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

Antioxidant effect of lemon balm (Melissa officinalis) and mate tea (Ilex paraguensys) on quality, lipid peroxidation and DNA oxidation of cryopreserved boar epididymal spermatozoa

In this study, we investigated the protective ability of the addition of two antioxidant herb extracts, mate tea and lemon balm, on boar epididymal frozen–thawed spermatozoa quality. Testes from mature boars were collected at local slaughterhouse, and sperm samples from epididymis were recovered by flushing. Spermatozoa were cryopreserved in lactose–egg yolk buffer supplemented with various concentrations of lemon balm and mate tea (0, 2.5, 5 and 10 g l−1) using the straw‐freezing procedure. Motion parameters, acrosome and plasma membrane integrity, lipoperoxidation levels and DNA oxidative damage (8‐hydroxy‐2′‐deoxyguanosine base lesion) were evaluated. There were no differences among experimental groups with regard to motility characteristics, viability, acrosome and plasma membrane integrity; however, the highest concentration of lemon balm produced significant (P < 0.05) improvement in curvilinear trajectory, straightness and amplitude of lateral head displacement after thawing. The supplementation of freezing extender with mate tea and lemon balm reduced sperm lipid membrane peroxidation, and only mate tea protected DNA against oxidative damage during cryopreservation at 120 min post‐thawing (P < 0.05). Mate tea experimental extender at concentration of 10 g l−1 showed the lowest percentage of sperm oxidised DNA and malondialdehyde generation; thus, mate tea is a potential candidate such as antioxidant compound on boar sperm cryopreservation medium.EEA BalcarceFil: Luño, Victoria. Universidad de Zaragoza. Facultad de Veterinaria. Departamento de Patología Animal; EspañaFil: Gil, María Lydia. Universidad de Zaragoza. Facultad de Veterinaria. Departamento de Patología Animal; EspañaFil: Olaciregui, Maite. Universidad de Zaragoza. Facultad de Veterinaria. Departamento de Patología Animal; EspañaFil: Jerez, Rodrigo A. Universidad de Zaragoza. Facultad de Veterinaria. Departamento de Patología Animal; EspañaFil: de Blas, Ignacio. Universidad de Zaragoza. Facultad de Veterinaria. Departamento de Patología Animal; EspañaFil: Hozbor, Federico Andres. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Departamento de Producción Animal. Biotecnología de la Reproducción; Argentin