Influence of culture conditions on the secretome of mesenchymal stem cells derived from feline adipose tissue: Proteomics approach.

This study aimed to describe the secretome of mesenchymal stem cells derived from feline adipose tissue (AD-MSCs) and compare the effects of different culture conditions on AD-MSC proteomics using a shotgun approach. Adipose tissue was collected from 5 female cats and prepared to culture. Conditioned media was collected at third passage, in which the cells were cultured under 4 conditions, normoxia with fetal bovine serum (N + FBS), hypoxia with FBS (H + FBS), normoxia without FBS (N - FBS), and hypoxia without FBS (H - FBS). Then, the secretome was concentrated and prepared for proteomic approaches. Secretomes cultured with FBS-free medium had more than twice identified proteins in comparison with the secretomes cultured with FBS. In contrast, hypoxic conditions did not increase protein amount and affected only a small proteome fraction. Relevant proteins were related to the extracellular matrix promoting environmental modulation, influencing cell signaling pathways, and providing a suitable environment for cell proliferation and maintenance. Moreover, other proteins were also related to cell adhesion, migration and morphogenesis. Culture conditions can influence protein abundance in AD-MSC secretome, and can give also more specificity to cell and cell-free treatments for different diseases.

Growth performance, reproductive parameters and fertility measures in young Nellore bulls with divergent feed efficiency.

The growth, sexual maturity and fertility-related parameters related of young Nellore bulls with divergent residual feed intake (RFI) raised on pasture were evaluated. After classification of 48 young males as low and high RFI (more and less efficient, respectively), the animals were evaluated for growth and reproductive parameters at 28-day intervals from 14.3 to 24.6 months of age. The semen was cryopreserved in the last sampling and fresh and post-thaw semen samples were evaluated. Low RFI bulls exhibited higher initial and final body weight (P < 0.05), but feed intake, body condition score and growth measures evaluated by carcass ultrasound were unaffected by RFI (P > 0.05). The scrotal circumference, sperm concentration, defects, and quality of fresh semen, and ultrasonographic testicular characteristics were unaffected by RFI (P > 0.05). However, velocity parameters such as average path and curvilinear velocities determined by computer-assisted sperm analysis of thawed semen submitted to the rapid thermoresistance test were improved (P < 0.05) in low RFI bulls, but this improvement in quality did not enhance in vitro sperm fertilizing ability. Our results demonstrated significant differences in metabolism and growth performance between bulls of divergent RFI. In addition, there was slight improvement in the semen quality of bulls with low RFI bulls, but this did not enhance in vitro fertilizing ability. Selection of beef bulls for RFI can be performed, which will result in economic benefits by improving the growth performance of the animals without affecting reproductive parameters.

Cholesterol-Loaded Cyclodextrin Addition to Skim Milk-Based Extender Enhances Donkey Semen Cooling and Fertility in Horse Mares.

The present study aimed to compare semen parameters and fertility of cooled donkey semen extended in a commercially available skim milk (SKM) based extender and the same extender with cholesterol-loaded cyclodextrin (SKM-CLC). In Experiment 1, thirty-five ejaculates from seven jacks were split in SKM and SKM-CLC, extended at 50 million sperm/mL and stored at 5°C for 48 hours. Total motility (TM), progressive motility (PM), percentage of sperm with rapid motility (RAP) were assessed with CASA. Plasma membrane stability (PMS), and high mitochondrial membrane potential (HMP) were assessed with the combination of Yo-Pro and MitoStatusRed with flow cytometry. Semen was assessed before (0), 24 and 48h after cooling. In Experiment 2, two estrous cycles of 15 mares were used for fertility assessment. Mares were examined every other day by transrectal ultrasonography and had ovulation induced with 250 µg of histrelin acetate when a ≥35 mm follicle was first detected. Mares were randomly inseminated with semen obtained from one jack. Semen was extended in either SKM or SKM-CLC and cooled-stored for 24 hours. Pregnancy diagnosis was carried out 15-day post-ovulation. Data were analyzed with a mix model and Tukey's as posthoc and logistic regression model. Significance was set at P ≤ .05. There were no differences in TM, PM, RAP, PMS, and HMP for semen extended in either extender immediately before cooling (P > .05). There was a reduction in TM, PM, RAP, PMS, and HMP overtime across groups (P < .05); however, semen extended with SKM-CLC had superior TM, PM, RAP, PMS, and HMP than semen extended in SKM at 24- and 48-hours post-cooling (P < .05). Mares bred with semen extended in SKM had a lower conception rate (13%, 2/15 cycles) than cycles bred with SKM-CLC (47%, 7/15 cycles; P < .05). In conclusion, incorporating CLC into SKM extender improved cooling ability and fertility of donkey semen in horse mares. It remains to be determined if similar results can be obtained in clinical practice with mares and jennies.

Effects of the cryopreservation process on dog sperm integrity.

Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

Clinical safety of intratesticular transplantation of allogeneic bone marrow multipotent stromal cells in stallions.

Although stem cell therapy is a promising alternative for treatment of degenerative diseases, there are just few reports on the use of stem cells therapy in horse's reproductive system. This study aims to evaluate the effect of intratesticular injection of bone marrow mesenchymal stromal/stem cells (MSCs) in healthy stallions, and its outcome on seminal parameters and fertility. In Experiment 1, 24 stallions were divided into treatment group (TG) and control group (CG). In the TG, an intratesticular application of MSC was performed, and in the CG, only PBS was used. Measurements of testicular volume, surface temperature and Doppler ultrasonography were performed 24 and 48 hr after treatments. Fifteen days after application, the testicles were removed and submitted to histological analysis. In Experiment 2, 3 fertile stallions received similarly treatment with MSCs. Physical examination and sperm analysis were performed weekly during 60 days after treatment, and at the end, semen from one of them was used for artificial inseminations of 6 healthy mares. In Experiment 1, clinical examinations showed no signals of acute inflammation on both groups according to the analysed variables (p > .05). Also, no signal of chronic inflammation was observed on histological evaluation. In Experiment 2, stallions presented no physical alterations or changes in sperm parameters, and a satisfactory fertility rate (83%; 5/6) was observed after AI. The results support the hypothesis that intratesticular application of bone marrow MSCs is a safe procedure, and this could be a promising alternative to treat testicular degenerative conditions.

Evaluation of cooling and freezing systems of bovine semen.

Semen cryopreservation comprises different steps, among them are the cooling and freezing rates which significantly influence the quality of thawed sperm. Different systems with variable freezing rates are used for freezing bull semen in the field, with a consequence of variable success rates. The objective of this study was to compare different systems for freezing bull semen in the field. Five cooling methods of semen and two methods for the subsequent freezing phase (5 × 2 factorial scheme) were used. Two to four ejaculates were collected from 12 bulls with an electroejaculator. The ejaculates were diluted in BotuBov® to a concentration of 50 × 106 spermatozoa/mL in 0.5-mL straws. After dilution, the straws were cooled to 5 °C in five cooling systems: TK 4000® at a cooling rate of -0.25 °C/min (R1); TK 4000® at a rate of -0.5 °C/min (R2); Minitube® refrigerator at a rate of -2.8 °C/min (R3); Botutainer® at a rate of -0.65 °C (R4), and domestic refrigerator at a rate of -2.0 °C/min (R5). After stabilization at 5 °C for 4 h, these straws were then submitted to two freezing systems: TK 4000® at a freezing rate of -15 °C/min (C1) and Styrofoam box with liquid nitrogen at a rate of -19 °C/min (C2). Sperm kinetics were evaluated by computer-assisted sperm analysis at four time points: in fresh semen, after cooling, post-thawing, and after the rapid thermal resistance test (TRT). In addition, plasma and acrosomal membrane integrity, mitochondrial potential and intracellular H2O2 were analyzed after thawing by flow cytometry. The R1, R2 and R4 cooling systems were the most efficient in preserving sperm viability, membrane integrity and intracellular H2O2. Samples frozen in the C1 system exhibited better post-thaw and post-TRT kinetics than C2 samples. In conclusion, slower cooling curves in conjunction with a constant freezing rate obtained with the programmable unit were more efficient for freezing bull semen in the field.