Protocols for cooling or freezing goat semen usually recommend centrifugation for seminal plasma removal. However, little is known about the effect of this process on goat sperm viability and functionality. The present study evaluated the effects of centrifugation force on the plasma membrane, acrosomes, and DNA integrity of goat semen. Four ejaculates from each of the four different Anglo Nubian male goats were used. Semen samples were obtained using artificial vagina, and immediately after collection, ejaculates were diluted using Ringer’s sodium lactate solution and split into three groups: Control (CG, without centrifugation), G1 (centrifugation 600 x g/10 min), G2 (centrifugation 1200 x g/10 min). After centrifugation, seminal plasma was removed, the sperm pellets were resuspended using Tris-egg yolk extender (80 x 106 spermatozoa/mL) and the sperm morphology was analyzed. Samples were cooled at 5°C for 5, 24, 36, and 48 h and then sperm plasma membrane and acrosome integrity (PMAI, %) and sperm DNA fragmentation index (SDF, %) were evaluated at each time-point, using a flow cytometer. Additionally, sperm movement was determined using computer semen analysis (CASA) after 5, 24, and 48 h of refrigeration period. The semen centrifugation did not induce additional sperm morphology defect or reduction in sperm kinetics in the experimental groups. Differences were not observed (p > 0.05) in PMAI and SDF among different groups, in any of each timepointof the cooling process. In conclusion, centrifugation, even at high speeds, did not affect goat sperm integrity and functionality when submitted to refrigeration process.

Introduction The addition of low-molecular-weight antioxidants during the freezing process improves post-thaw sperm quality. The high antioxidant potential of cryopreserved semen could have a positive effect on the motility, viability, and energy status of sperm cells and their ability to bind to the zona pellucida of oocytes. The aim of the study was to determine the effects of different concentrations and combinations of vitamins E and C in a semen extender on selected quality parameters of frozen-thawed canine spermatozoa. Material and Methods The experimental material was the semen of four mixed-breed dogs. Sperm viability (motility, plasma membrane integrity, and mitochondrial function) was examined at 0, 60, and 120 min in semen samples supplemented with the extender and in the controls. Results Combined supplementation with vitamins C + E at a concentration of 200 + 200 μM /1 × 10⁹ spermatozoa had the most profound effect on total sperm motility, linear motility, and the percentage of spermatozoa with intact plasma membrane and active mitochondria. Conclusion The synergistic activity of vitamins E and C had a more beneficial influence on the quality of frozen–thawed sperm than these non-enzymatic antioxidants applied separately.

OBJECTIVE To determine the effect of subchronic oral exposure to zearalenone (ZEA) at a daily dose of 50 μg of ZEA/kg of body weight (an environmentally relevant concentration) on the reproductive system of rabbit bucks. ANIMALS 8 healthy sexually mature New Zealand White rabbits. PROCEDURES During the experimental period (March to June), each rabbit underwent a 7-week control protocol and then a 7-week treatment protocol. Water (0.5 mL) or ZEA solution (50 μg/kg [0.5 mL]) was administered orally once daily during the control and treatment period, respectively; ejaculates were collected weekly. Studied end points included semen quality variables (spermatozoa kinetics, morphology, viability, and DNA fragmentation), serum testosterone concentration, and results of histologic examination of the testes and epididymides following euthanasia at the end of the experimental period. RESULTS Treatment with ZEA solution resulted in significant increases in spermatozoa beat-cross frequency, in the percentages of spermatozoa with head and midpiece abnormalities, and in the percentages of DNA-fragmented spermatozoa, compared with effects of the control treatment. Serum testosterone concentration, other spermatozoa velocity variables, and percentages of progressive and total motility, rapidly or slowly moving spermatozoa, and live spermatozoa did not differ significantly between the 2 periods. Histologic examination revealed no patterns of abnormal findings in the testes and epididymides. CONCLUSIONS AND CLINICAL RELEVANCE Oral treatment with ZEA solution at an environmentally relevant concentration caused minor interference with rabbit bucks' sperm quality. Although mostly considered mild, the sperm quality changes warrant further investigation in terms of fertilizing capacity impairment.

OBJECTIVE To determine the in vitro effect of calcitriol on indicators of immune system function in endotoxin-primed blood samples from healthy dogs. SAMPLE Blood samples from 6 healthy adult dogs. PROCEDURES Leukocytes were primed by incubation of blood samples with lipopolysaccharide (LPS; endotoxin) or PBS solution (unprimed control group) for 1 hour. Following priming, blood samples were incubated with calcitriol (2 × 10(−7)M) or ethanol (control substance) for 24 hours. After sample incubation, LPS-stimulated leukocyte production of tumor necrosis factor (TNF) and interleukin-10 (IL10) was measured with a canine-specific multiplex assay, and apoptosis and toll-like receptor 4 (TLR4) expression were evaluated via flow cytometry. RESULTS LPS stimulation of unprimed leukocytes but not endotoxin-primed leukocytes resulted in a significant increase in TNF and IL10 production, confirming the presence of endotoxin tolerance in dogs in vitro. Endotoxin priming significantly increased neutrophil viability with no effect on lymphocyte viability or TLR4 expression by neutrophils and monocytes. Calcitriol exposure significantly decreased LPS-stimulated production of TNF by unprimed and endotoxin-primed leukocytes. Conversely, calcitriol exposure had no effect on IL10 production by unprimed leukocytes but did significantly increase IL10 production by endotoxin-primed leukocytes. Calcitriol had no significant effect on the degree of neutrophil or lymphocyte apoptosis, nor was neutrophil and monocyte TLR4 expression affected in unprimed or endotoxin-primed leukocytes. CONCLUSIONS AND CLINICAL RELEVANCE These data indicated that calcitriol induced an anti-inflammatory shift in unprimed and endotoxin-primed canine leukocytes in vitro, without compromising neutrophil and monocyte TLR4 expression or altering the viability of neutrophils and lymphocytes in canine blood samples.

OBJECTIVE To create a bioactive synovium scaffold by infusing decellularized synovial-derived extracellular matrix (synECM) with synovial-derived mesenchymal stem cells (synMSCs). SAMPLE Synovium from the femoropatellar and medial femorotibial joints of equine cadavers. PROCEDURES The synMSCs were cultured in monolayer and not treated or cotransduced to enhance expression of green fluorescent protein (GFP) and human bone morphogenetic protein (BMP)-2. The synECM was decellularized with 0.1% peracetic acid and then seeded with synMSCs (0.5 × 10(6) cells/0.5 mL) by use of a 30% serum gradient. Samples were evaluated on days 0, 3, 7, and 14. Cell migration, differentiation, and distribution into the synECMs were determined by cell surface marker CD90, viability, histologic morphology, and fluorescence microscopy results and expression of GFP, BMP-2, hyaluronan (HA), and proteoglycan (PG). RESULTS At day 14, synMSCs were viable and had multiplied 2.5-fold in the synECMs. The synECMs seeded with synMSCs had a significant decrease in CD90 expression and significant increases in HA and PG expression. The synECMs seeded with synMSCs cotransduced with GFP, or BMP-2 had a significant increase in BMP-2 expression. CONCLUSIONS AND CLINICAL RELEVANCE The synECM seeded with synMSCs or synMSCs cotransduced with GFP, or BMP-2 yielded a bioactive synovial scaffold. Expression of BMP-2 by synMSCs cotransduced to enhance expression of BMP-2 or GFP and an accompanying increase in both HA and PG expression indicated production of anabolic agents and synoviocyte differentiation in the scaffold. Because BMP-2 can promote repair of damaged cartilage, such a bioactive scaffold could be useful for treatment of injured cartilage.