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

Abstract

21The aim of our research was to examine the effects of crocin (0.5 (C0.5), 1 (C1) and 1.5 (C1.5) 22 mM) and naringenin (50 (N50), 100 (N100) and 150 (N150) µM) in cryopreservation extender 23 for freezing rooster semen. Sperm motility, viability, abnormalities, membrane integrity, 24 mitochondrial activity, apoptosis status, lipid peroxidation (LP), GPX, SOD, TAC, the mRNA 25 expression of pro-apoptotic (CASPASE 3) and anti-apoptotic (Bcl-2) genes, fertility and 26 hatchability rate were investigated following freeze-thawing. C1 and N100 resulted in the higher 27 (P < 0.05) total motility and progressive motility in comparison to the control group. C1 and 28 N100 improved viability, membrane integrity and reduced lipid peroxidation. We found much 29 higher values for mitochondria activity with C1 and N100 respect to the control group. The C1 30 and N100 showed lower percentages of early apoptosis when compared with control group. 31Also, C1 and N100 had higher TAC when compared with control group. The mRNA expression 32 of BCL-2 in the C1 and N100 group were significantly higher than that of other treatments. The 33 expression of CASPASES 3 was significantly reduced in C1 and N100 group (P < 0.05) when 34 compared to control group. Significantly higher percentage of fertility and hatching rate were 35 observed in C1 and N100 compared to the control group. In conclusion, crocin at 1 mM and 36 naringenin at 100 µM seem to improve the post-thawing rooster semen quality, fertility and 37 could protect the sperm against excessive ROS generation by reducing the pro-apoptotic 38 (CASPASE 3) and increasing anti-apoptotic (Bcl-2) genes. 39 40 41 3 42 1. Introduction 43 Despite its utilization over 70 years ago [1], cryopreservation of bird sperm causes low 44 fertility, which limits its applying in genetic stock preservation [2]. Cryopreservation causes 45 harmful effects on sperm that decrease sperm viability and motility [3-5]. Avian sperm are 46 particularly susceptible to oxidative stress [6], though reactive oxygen species (ROS), in 47 physiological quantities, are necessary for important sperm events leading to successful 48 fertilization [7]. In sperm, oxidative stress disturb motility and mitochondrial activity [8]; 49 induces lipid peroxidation of the membrane [9]; and the oxidation and DNA fragmentation [10]. 50Adding antioxidant compounds to the freezing medium is known as one of the ways to defeat 51 the deleterious effects of ROS on sperm fertility after thawing, because it blocks or inhibits 52 oxidative stress. Antioxidants provide a positive effect on semen, leading to an improvement in 53 some sperm parameters containing motility and membrane integrity [11][12][13]. 54Naringenin is known as a natural flavonoid that has been studied for some of the most 55 prominent properties containing antioxidant, antiproliferative, anti-inflammatory, and 56 antimutagenic ones [14]. It was observed in previous experimental studies that naringain protects 57 the cells from lead and arsenic-induced oxidative damage [1...

Quercetin and Naringenin Provide Functional and Antioxidant Protection to Stored Boar Semen

“…This property may be directly associated with the polar nature of NAR, which may facilitate its adherence to the lipid bilayer, and thus protect the sperm cell membrane [ 44 ]. Similarly to our findings, earlier studies have reported a significant decrease of LPO in male reproductive tissues and cells following NAR supplementation [ 18 , 48 , 49 ]. A similar hypothesis may be applied in the case of QUE, which has been partially confirmed in bovine and human spermatozoa [ 18 , 38 , 42 ].…”

“…As hypothesized by Adana et al [ 39 ] NAR could also affect enzymes responsible for the repair of sperm DNA strand breaks, or to upregulate the antioxidant pathway to subsequently neutralize excessive ROS. A direct beneficial effect of NAR on genes responsible for DNA-associated apoptotic changes was furthermore detected by Mehdipour et al [ 18 ], which was well-reflected in a higher fertilization potential of frozen-thawed rooster spermatozoa.…”

“…In the case of NAR, it may be hypothesized that the molecule exhibits its mitochondrial-protecting effects through a different mechanism of action. As revealed by Mehdipour et al [ 18 ], NAR supplementation to frozen rooster spermatozoa led to significant reduction of mitochondrial ruptures and a subsequent activation of the mitochondrial-mediated apoptotic pathway. A more favorable ratio of pro-apoptotic Bax/anti-apoptotic Bcl-2 genes was observed following exposure to NAR, which lead to a decreased release of cytochrome C and a subsequent deactivation of caspase-3 [ 35 ].…”

“…Preliminary reports suggest that both flavonoids have the ability to protect the sperm plasma membrane and acrosome structures, mitochondrial function, and DNA stability in rabbit [ 13 ] and bovine [ 14 ] spermatozoa subjected to induced oxidative stress. Their potential to provide protection to the structure and function of cryopreserved goat [ 15 ], ram [ 16 ], bull [ 17 ], rooster [ 18 ] or stallion spermatozoa [ 19 ] subjected to cryopreservation has been indicated as well. Nevertheless, both biomolecules have received relatively little attention with respect to porcine semen.…”

Crocin Improves the Quality of Cryopreserved Goat Semen in Different Breeds

“…Similar results were reported in cattle, in which 1 mM of crocin was the most effective in improving motility [ 22 ]. Likewise, in other papers, the beneficial effect of saffron and its bioactive components, including crocin, on motility, viability and membrane integrity in the spermatozoa of human, rat, rooster and rabbit were reported [ 43 , 44 , 45 , 46 ]. In this study, however, no differences were recorded for sperm viability and membrane integrity that were high, regardless of breeds and treatment.…”