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Freeze-drying sperm seems to be the ultimate alternative to preserve sperm cells from endangered species because of its easiness in transportation and storage of samples. This technique has already been used for equine, bovine, murine, rabbit, canine, and feline sperm cells. However, it is still important to verify the DNA integrity of such samples to produce viable offspring. The objective of this study was to verify the reliability of acridine orange (AO) staining to assess DNA damage of feline freeze-dried sperm samples. Three normospermic cats were used as sperm donors, and sperm samples were collected with the use of an artificial vagina without the presence of an oestrous queen. The control group (G1) used only fresh semen. To increase the range of sperm variability before freeze-drying, the sperm samples were supplemented with 2 extenders, namely SOF (G2) and human tubal fluid (G3). These extenders were selected because of their lack of cryoprotectant agents with a view to producing membrane damage and thus putting DNA integrity at risk. Samples were taken to a freeze-dryer (Edwards do Brasil, Brazil) to produce stable free-dried sperm. Over 50 samples were assessed for DNA damage by using AO and alkaline comet assay (CA). Acridine orange taints red/orange the sperm cells that present sperm damage, and CA shows long comet tails for cells with DNA damage. The CA was selected to countercheck the AO results owing to its higher accuracy. Nevertheless, both AO and CA count 100 cells per analysis, and a total of 3 repetitions per sample were sufficient. For the statistical analysis, SAS PROC GLM was used, and the significance level was set at 0.05. The AO staining showed that the G1 samples had a greater DNA damage (27.1%) if compared with G2 (15.2%) and G3 (23.1%; P < 0.0001). Nevertheless, G2 and G3 displayed sufficient DNA integrity throughout the whole lyophilization process. The CA, which is a more precise evaluation technique, proved that the groups had distinguished results but still were ordered in the same way: G1 was the one with more DNA damage (17.7%), G3 was second (14.4%), and G2 was third (3.2%) in DNA alterations. Just like AO, the CA showed difference among treatments (P < 0.0001). Since AO is a technique that requires very little sophistication and presented about the same results as the very elaborate CA method, it might be used on a daily basis to assess feline sperm DNA that underwent the freeze-drying process.

Alterations to a sperm cell that are similar to apoptosis reduce the sperm cell longevity in the female reproductive tract. The aim of the present study was to evaluate the effect of sperm motility factors [caffeine, heparin, and penicillamine, hypotaurine, and epinephrine (PHE)] on phosphatidylserine translocation in equine epididymal semen. Equine epididymal samples were obtained from 10 stallions using retrograde flush with 40 mL of BotuSemen® (BS) per epididymal cauda. The samples were divided into 3 groups: BS (control), Fert-TALP (Fert), and Sperm-TALP (SP), and diluted 1 : 1 with these media. Samples containing 100 × 106 sperm cells were loaded into straws, and then incubated at room temperature (25°C) for 15 min. The samples were centrifuged (1000g for 10 min), the supernatant was removed and the pellets were resuspended in BotuCrio®. Samples were loaded into 0.5-mL straws and cooled at 5°C for 20 min. Then, they were frozen in nitrogen vapor (6 cm above the level of LN) for an additional 20 min and finally plunged into nitrogen and stored. The straws were thawed at 46°C/20 min. Sperm motility was evaluated by computer-assisted semen analysis (CASA; HTM-IVOS 12). To evaluate the pancreatic secretory trypsin inhibitor (PSTI), the Annexin V-FITC Apoptosis Detection Kit (6710KK; Pharmingen, San Jose, CA, USA) was used according to the manufacturer’s recommendations. The samples were diluted in Annexin V buffer solution to a concentration of 2 × 106 spermatozoa mL–1 and then 5 mL of Annexin V-FITC, 5 mL of propidium iodide (PI, 50 mg mL–1), and 2 mL of Hoechst 33342 dye (H342, 40 mg mL–1) were added. The samples were then homogenized and incubated for 15 min. Then, 400 µL of Annexin V buffer solution was added to obtain a final concentration of 1 × 106 spermatozoa mL–1. The samples were evaluated. Flow cytometry was carried out at BD LSR II (Becton Dickinson, Mountain View, CA, USA). Mean and standard deviation were calculated, and then the normality test was evaluated by the Kolmogorov-Smirnov test. An analysis of variance and Tukey test with a P < 0.05 significance level were used to compare the mean values. Statistical analysis was performed using ANOVA followed by the Tukey test (P < 0.05). There was no significant difference in the percentage of non-translocated viable cells. Total motility (36.2 ± 18.18, 52.3 ± 18.40, and 51.4 ± 22.22) and progressive motility (13.8 ± 9.27, 27.7 ± 13.48, and 28.1 ± 15.29) were assessed for BS, Fert, and SP samples, respectively. Mean values of phosphatidylserine translocation index (±SD) of dead cells (8.40 ± 5.1, 6.12 ± 3.9, and 18.6 ± 22.3), viable cells with translocation of phospholipids (61.5 ± 21.3, 63.9 ± 20.6, and 52.2 ± 27.2), viable (28.3 ± 13.6, 28.7 ± 13.7, and 27.7 ± 15.4), and dead cells with translocation of phospholipids (1.8 ± 1.1, 1.2 ± 1.9, and 1.4 ± 2.6) were registered for BS, SP, and Fert, respectively. No difference was observed among incubation media. The results of the present experiment reinforce the idea that the incubation of equine epididymal semen with either SP or Fert medium has a beneficial effect on sperm parameters without being deleterious to sperm fertility.

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Effect of refrigeration systems upon frozen bull sperm viability assessed by computer-assisted sperm analysis and fluorescent probes AbstractSperm cryopreservation success depends upon the maintenance of spermatozoa fertility potential. Sperm cells must preserve both integrity and functionality of several cell structures. The stabilization phase must allow the exit of water from the sperm cells via osmosis. This study aimed to compare the effect of refrigeration in the commercial refrigerator (CR) and the transport/refrigeration box (TRB) upon the viability of frozen bull sperm diluted in three different extenders (A, B and C). Ten Nellore bulls, Bos taurus indicus maintained in Artificial Insemination Center were used and the spermatozoa samples was assessed for Plasma Membrane Integrity and CASA evaluation. The stabilization phase (5°C/4 hours) was performed in the CR as well as in the TRB, and then samples were exposed to nitrogen vapor during 20 minutes and then plunged into nitrogen. The statistical analysis was done using the variance analysis and the significance level was set at 5%. In the CR the post-thawing parameters for PM and ALH were higher (p < 0.05) in the extender A (glicine egg-yolk) and extender B (glicine egg-free) when compared with extender C (TRIS egg-yolk). As for BCF, STR and LIN, the parameters were higher (p < 0.05) in extender B than in C. Samples that were stabilized in the TRB presented higher post-thawing parameters (p < 0.05) for PM and LIN in extender A and extender B when compared with C. BCF and STR parameters were higher (p < 0.05) in extemder B when compared with C. Extender B samples had higher (p < 0.05) PMI when stabilized in CR. The findings in this experiment enable us to say that both CR and TRB were effective in keeping the viability of post-thawing bull semen. membranas plasmáticas e por meio da análise computadorizada (CASA). A fase de estabilização (5°C/4 horas) foi realizada em RC e em CTR, sendo as amostras expostas ao vapor de nitrogênio durante 20 minutos e após mergulhadas no nitrogênio. A estatística foi feita com a análise de variância com nível de significância a 5%. No RC os parâmetros pós-descongelação para PM e ALH foram superiores (p < 0,05) no meio B em relação ao C. Amostras que foram estabilizadas na CTR apresentaram parâmetros superiores (p < 0,05) para PM e LIN nos meios A e B, em relação ao C. Os parâmetros BCF e STR foram superiores (p < 0,05) no meio B em relação ao C. As amostras do meio B tiveram maior (p < 0,05) PMI quando estabilizadas no RC. No presente estudo, conclui-se que o RC e a CTR foram efetivos na manutenção da viabilidade pós-descongelação do sêmen bovino. Palavras-chave: Touros zebu, sêmen refrigerado, sêmen congelado, CASA, integridade de membrana

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