EFFECTS OF PHOENIX DACTYLIFERA POLLEN GRAINS EXTRACT SUPPLEMENTATION ON POST-THAW QUALITY OF ARABIAN STALLION SEMEN
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Bulgarian Journal of Veterinary Medicine, 2018, 21, No 1, 40–49
ISSN 1311-1477; DOI: 10.15547/bjvm.1044
Original article
EFFECTS OF PHOENIX DACTYLIFERA POLLEN GRAINS
EXTRACT SUPPLEMENTATION ON POST-THAW
QUALITY OF ARABIAN STALLION SEMEN
G. A. EL-SISY1, D. A. EL-BADRY2, R. I. EL-SHESHTAWY1 & W. S. EL-NATTAT1
1
Animal Reproduction and AI Department, Veterinary Researches Division,
National Research Centre, Dokki, Egypt; 2Department of Artificial Insemina-
tion and Embryo Transfer, Animal Reproduction Research Institute,
Agriculture Research Center, Giza, Egypt
Summary
El-Sisy, G. A., D. A. El-Badry, R. I. El-Sheshtawy & W. S. El-Nattat, 2018. Effects of
Phoenix dactylifera pollen grains extract supplementation on post-thaw quality of Arabian
stallion semen. Bulg. J. Vet. Med., 21, No 1, 40–49.
This study explored the effect of extender supplementation with different concentrations of date palm
pollen grain (DPPG) on post-thawed sperm motility, viability index, membrane and acrosome integri-
ties in Arabian stallions. Five ejaculates from each of four Arabian stallions were subjected to cryo-
preservation with a modified INRA-82, without any supplementation (control) or supplemented with
50, 100, 150, 200 and 250 mg DPPG. After thawing, all samples were maintained at 37 oC, while
analyses were performed at 0, 1, and 2 and 3 hours. Sperm motility percentage, viability index, mem-
brane integrity percentage and acrosome integrity of each sample were determined by conventional
laboratory methods. The addition of 100 mg DPPG resulted in improved maintenance of sperm moti-
lity after 0 and 60 min post-thawing, as compared to the control and other treatment groups. Non-
significant effects on viability index were observed after enrichment of extender with 100 and 150 mg
DPPG. The addition of 100 and 150 mg DPPG resulted in significant (PG. A. El-Sisy, D. A. El-Badry, R. I. El-Sheshtawy & W. S. El-Nattat
action of reactive oxygen species (ROS) intracellular ATP levels causing decrease
and oxidative stress on body cells and in the viability and motility of cryopre-
tissues has attracted research attention. served sperm (Baumber et al., 2000;
From ancient times, date palm pollen Agarwall & Said, 2005). To alleviate the
grain (DPPG, male germ powder of palm harmful effects of ROS, seminal plasma
flowers) was used to improve reproduc- possesses powerful source of ROS scav-
tive performances in men and woman as engers which offer protection for equine
dietary supplement. DPPG has a potent sperm, including enzymes such as super-
nutritive importance as it is rich in phyto- oxide dismutase, catalase, glutathione
chemicals such as estrone, α-amirin, peroxidase, and small molecular antioxi-
triterpenoidal saponins, flavonoids es- dants such as ascorbic acid and α-
trone, estradiol, estriol and a crude go- tocopherol (Aitken & Baker, 2004; Sikka,
nadotrophic substance (Abedi et al., 2004). Unfortunately, removal of seminal
2014). plasma during equine semen processing
Various studies have concluded that and consequent loss of these antioxidants
dietary supplementation could improve may increase the vulnerability of sperm
spermatogenesis, increase sperm counts, oxidative stress (Ball, 2008). Owing to the
motility, and increase the concentration of evidenced damaging effect of ROS to
testosterone, FSH and LH (Salman et al., genomic material and sperm membranes,
2014; Baharara et al., 2015; El-Kashlan et it is necessary to establish whether semen
al., 2015; Rasekh et al., 2015). Also, extender enriched with various antioxi-
DPPG is a rich source of natural antioxi- dants could improve the survival and fer-
dants. El-Kashlan et al. (2015) observed tility of cryopreserved equine sperm (Sin-
that DPPG extract possessed a powerful clair, 2000). The addition of natural ex-
free radical scavenging capacity. Hassan tracts and infusions from plant sources to
(2011) and Bishr & Desoukey (2012) at- semen extenders was indicated in cattle
tributed the powerful antioxidant capacity and caprine sperm preservation for their
of pollen grain to its high content of phe- protective, nutritive, antioxidant and anti-
nolic, carotenoid, flavonoid compounds, bacterial properties (Sansone, 2000). Al-
and considerable amount of vitamins A, E Dujaily et al. (2012) concluded that the
and C. Moreover, pollens have antibacte- addition of 20% P. dactylifera pollen ex-
rial, antifungal and antiviral activity tract to the culture medium of the in vitro
(Abedi et al., 2012; Mallhi et al., 2014). sperm activation improved sperm motility.
Cryopreservation causes a wide range El-Sheshtawy et al. (2014) found out that
of chemical, physical and mechanical in- the addition of aqueous extract of the
jures to sperm membranes of all mammal- DPPG to TRIS-citrate-fructose extender
ian species (Watson, 2000), which are improved survival rates and semen quality
attributed to temperature changes, altera- after chilling and cryopreservation in
tions in the transition from the lipid phase, bulls. To the best of our knowledge, only
production of reactive oxygen species few studies have been conducted to evalu-
(ROS) and osmotic stress (Ortega Ferru- ate the potency DPPG extract added to
sola et al., 2009; Câmara et al., 2011). semen extender in preserving and main-
Moreover, the overproduction of ROS taining sperm quality.
causes oxidative stress that involves struc- Despite the substantial flow of data on
tural damage of sperm membranes, fall of the promising quality and characteristics
BJVM, 21, No 1 41Effects of Phoenix dactylifera pollen grains extract supplementation on post-thaw quality of ….
of DPPG, few studies have so far been Semen collection and processing
conducted to investigate the effect of en-
On a once weekly collection schedule,
richment of semen freezing extender with
five ejaculates per stallion were obtained
DPPG extracts on sperm viability after
from 4 Arabian stallions, aged 8–14 years,
freeze thawing. The tendency for using
and individually housed at Police Aca-
natural antioxidants of plant origin is con-
demy stud, Cairo, Egypt. At the time of
tinuously increasing; therefore, the aim of
collection, early in the morning, a teaser
this study was to investigate the efficacy
estrus mare was used for the stallion to
of addition of aqueous extract of the
mount. Semen was collected using a lubri-
DPPG to semen extender (modified
cated and pre-warmed (45 to 50 °C) Colo-
INRA-82) on the stallion sperm’s survival
rado model artificial vagina with an inline
rates after freezing/thawing.
filter to separate the gel fraction.
Immediately following collection, the
MATERIALS AND METHODS gel-free portion of the ejaculate was
evaluated for volume and progressive
Preparation of extender motility. Sperm concentration was deter-
Modified INRA-82 (mINRA-82) de- mined with a haemocytometer. Only
scribed by El-Badry et al. (2014) was ejaculates with at least 60% progressively
used as base and control extender. This motile sperm and 250×106 sperm cell/mL
extender consists of 25 g/L glucose were used for freezing. The semen was
monohydrate, 1.5 g/L lactose monohy- extended 1:1 (semen:extender) in mINRA-
drate, 1.5 g/L raffinose pentahydrate, 0.4 82 warmed to 38 oC. The diluted samples
g/L potassium citrate monohydrate, 0.3 were placed into 15-mL tubes and centri-
g/L sodium citrate dihydrate, 4.76 g fuged for 10 min at 400×g. (Cochran et
HEPES, pH 7.0, 500 mg/L gentamycin, al., 1984). At least 95% of the supernatant
0.035% SDS and 0.15% skimmed milk. was removed (Loomis, 2006) and each
All ingredients were dissolved in one liter pellet was re-suspended with mINRA-82
of distilled water. (containing 5% glycerol and 15% egg
yolk) enriched with DPPG (mIPG-50,
Preparation of extract mIPG-100, mIPG-150, mIPG-200, mIPG-
Pollen grain extract was prepared from the 250) and mINRA-82 (control without
chafe of DPPG obtained from the palm DPPG) to a final sperm concentration of
tree pollen at El-Fayoum governorate. To 100×106 motile sperm/mL. Each aliquot
5 test tubes each containing 5 mL of was cooled slowly to 5 oC over one hour
mINRA-82 50, 100, 150, 200 and 250 mg under aerobic conditions, and then incu-
of this powder (El-Shehstawy et al., 2014) bated at 5 oC for 30 min (Crockett et al.,
were added. All tubes were put in cooling 2001). The extended semen was drawn
incubator (adjusted at 5 °C) for three days into 0.5 mL straws (Minitube, Germany)
with daily vortexing and finally centri- and sealed thermally and placed 4 cm
fuged to get the supernatant extenders above liquid nitrogen in the vapour phase
(IPG-50, mIPG-100, mIPG-150, mIPG- in foam box for 10 min before being
200 and mIPG-250, respectively). plunged into the liquid phase (Cristanelli
et al., 1985). The straws were then stored
in goblets on canes and kept immersed in
liquid nitrogen. For thawing, two straws
42 BJVM, 21, No 1G. A. El-Sisy, D. A. El-Badry, R. I. El-Sheshtawy & W. S. El-Nattat
per treatment were warmed in a water bath Sperm acrosomal integrity was esti-
at 38 oC for 30 s. mated using fast green stain (Wells &
Awa, 1970).
Evaluation of frozen-thawed semen
Statistical analysis
Sperm motility was examined and re-
corded using a pre-warmed stage of phase One way analysis of variance and Dun-
contrast microscope (200×) just after can’s multiple range tests (using SPSS
thawing, 1, 2 and 3 h post thawing. The program version 16.0) were done for the
post-thawing viability indices were calcu- obtained data of frozen-thawed semen
lated according to Milovanov (1962) to be qualities after transformation of percent-
equal to half of the post-thaw motility in ages to their corresponding arc-sin values
addition to the summation of recorded (Snedecor & Cochran, 1989). PEffects of Phoenix dactylifera pollen grains extract supplementation on post-thaw quality of ….
Table 2. Effect of date palm pollen grain (DPPG) addition to modified INRA-82 on post-thaw stal-
lion sperm parameters. Data are presented as mean ± SEM, n=15
Sperm parameters (arc-sin values)
Pollen enrichment
Viability index HOS (%) Acrosome integrity (%)
a * b
mINRA-82 (control) 114.00 ± 1.09 34.33 ± 1.55 33.33b ± 1.12
mINRA-82+50 mg DPPG 99.17b ± 0.63 32.33bc ± 0.45 31.00bc ± 0.22
mINRA-82+100 mg DPPG 125.83a ± 1.26 41.33a ± 0.83 38.33a ± 1.01
mINRA-82+150 mg DPPG 115.00a ± 1.09 41.33a ± 2.33 38.67a ± 1.64
mINRA-82+200 mg DPPG 102.50b ± 1.26 34.00b ± 1.57 32.00bc ± 1.43
mINRA-82+250 mg DPPG 72.50c ± 0.63 29.67c ± 0.98 29.00c ± 1.15
Different superscript letters within columns (a, b, c) are significantly different (PG. A. El-Sisy, D. A. El-Badry, R. I. El-Sheshtawy & W. S. El-Nattat
tract resulted in improvement in the sperm tase, and catalase. Removal of seminal
motility. In a previous study, El-Shesh- plasma during semen processing before
tawy et al. (2014) noticed significant im- freezing discard antioxidants present in
provement in bull sperm motility when semen and subjecting spermatozoa to ex-
250 mg aqueous extract of the DPPG in- treme ROS injury (Aitken et al., 2007).
cluded to the Tris-citrate-fructose ex- Oxidative stress has deleterious effects on
tender for chilling and cryopreservation. sperm membrane fluidity, integrity, and
The current results disagreed with our pre- flexibility, characteristics linked with fer-
vious study in bulls which may be related tilising capacity (Said et al., 2005). Al-
to species-specific differences. The capa- Farasi et al. (2005) and Mansouri et al.
bility of sperm from different species to (2005) reported the potent antioxidant
withstand the freezing process is variable, activity of the aqueous extract of dates
bovine sperm spermatozoa are more toler- which was attributed its inclusion to the
ant to freezing-thawing than the equine wide range of phenolic compounds in-
spermatozoa (Rodgers et al., 2014). cluding p-coumaric, ferulic and sinapic
Motility is the most essential indicator acids, flavonoids and procyanidins. Palm
of the in vivo sperm fertilising ability pollen has also been recognised as a rich
(Vijayaraghavan, 2003). The beneficial source of phytochemicals and nutrients
effect of pollen grain, on motility and vi- mainly carotenoids and flavonoids (Daoud
ability has been proved in rabbits, rats, et al., 2015). Fresh pollen has a highly
mice, and humans (Marbeen et al., 2005; nutritive value as it is rich in mineral,
Al-Sanafi et al., 2006; Bahmanpour, multi-vitamins, sugars, lipids, growth fac-
2006; Faleh & Sawad, 2006; Rasekh et tors and more than 100 enzymes and co-
al., 2015). factors (Hassan, 2011). Moreover, it is a
Our results showed that enriching stal- superior source of protein, amino acid,
lion freezing extender with 100 and 150 fatty acid, enzymes, hormones (estrone,
mg pollen grain has advantageous effect estradiol and estriol) and mineral salts
on viability index, membrane and acro- (Alferz & Campos, 2000).
some integrity compared to the control Palm pollen contains an efficient mate-
group. During cryopreservation, mammal- rial for improving semen quality and male
ian sperm is subjected to tremendous fertility because it is rich in vitamins A, E
chemical and physical damage as a result and C and it is a good source of minerals
of phase transition changes, osmotic stress such as zinc, selenium, iron, cooper and
and overproduction of reactive oxygen cobalt (Hassan, 2011) that stimulates
species (ROS) which results in increase sperm motility and the progressive for-
lipid peroxidation of the membrane (Or- ward movement. All of these above men-
tega Ferrusola et al., 2009; Câmara et al., tioned components could improve sperm
2011). In various animal species, includ- motility. Vitamin C is a naturally occur-
ing horses, lipid peroxidation has been ring antioxidant in seminal plasma (Anane
documented as an important marker of & Creppy, 2001) and is an essential cofac-
oxidative stress and cryonic injury in tor of enzymes that are free radicals scav-
spermatozoa (Aitken et al., 2007; Ortega engers (Michael et al., 2008). It could
Ferrusola et al., 2009). The main ROS possibly improve sperm function by re-
scavengers in equine semen are glu- ducing cell damage (Gangwar et al.,
tathione peroxidase, superoxide dismu- 2015). On the other hand, α-tocopherol,
BJVM, 21, No 1 45Effects of Phoenix dactylifera pollen grains extract supplementation on post-thaw quality of ….
one of the major sperm antioxidants, was infertility: a clinical approach. BJU Inter-
established to be abundant in spermatozoa national, 95, 503–507.
membrane (Suleiman et al., 1996; Surai et Aitken, R. J. & M. A. Baker, 2004. Oxidative
al., 2000) and protect sperm motility from stress and male reproductive biology. Re-
oxidative harm (Brzezińska-Ślebodzińska production, Fertility Development, 16,
et al., 1995). Besides, date palm pollen 581-588.
suspension was reported to have potent Aitken, R. J., J. K. Wingate, G. N. De Iuliis &
antimicrobial, antifungal and anti-toxicant E. A. McLaughlin, 2007. Analysis of lipid
activities (Aba Al-Khail et al., 2003; peroxidation in human spermatozoa using
BODIPY C11. Molecular Human Repro-
Shakibaa et al., 2011; Daoud et al., 2015)
duction, 13, 203–211.
and these effects could improve post-
thawing semen quality through reduction Al-Dujaily, S. S., N. J. Al-Shahery & A. A.
Zabbon, 2012. Effect of Phoenix dactylif-
of bacterial growth in the semen extender. era pollen on in vitro sperm activation of
In conclusion, the inclusion of 100 and infertile men. Al-Mustansiriyah Journal of
150 mg date palm pollen grain extract in Science, 23, 27–34.
modified INRA-82 had a beneficial effect Al-Farasi, M., C. Alasalvar, A. Morris, M.
on the chilling and freezing process of Baron & F. Shahidi, 2005. Compositional
Arabian stallion sperm. and sensory characteristics of three native
sun dried date (Phoenix dactylifera L.) va-
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ACKNOWLEDGEMENTS ture and Food Chemistry, 53, 7586–7591.
The authors are greatly indebted to the Na- Alferz, M. J. M. & M. S. Campos, 2000. Bene-
tional Research Center and the Police Acad- ficial effect of pollen and or propels on the
emy stud for their helpful and valuable aids iron, calcium, phosphorus and magnesium
and all the facilities they presented to achieve in rats with nutritional ferropenic anemia.
the present article. Journal of Agriculture and Food Chemis-
try, 48, 5715–5722.
Al-Sanafi, A. I., E. F. Bahaaldeen, M. I.
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