Effect of crocin and naringenin supplementation in cryopreservation medium on post-thawed rooster sperm quality and expression of apoptosis associated genes

The aim of our research was to examine the effects of crocin (0.5 (C0.5), 1 (C1) and 1.5 (C1.5) mM) and naringenin (50 (N50), 100 (N100) and 150 (N150) µM) in cryopreservation extender for freezing rooster semen. Sperm motility, viability, abnormalities, membrane integrity, mitochondrial activity, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC, the mRNA expression of pro-apoptotic (CASPASE 3) and anti-apoptotic (Bcl-2) genes, fertility and hatchability rate were investigated following freeze-thawing. C1 and N100 resulted in the higher (P < 0.05) total motility and progressive motility in comparison to the control group. C1 and N100 improved viability, membrane integrity and reduced lipid peroxidation. We found much higher values for mitochondria activity with C1 and N100 respect to the control group. The C1 and N100 showed lower percentages of early apoptosis when compared with control group. Also, C1 and N100 had higher TAC when compared with control group. The mRNA expression of BCL-2 in the C1 and N100 group were significantly higher than that of other treatments. The expression of CASPASES 3 was significantly reduced in C1 and N100 group (P < 0.05) when compared to control group. Significantly higher percentage of fertility and hatching rate were observed in C1 and N100 compared to the control group. In conclusion, crocin at 1 mM and naringenin at 100 µM seem to improve the post-thawing rooster semen quality, fertility and could protect the sperm against excessive ROS generation by reducing the pro-apoptotic (CASPASE 3) and increasing anti-apoptotic (Bcl-2) genes.

extender on post-thawed rooster sperm quality and expression of apoptosis associated genes. 66 Quality and fertility analyses of the post-thaw sperm integrated with naringenin and crocin were 67 also performed after the freezing and thawing process.   75 This study was performed on ten adult Ross 308 broiler breeder roosters (30 week old) which 76 were kept individually in cages (diet compositions were included: 12% crude protein and 2,750 77 kcal maintenance energy/kg). Semen was collected twice a week from individual birds in a 78 graduated plastic tube [19]. Semen samples from each rooster were analyzed individually. The 79 samples that had the standard criteria motility of >80% concentration of >3 × 10 9 sperm/mL and 80 volume of >0.2 mL were used in the present study. Next, to remove individual differences, 81 semen samples were pooled and then assigned into 7 equal aliquots.  83 Seven experimental groups were applied in this study for semen dilution (Table 1)  Ltd.) and an Olympus AU 400 automatic biochemistry analyzer (Olympus, Tokyo, Japan).   Table 2. The specificity of the primers was checked by a 156 BLAST analysis of the National Center for Biotechnology information's database. At the 157 meantime, GAPDH was amplified as an endogenous control gene.

Extender preparation and cryopreservation
158 Table 2 159 Primer sequences used for quantitative real-time polymerase chain reaction

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Motility and velocity variables of frozen-thawed of rooster sperm supplemented with 193 different levels of crocin and naringenin are depicted in Table 3. C1 and N100 resulted in higher 194 (P < 0.05) total motility and progressive motility compared to the control group. The analysis did 195 not reveal any significant differences among different concentrations of crocin and naringenin on 196 the VCL, VAP, VSL, ALH, LIN, BCF and STR parameters.
197 Table 3 198 Effect of different levels of crocin and naringenin on motility parameters of rooster thawed semen, 199 analyzed by CASA (n = 5). and naringenin does not seem to impact the abnormal forms after freeze-thawing (Fig. 5).

215
Superior results were observed for viable sperm in C1 and N100 compared with control group 216 (Fig. 6).
217 Table 4 details the data on apoptosis status analysis. The most remarkable result is that the 218 percentage of live sperm was emerged to be higher in 1 mM crocin and 100 µM naringenin in 219 comparison with the control. Apoptotic spermatozoa were significantly reduced in the C1 and 220 N100 levels when compared to control group.
221 Table 4 222 Effect of different levels of crocin and naringenin on viable, apoptotic and dead sperm in rooster thawed 223 semen, as assessed by flow cytometry (n = 5).   Table 5 reports the data on effects of various levels of crocin and naringenin on the oxidative 231 parameters status of rooster sperm following freeze-thawing. We can note from the table that the 232 highest values for TAC activity were achieved in the C1 and N100 groups compared with control group. Also, malondialdehyde was significantly (P < 0.05) lower in C1 and N100 than the 234 control. The analysis did not reveal any significant differences for SOD and GPx parameters.  Table 5 237 Effect of different levels of crocin and naringenin on malondialdehyde concentration (MDA), glutathione 238 peroxidase (GPx) and superoxide dismutase (SOD) activities and total antioxidant capacity (TAC) of 239 rooster thawed semen (n = 5).  The results of mRNA expressions of BCL-2 and CASPASE 3 are showed in Fig.7 and Fig. 8.

247
The mRNA expressions of BCL-2 in the C1 and N100 group were significantly higher than that 248 in other treatments. The expression of CASPASES 3 was significantly reduced in C1 and N100 249 group (P < 0.05) when compared to control group.

250
The findings of the fertility trial (Table 6) revealed a significantly higher (P < 0.05) percentage 251 of fertility and hatching rate in C1 and N100 compared to the control group.
252 Table 6 255 Effect of crocin and naringenin on fertility and hatchability rates of rooster semen after freeze-thawing It is shown that crocin can reduce the levels of superoxide anion and hydrogen peroxide. The

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Our previous studies has adopted an approach in the study of the associations between sperm proportional to the function of sperm [32,44]. These data were again confirmed in the present 297 investigation, in which the MDA level was evaluated because it is known as a gold marker for 298 oxidative stress, a phenomenon extremely associated to the antioxidant system. In line with our study, Sapanidou, Taitzoglou (17), showed that MDA production decreased while supplementing 300 1 mM crocin in sperm.

301
According to our results, naringenin 100 µM reduced the MDA level. A satisfactory 302 explanation for this may be related to its structure-activity. Naringenin can give hydrogen to 303 ROS that allows the acquisition of a stable composition, allowing the elimination of these free 304 radicals. Another interesting reason is the existence of phenolic rings in naringenin which act as 305 electron barriers to remove superoxide anions characteristic known as free radicals [45].  Caspase-3 is known as the critical effector caspase responsible for the execution of apoptotic cell 341 death by cleaving numerous cellular substrates [56].

342
The results of this study show that the enhancement in fertility result using thawed sperm 343 stored in C1 and N100 was consistent with the other sperm functional parameters. The freezing and thawing process dramatically reduces the fertilization capacity of the rooster sperm.  Effect of glutamine and sugars after bull spermatozoa cryopreservation. Theriogenology.