Degenerative changes of the endometrium are directly related to age and fertility in mares. Chronic degenerative endometritis (CDE) is correlated with uterine fluid retention and reduced ability to clear uterine inflammation. Recent research in the areas of equine surgery and sports medicine has shown that platelet-rich plasma (PRP) treatment acts as an immunomodulator of the inflammatory response. Therefore, the aim of this study was to determine if the uterine infusion of PRP could modulate the local inflammatory response and modify the intrauterine NO concentrations after artificial insemination (AI) in both normal mares and those with CDE. Thirteen mares with endometrium classified as grade III on the histology (mares with CDE) and eight mares with endometrial histological classification I or II-a normal mares were selected to investigate the effect of PRP therapy. The mares were inseminated with fresh semen in two consecutive cycles in a crossover study design. Thereby, each mare served as its own control and the treatment was performed with intrauterine PRP infusion four hours after AI. The percentage of neutrophils in uterine cytology (CIT, %), uterine fluid accumulation observed on ultrasonography (FLU, mm) and nitric oxide concentration of uterine fluid (NO, μM) were analyzed before and 24 hours after AI. The results reported that mares with CDE (CIT, 68.3 ± 3.27, FLU, 10.7 ± 1.61) have a higher (P < 0.05) intrauterine inflammatory response after AI than normal mares (CIT, 24.4 ± 3.56, FLU, 0), but NO concentrations did not differ (P > 0.05) between categories of mares. In treated cycles with PRP, the intrauterine inflammatory response decrease (P < 0.05) in CDE mares (CDE: CIT, 31.4 ± 6.48, FLU, 5.5 ± 1.28; normal mares: CIT, 13.5 ± 4.31, FLU, 0) when compared with nontreated cycle (CDE: CIT, 68.3 ± 3.27, FLU, 10.7 ± 1.61; NM: CIT, 24.4 ± 3.56, FLU, 0), but did not modify NO concentrations in uterine fluid. Thus, we can conclude that PRP was effective in modulating the exacerbated uterine inflammatory response to semen in mares with CDE but did not reduce NO concentrations in intrauterine fluid.

Cryopreservation of bull semen is a common biotechnology procedure in cattle breeding. However, when the ejaculate is obtained by electroejaculation, wide variation is observed in the sperm/seminal plasma (SP) ratio that can affect the freezability of semen in this species. The removal of SP may improve the quality of frozen bull semen. The objective of this study was to evaluate the effect of SP removal from the ejaculate on the cryopreservation of semen from 38 Nellore bulls collected by electroejaculation. After collection, the ejaculate was divided into three aliquots: (1) control (N) diluted to a concentration of 60 × 10 spermatozoa/mL and frozen with SP; (2) centrifugation (C) at ×600g for 10 minutes and the pellet resuspended and frozen at the same concentration as N; and (3) filtration (F) through SpermFilter and sperm recovered and frozen at the same concentration as N. After thawing, sperm kinetics, plasma and acrosome membrane integrity, mitochondrial membrane potential, oxidative stress, and in vitro fertility were evaluated. Statistical analysis was performed using the SAS 9.2 package, and differences were considered significant when P < 0.05. Higher average path velocity and straight-line velocity were observed in the groups submitted to SP removal compared to the control group (P < 0.01). In contrast, filtered samples exhibited higher beat cross frequency, straightness, and linearity compared to the other groups. Plasma membrane integrity was reduced when SP was removed, but lower oxidative stress was observed in groups C and F (34.91 ± 2.95% and 31.63 ± 2.95%, respectively) compared to group N (57.39 ± 2.95%). However, the percentage of hatched blastocysts was similar in the N and F groups (21.22 ± 1.05% and 24.00 ± 1.05%, respectively) and higher compared to group C (18.83 ± 1.05%). In conclusion, removal of SP by centrifugation for bull semen freezing reduced the rate of in vitro-produced embryos, whereas filtration of prefrozen semen was found to be an efficient alternative in terms of semen freezability and in vitro production of bovine embryos.

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 × 10 6 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 H 2 O 2 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 H 2 O 2. 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.

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The use of the conditioned medium (CM) for diseases treatment is based on its enrichment with biomolecules with therapeutic properties and themselves have a beneficial effect. Secretome of bovine endometrial mesenchymal progenitor/stem cells (eMSCs) using a proteomics approach is until now unknown. This work aimed to evaluate the secretome of bovine eMSCs-CM challenged or not with lipopolysaccharide (LPS). For this, eMSCs characterized were challenged (TG) or not (CG). The CM was collected 12h after stimulation and submitted to mass spectrometry analysis. The classification of identified proteins was done by PANTHER according to biological processes, molecular function, cellular component and protein class. 397 protein groups were identified in TG and 302 in CG. We observed positive enrichment for antibacterial response proteins, macrophage activation function, receptor-mediated endocytosis, hydrolase activity, inhibitory enzyme in TG, and for activity structural molecule and intermediate filament cytoskeleton in the CG. Our experimental model shows that eMSCs respond to LPS in the concentration used and can be used to study immune-inflammatory response, besides of the secretion of proteins mainly related to tissue remodeling, immune response and angiogenesis which is an interesting feature for use in cell therapy.