Swine spermatozoa trigger aggregated neutrophil extracellular traps leading to adverse effects on sperm function.

In pigs, the number of PMN in uterus lumen increases within few hours after natural or artificial AI resulting in early PMN-derived innate immune reactions. Sperm-NETs formation was recently reported to occur in various mammalian species. Aim of this study was to investigate direct interactions of boar spermatozoa with swine PMN, the release of sperm-mediated NETs, and to assess NET-derived effects on sperm functionality. Sperm-triggered NETs were visualized by SEM- and immunofluorescence analyses. Sperm-mediated NETosis was confirmed by presence of extruded DNA with global histones and NE. Largest sizes of sperm-mediated aggNETs were detected after 5 h thereby resulting in effective massive sperm entrapment. The number of aggNETs increased from 3 h onwards. Kinetic studies of swine sperm-mediated NETosis showed to be a time-dependent cellular process. In addition, number of NETs-entrapped spermatozoa increased at 3 h of exposure whilst few free spermatozoa were detected after 3 h. Anchored NETs also increased from 3 h onwards. The cytotoxicity of NETs was confirmed by diminution of the total motility and the progressive motility. Spermatozoa membrane integrity and function loss exposed to NETs was confirmed from 3 h. Experiments revealed NETs-derived damaging effects on swine spermatozoa in membrane integrity, motility and functionality. We hypothesize that swine sperm-triggered aggNETs might play a critical role in reduced fertility potential in swine reproductive technique. Thus, aggNETs formation needs to be considered in future studies about uterine environment as well as advance of sperm in the porcine female reproductive tract.

Freezing dog semen using -80 °C ultra-freezer: Sperm function and in vivo fertility.

Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN2). However, previous works in freezing canine semen using a -80 °C ultra-freezer (-80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN2 or -80°C-UF on sperm function and in vivo fertility of frozen-thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN2 or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN2 frozen-thawed semen = 5 and -80°C-UF frozen-thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using -80 °C UF is an effective technique for long-term preservation of canine spermatozoa.