Specific phosphodiesterase type-10 inhibitor, papaverine, added after the cooling period improves canine sperm quality.

The use of chilled semen has gained increasing interest in canine reproductive services. The addition of phosphodiesterase (PDE) inhibitors that increase the intracellular cyclic adenosine monophosphate levels may improve sperm motility. The purpose of this study was to examine the quality of sperm under the effect of the specific PDE-10 inhibitor (papaverine) added after storage for 1, 2, and 3 days at 5 °C. The ejaculates were obtained from 5 healthy Beagle dogs by digital manipulation. After collection, ejaculates were pooled, extended and cooled at 5 °C during 3 days. Sperm parameters were tested 30 min after the addition of different papaverine (PA) concentrations: 0, 5, 10 and 20 µM. Sperm motility (CASA), viability (PI/FITC-PNA) and capacitation status (chlortetracycline assay) were evaluated. The results showed that the addition of PA has no effect on sperm samples at day 0. However, concentrations of 5 and 10 µM increased (p < .05) sperm motility kinetics and viability significantly compared to the control at day 1, day 2 and day 3 of cooling. The addition of 20 µM PA decreased (p < .05) sperm quality parameters significantly and increased the percentage of capacitated/acrosome-reacted spermatozoa. In conclusion, the addition of 5 and 10 µM PA concentrations after cooled storage improved canine sperm quality.

Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C.

Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

Effects of seminal plasma and different cryoprotectants on rabbit sperm preservation at 16°C.

The purpose of this research was to assess whether the presence of seminal plasma (SP) can improve sperm quality of rabbit spermatozoa stored at 16°C for 72 h and moreover evaluate the cryoprotectant effects of glycerol, N-N-Dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP). Semen samples were pooled and divided in eight fractions. Four of them were diluted with INRA (extender A), INRA with 6% glycerol (extender B), INRA with 6% DMF (extender C), or INRA with 6% NMP (extender D), respectively. The other four fractions were centrifuged, and the supernatant was removed in order to eliminate SP. Each sample was then resuspended with extender A, B, C, or D, respectively. All samples were stored at 16°C and analysed at 4, 24, 48, and 72 h by ISAS®, vitality test, HOS test, and acrosome integrity test. After analyse of the results, SP samples showed a significantly higher percentage (P=0.020) in the HOS test (71.9 ± 1.6%) than non-SP samples (66.5 ± 1.6%). Non-SP samples had better results for kinematic parameters. Extenders A and C showed great results for the percentage of motile spermatozoa (63.1 ± 4.3% and 63.4 ± 3.7%, respectively), vitality (88.9 ± 2.6% and 87.7 ± 2.7%, respectively), and HOS test (68.9 ± 1.4% and 75.2 ± 1.4%, respectively). Extenders B and D showed worse data for sperm quality. These results suggest that SP has a protective effect on rabbit sperm membranes and maintains better sperm motility. The addition of glycerol and NMP to INRA does not improve rabbit sperm quality; nevertheless, the DMF cryoprotectant exerts a protective effect on the membrane of spermatozoa, improving seminal quality during rabbit sperm preservation at 16°C.

Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents.

Freeze-drying (FD) technique has been applied as an alternative technology to preserve gene resources to allow simple sperm preservation and shipment at 4 °C. Nevertheless, DNA sperm might be damaged by mechanical or oxidative stress throughout FD procedure. Therefore, suitable protection to maintain DNA integrity is required. The aim of this study was to determine the effect of rosmarinic acid (RA) as an antioxidant and two chelating agents (EGTA and EDTA) on the DNA integrity of freeze-dried rabbit sperm after storage of the samples at 4 °C and room temperature for 8 months. Rabbit sperm were freeze-dried in basic medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with 50 mM EGTA (1), 50 mM EGTA plus 105 µM RA (2), 50 mM EDTA (3) or 50 mM EDTA plus 105 µM RA (4). Semen samples were kept at 4 °C and room temperature during 8 months. After rehydration, DNA integrity was evaluated with Sperm Chromatin Dispersion test observing that DNA fragmentation was higher when semen samples were freeze-dried with EGTA (10.9%) than with EDTA (4.1%) (p < 0.01). Furthermore, RA acted better under adverse conditions and no significant differences were found in temperature storage. Summarizing, FD is a method that can allow simple gene resources preservation among 4 °C to 25 °C during 8 months and transportation without the need for liquid nitrogen or dry ice. EDTA chelating agent is the most suitable media for freeze-dried rabbit sperm and the addition of RA protects the DNA against the oxidative stress caused during FD procedure.

Chelating agents in combination with rosmarinic acid for boar sperm freeze-drying.

The presence of DNA protective agents in the medium is necessary to maintain sperm functionality after freeze-drying procedure. The objective of this study was to investigate the effect of chelating agents, ethylene diaminetetraacetic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA), in combination with rosmarinic acid (RA) on DNA integrity of freeze-dried boar sperm. We also examined the effect of these agents on the in vitro developmental ability of porcine oocytes following sperm injection (ICSI). Heterospermic mix, obtained from ejaculated sperm of three boars, was freeze-dried in two different chelating agents' media: 50mM EDTA or 50mM EGTA, and in these media supplemented with 105µM of rosmarinic acid. Frozen-thawed sperm was used as control. After rehydration, samples were subjected to DNA damage detection using Sperm Chromatin Dispersion test. ICSI was performed to verify the ability of freeze-dried sperm to participate in embryonic development. Five replicated trials were carried out for each group. In the presence of rosmarinic acid, the percentage of spermatozoa with DNA damage decreased significantly (p=0.010), without differences between the two chelating agents combination. EDTA solution preserves more efficiently DNA integrity of boar sperm than EGTA solution (p=0.002). There were no significant differences among the studied groups related to the blastocyst formation rate. Results suggested that the addition of rosmarinic acid to the medium improves sperm DNA integrity after freeze-drying, but does not promote fertilization and blastocyst development. We also observed a similar percentage of embryos production with freeze-dried and with frozen-thawed sperm.

In vitro developmental ability of ovine oocytes following intracytoplasmic injection with freeze-dried spermatozoa.

Freeze-drying (FD) is a new and alternative method to preserve spermatozoa in refrigeration or at room temperature. Suitable protection is required to maintain the sperm DNA integrity during the whole process and storage. The aim of this study was to examine the effect of rosmarinic acid and storage temperature on the DNA integrity of freeze-dried ram sperm. In addition, we evaluated the in vitro developmental ability to the blastocyst stage of oocytes injected with freeze-dried sperm. Ram sperm was freeze-dried in basic medium and in this medium supplemented with 105 µM rosmarinic acid. The vials were stored for 1 year at 4 °C and at room temperature. Frozen sperm was used as control. After rehydration, sperm DNA damage was evaluated, observing that the percentage of spermatozoa with DNA damage decreased significantly in the presence of rosmarinic acid, without differences between the two storage temperatures. Moreover, no differences were observed between the freeze-dried group and the frozen-thawed group in terms of blastocyst formation rate. We proved for the first time that ovine spermatozoa can be lyophilized effectively, stored at room temperature for long term, reconstituted and further injected into oocytes with initial embryo development.

Fertilisation rate obtained with frozen-thawed boar semen supplemented with rosmarinic acid using a single insemination timed according to vulvar skin temperature changes.

Artificial insemination (AI) of sows with frozen-thawed semen usually results in lower pregnancy rates and litter sizes than the use of liquid preserved semen. The present study evaluated the effectiveness of vulvar skin temperature changes as a predictor of ovulation in sows and determined the fertility rates obtained after AI with frozen-thawed semen supplemented with rosmarinic acid (RA). Semen was collected from mature boars and cryopreserved in experimental extenders supplemented with or without 105 µM of RA. Multiparous sows were inseminated with a single dose of semen when vulvar skin temperature decreased to a value below 35 °C. Intrauterine insemination was performed using 1.5 × 109 spermatozoa. The sows were slaughtered 48 h after AI and the embryos and oocytes were recovered from the oviducts. Total and progressive motility, viability and acrosome integrity were significantly (P < 0.05) higher in RA-supplemented semen samples compared with the control. Fertilisation occurred in all sows inseminated in the study, although there were no significant differences between the experimental groups. Sows inseminated with RA-supplemented semen showed a slight increase in the number of embryos recovered as compared to sows inseminated with control semen. In conclusion, insemination according to vulvar skin temperature changes resulted in successful fertilisation in all sows, although supplementation of the freezing media with RA did not improve the fertilising ability of frozen-thawed boar sperm.

Rosmarinic acid improves function and in vitro fertilising ability of boar sperm after cryopreservation.

During cryopreservation, oxidative stress exerts physical and chemical changes on sperm functionality. In the present study we investigated the antioxidant effect of rosmarinic acid (RA) on quality and fertilising ability of frozen-thawed boar spermatozoa. Ejaculates collected from mature boar were cryopreserved in lactose-egg yolk buffer supplemented with different concentrations of RA (0 µM, 26.25 µM, 52.5 µM and 105 µM). Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels, DNA oxidative damage (8-hydroxy-2-deoxyguanosine base lesion) and in vitro fertilisation ability were evaluated. Total and progressive motility were significantly higher in experimental extenders with RA than in the control (P<0.05) at 0 and 120 min post-thawing. The plasma and acrosomal membrane integrity were improved by supplementation with 105 µMRA (P<0.05). Negative correlation between RA and malondialdehyde (MDA) concentration were determined (P<0.05). After thawing, the percentage of spermatozoa with oxidised DNA did not differ between extenders, however, at 120 and 240 min post-thawing, the samples supplemented with 105 µMRA showed the lowest DNA oxidation rate (P<0.05). The penetration rate was significantly higher on spermatozoa cryopreserved with 105 µMRA (P<0.05). The results suggest that RA provides a protection for boar spermatozoa against oxidative stress during cryopreservation by their antioxidant properties.