Cinnamtannin B-1 (CNB-1) is a naturally occurring trimeric A-type proanthocyanidin contained in several plants such as cinnamon (Cinnamomum zeylanicum). It is considered to be a potent antioxidant. The protective effect of CNB-1 against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from six stags were thawed, pooled and extended to 400 × 106 sperm/ml in BGM (bovine gamete medium). After being aliquoted, the samples were supplemented with different concentrations of CNB-1 (0, 0.1, 1, 10 and 100 μg/mL), with or without induced oxidative stress (100 μM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. This experiment was replicated six times. Spermmotility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (TUNEL) were assessed. After 4 h of incubation, CNB-1 prevented the deleterious effects of oxidative stress, thus improved sperm progressivity and velocity (P<0.05). Furthermore, 1 and 10 μM CNB-1 improved sperm linearity, even when compared to those samples that had not been subjected to oxidative stress (P<0.05). The greatest concentration, 100 μM, prevented sperm lipoperoxidation and reduced ROS production in samples subjected to oxidative stress.
- MeSH
- antioxidancia farmakologie MeSH
- fragmentace DNA MeSH
- motilita spermií účinky léků MeSH
- oxidační stres účinky léků MeSH
- peroxidace lipidů MeSH
- proantokyanidiny farmakologie MeSH
- reaktivní formy kyslíku metabolismus MeSH
- spermie účinky léků MeSH
- uchování spermatu veterinární MeSH
- viabilita buněk účinky léků MeSH
- vysoká zvěř * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm.
- MeSH
- fragmentace DNA MeSH
- kryoprezervace metody veterinární MeSH
- motilita spermií MeSH
- poměr pohlaví MeSH
- předvýběr pohlaví veterinární MeSH
- průtoková cytometrie veterinární MeSH
- spermie fyziologie MeSH
- uchování spermatu metody veterinární MeSH
- umělá inseminace veterinární MeSH
- vysoká zvěř * MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll(®) , Puresperm(®) and Bovipure(™) , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure(™) yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll(®) and Puresperm(®) considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll(®) selected less live and more apoptotic spermatozoa than Puresperm(®) and Bovipure(™) for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.
- MeSH
- analýza spermatu MeSH
- centrifugace - gradient hustoty veterinární MeSH
- kryoprezervace veterinární MeSH
- separace buněk metody veterinární MeSH
- uchování spermatu veterinární MeSH
- vysoká zvěř fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 μM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome-reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca(2+) levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.
- MeSH
- estrus krev fyziologie MeSH
- kapacitace spermií fyziologie MeSH
- kaspasy farmakologie MeSH
- motilita spermií MeSH
- ovce krev fyziologie MeSH
- reaktivní formy kyslíku MeSH
- spermie fyziologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
