24 g/kg FO), and the trial lasted 10 weeks.
The results showed that the addition of FO independently can increase sperm motion parameters, the levels of catalase (CAT), glutathione peroxidase (GSH-Px), testosterone (T) and estradiol (E2), while reduce oxidative stress in seminal plasma (P < 0.05). Supplement of VE independently can increased the motility, motility parameters, CAT and superoxide dismutase (SOD) levels, and reduce oxidative stress in seminal plasma (P < 0.05). There was an interaction effect of FO × VE on motility and reactive oxygen species (ROS), while GSH-Px and ROS were affected by week × VE 2-way interaction, levels of T and E2 were also affected by the dietary FO × week interaction (P < 0.05). The triple interaction effects of FO, VE and week were significant for malondialdehyde (MDA) (P < 0.05). Compared with the control group, sperm from the FOVE group had a significantly higher in vitro fertilization (IVF) rate, and subsequent embryos had increased developmental ability with reduced ROS levels at the eight-cell stage, then increased adenosine triphosphate (ATP) content and gene expression levels of CAT, CDX2, Nanog, and SOD at the blastocyst stage (P < 0.05). Metabolomic and transcriptomic results indicated that dietary supplementation of FO and VE increased the expression of the metabolite aconitic acid, as well as the expression of ABAT and AHDHA genes.
With in-silico analysis, it can be concluded that the effects of dietary FO and VE on improving semen quality and embryo development may be related to increased aconitic acid via the ABAT and AHDHA genes involved in the propionic acid metabolism pathway.

The rapid emergence of antibacterial resistance requires alternatives to antibiotics to be found, including for semen preservation. One of the possible alternatives would be to use plant-based substances with known antimicrobial effects. The objective of this study was to test the antimicrobial effect of pomegranate powder, ginger, and curcumin extract in two concentrations on bull semen microbiota after exposure for <2 h and 24 h. An additional aim was to evaluate the effect of these substances on sperm quality parameters. The bacterial count in semen was low from the beginning; however, a reduction was present for all tested substances compared with control. A reduction in bacterial count in control samples was also observed with time. Curcumin at a concentration of 5%, reduced bacterial count by 32% and was the only substance that had a slight positive effect on sperm kinematics. The other substances were associated with a decline in sperm kinematics and viability. Neither concentration of curcumin had a deleterious effect on sperm viability parameters measured by flow cytometry. The results of this study indicate that curcumin extract at a concentration of 5% can reduce the bacterial count and does not have a negative influence on bull sperm quality.

Low fertility rates in lactating dairy cows as well as restricted availability of semen doses of young bulls with high genetic merit are two major problems in the reproduction of dairy cows. By using liquid semen (LS), the number of doses per ejaculate can be increased. One of the challenges of optimizing the reproductive performance of dairy cows is the phenomenon of variable estrus lengths. The objective of this study was to determine whether the use of LS affects pregnancy outcome of dairy cows with delayed ovulation, when compared with frozen semen (FS). A randomized controlled clinical trial was implemented. In a split-sample procedure, 131 ejaculates were processed into LS (Caprogen, LIC, New Zealand) and FS (BioXcell, IMV, France). Both semen types of each ejaculate were inseminated under the same field conditions to cows showing natural or induced heat. Cows and semen type were allocated according to the last digit of the cows identification number (even = frozen semen, odd = liquid semen). Inseminations (n = 667) were conducted after localization of the pre-ovulatory follicle. Determination of ovulation was performed 24 h post AI per transrectal ultrasonographic examination. Ovulations were classified as delayed when the pre-ovulatory follicle was still present at ovulation control. The prevalence of delayed ovulations was 25.2%. Data of 667 inseminations were analyzed with a generalized linear mixed model including semen type (P = 0.016), parity (P = 0.014), backfat thickness (P = 0.006), estrus induction (P = 0.010), ovulation (P = 0.265) and the interaction term \'semen type by ovulation\' (P = 0.094). Overall, a higher pregnancy per AI (P/AI) of LS (45.4%) than P/AI of FS (33.7%) was found. In cases of delayed ovulations, use of LS resulted in higher P/AI (46.8%) compared with FS (27.7%; P = 0.017). We concluded that the fertilizing capacity of LS in prolonged intervals from AI to ovulation might be greater when compared with FS and could be an efficient tool to improve fertility of lactating dairy cows with delayed ovulations.