'RFWRUDO7KHVLV





6WXG\RI/LSLG&RPSRQHQWVLQ%RYLQH)UR]HQ6SHUP





0G0D]KDUXO,VODP*UDGXDWH6FKRRORI%LRVSKHUH6FLHQFH
+LURVKLPD8QLYHUVLW\
6HSWHPEHU







'RFWRUDO7KHVLV





6WXG\RI/LSLG&RPSRQHQWVLQ%RYLQH)UR]HQ6SHUP





0G0D]KDUXO,VODP'HSDUWPHQWRI%LRUHVRXUFH6FLHQFH
*UDGXDWH6FKRRORI%LRVSKHUH6FLHQFH
+LURVKLPD8QLYHUVLW\
6HSWHPEHUA CKNOWLEDGEMENT
A ll praises t o “Almighty A llah”, the supreme ruler of the universe, t he greatest, the most gracious
an d the most merciful, who has created and e nabled the author t o c arry out the research work
an d complete this t hesis successfully.
It i s privilege to express t he sincere gratitude, heartfelt respect, kind regards, profound sense of
appreciation an d i ndebtedness t o my r e spected supervisor D r . Shimada Masayuki, Professor,
Laboratory of R eproductive Endocrinology, Gr aduate Sc hool of Bi osphere S cience, Hiroshima
U n iversity for his scholastic guidance, untiring assistance, keen i nterest, constructive criticism,
valuable suggestions, continuous encouragement and affectionate feelings throughout the courseof t he research work and in the preparation of t his manuscript.
I fe el pr oud t o acknowledge my gr atefulness, boundless gr atitude and pr ofound regards to mysupervisory committee members, Professor T aketo Obitsu, Laboratory of A nimal Nutrition andPhysiology, Professor Naoki Isobe, Laboratory of A nimal Histophysiology, Dr. Takashi Umehara(A ssistant Pr ofessor), Laboratory of R eproductive En docrinology, Gr aduate Sc hool of Biosphere
Sc ience, Hiroshima University, for their kind cooperation, valuable advice, constructive criticism
an d helpful comments that have made e asy to c omplete this thesis.
I would like t o e xpress my he artfelt gr atitude, indebtedness, and profound appreciation to Dr.
T omoko Kawai (Assistant Professor), D r. Natsumi T sujita, Dr. Masanori Koyago, Laboratory of
R e productive En docrinology, Hi r oshima U n iversity, for t heir c ooperation and learninge x periments step by step. I am so much grateful to t hem.
I gr atefully express my best r egards and i mmense indebtedness t o my respected senior fellow Dr.
S.A .M Hoque for his unmeasurable cooperation, support, and inspiration from the very beginningof my PhD journey t o t he e nd. I must acknowledge his academic ad mental support to become
adjusted in the Japanese c ulture and life.
I would like t o e xpress my love and gr atitude t o my lab mates Ishi, Fu jita, T omiya, Koizumi,
Yanagawa, Ade, Zhu, Hibi, Tawara, Chika, Creese, Enoc, A rai for their friendly help, inspiration,an d encouragement during the study period.
I would like t o acknowledge my ac ademic i nstitution, Bangabandhu Sheikh Mujibur Rahman
A gricultural U niversity t o allow me st udy leave t o c arry out t he doctoral degree. My special
appreciations go t o the Graduate School of Biosphere Science, Hiroshima University, Hiroshima,
Japan; and t o t he Japanese Ministry of Education, Culture, Sports, Scienc e and Technology for
sc holarship and support for this study.
I would like t o express heartiest gratitude t o the friends and family from Bangladesh Community
who gave me assistance with care.
Fi nally, I would like t o express my sincere profound gratitude an d deepest appreciation to myparents and my wi fe Mashruba R ahman for t heir n ever-ending pr ayer, affections, supports,
sac rifice, i n spiration, e n couragement, an d c ontinuous ble ssings t o c omplete t his journey
suc cessfully. I would like to dedicate my doctoral t hesis t o my innocent and adorable daughters
T abiba Mazhar and Tanisha Mazhar who sacrificed t heir father’s accompany during this study
pe riod.
T he A uthor

/LVWRIFRQWHQWV
7RSLFV
3DJH1R&KDSWHU *HQHUDO,QWURGXFWLRQ
1.1
Spermatogenesis and Spermiogenesis
1
1.2
Sperm Maturation
2
1.3
Changes of Sperm during Ejaculation
4
1.4
Sperm Motility Pattern and ATP Production
5
1.5
Changes of Sperm during Cryopreservation7
1.6
Known fact and unknown maiden discovery of present study
8&KDSWHU ([SHULPHQW
Study on variation of lipid components in bovine frozen sperm andtheir role on accelerating motility and quality parameters as mixture
2.1
2.2
2.3
2.4
&KDSWHU
3.1
3.2
3.3
3.4
&KDSWHU

4.1
4.2
4.3
4.4
&KDSWHU&KDSWHU
5HIHUHQFHVIntroduction
Materials and Methods
Results
Discussion
Abstract([SHULPHQW
Study on effect of fatty acids on sperm linear motility throughmitochondrial ATP production
Introduction
Materials and Methods
Results
Discussion
Abstract([SHULPHQW
Study on effect of cholesterol on recovery of mitochondrial potential
to keep linear motility in bovine frozen sperm
Introduction
Materials and Methods
Results
Discussion
Abstract*HQHUDO'LVFXVVLRQ
6XPPDU\




i

10
11
14
16
19

31
33
34
37
41

52
53
54
56
59
68
73
76


/LVWRI7DEOHV
7DEOH
7DEOH

7LWOHRI7DEOHV
Modified Bracket-Oliphant (BO) medium for thawing and incubationof spermDifferent fatty acid amount (pmol/sperm) calculated by GC-MS fromdifferent stocks of bull semen straw



















ii

3DJH1R
20
20

/LVWRI)LJXUHVFigure 1

Figure 2

Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10

Figure 11
Figure 12
Figure 13
Figure 14
Figure 15
Figure 16

Title of the figures
Page No.
Correlation between different saturated fatty acids with total sperm
21
motility. The X-axis indicates the amount of different fatty acids,
and the Y-axis indicates the total sperm motility of different bull
straws.
Correlation between different unsaturated fatty acids with the total
22
sperm motility. The X-axis indicates the amount of different fatty
acids, and the Y-axis indicates the total sperm motility of differentbull straws.
Chromatogram of different fatty acid components present in sperm
23
thawed with LM found by GC-MS.
Chromatogram of different fatty acid components present in sperm
24
thawed without LM found by GC-MS.
Total fatty acids amount (pmol/sperm) after thawing the sperm in
25
LM (L+) compared to control (L-) found by GC-MS study.
LM improves sperm motility pattern
26
CASA motility track of different doses of LM (LM)27
Correlation between ALH and VCL (A.) and BCF and VSL(B.) of
28
each sperm treated with LM (LM+) in thawing media comparedwith control (LM-).
Correlation between BCF and VCL (A.) and VCL and VSL(B.) of
29
each sperm treated with LM (LM+) in thawing media comparedwith control (LM-).
LM improves sperm quality. Where; (A.) Mean fluorescence30
intensity (MFI) observed by JC-1 stain for mitochondrial activity(B.) Membrane integrity detected by SYBR-14/PI stain and (C.)
Live sperm (%) measured by Annexin V stain.
Fatty acids improve sperm motility parameter.
42
CASA derived motility track of different fatty acids at 50nM
43
concentration.
Correlation between ALH and VCL (A.) and BCF and VSL(B.) of
44
each sperm treated with Myristic Acid (MA) in thawing media
compared with control.
Correlation between BCF and VCL (A.) and VCL and VSL(B.) of
45
each sperm treated with Myristic Acid (MA) in thawing media
compared with control.
Correlation between ALH and VCL (A.) and BCF and VSL(B.) of46
each sperm treated with Palmitic Acid (PA) in thawing media
compared with control.
Correlation between BCF and VCL (A.) and VCL and VSL(B.) of
47
each sperm treated with Palmitic Acid (PA) in thawing media
compared with control.

iii

Figure 17
Figure 18
Figure 19

Figure 20

Figure 21
Figure 22
Figure 23
Figure 24
Figure 25
Figure 26

Figure 27

Title of the figures
Page No.
Total fatty acids amount measured by GC-MS after thawing the48
sperm in respective fatty acids and incubation for 30 min with BSA
at 37˚C. Y axis indicating the amount of fatty acids (pmol/sperm).
Fatty acids changes sperm mitochondrial activity.
49
Fluorescence intensity of sperm and localization of fatty acid50transporter where; (A)Fluorescent signal of FITC palmitic acid,(B). overlay of different doses of FITC palmitic acid (C). Mean
fluorescence intensity of different doses of FITC palmitic acid and(D.) Localization of fatty acid transporters.
Metabolic effect of MA or Etomoxir, the inhibitor of β-oxidation,51
on frozen bovine sperm where; (A.) Trace of OCR before/after thetreatment of several concentration of myristic acid (MA)(B.)
Increase ratio of OCR after the treatment of MA (C.) Trace of OCR
before/after the treatment of 50 nM MA or MA + Etomoxir (D.)
Increase ratio of OCR after the treatment of 50 nM MA or MA +Etomoxir.
CASA motility parameters of sperm incubated with different dosesof cholesterol for 30 min.
CASA motility track at different doses of cholesterol (CHL).
Correlation between ALH and VCL (A.) and BCF and VSL(B.) of
each sperm treated with Cholesterol (CHL) in thawing media
compared with control.
Correlation between BCF and VCL (A.) and VCL and VSL(B.) of
each sperm treated with Cholesterol (CHL) in thawing media
compared with control.
Cholesterol improves sperm survivability, where; (A.) Membrane
integrity (B) Annexin v Staining
Fluorescence intensity of sperm and localization of cholesterol
uptake, where; (A.) Overlay of fluorescence intensity at different
doses of fluorescence tagged cholesterol (B.) Mean fluorescenceintensity at different doses of fluorescence tagged cholesterol (C.)
Localization of cholesterol uptake signal in sperm.
Effect of Cholesterol on oxygen consumption rate (OCR) of sperm,
where; (A) indicating the effect of oligomycin injection (B)indicating the ATP production rate of sperm (C) &(D) indicating
the effect of FCCP injection (E) indicating the mitochondrial
activity of sperm



iv

61
62
63
64
65
66

67


&KDSWHU*HQHUDO,QWURGXFWLRQ







&RQWHQWV
1.1
1.2
1.3
1.4
1.5
1.6



TopicsSpermatogenesis and Spermiogenesis
Sperm Maturation
Changes of Sperm during Ejaculation
Sperm Motility Pattern and ATP Production
Changes of Sperm during Cryopreservation
Known fact and unknown maiden discovery of present study

Page No.
1
2
4
5
7
8

Reproduction is the process of producing new generation that are more or less similar to it’sancestors. In the nature, there are two types of reproduction- sexual and asexual. In the sexual
reproduction two types of gametes (sperm cell and oocyte) are fused together to produce thediploid zygote in the process of fertilization (Frits & Gadella, 2000). The spermatozoa areproduced by the process of spermatogenesis inside the male gonads (testicles) via meiotic divisionin the seminiferous tubules (Barth & Oko, 1989).
For the last 50 years, in the modern reproductive biotechnology, semen cryopreservation is being
used as assisted reproductive technology (ART) for genetic improvement, control of venereal
diseases and facilitates the management of beef and dairy cattle (Khalil et al., 2017). Although infrozen semen technology, almost 50% sperm is died during cryopreservation due to cryoinjury or
cryodamage and thawing of the semen, artificial insemination (AI) with frozen semen is
predominantly used in animal husbandry sectors especially in dairy cattle production to improve
the genetic potentiality and to increase the productivity (Perumal, 2018).
6SHUPDWRJHQHVLV DQGVSHUPLRJHQHVLVMale germ cells start to develop from a self-renewing stem cell pool, in the seminiferous tubulesof the testes throughout life from puberty to old age and the complete process of germ cell
development is termed as spermatogenesis. Spermatogenesis is a complex process which produce
large numbers of normal spermatozoa that is required for male fertility. Spermatogenesis
commences in the fetus at puberty after a long preparatory period of "prespermatogenesis". There
are three major stages of spermatogenesis; spermatogoniogenesis, maturation of spermatocytes
and spermiogenesis i.e cytodifferentiation of spermatids. Depending on the position in the basal
part of the germinal epithelium, their morphology and stainability of nuclei spermatogonia arevaried into three types: A pale type-, A dark type- and B type-spermatogonia (Clermont, 1966).
Both pale and dark A type spermatogonia are necessary for intact spermatogenesis and A typespermatogonia belong to the stem cell pool of spermatogenesis whereas B type-spermatogonia
represent the starting of germ cell development up to spermatids.
The meiosis stage of spermatogenesis is revealed by the changes in chromatin configuration in the
nucleus after the last spermatogonial division and the cells in this stage are called spermatocytes.
Due to the two divisions of meiosis process, cells before the first meiotic division are named
primary spermatocytes and before the second meiotic division secondary spermatocytes. The
primary spermatocytes are the largest germ cells of the germinal epithelium. In the basal
Page | 1

compartment of the germinal epithelium meiosis of spermatocytes begins with the leptotene stageof prophase cell division.
The round spermatid, which evolves from the second meiotic division, go through a series ofcomplex cytological incidents which transmute it into the spermatozoon (de Kretser and Kerr,
1994). Several events like doubling of DNA, condensation of chromosomes, pairing ofhomologuous chromosomes and finally the "crossing over" take place during the prophase. After
division, the germ cells turned into secondary spermatocytes. Secondary spermatocytes divide
quickly to the spermatids without DNA replication. Two maturation divisions of each
spermatocyte result in four haploid cells, the spermatids and these distinguish into mature
spermatids through a process called spermiogenesis. During the spermiogenesis three processes
take place; condensation of the nuclear chromatin in volume to about one tenth of an immature
spermatid, development of the enzyme filled acrosome cap by the golgi apparatus and its
attachment to the nucleus and expansion of flagellum structures and their implantation to the
nucleus (Holstein & Roosen-Runge,1981). The spermatids develop such a configuration whichenables them to depart the germinal epithelium during a complex process, called spermiation. Afterthe spermiogenesis the cells are released from the germinal epithelium and during this moment thefree cells are known as spermatozoa.
6SHUP0DWXUDWLRQ
After completing spermatogenesis and spermiogenesis in the testis, the spermatozoa are notcapable of fertilizing an oocyte. The immature sperm in testis do not have a moving ability andmaturation process in epididymis is required for sperm mobility. The epididymal sperm maturation
includes the attainment of the forward motility and the fertilizing capability (Cooper & Yeung,2006). Epididymis is the male reproductive organ, anatomically characterized as three different
regions as the head (caput), the body (corpus), and the tail (cauda), responsible for the spermtransport, concentration, storage, and maturation, (Frenette et al., 2004). Each epididymal segment
demonstrated great variability from one epididymal segment to the other in fluid composition(Dacheux, 2002; Dacheux & Belghazi, 2006) as well as the pattern of gene expression (Kirchhoff,
1999; Rodriguez et al., 2001).
Spermatozoa are transported through the caput and corpus epididymal regions by nonstop
peristaltic contractions created in the smooth muscles present in the wall of the duct, whereas thecauda is maintained calm unless it can be stimulated to contract, and cauda is the main region
Page | 2

responsible for the sperm storage and survival (Jones & Murdoch, 1996). During this journey,
spermatozoa undergo many biochemical alterations including changes in the plasma membraneprotein and lipid composition (phospholipids composition, cholesterol/phospholipids ratio),
relocalization of surface antigens, removal, and modifications of surface proteins (Cuasnicu et al.,
2002; Sullivan, 1999), structural modifications of the cytoplasmic perinuclear theca (PT) (Mujica
et al., 2003), and the ability to respond to hypoosmotic stress (Sahin et al., 2009). Biochemical andmorphological modifications during sperm maturation include the addition of epididymal proteinsto the maturating sperm, and some molecular reorganizational changes at the sperm membrane(Cooper, 1998). Furthermore, changes in membrane fluidity as a consequence of cholesterol effluxand protein dissociation from raft domain seems to be also a mechanism that occurs both in thematurational processes occurring at the time of ejaculation (Girouard et al., 2008).
Sperm undergo changes in their protein, lipid, and sugar content during epididymal shipment. The
mechanisms involved in the transfer of epididymal proteins to the sperm are not fully understood.
The epididymal epithelium consist of different epithelial cell types, notably principal cells, narrowcells (found only in the initial segment), clear cells and basal cells (Hermo et al., 2002). Each ofthese cell categories has a certain structure and function that fluctuates depending on theirlocalization along the epididymis (Breton et al., 2016). Additionally, interaction between thesedifferent cellular types creates a luminal microenvironment suitable for sperm maturation andstorage in the epididymis (Shum et al., 2011). Principal cells secrete proteins by both merocrine
and apocrine mechanisms to the epididymal lumen (Hermo et al., 2002). Merocrine secretion
includes membrane fusion between Golgi-derived vesicles and the cell plasma membrane
permitting the release of the vesicle contents to the extracellular space (Farkas, 2015). In apocrine
secretion epididymosomes are believed to be released by principal cells into the lumen of theepididymis (Sullivan et al., 2007). Epididymosomes are tiny membranous vesicles (25–300 nm indiameter) that contain different proteins, lipids, and non-coding RNAs (Belleannee et al., 2013,
Martin-DeLeon, 2015). Proteins transmitted from epididymosomes to sperm can either beincorporated into the plasma membrane of sperm (Kirchhoff et al., 1996) or to intracellular
structures (Eickhoff et al., 2001, Frenette et al., 2005). Epididymosome may transfer protein tosperm by fusion between sperm and these membrane vesicles through ADAM7, a member oftransmembrane protein family, has biochemical characteristics of an integral membrane protein(Oh et al. 2009).
Page | 3&KDQJHVRI6SHUPGXULQJ(MDFXODWLRQ
During ejaculation, the seminal plasma components are mixed with the spermatozoa. In the bovine,
the seminal vesicles produce a family of proteins called bovine seminal plasma or BSP proteins
which contain two fibronectin type II domains in tandem (Seidah et al., 1987, Calvete et al., 1996)
that bind to choline phospholipids (Desnoyers & Manjunath, 1992) on the sperm membrane andexcite their phospholipid and cholesterol efflux (Therien et al., 1998).
Bovine cauda epididymal spermatozoa have proteins associated to rafts (P25b and AK1) and nonrafts domains (aldose reductase and MIF) and these raft domain proteins are displaced to non rafts domains as early as 15 min and 30 min after ejaculation respectively (Girouard et al., 2008).
These changes are accompanied by a decrease in the cholesterol content in ejaculated spermcomparedd with cauda epididymal spermatozoa (Caballero et al., 2011) and PDC-109, a protein
responsible for the cholesterol efflux in the bovine sperm at the ejaculation (Esch & Ling, 1983).
Niemann-Pick C2 (NPC2), also found in the seminal plasma of various species capable ofdissociating the P25b from the raft domains after 4 hours of incubation (Okamura et al., 1999;
Fouchecourt et al., 2000). The ejaculated spermatozoa plasma membrane reorganization could
have an important role in the correct positioning of molecules involved with the attachment to theoviductal epithelium and female gamete interactions and AK activity is important for biosynthesis
of ATP and then for flagellar motility (Schoff et al., 1989).
During ejaculation, the composition of epididymal sperm is altered, the proteome of ejaculated
sperm varies from epididymal sperm in ram (Pini et al., 2016) and boar (Perez et al., 2018).
Additionally, ejaculated sperm of ram contain lower concentrations of phospholipids andcholesterol than testicular sperm (Scott et al., 1967). Although variations between seminal andepididymal fluid are involve in freezing resistance in different specieses (Varisli et al., 2009),
epididymal sperm was found to be more resistant than ejaculated sperm to osmotic stress, cold
shock, and cryopreservation process in ram (Tsikis et al., 2018), bulls (Cunha et al., 2016), boars(Perez et al., 2019), and stallions (Braun et al., 1994). The luminal fluid microenvironment protects
the undermining processes related with sperm capacitation during sperm storage in the cauda ofthe epididymis (Fraser et al., 1990, Verma, 2001).
In mammals, for in vivo fertilization sperm motility is crucial for sperm movement from theejaculation sites to the oviduct and the motility patterns are continuously changed after ejaculation
to fertilization in the female tract (Suarez & Pacey,2006). Motility patterns are reliant on flagellar
Page | 4

motion and the linear motility pattern is induced by the symmetrical flagellar motion (Curtis et al.,2012). The linear motility, is essential for sperm migration from the cervix to the uterus and thento oviduct, characterized by low lateral amplitude and high straight-line velocity (Shalgi et al.,
1992; Ishijima,2015). In oviduct during fertilization process, hyperactivation is prerequisite forcapacitation of sperm (Suarez, 2008), where curvilinear velocity is high as well as high lateralamplitude (Stauss et al., 1995).
During capacitation, sperm go through a change in the motility pattern called hyperactivation andbecome competent to undergo a physiological secretory event known as acrosome reaction (AR)also known as acrosomal exocytosis (AE) (Yanagimachi, 1970). Hyperactivation is crucial for
successful fertilization because it simplifies the sperm release from the oviductal reservoir and thepenetration through the cumulus oophorus and the zona pellucida (ZP), the extracellular matrix
enveloping the egg (Demott and Suarez, 1992). It was demonstrated that sperm separated from thecauda of epididymis, which is ready to ejaculate demonstrate progressive motility, which ischaracterized by high velocities and symmetrical, low-amplitude flagellar bends (Fraser, 1977).
On the contrary, sperm isolated from the oviduct the fertilization site, show hyperactivated motility
characterized by more asymmetrical, higher-amplitude flagellar bends, leading to more vigorousand less progressive ‘‘whiplash’’ motility (Suarez & Osman, 1987) which is required forfertilization (Yanagimachi,1970). Another report on mice demonstrated that sperm in t h e caputof epid id ymis showed irregular flagellar beat w it h little progression whereas in t h eproximal corpus region circular mo v e me n t patterns in red uced linearity (LIN) andstraightness (STR) were reflected (Soler et al., 1994). In conclusion, epididymis sperm easily
induce capacitation whereas ejaculated sperm show linear motility pattern.
6SHUP0RWLOLW\ 3DWWHUQDQG$733URGXFWLRQSperm motility pattern is very peculiar characteristic which varies from ejaculation to fertilization.
Motility patterns are dependent on flagellar motion, and it was reported that the linear motility
pattern was induced by the symmetrical flagellar motion (Ho et al., 2002; Curtis et al., 2012). The
linear motility is prerequisite in the migration process of sperm from the cervix to the uterus andthen to oviduct, characterized by low lateral amplitude and high straight-line velocity (Shalgi etal., 1992; Ishijima,2015). Another motility pattern known as hyperactivated motility characterized
by asymmetrical, irregular flagellar beat with high lateral amplitude and high curvilinear

Page | 5velocity (Stauss et al., 1995). In oviduct during fertilization process, hyperactivation is basic
requirement for capacitation of sperm (Suarez, 2008).
Mammalian spermatozoa particularly use adenosine triphosphate (ATP) to maintain theintracellular milieu (Mishro & Ramya, 2012) and for cellular processes such as motility,
capacitation, hyperactivation and the acrosome reaction, all are required for successful fertilization(Mannowetz et al., 2012, Mukai et al., 2012). ATP, the leading chemical energy in cells, isgenerated mostly from two pathways: glycolysis in the cytosol and oxidative phosphorylation(OXPHOS) in the mitochondria (Du Plessis et al., 2015). The amount of ATP produced byglycolysis versus OXPHOS added to the total amount of ATP in the cell varies depending on thecell type, the growth stage, and the microenvironment (Zheng, 2012, Jose et al., 2011). Though, aspermatozoon can survive entirely on glycolytic energy (Spiropoulos et al., 2002), but it
necessitates OXPHOS for differentiation and maturation (Nakada et al., 2006, Ford, 2006). The
selected metabolic pathway chosen by spermatozoa relating to energy production is extremely
species specific (Storey, 2008). For example, glycolysis was required for capacitation, motility
hyperactivation, and oocyte-fusion in human (Hereng et al., 2011) hamster (Dravland & Meizel,
1981) and macaque sperm (VandeVoort & Overstreet, 1995). On contrary, glucose hinderedcapacitation in guinea pig (Hyne & Edwards,1985), bovine (Parrish et al., 1989) and dog sperm(Albarracın et al., 2004), where in vitro capacitation was carried out in glucose free medium.
Mitochondrial ATP is generated through transformation of ADP by the reduction of electron
carriers (Amaral et al., 2013). It is reported that under low glucose conditions, most cells can moveon the glycolytic pathway to mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP(du Plessis et al., 2015), for instance, report on boar sperm study (Zhu et al., 2019). The electrontransfer chain (ETC) comprises of several complexes that carry electrons attained from theoxidation of NADH and FADH2 moiety of succinate dehydrogenase, and ultimately decreased thefinal acceptor oxygen to water (Bratic & Trifunovic, 2010).
In reality, Ldh-c knock out mice model revealed that for hyperactivated motility the glycolysis was
crucial (Odet et al., 2008). Rotenone, an inhibitor of complex I, dropped the sperm mitochondrial
activity and ATP levels with progressive motility and straight-line velocity but not lateralamplitude and total motility in boar (Zhu et al., 2019), stallion (Plaza Davila et al.,2015) and human(Barbonetti et al.,2010). Additionally, rotenone suppressed the sperm capacitation, acrosome
reaction and fertilization rate in hamster (Rogers et al., 1977) and stallion (Plaza Davila etPage | 6

al.,2015), implying that mitochondrial ATP production is vital for sperm linear motility andhyperactivation. It has been reported that acetyl CoA is generated by mitochondrial β-oxidationfrom fatty acids (Aon et al., 2014). In human sperm it was described that a large proportion of themetabolic proteome (24%) comprised enzymes were engaged in lipid metabolism, including
enzymes for β-oxidation (Amaral et al., 2013). Excitingly, the mitochondrial β-oxidation is anactive regulator for sperm motility because inhibition of β-oxidation using etomoxir led to adecrease of sperm motility (Amaral et al., 2013). Moreover, it was reported in bulls that palmitic
acid and oleic acid advanced sperm progressive linear motion and viability when added to theextender (Kiernan et al., 2013). Another report on boar sperm stated that unsaturated fatty acids
enhanced progressive motility and viability during in vitro incubation (Kelso et al., 1997),
suggesting that mitochondrial β-oxidation is very essential for sperm progressive motility andsurvivability.
&KDQJHVRI6SHUPGXULQJ&U\RSUHVHUYDWLRQIn the present world artificial insemination (AI) of animals and assisted reproductive technology(ART) in human routinely use cryopreserved spermatozoa (Kopeika et al., 2015; Yeste, 2016)
although first semen cryopreservation record dates back approximately 250 years, when Lazaro
Spallanzani (1776) attempted to preserve spermatozoa by cooling it in snow (Royere et al.,1996).
Crucial factor in sperm cryobiology is that sperm are small cells with a substantial surface area
(John Morris et al., 2012; Morris, 2006). These physical characteristics makes them less vulnerable
to potential damage which is engaged by the viscosity and glass transition temperature of theintracellular cytosol in sperm cells (Isachenko et al., 2003). Although cryodamage in spermatozoa
varies among different species (Ozkavukcu et al., 2008; Yeste, 2016), common modifications are
acrosome disintegration and partial deletion of the outer acrosomal membrane with reduction ofacrosomal content that are attached to physical freezing events of sperm (Barthelemy et al., 1990).
On the Other hand, damage to the lipid membrane structure caused by osmotic changes, prominent
to tension changes in water canal proteins and ionic leakage in plasma membranes and resulting
in morphological changes (Sa-Ardrit et al., 2006).
The sperm plasma membrane is the first structure affected by cryopreservation (Bailey et al.,2000). It has been reported that sperm cryopreservation induced in enlarge plasma membrane
fluidity–permeability, overproduction of reactive oxygen species (ROS), reduction of acrosome
integrity, impairment of mitochondrial membrane potential and lower sperm motility in bull
Page | 7

(Khalil et al., 2018, Yoon et al., 2015), buffalo (Rasul et al., 2001, Kumar et al., 2016), buck
(Ahmad et al., 2014, Chauhan et al., 1994), ram (Nur et al., 2010, García-Álvarez et al., 2009) andred deer (Fernández-Santos et al., 2006). The sperm plasma membrane of ruminants contains highlevels of unsaturated phospholipids and low levels of cholesterol that declines the resistance of
sperm to the freezing–thawing process (Darin-Bennett et al., 1977). During freezing process,phospholipids of sperm membrane undergo a reallocation throughout the membrane, and some ofthem switch from liquid to gel state quicker than others due to structural differences, subsequently
a lipid phase separation occurred (Grötter et al., 2019). As a result, some sperm surface proteins
as well as membrane proteins are lost or translocated and the lipid–protein interactions are
disturbed which is required for a proper membrane activity (Lemma, 2011).
.QRZQ)DFWDQG8QNQRZQ0DLGHQ'LVFRYHU\RI3UHVHQW6WXG\In the present time this is well established that sperm are very unique male reproductive unit thathas a distinctive membrane configuration and unusual nucleus, mitochondria, cytoplasm &cytoskeleton structure compared to somatic cells. These characteristics are formed during
spermatogenesis in testis. Different maturational phases occur in the epididymis. Epididymal
sperm are more effective in capacitation than fresh sperm immediately after ejaculation. For theeffective use of potentialities of breeding animal specially in cattle industry artificial insemination(AI) is used for the last 5/6 decades using frozen sperm. Cryopreservation although can keep thesperm for long time future use even the animal is dead, but unfortunate it deteriorates the qualityof sperm. There are so many studies regarding the cryodamage of bovine sperm for example,
Khalil et al. (2018) stated that sperm cryopreservation is responsible to enhance plasma membrane
fluidity–permeability, excessive production of reactive oxygen species (ROS), decline ofacrosome integrity, deficiency of mitochondrial membrane potential and lower sperm motility inbull (Khalil et al., 2018). Though some researcher reported on prevention of cryodamage by
supplying cholesterol or other cryoprotectant during freezing of bovine sperm (Purdy & Graham,
2004) but unfortunately, there are lack of published reports on recovery of bovine frozen sperm
after thawing and washing using an appropriate media. Many researchers described that fatty acidsin thawing media can improve sperm motility, acrosome reaction and fertility in case of boar sperm(Hossain et al., 2007). Again, some researcher stated that sperm can utilize energy substrate
supplied in the thawing media of boar sperm (Zhu et al., 2020). Sperm are changed during
maturation and ejaculation stages. As far author knowledge, nobody described what is the actual
Page | 8

molecular mechanism of sperm to use the fatty acid as energy source and cholesterol to recover
the damages of sperm during freezing and thawing process.
Therefore, the present study was undertaken to understand the components of LM (LM) in bovine
frozen sperm membrane with the amount of fatty acids on sperm membrane and their role inthawing media to improve the quality of bovine frozen sperm were investigated. Moreover, theindividual effect of cholesterol and fatty acids combined in the LM on bovine frozen sperm werealso analyzed. To clear the precise hypothesis, three (3) individual experiments were conducted.
In Experiment 1, it was hypothesized that sperm contain lipid components in variable quantity inits membrane and sperm may uptake fatty acids to their membrane from LM during thawing.
Another assumption was LM in thawing media of bovine frozen sperm can improve sperm quality
like motility, membrane integrity, survivability and ultimately invitro fertilization (IVF) rate. To
clear the hypothesis, computer assisted semen analysis (CASA), Gas chromatography and mass
spectrometry (GC-MS), and flow cytometry (FCM) with different stains were performed using
bovine frozen sperm.
In Experiment 2, it was assumed that sperm can transport incorporated the fatty acid to themitochondria for energy production to keep the linear motility pattern during incubation. To clarify
the assumption, CASA, GC-MS, FCM with different stains, oxygen consumption rate (OCR) andimmunofluorescence (IF) were performed using bovine frozen sperm.
In Experiment 3, it was supposed that sperm can recover their membrane integrity using thecholesterol supplied in thawing media and improve quality parameters like sperm motility andsperm membrane integrity. To make understand the hypothesis, CASA, FCM with different stains,oxygen consumption rate (OCR) was performed using bovine frozen sperm.






Page | 9

&KDSWHU([SHULPHQW
6WXG\RQYDULDWLRQRIOLSLGFRPSRQHQWVLQERYLQHIUR]HQVSHUP DQGWKHLUUROHRQDFFHOHUDWLQJPRWLOLW\DQGTXDOLW\SDUDPHWHUVDVPL[WXUH&RQWHQWV

2.1
2.2
2.3
2.4









7RSLFV
Introduction
Materials and Methods
Results
Discussion
Abstract

3DJH1R
10
11
14
16
19

,QWURGXFWLRQSperm are the biological and functional male reproductive unit and have a unique membraneconfiguration and unusual nuclear, mitochondrial, cytoplasmic, and cytoskeletal structurescompared to those of somatic cells (Flesch & Gadella, 2000). These characteristics are formed
during spermatogenesis in the testis. However, the plasma membrane structure of sperm is not
fully mature when they are released from the testis, and the membrane structure is further changed
during the maturation process in the epididymis (Eddy et al., 1994). The biochemical changes inthe sperm plasma membrane in the epididymis include release, modification, and adsorption ofproteins and lipids (Frits & Gadella, 2000). Although the role of these surface alterations has notbeen clearly described, it has been investigated that some adsorbed proteins are involved in spermoocyte binding during fertilization (Flesch & Gadella, 2000). Another report indicated that theexpression of membrane receptors, e.g., C-met receptors, varies due to changes in the lipid
structure of the sperm membrane (Depuydt et al., 1996).
The lipid composition of the sperm membrane significantly affects the functional characteristics
of spermatozoa (Zalata et al., 1998). This composition is different among species, but normally it
contains approximately 70% phospholipids, 25% neutral lipids and 5% glycolipids (on a molar
basis) (Mann et al., 1981). Biophysical studies reported that just after leaving the testes, sperm
cells stop the synthesis of plasma membrane lipids and proteins, which means that components ofthe plasma membrane cannot be newly synthesized (Frits & Gadella, 2000). During epididymal
maturation, the phospholipid (PL) composition of the membrane may change due to a high content
of unsaturated fatty acids in epididymis fluid (Hall et al., 1991). These changes increase membrane
fluidity because lipids in the sperm plasma membrane regulate the permeability of the sperm cell
membrane (Frits & Gadella, 2000). The increased permeability returns to a low level after sperm
are mixed with seminal plasma. The change of membrane permeability is explained by different
stability of cholesterol that is taken to the sperm membrane being different from that of seminal
plasma, imposing a conformational order of lipids in the membrane (Leahy & Gadella, 2015).
Therefore, the sperm membrane would be changed by different lipid components under epididymal
and semen conditions (Towhidi & Parks, 2012).
In the relationship between exogenous fatty acids and sperm quality, an experiment on a feeding
trial of essential fatty acids in rats implied that membrane-associated fatty acids are important for
10 | P a g e

sperm motility, sperm viability and the fusion process between spermatozoa and oocytes (Lenzi etal., 1996; Lewis SE & Maccarrone, 2009). It was observed that the incubation of human spermwith glycerophospholipids improves sperm viability, motility, and resistance to oxidizing agentslike H 2 O 2 (Ferreira et al.,2018). In the boar sperm study, it was reported that fatty acids, especiallyarachidonic acid, play an important role in sperm movement and a combination of arachidonic andoleic acid is important for inducing acrosomal reaction (AR) (Hossain et al., 2007). In the Brown
Swiss bull sperm study, it was shown that the optimal level of n-3 FA (10 ng mL−1) combinedwith the highest level of vitamin E (0.4 mM) in sperm handling extender changed the membrane
fatty acid composition and improved the freezability of sperm (Towhidi & Parks, 2012).
Against the positive effects of exogenous fatty acids, sperm plasma and acrosome membranes aredamaged during the freezing and thawing process. The process increases oxidative stress in sperm,
which changes sperm membrane permeability and might lead to reducing the spermatozoa
functional integrity and decreasing its fertilizing ability (Gillan & Maxwell, 1999). However, tothe best of our knowledge, the changes in fatty acid components in the sperm structure during
sperm freezing and thawing process is unrevealed. Moreover, the relationship of different fatty
acid components of the sperm membrane with the freeze-thaw process and sperm motility is still
unknown in bovine species. Therefore, firstly we focused on the variations in membrane fatty acidcomponents among the individuals of bovine frozen sperm from different animals. Secondly, we
tried to correlate the relationship between sperm motility with the different membrane incorporated
fatty acids of bovine frozen sperm. Finally, we studied on sperm quality parameters like motility,
membrane integrity, hexokinase activity and mitochondrial activity by adding a LM to the basic
thawing media.
0DWHULDOV DQGPHWKRGV0DWHULDOV
Routine chemicals were obtained from FUJIFILM Wako Pure Chemical Industries, Ltd., (Osaka,
Japan) and Nakarai Chemical Co. (Osaka, Japan). Frozen straws of bovine sperm (Holstein
Friesian) were kindly gifted from Japan and Livestock improvement association of Japan. INC(Maebashi, Japan).

11 | P a g e6SHUPSUHSDUDWLRQDQGLQFXEDWLRQ
Frozen bull semen was thawed in water at 37 °C and washed by centrifugation twice (600 g, 37 °C,
4 min) with 6 mL of Bracket-Oliphant (BO) medium (Table-1) (Brackett et al. 1978) using theheated and refrigerated centrifuge machine (Kubota 5910, Japan). The sperm pellet was
resuspended with BSA containing BO medium without LM (0.1%) (L0288, Sigma-Aldrich, USA)
that were used in thawing media, and then the sperm were incubated without LM for 30 min at
37 °C under a humidified atmosphere of 5% CO2 in the air.
6DPSOHSUHSDUDWLRQIRUJDVFKURPDWRJUDSK\PDVV VSHFWURPHWU\ DQDO\VLVFatty acids in sperm were extracted and methylated by the Fatty Acid Methylation Kit (06482-04,
Nacalai Tesque Inc. Japan), and then were purified by the Fatty Acid Methyl Ester Purification Kit(06483-94, Nacalai Tesque Inc. Japan) according to manufacturer’s instructions. The methylated
samples (3.0 mL) were dried, and then dissolved in 50 μL of the eluting solution. The samples
were subjected to GC-MS.
*DVFKURPDWRJUDSK\PDVV VSHFWURPHWU\DQDO\VLV
Agilent 7890A GC System coupled to JMS-T 100 GCv mass detector, an Agilent 7693A
autosampler was injected with 1.0 μL aliquot of samples. HP-5 capillary column (60 m × 0.32 mm
i.d. × 0.25 μm film thickness; 19091J-413, Agilent Technologies, Santa Clara, CA) was used toanalyze the samples and reference standards (10, 1.0, 0.1, 0.01, 0.001mM). Helium was used as
the carrier gas at a constant flow rate of 2.0 mL/min. The temperature for the GC oven varied
starting at 100 °C to gradually increasing up to 300 °C at the rate of 10 °C / min and held at 300°C for 20 min. Ionization was performed using electron ionization (EI) at electron energy of 70
eV. The operation of mass spectrum was carried out in the scan mode (mass scanning range of 29800 m/z). NIST MS Search v. 2.0 was used to detect and identify the fatty acids.
'HWHFWLRQRI6SHUP0RWLOLW\ E\&RPSXWHU$VVLVWHG6SHUP$QDO\VLV &$6$ 6\VWHPSperm motility was evaluated using CASA according to our previous study (Zhu et.al. 2019). A
10 μL of the sample was placed in a pre-warmed counting chamber to take the CASA reading after
incubation of sperm at different time intervals. Sperm tracks (0.5s, 45 frames) were captured at 60
12 | P a g e

Hz according to our previous study (Zhu et.al. 2019), using a CASA system (HT CASA-CerosII;
Hamilton Thorne, MA, United States). Minimum three replications were used for each sample andmore than 200 trajectories were recorded.
0LWRFKRQGULDO $FWLYLW\Mitochondrial activity of sperm was measured using MitoPT® JC-1 Assay Kit (911, Immuno
Chemistry Technologies, LLC.) according to Zhu et.al. 2019. Briefly, sperm samples wereincubated with 200 μL of 1x working solution at 37ºC for 30 min in dark. The mitochondrial
activity was analyzed by flow cytometry using a filter with a bandwidth of 574/26 nm (Attune®
NxT Acoustic Focusing Cytometer, Invitrogen) and measured as mean fluorescence intensity(MFI) of JC-1 orange aggregates. A total of 20,000 sperm events were analyzed.
0HPEUDQHLQWHJULW\The sperm membrane integrity was measured using a LIVE/DEAD Sperm Viability Kit (L7011;
Thermo Fisher Scientific) according to manufacturer protocol. Briefly, Sperm were stained withSYBR-14/PI and incubated with 200 μL 1x working solution at 37ºC for 10 min in dark. The
staining was analyzed by flow cytometer (Attune® NxT Acoustic Focusing Cytometer,
Invitrogen) with Excitation/ Emission= 515/545 nm for SYBR-14 fluorescence, Excitation/
Emission= 675/715 nm for PI fluorescence. A total of 20,000 sperm events were analyzed. There
were four quadrants on the specific events gated area. Q1 (-/+) indicating the percentage of SYBR14 containing sperm cells, Q2 (+/+) indicating the percentage of both SYBR-14 and PI containing
sperm cells, Q3 (+/-) indicating the percentage of PI containing sperm cells and Q4 (-/-) indicating
the percentage of sperm cells neither containing PI nor SYBR-14.
$QQH[LQYVWDLQLQJThe sperm membrane apoptosis was measured using an Annexin v conjugate: Alexa Fluor®647
Kit (A23204; Invitrogen, Molecular probes) according to manufacturer protocol. Briefly, Spermwere stained with Alexa Fluor®647 /PI and incubated with 200 μL 1x working solution at 37ºC
for 30 min in dark. The staining was analyzed by flow cytometer (Attune® NxT Acoustic Focusing
Cytometer, Invitrogen) with Excitation/ Emission= 663/677 nm for Alexa Fluor®647
fluorescence, Excitation/ Emission= 561/587 nm for PI fluorescence. A total of 20,000 sperm
events were analyzed. There were four quadrants on the specific events gated area. Q1 (-/+)
13 | P a g e

indicating the percentage of necrotic sperm cells, Q2 (+/+) indicating the percentage of earlynecrotic sperm cells, Q3 (+/-) indicating the percentage of apoptotic sperm cells and Q4 (-/-)
indicating the percentage of live sperm cells.
6WDWLVWLFDO DQDO\VLV
Statistical analyses of data from three or four replicates for comparison were carried out by either
Student's t-test or one-way ANOVA followed by Student's t-test (Statview; Abacus Concepts, Inc.,
Berkeley, CA).
5HVXOWV9DULDWLRQVRIPHPEUDQHIDWW\DFLGVE\*&06
The variations of fatty acid components of the sperm membrane were analyzed by GC-MS using
frozen semen samples from ten different bulls (Table- 2). Both saturated and unsaturated fatty
acids were detected at a significant (P<0.05) level of difference in d ifferent bull’s straws. Amongthe saturated fatty acids, palmitic acid (C16:0), margaric acid (C17:0), stearic acid (C18:0) anddocosanoic acid, commonly known as behenic acid (C22:0), were present in all bull straws.
Myristic acid (C14:0), pentadecylic acid (C15:0), and arachidic acid (C20:0) were present in allbull straws except in those of bulls (Bull ID 656 & 729); (Bull ID 445, 612, 656 & 729), and (BullID 445, 612, 656,729 & 250), respectively. In the case of unsaturated fatty acids, docosahexaenoic
acid (C22:6) was detected in all bull’s straws. Linoleic acid (C18:2) and arachidonic acid (C20:4)
were present in all bull’s straws except those of bulls (Bull ID 445, 612, 656 & 729) and (Bull ID
408 & 656), respectively.
5HODWLRQVKLSEHWZHHQVSHUPWRWDOPRWLOLW\ DQGGLIIHUHQWIDWW\DFLGVThere were positive correlations with some fatty acids and total sperm motility after 30 min of
incubation, and others were negatively correlated (Figure 1 & 2). Total sperm motility wascalculated by CASA derived motility tracks after 30 min of incubation at 37ºC condition. Myristic
acid and stearic acid showed a positive correlation with the total motility of sperm (Y-axis of thegraph), whereas docosanoic acid (behenic acid) was negatively correlated, although all correlations
were statistically non-significant at P= 0.28, P= 0.06 and P= 0.10, respectively.

14 | P a g e(IIHFWRI/0RQVSHUPPHPEUDQHIDWW\DFLGVTo understand whether sperm selectively uptake fatty acids from exogenous source or not, sperm
were thawed with 0.1 % (v/v) of LM containing fatty acids (2 μg/mL arachidonic acid and 10
μg/mL each linoleic, linolenic, myristic, palmitic and stearic acid) and 0.22 mg/mL cholesterol.
Fourteen kinds of fatty acids were detected by GC-MS (Figure 3 & 4) and 6 of them weresignificantly (P < 0.05) increased in sperm thawed with LM as compared with sperm without LM
condition (Figure 5).
6SHUPPRWLOLW\ SDUDPHWHUVZLWKWKH/0
Sperm motility parameters were calculated by CASA-derived motility tracks after 30-min
incubation. BCF, VCL, VSL and TM were significantly (P<0.05) higher in sperm thawed with
0.1% (v/v) LM condition (0.1% LM) compared to sperm thawed without LM condition (control)and other doses of LM (Figure 6 & 7). Lateral amplitude (ALH) was not significantly (P<0.05)
changed in all conditions except 1% LM condition. Therefore, 0.1% LM was selected as optimum
dose for further experiments.
Scatter plot was made considering the parameters of individual sperm data that were significantly
increased by LM treatment, BCF (Y Axis of graph) and VSL (X axis of graph) in figure 8; BCF(Y Axis of graph) and VCL (X axis of graph), VSL (Y Axis of graph) and VCL (X axis of graph)in figure 9 to understand the effect of LM on individual sperm (Figure 8 & 9). In each figure, thelines were made in scatter plot considering the mean value of each motility parameter in control
group of sperm. The percentage of sperm in the upper right quadrant of scatter plot wassignificantly (P<0.05) higher in LM treatment group rather than control group in both figures.
/0DIIHFWVSHUPPLWRFKRQGULDO DFWLYLW\Mitochondrial membrane activity was analyzed by JC-1 staining. Mean fluorescence intensity(MFI) of the sperm incubated for 30 min at 37ºC thawed in the LM condition significantly (P<0.05)
differed from the sperm incubated for the same time and under the same conditions but underwent
thawing without LM condition (Figure 10-A). Sperm showed a significantly lower MFI in without
LM condition than with LM condition.

15 | P a g e/0DIIHFWVSHUPPHPEUDQHLQWHJULW\The sperm membrane integrity was analyzed by SYBR-14/PI staining. The intact membrane
integrity of the sperm incubated for 30 min at 37ºC thawed in the LM condition, stained by SYBR14 showed significantly (P<0.05) higher percentages in the LM condition than without LM
condition thawing (Figure 10-B). Damages of sperm membrane integrity stained by PI was
significantly (P<0.05) lower in the LM condition group than in the group without LM condition
thawing (Figure 10-B).
/0VXSSUHVVVSHUPDSRSWRVLVLive, apoptotic, necrotic, and early necrotic cells were analyzed by Annexin V staining. The
number of live sperm cells was significantly (P<0.05) higher in the LM condition group than
without LM condition thawing (Figure 10-C). The numbers of necrotic and early necrotic sperm
cells were significantly (P<0.05) lower in the LM group than without LM condition thawing(Figure 10-C).
'LVFXVVLRQDuring the freezing and/or thawing process, the levels of some fatty acids, such as the PUFAs
C18:2, C20:3, C20:4, and docosahexaenoic acid (C22:6 n-3; DHA) significantly decreased, andthe levels of saturated fatty acids (SFAs) C:16 and C:18 increased in human sperm (Alvarez &Storey,1992). The increase of the fatty acids might be explained by the incorporation orcontamination of them from egg-yolk based freezing extender to sperm. However, the reductionof them during the freezing and thawing process is induced by the release of polyunsaturated fatty
acids from the plasma membrane (Aitken,1995). The alteration of the membrane is thought to be
the lipid peroxidation process induced by ROS (Reactive oxygen species) that is generated insperm during both the freezing and thawing process (Alvarez et al., 1987). It has also been known
that oxidative stress decreases sperm survival and fertility (Aitken,1995). In the present study, 10
lots of cryopreserved bull semen were used for GC-MS analysis, and among the saturated fatty
acids, except docosanoic acid (behenic acid) (C22:0), a positive correlation between the level offatty acids and total sperm motility was observed as similar to those in human sperm study(Martinez et al., 2013). Although we did not determine whether the low levels of sperm werecaused by the freezing and/or thawing process or were dependent on the individual’s samples
16 | P a g e

before freezing, the different components of fatty acids would affect the sperm motility of frozenthawed bull sperm.
Moreover, it was reported in bulls that palmitic acid and oleic acid advanced sperm progressive
linear motion and viability when added to the extender (Kiernan et al., 2013). Another report on
boar sperm stated that unsaturated fatty acids enhanced progressive motility and viability duringin vitro incubation (Kelso et al., 1997). The treatment with unsaturated fatty acids enhances ATP(Adenosine triphosphate) production and increases intracellular Ca2+ and cAMP (Cyclic
adenosine monophosphate) concentrations, which in turn induces the acrosome reaction (AR) in
boar spermatozoa (Hossain et al., 2007). The process of induction of AR is called as capacitation,and sperm undergo a change in the motility pattern called as hyper activation during thecapacitation process (Yanagimachi,1970). Moreover, Abavisani et al. (2013) reported that theaddition of Ω-3 PUFAs (Poly unsaturated fatty acids) directly to semen extenders was not effective
in protecting the bull sperm membrane (Abavisani et al., 2013). It was shown that a long-chain
SFA (palmitic acid) can preserve progressive linear motility and viability whilst keeping ROS
levels at a minimum level in boar sperm (Kiernan et al., 2013). On the other hand, the very longchain SFAs C22:0, and C24:0 were inversely related to the total antioxidant capacity (TAC), which
is directly correlated with motion parameters after thawing of human sperm (Martinez et al., 2012).
Some studies on bovine species (Neill & Masters,1972) and ovine species (Neill & Masters,1973)
reported that sperm cells are able to take up lipid components or fatty acids from the surrounding
environment during incubation (Cerolini et al., 2001). We used a LM in the thawing media withthe hypothesis that sperm could selectively uptake some kinds of fatty acids from a LM containing
both saturated and unsaturated fatty acids to improve their motility. In our GC-MS study, the status
of different fatty acids was significantly (P<0.05) higher in the LM condition rather than without
LM condition (Figure 5). The most up taken saturated fatty acid was myristic acid (C14: 0), andin the case of unsaturated fatty acids, linoleic acid (C18:2) was most significant. It has been
described that fatty acids were incorporated into the lipids of sperm cell membranes within twohours of addition to cell cultures (Neill & Masters,1971). From the perspective of fatty acidcomposition, it was reported that diglycerides contained myristic acid (C14: 0) as the major
component of bull sperm lipids (Neill & Masters,1972). It is suggesting that myristic acid (C14:0) was incorporated into sperm membrane lipids and improved sperm motility and otherparameters. Boar sperm study described that the addition of exogenous fatty acid s (oleic acid and
17 | P a g e

palmitic acid) to the dilution medium improved sperm quality by utilizing them as energy
substrates for ATP production via β-oxidation (Zhu et al., 2020).
The metabolic activity of the sperm including motility, vigor, and wave motion decreases with thedamage of the sperm membrane due to cold shock followed by increasing the permeability thatcauses the loss of intracellular components (Amann et al., 1987, Graham et al.,1996). Cholesterol
controls membrane structure by decreasing cold shock sensitivity through extending the phasetransition of the sperm membranes (Quinn, 1989), thus decreasing lipid from accumulating into
particular domains within the membrane (Drobnis et al., 1993) and increasing membrane stability
at lower temperature (Purdy et al., 2005). Therefore, cryo-damage occur at freeze thaw process
can be reduced by supplying cholesterol in freezing diluents (Wessel and Ball, 2004). Additionally,
it was described that cholesterol loaded methyle β-cyclodextrin in extender advanced survivabilityof stallion (Combes et al., 2000; Graham, 1998) and bull spermatozoa (Amorim et al., 2009) after
cryopreservation.
In conclusion it can be said that, as LM is composed of both saturated and unsaturated fatty acids
with cholesterol thus, it affects the sperm motility pattern, membrane structure. This is actually
preliminary findings about the effect of LM on bovine frozen sperm. Mechanism of sperm energy
production for linear motility and retain of sperm membrane integrity is still unknown. Therefore,
our next study will focus on molecular mechanism of sperm energy production using fatty acidsin thawing media and sperm membrane potentials using cholesterol separately.

18 | P a g e

$EVWUDFWSperm is very unique in their membrane structure and cryopreserved sperm is very sensitive tocold shock damage. Cryodamage affect sperm motility and membrane structure highly. In thisstudy it was hypothesized that Lipid Mixture (LM) in thawing media maybe beneficial for bovine
frozen sperm to keep good motility with sound membrane structure by maintaining their membrane
lipid composition healthy. To clarify the hypothesis CASA, GC-MS and FCM with different stains
were performed. In this study it was revealed that LM thawed bovine frozen sperm showedsignificantly (P<0.05) good motility parameters compared to control group. It was observed thatfatty acids from LM was up taken by LM thawed sperm compared to control and there was apositive correlation between fatty acids amount and total sperm motility. From this study it wasalso uncovered that membrane integrity, mitochondrial activity and live sperm was significantly(P<0.05) higher in LM thawed sperm compared to without LM thawed sperm used as control.
Therefore, it can be concluded that LM in thawing media is beneficial for bovine frozen sperm tokeep their motility with other quality parameters in healthy status.

19 | P a g e

7DEOH: Modified Bracket-Oliphant (BO) medium for thawing and incubation of sperm
Component

Amount65.5 mg/mL
3.0 mg/mL
3.3 mg/mL
1.28 mg/mL
1.06 mg/mL
3.104 mg/mL
0.1376 mg/mL
0.05 mg/mL
2.502 mg/mL
0.9 mg/mL
0.015 mg/mL
5.0 mg/mL

NaCl
KCl
CaCl2 -2H 2 O
NaH 2 PO 4 -2H 2 O
MgCl2 -6H2 O
NaHCO 3Pyruvic acid
Streptomycine
GlucoseCaffeine
HeparinBSA


7DEOH Different fatty acid amount (pmol/sperm) calculated by GC-MS from different stocks ofbull semen straw. Different superscripts differ significantly at P < 0.05.
Bull

MA

PA

PA

MA

SA

AA

BA

LA

AA

DHA

ID

(C14:0)(C15:0)(C16:0)(C17:0)(C18:0)(C20:0)(C22:0)(C18:2)(C20:4)(C22:6)408

116.16 e

69.66 d5628.22 h

28.33 h1256.43 f

12.32 e4965.64 e

91.41 f0.00 i4525.84 g

445

53.58 f0.00 g14483.84c

59.08 f65.98 j0.00 f

8122.40 a

0.00 g128.55 f

13661.29b612

11.71 h0.00 g11595.12e

41.19 g145.88 h

0.00 f

5428.30d

0.00 g31.52 h10337.20d

656

0.00 i0.00 g3007.02 j

72.77 e86.33 i0.00 f729.35 j

0.00 g0.00 i799.70 j729

0.00 i0.00 g3930.35 i

24.85 i151.97 g

0.00 f3568.45 g 0.00g

71.38 g2166.90 h

423

934.76 d1438.17a

20348.23a

1662.61b

2280.13 c

852.70 a

6600.56b

1717.67 a

1418.16b

14866.99a

389

3160.63 a

31.14 f

14501.66b

1938.53 a

1806.41d

829.97 b

4064.14 f

1173.42 e

816.55 c

8911.47 e

9501452.53b

621.39 c

10764.49 f

1175.23d

2838.80b13.74 d934.84 i

1292.40d

673.60 d

804.42 i

250

18.76 g935.91 b

12072.47d

5.73 j1714.67 e

0.00 f5533.74 c

1334.86 c

1568.16 a

10946.61c1481228.41 c

62.77 e7377.45 g

1226.70 c

3505.65 a

44.22 c2595.73h

1475.46b

554.88 e462.31 f

20 | P a g e)LJXUH Correlation between different saturated fatty acids with total sperm
motility. Here, X-axis indicates the amount of different fatty acids, and the Yaxis indicates the total sperm motility of different bull straws.

21 | P a g e)LJXUH Correlation between different unsaturated fatty acids with the total sperm motility.
Here, X-axis indicates the amount of different fatty acids, and the Y-axis indicates the total
sperm motility of different bull straws.

22 | P a g e)LJXUHChromatogram of different fatty acid components present in sperm thawed withlipid mixture found by GC-MS.

23 | P a g e)LJXUHChromatogram of different fatty acid components present in sperm thawed without
lipid mixture found by GC-MS.

24 | P a g e)LJXUHTotal fatty acids amount (pmol/ Sperm) after thawing the sperm in lipid mixture (L+)
compared to control (L-) found by GC-MS study.
Here, L+ means thawing sperm with lipid mixture and L- means thawing sperm without lipid
mixture condition. Y axis indicating the amount of total fatty acid.
Values are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
Here, (A.) MA (C14 :0) = Myristic acid; (B.) PA (C16 :0) = Palmitic acid; (C.) MA (C17 :0) =Margaric acid (D.) SA (C18 :0) = Stearic acid (E.) LA (C18 :2) = Linoleic acid; (F.) DHA (C22:6) = Docosahexaenoic acid25 | P a g e)LJXUHLipid mixture improves sperm motility pattern. Here, LM means thawing sperm withlipid mixture and control means thawing sperm without lipid mixture condition. 
Values are specified (Mean±SD) with three replications. Different superscripts differ significantly
at P < 0.05.
ALH indicating amplitude of lateral head displacement
BCF indicating beat cross frequency
VCL indicating curvilinear velocity
VSL indicating straight line velocity
TM indicating total motility

26 | P a g e)LJXUH CASA motility track of different doses of lipid mixture (LM). Here, 0.1% LM shows
Highest linear motility track compared to other tracks.

27 | P a g e)LJXUH  Correlation between ALH and VCL (A.) and BCF and VSL(B.) of each sperm treatedwith Lipid mixture (LM+) in thawing media compared with control (LM-). The lines in scatterplot were made considering the mean value of each motility parameter in control group of sperm.
In each correlation figure the second quadrant (ii) region, indicating the lower value of bothparameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
different in LM treated group compared to control.

28 | P a g e)LJXUH   Correlation between BCF and VCL (A.) and VCL and VSL(B.) of each sperm treatedwith Lipid mixture (LM+) in thawing media compared with control (LM-). The lines in scatterplot were made considering the mean value of each motility parameter in control group of
sperm.
In each correlation figure the second quadrant (ii) region, indicating the lower value of bothparameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
different in LM treated group compared to control.

29 | P a g e)LJXUHLipid mixture improves sperm quality. Here, LM means thawing sperm with lipid
mixture and control means thawing sperm without lipid mixture condition.
Values are specified (Mean±SD) with three replications. Different superscripts in various
parameters differ significantly at P < 0.05 in One way ANOVA with t-test.
A. Mean fluorescence intensity (MFI) observed by JC-1 stain for mitochondrial activity
B. Membrane integrity detected by SYBR-14/PI stain
C. Live sperm (%) measured by Annexin V stain

30 | P a g e

&KDSWHU([SHULPHQW
6WXG\RQHIIHFWRIIDWW\DFLGVRQVSHUPOLQHDUPRWLOLW\WKURXJKPLWRFKRQGULDO$73SURGXFWLRQ&RQWHQWV

3.1
3.2
3.3
3.4


7RSLFV
Introduction
Materials and Methods
Results
Discussion
Abstract

3DJH1R
31
33
34
37
41



,QWURGXFWLRQ 
Each living cell need energy for both growth and function and sperm, the male gametes, are notexception. Sperm motility is induced by ATP production (Brokaw, 1972) and indispensable forsperm migration to the oocyte for successful fertilization (Suarez & Pacey, 2006). Motility patterns
are reliant on flagellar motion and the linear motility pattern is induced by the symmetrical flagellar
motion (Ho et al., 2002; Curtis et al., 2012). The linear motility, is essential for sperm migrationfrom the cervix to the uterus and then to oviduct, characterized by low lateral amplitude and highstraight-line velocity (Shalgi et al., 1992; Ishijima,2015). In oviduct during fertilization process,hyperactivation is prerequisite for capacitation of sperm (Suarez, 2008), where curvilinear velocity
is high as well as high lateral amplitude (Stauss et al., 1995).
For sperm motility, two main metabolic pathways are required for ATP production namely
glycolysis which is occurred in sperm tail and mitochondrial oxidative phosphorylation(OXPHOS) is activated in midpiece of sperm (Du Plessis et al., 2015). Generally, glycolysis iscarried out in the cytoplasm where pyruvate is produced from glucose and then it enters the
mitochondria to be oxidized to acetyl CoA which combines with oxaloacetate to start thetricarboxylic acid (TCA) cycle (Zheng, 2012). However, in sperm tail as mitochondria areinadequate, so mitochondria ATP production is not highly increased although high dose of glucose
rapidly induces temporary hyperactivation status (Suarez et al., 1991). Indeed, Ldh-c knock out
mice model demonstrated that for hyperactivated motility the glycolysis was essential (Odet et al.,2008). Rotenone, an inhibitor of complex I, declined the sperm mitochondrial activity and ATP
levels with progressive motility and straight-line velocity but not lateral amplitude and total
motility in boar (Zhu et al., 2019), stallion (Plaza Davila et al., 2015) and human (Barbonetti et al.,2010). Further reports revealed that rotenone suppressed the sperm capacitation, acrosome reaction
and fertilization rate in hamster (Rogers et al., 1977) and stallion (Plaza Davila et al., 2015),
indicating that mitochondrial ATP production is crucial for sperm linear motility andhyperactivation.
Remarkably, it was reported in boar sperm decreasing the glucose level in extender induced linear
motility and boosted OXPHOS, again completely removal of glucose from the medium failed tomaintain the high linear motility and mitochondrial activity (Zhu et al., 2019). The glucose is used
31 | P a g e

as substrate not only in glycolysis but also in pentose phosphate pathway (PPP) to generate
NADPH, which impacts on redox homeostasis to maintain sperm motility (Stincone et al., 2015;
Gruning et al., 2014) suggesting that the pyruvate transformed from glucose would not be main
sources of acetyl CoA to activate OXPHOS in sperm. It has been reported that acetyl CoA is
produced by mitochondrial β-oxidation from fatty acids (Aon et al., 2014). The oxygen availability
and the composition of metabolic substrates causes variation in metabolic pathways of cells (Gohil
et al., 2010; Potter et al., 2016).
Interestingly, LM study (Chapter 2) showed that the saturated fatty acids have a positive
correlation between the level of fatty acids and total sperm motility, similar result was observed inhuman sperm study (Martinez et al., 2013). From GC-MS study of previous chapter, the status ofdifferent fatty acids was significantly (P<0.05) higher in the LM condition and the most up taken
fatty acid was myristic acid (C14: 0), a long chain saturated fatty acid. It was reported that a longchain SFA (palmitic acid) can preserve progressive linear motility and viability whilst keeping
ROS levels at a minimum level in boar sperm (Kiernan et al., 2013). Additionally, it was reported
that the addition of Ω-3 PUFAs (Poly unsaturated fatty acids) directly to semen extenders was noteffective in protecting the bull sperm membrane (Abavisani et al., 2013). Alternatively, the very
long-chain SFAs C22:0, and C24:0 were inversely related to the total antioxidant capacity (TAC),
which is directly correlated with motion parameters after thawing of human sperm (Martinez etal., 2012). Several reports on bovine species (Neill & Masters,1972) and ovine species (Neill &Masters,1973) revealed that sperm cells are able to take up lipid components or fatty acids fromthe surrounding environment during incubation (Cerolini et al., 2001). From the perspective of
fatty acid composition, it was described that diglyceride contained myristic acid (C14: 0) as the
major component of bull sperm lipids (Neill & Masters,1972) suggesting that myristic acid (C14:0) can incorporated into sperm lipids and might improve sperm motility parameters.
In human sperm it is reported that a large proportion of the metabolic proteome was involved inlipid metabolism, including enzymes for β-oxidation (Amaral et al., 2013). Excitingly, themitochondrial β-oxidation is an active regulator for sperm motility because inhibition of βoxidation using etomoxir led to a decrease of sperm motility (Amaral et al., 2013). Moreover, it
was reported in bulls that palmitic acid of saturated fatty acid class improved sperm progressive
linear motion and viability when supplemented to the extender (Kiernan et al., 2013), suggesting

32 | P a g ethat mitochondrial β-oxidation is very essential for sperm progressive motility and survivability
where exogenous fatty acids are the main source of energy.
However, the mechanism by which saturated fatty acid promotes sperm progressive motility andviability is still ambiguous. Therefore, in this present study, it was assumed that saturated fatty
acid might contribute to ATP production via mitochondrial β-oxidation to protect sperm in vitro.
To clarify the hypothesis sperm motility was focused with exogenous different saturated fatty acids
to the thawing media by CASA and the level of the different incorporated fatty acids was analyzed
by GC-MS in bovine frozen sperm. Finally, sperm mitochondrial activity and oxygen consumption
rate was studied by the treatment with saturated fatty acid.
0DWHULDOV DQGPHWKRGV0DWHULDOV
Routine chemicals were obtained from FUJIFILM Wako Pure Chemical Industries, Ltd., (Osaka,
Japan) and Nakarai Chemical Co. (Osaka, Japan). Frozen straws of bovine sperm were kindly
gifted from Japan and Livestock improvement association of Japan. INC (Maebashi, Japan).
6SHUPSUHSDUDWLRQDQGLQFXEDWLRQ
Frozen bull semen was thawed in water at 37 °C and washed by centrifugation twice (600 g, 4min) with 6 mL of Bracket-Oliphant (BO) medium (Table-1) (Brackett et al. 1978) using the heated
and refrigerated centrifuge machine (Kubota 5910, Japan). The sperm pellet was resuspended with
BSA containing BO medium without 50 nM of myristic acid (MA) (130-03432, FUJIFILM Wako
Pure Chemical Industries, Ltd., Japan), palmitic acid (PA) (165-00102, FUJIFILM Wako Pure
Chemical Industries, Ltd., Japan,), and behenic acid (BA) (216941-5G, Sigma- Aldrich, USA),
that were used in thawing media, and then the sperm were incubated for 30 min at 37 °C under ahumidified atmosphere of 5% CO2 in the air. Before using these FAs in thawing media, they weredissolved in 99.5% ethanol (09-0770-3, Sigma- Aldrich, USA) and diluted with BO media to makethe 50 nM final concentration.
6DPSOHSUHSDUDWLRQIRUJDVFKURPDWRJUDSK\PDVV VSHFWURPHWU\ DQDO\VLVFatty acids in sperm were extracted and methylated by the Fatty Acid Methylation Kit (06482-04,
Nacalai Tesque Inc. Japan), and then were purified by the Fatty Acid Methyl Ester Purification Kit(06483-94, Nacalai Tesque Inc. Japan) according to manufacturer’s instructions. The methylated
33 | P a g e

samples (3.0 mL) were dried, and then dissolved in 50 μL of the elution solution. The samples
were subjected to GC-MS.
*DVFKURPDWRJUDSK\PDVV VSHFWURPHWU\DQDO\VLV
Agilent 7890A GC System coupled to JMS-T 100 GCv mass detector, an Agilent 7693A
autosampler was injected with 1.0 μL aliquot of samples. HP-5 capillary column (60 m × 0.32 mm
i.d. × 0.25 μm film thickness; 19091J-413, Agilent Technologies, Santa Clara, CA) was used toanalyze the samples and reference standards. Helium was used as the carrier gas at a constant flowrate of 2.0 mL/min. The temperature for the GC oven varied starting at 100 °C to gradually
increasing up to 300 °C at the rate of 10 °C / min and held at 300 °C for 20 min. Ionization was
performed using electron ionization (EI) at electron energy of 70 eV. The operation of mass
spectrum was carried out in the scan mode (mass scanning range of 29-800 m/z). NIST MS Searchv. 2.0 was used to detect and identify the fatty acids.
'HWHFWLRQRI6SHUP0RWLOLW\ E\&RPSXWHU$VVLVWHG6SHUP$QDO\VLV &$6$ 6\VWHPSperm motility was evaluated using CASA according to our previous study (Zhu et al., 2019). A
10μL of the sample was placed in a pre-warmed counting chamber to take the CASA reading after
incubation of sperm at different time intervals. Sperm tracks (0.5s, 45 frames) were captured at 60
Hz according to our previous study (Zhu et al., 2019), using a CASA system (HT CASA-CerosII;
Hamilton Thorne, MA, United States). Minimum three replications were used for each sample andmore than 200 trajectories were recorded.
0LWRFKRQGULDO $FWLYLW\Mitochondrial activity of sperm was measured using MitoPT® JC-1 Assay Kit (911, Immuno
Chemistry Technologies, LLC.) according to previous study (Zhu et al., 2019). Briefly, sperm
samples were incubated with 200 μL of 1x working solution at 37ºC for 30 min in dark. The
mitochondrial activity was analyzed by flow cytometry using a filter with a bandwidth of 574/26
nm (Attune® NxT Acoustic Focusing Cytometer, Invitrogen) and measured as mean fluorescenceintensity (MFI) of JC-1 orange aggregates. A total of 20,000 sperm events were analyzed.

34 | P a g e2[\JHQ&RQVXPSWLRQ$VVD\Cellular oxygen consumption rate (OCR) was monitored in real time using a Seahorse Bioscience
Extracellular Flux Analyzer (XF HS Mini, Agilent, Santa Clara, CA). For flux analyzer, NaHCO 3Free HTF medium was made according to previous study (Balbach et al., 2020). Frozen bovine
sperm were thawed as above. After washing by NaHCO 3 -Free HTF medium, cell numbers were
quantified and diluted to 3,000,000 sperms/180 μL of NaHCO 3 -Free HTF medium. Assay plates
were coated with concanavalin A (0.5 mg/mL, Fujifilm Wako Chemicals, Osaka, Japan) overnightthe day before the assay. Each well was then seeded with 180 μL of NaHCO 3 -Free HTF medium
containing 3,000,000 sperm. The assay was performed in 6 min cycles of mix (3 min) and measure(3 min) as per manufacturer’s recommendations. Cells were treated as indicated with myristic acid(final concentration: 5, 50 or 500 nM) and/or etomoxir (final concentration: 40 nM).
)ORZF\WRPHWU\IRUTXDQWLI\LQJWKHLQWDNHRISDOPLWLF DFLGTo detect the fluorescence intensity in each sperm by flow cytometry, frozen-thawed bovine sperm
were incubated with fluorescence labeled palmitic acid (BODIPY TM FL C16 , D3821 Invitrogen,USA) in a dose dependent manner (1 μM, 10 μM, 100 μM). After washing, the fluorescence level
was analyzed by flow cytometry using a filter with a bandwidth of 530/30 nm (Attune® NxT
Acoustic Focusing Cytometer, Invitrogen) and measured as the intensity of the average value. A
total of 20,000 sperm events were analyzed.
,PPXQRIOXRUHVFHQFH
Sperm were thawed and washed by BSA-free BO medium and mounted on glass slides and airdried. The sperm were fixed with 100% methanol for 10 min at room temperature. Sperm wereprobed with the 1:100 diluted primary antibody (Anti-CD36 antibody; NB 110-59724, Novus
Biologicals, USA and anti-GOT2 antibody; NBP2-16708, Novus Biologicals). After washing by
PBS (-), the antigens were visualized with Cy3-conjugated goat anti-mouse IgG (1:100, Sigma),
FITC-conjugated goat anti-rabbit IgG (1:100, sigma) and DAPI (VECTESHIELD Mounting
Medium with DAPI, Vector Laboratories, Burlingame, CA, USA). Digital images were captured
using a Keyence BZ-9000 microscope (Keyence Co., Osaka, Japan).

35 | P a g e6WDWLVWLFDO DQDO\VLV
Statistical analyses of data from three or four replicates for comparison were carried out by either
Student's t-test or one-way ANOVA followed by Student's t-test (Statview; Abacus Concepts, Inc.,
Berkeley, CA).
5HVXOWV6SHUPPRWLOLW\ SDUDPHWHUVZLWKIDWW\DFLGVSperm motility parameters were calculated by CASA-derived motility tracks after 30-min
incubation. In the sperm treated with MA or PA, BCF, VCL, VSL and TM were significantly (P< 0.05) higher than the control (F-) group (Figure 11). The values of VCL, VSL and TM werehighest in MA group. Scatter plot was made considering the parameters of individual sperm datathat were significantly increased by fatty acid treatment, BCF (Y Axis of graph) and VSL (X axisof graph), between BCF (Y Axis of graph) and VCL (X axis of graph), and between VSL (Y Axisof graph) and VCL (X axis of graph) in MA group (Figure 13 & 14) and PA group (Figure 15 &16) respectively. The lines were made in scatter plot considering the mean value of each motility
parameter in control group of sperm. The percentage of the number of sperm in the upper rightquadrant of scatter plot was significantly (P<0.05) higher in fatty acid treatment group rather thancontrol group in both fatty acid’s figures (Figure 13,14,15,16).
&KDQJHVRIGLIIHUHQWIDWW\DFLGOHYHOGXULQJLQFXEDWLRQ
To know whether the exogenous fatty acids of sperm were incorporated to sperm and then used asenergy sources during 30-min incubation to increase the sperm velocity or not. GC-MS study
showed that the significant increase levels of myristic acid (MA), palmitic acid (PA) and stearicacid (SA) but not behenic acid (BA) were observed in the treated sperm with each fatty acid (Figure17). Interestingly, the increasing levels were not maintained during incubation and the levels weresignificantly (P<0.05) decreased to the basal levels. However, BA had no significant changes
during incubation (Figure 17).
)DWW\DFLGVDIIHFWVSHUPPLWRFKRQGULDO DFWLYLW\Mitochondrial membrane activity was analyzed by JC-1 staining. Mean fluorescence intensity(MFI) of the sperm incubated for 30 min at 37ºC thawed with the different fatty condition
36 | P a g e

significantly (P<0.05) differed from the sperm incubated for the same time and under the same
conditions but underwent thawing without fatty condition (Figure 18). Sperm showed asignificantly lower MFI in without fatty acid condition and BA condition than with MA and PA
condition.
/RFDOL]DWLRQRIIDWW\DFLGWUDQVSRUWHULQVSHUP
When frozen-thawed sperm were incubated with fluorescence labeled palmitic acid, thefluorescence signals were selectively observed in midpiece of sperm. The fluorescence intensity
in each sperm detected by FACS was significantly (P<0.05) increased in dose dependent manner(Figure 19-A, B & C). Furthermore, fatty acid transporters (CD36 and GOT2) were localized in
the joint region between head and midpiece of sperm (Figure 19-D).
(IIHFWRIIDWW\DFLGLQVSHUP PHWDEROLFFKDQJHVTo quantify the metabolic changes of mitochondrial activity, real-time changes were monitored in
mitochondrial β-oxidation using a Seahorse Bioscience Extracellular Flux Analyzer (XF HS Mini,
Agilent, Santa Clara, CA). Firstly, the oxygen consumption rate (OCR) was traced at different
concentration of MA (Figure 20-A & B). The significant increase of OCR was induced by the
injection of 50 nM of MA. Etomoxir, an inhibitor of β-oxidation significantly suppressed the MAinduced OCR rate (Figure 20-C & D).
'LVFXVVLRQSperm motility pattern are changed from ejaculation up to fertilization process. In vivo
fertilization, the motility pattern of sperm is altered from linear motility in the uterus to zigzag
motility in the oviduct during the journey of sperm in female reproductive tracts (De Lamirande
et al., 1997). It is generally accepted that the sperm motility is regulated by ATP produced either
from glycolytic pathway in cytoplasm or OXPHOS in mitochondria or both (Storey, 2008; Mukai& Travis, 2012). It was revealed in boar sperm study that mitochondrial activity was increased
under low glucose condition, and the ATP produced in mitochondria was associated with a highspeed linear motility (Zhu et al., 2019), which is characterized by high straight-line velocity (VSL)and low lateral amplitude (ALH) (Shalgi et al., 1992), indicating that sperm linear motility is
directly associated with mitochondrial activity.

37 | P a g e

As LM is composed of both saturated and unsaturated fatty acids and cholesterol, in previous
chapter it was described that the levels of saturated long fatty acid including myristic acid (C14);
palmitic acid (C16); margaric acid (C17) and stearic acid (C18) were increased in sperm after thetreatment with LM. Excitingly, present study showed that after the treatment of each fatty acid theincreasing levels of myristic acid (MA), palmitic acid (PA) and stearic acid (SA) but not behenic
acid (BA), were not maintained during incubation and the levels were significantly (P<0.05)
decreased to the basal levels. However, BA had no significant changes during incubation (Figure17). It is suggesting that during incubation period the long chain fatty acids (MA, PA, SA) wereincorporated to sperm and used as energy sources to keep motility sound.
It was reported that unsaturated fatty acids reduced quality of frozen thawed bull sperm in citrateextender (Kandelousi et al., 2013). Similarly, other experiments revealed that unsaturated fatty
acids supplementation lowered sperm viability; motility and morphology (Abavisani et al., 2013).
Therefore, in this study saturated fatty acids were added to sperm thawing medium to determine
whether exogenous saturated fatty acids would be incorporated into frozen-thawed bull sperm andimprove the motility of frozen-thawed sperm or not. The addition of long-chain saturated fatty
acids (C14) but not ultralong one (C22) to thawing medium significantly (P<0.05) induced linear
motility pattern. It has been also reported that the long chain saturated fatty acid (C16) can preserve
progressive linear motility and viability whilst keeping ROS levels at a minimum level in boar
sperm (Kiernan et al., 2013). On the other hand, the very long-chain saturated fatty acids (C22:0
and C24:0) were inversely related to the total antioxidant capacity (TAC), which is directly
correlated with motion parameters after thawing of human sperm (Martinez et al., 2012).
In this study with fatty acid, the progressive linear motility was improved in bovine frozen spermwith high straight-line velocity (Figure11). The high curvilinear velocity (VCL) was also observedin fatty acid treatment compared to control (Figure 11); however, ALH was not increased,suggesting that hyperactivation was not induced by the treatment with fatty acid because both of
ALH and VCL are increased whereas VSL is decreased in hyper-activated sperm (Zhu et al., 2019).
Individual sperm effect of fatty acid on sperm motility parameters also revealed that fatty acidaffected significantly (P< 0.05) individual sperm VSL and VCL (Figure 13,14,15,16), indicatingthat linear motility pattern was induced after the treatment of fatty acid. Therefore, it is suggestingthat saturated fatty acids would be utilized in the mitochondria of sperm for the production of ATP.

38 | P a g e

Although fatty acids are essential nutrients for cellular functions (McArthur et al., 1999), but how
they are transported across the plasma membrane in sperm, is not well understood. There wasevidence that the uptake of protein-facilitated fatty acids is the key pathway in metabolic tissues
including liver, adipose tissue, and muscle (Hajri et al., 2002; Ehehalt et al., 2006). CD36 was amajor participant in metabolic tissues including the proteins involved in fatty acids uptake (Pepino
et al., 2014; Su & Abumrad, 2009). The CD36 forms heterodimer with plasma membrane fatty
acid binding protein (FABPpm/GOT2), which transports the vital fatty acid in the heart of human(Gimeno et al., 2003). Another study on liver cell of rat revealed that FABPpm/GOT2 was locatedin the plasma membrane and has been detected as fatty acid transporters to regulate the uptake oflong chain fatty acids (Schwieterman et al., 1988). Immunofluorescence study showed that both
CD36 and GOT2 are localized in the joint of head and midpiece of sperm (Figure 19) where
actually mitochondria are located. Moreover, the fluorescence-tagged palmitic acid was observedin sperm midpiece, indicating that saturated fatty acid was up taken to sperm midpiece via a CD36GOT2 dependent manner for using ATP production in mitochondria.
Mitochondrial ATP is generated through transformation of ADP by the reduction of electron
carriers (Amaral et al., 2013). The electron transfer chain (ETC) comprises of several complexes
that carry electrons attained from the oxidation of NADH and FADH2 moiety of succinate
dehydrogenase, and ultimately decreased the final acceptor oxygen to water (Bratic & Trifunovic,
2010). It has been reported that exogenous fatty acids can be used as energy substrate for sperm.
For example, boar sperm study reported that the addition of exogenous fatty acids (oleic acid andpalmitic acid) to the dilution medium improved sperm quality by utilizing them as energy
substrates for ATP production via β-oxidation (Zhu et al., 2020). In this study, myristic acidshowed good result in mitochondrial β-oxidation (Figure 20), suggesting that long chain saturated
fatty acids in thawing media of frozen bovine sperm can be used as energy substrate for ATP
production in mitochondria in sperm midpiece to induce and maintain their linear motility.
It can be concluded that saturated fatty acids were transported to the sperm mitochondria throughfatty acid transporters, where they acted as energy substrate for mitochondrial β-oxidation to
produce energy for the sperm to keep linear motility. Thus, to increase and keep sperm fertilization
ability, exogenous fatty acid is a beneficial factor to induce and keep sperm linier motility. It may
possible that thawing sperm with saturated fatty acids containing insemination medium will boost

39 | P a g e

sperm linear motility in the female genital tracts to improve fertilization as a simple and low-costtactic.
Although present study was deeply concerned with motility parameters, especially the motility
pattern of sperm induced by fatty acid treatment with mitochondrial activity as energy production
unit alongside the fatty acid transporter of sperm molecular mechanism, activities of cholesterol
another vital part of LM on the sperm membrane is still unknown. Further studies on the actual
mechanism of cholesterol on the sperm membrane will be discussed in the next chapter.

40 | P a g e

$EVWUDFWSperm motility patterns are dependent on energy substrate and their effective utilization in energy
producing segments of sperm. For successful fertilization sperm linear motility is very crucial andthis is induced by mitochondrial ATP production. Present study was undertaken to clarify whether
bovine sperm could uptake fatty acids (FAs) in thawing media and produce ATP in mitochondria
for maintaining linear motility. Bovine frozen semen was thawed in FAs contained in thawing
media and the sperm motility was analyzed by commuter assisted sperm analysis (CASA). The
kinetic changes of FAs level in sperm were detected by GC-MS. The mitochondrial activity ofsperm treated with FAs was analyzed as fluorescence intensity of JC-1 staining and oxygen
consumption rate (OCR). FA transporter (CD36 and GOT2) was observed by whole-mounted
immunofluorescence. Sperm linear motility was significantly (P<0.05) increased by the thawing
with FA. Moreover, saturated fatty acids (SFAs) were predominant in sperm thawed with LM
described in previous study. Interestingly, present study revealed that long chain saturated fatty
acids (LCSFA) (C14, C16, C18) were decreased during 30 min incubation, except very long chain
saturated fatty acid (C22). Another crucial factor revealed from this study that the bovine frozen
sperm possessed FA transporter in midpiece where the fluorescence signals were detected after thetreatment with fluorescence-tagged FA. The treatment with FA activated electron transportationin mitochondria through β-oxidation. Sperm linear motility is prompted by FAs in thawing media
of bovine frozen sperm might provide a new approach for upgrading the artificial insemination
technique of both livestock animals and human infertility care.

41 | P a g e)LJXUHFatty acids improve sperm motility parameter. Values are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
Here, MA= Myristic Acid (C14:0), PA= Palmitic Acid (C16:0), BA= Behenic Acid (C22:0) and
F- = Without any fatty acid.








42 | P a g e











43 | P a g e )LJXUH  Correlation between ALH and VCL (A.) and BCF and VSL(B.) of each sperm
treated with Myristic Acid (MA) in thawing media compared with control. The lines in scatter plot were made considering the mean value of each motility parameter in control group of

sperm.

In each correlation figure the second quadrant (ii) region, indicating the lower value of both parameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
different in MA treated group compared to control.


44 | P a g e






)LJXUH Correlation between BCF and VCL (A.) and VCL and VSL(B.) of each sperm treatedwith Myristic Acid (MA) in thawing media compared with control. The lines in scatter plot weremade considering the mean value of each motility parameter in control group of sperm.



In each correlation figure the second quadrant (ii) region, indicating the lower value of both parameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
 different in MA treated group compared to control.

45 | P a g e






)LJXUH  Correlation between ALH and VCL (A.) and BCF and VSL(B.) of each sperm
treated with Palmitic Acid (PA) in thawing media compared with control. The lines in scatterplot were made considering the mean value of each motility parameter in control group of sperm.


In each correlation figure the second quadrant (ii) region, indicating the lower value of bothparameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
 different in PA treated group compared to control.

46 | P a g e )LJXUH Correlation between BCF and VCL (A.) and VCL and VSL(B.) of each sperm treated with
Palmitic Acid (PA) in thawing media compared with control. The lines in scatter plot were madeconsidering the mean value of each motility parameter in control group of sperm.
In each correlation figure the second quadrant (ii) region, indicating the lower value of bothparameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
different in PA treated group compared to control.

47 | P a g e)LJXUH  Total fatty acids amount measured by GC-MS after thawing the sperm in
respective fatty acids and incubation for 30 minutes with BSA at 37˚C. Y axis indicating theamount of fatty acids (pmol/ Sperm).
Values are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
Here, Control = Without any fatty acids; (A) MA= Myristic acid; (B) PA= Palmitic acid; (C)SA= Stearic acid; and (D) BA= Behenic (Docosanoic acid) acid.

48 | P a g e)LJXUHFatty acids changes sperm mitochondrial activity.
Values are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
Here, MA= Myristic Acid (C14:0), PA= Palmitic Acid (C16:0), BA= Behenic Acid (C22:0),
and control = Without fatty acids.

49 | P a g e)LJXUHFluorescence intensity of sperm and localization of fatty acid transporter.
A. Fluorescent signal of FITC palmitic acid,B. overlay of different doses of FITC palmitic acid
C. Mean fluorescence intensity of different doses of FITC palmitic acid and
D. Localization of fatty acid transporters

50 | P a g e)LJXUHMetabolic effect of MA or Etomoxir, the inhibitor of β-oxidation, on frozen bovine
sperm.
Values are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
A. Trace of OCR before/after the treatment of several concentration of myristic acid (MA)B. Increase ratio of OCR after the treatment of MA
C. Trace of OCR before/after the treatment of 50 nM MA or MA + Etomoxir
D. Increase ratio of OCR after the treatment of 50 nM MA or MA + Etomoxir

51 | P a g e

&KDSWHU([SHULPHQW
6WXG\RQHIIHFWRIFKROHVWHURORQUHFRYHU\RIPLWRFKRQGULDOSRWHQWLDO
WRNHHSOLQHDUPRWLOLW\LQERYLQHIUR]HQVSHUP&RQWHQWV

7RSLFV

4.1
4.2
4.3
4.4

Introduction
Materials and Methods
Results
Discussion
Abstract

3DJH1R
52
53
55
57
60

,QWURGXFWLRQSperm are greatly polarized and specialized male reproductive unit that have a unique membraneconfiguration and unusual nuclear, mitochondrial, cytoplasmic, and cytoskeletal structurescomparedd to those of somatic cells (Kaka et al., 2015). The sperm membranes consist ofphospholipids and sphingolipids in large proportions, along with cholesterol (Cincik et al., 2007).
Plasma membrane structure of sperm is not fully mature when they are released from the testis,and it is further changed during the maturation process in the epididymis (Eddy et al., 1994). Thechange of membrane permeability is explained by different cholesterol stability, also changed in
the sperm plasma membrane by imposing conformational order on lipids in the seminal plasma
during ejaculation (Leahy & Gadella, 2015). Furthermore, changes in membrane fluidity as aconsequence of cholesterol efflux and protein dissociation from raft domain seems to be also amechanism that occurs both in the maturational processes occurring at the time of ejaculation(Girouard et al., 2008) and in capacitation (Sleight et al., 2005; Cross, 2004).
Mammalian spermatozoa can be damaged during the semen cryopreservation process (Watson,2000). During cryopreservation sperm damage arise due to cold shock, low-temperature exposure,osmotic stress, oxidative stress, and combinations of these circumstances (Purdy, 2006). It has
been proposed that the sperm plasma membrane is the leading site of damage induced bycryopreservation and is one of the major causes to reduced motility and fertility of sperm during
cryopreservation (Moore et al., 2005; Chakrabarty et al., 2007). Cold shock induces plasma
membrane lipid phase transitions, which can be reduced by increasing the proportion of cholesterol
within the plasma membrane (Drobnis et al., 1993). In addition, cryopreservation inducescholesterol depletion from plasma membrane, which in turn causes membrane destabilization(Bailey et al., 2008). Damages occur in spermatozoa due to cryopreservation includes themetabolism and mitochondrial bioenergetic processes of sperm (Oberoi et al., 2014).
Surprisingly, in LM study (Chapter 2) it was observed that mitochondrial activity, membrane
integrity and rescue from cell apoptosis was promisingly higher in LM treated sperm compared tocontrol. Additionally, in the fatty acid study (Chapter 3) it was described that saturated fatty acids
are used for energy sources in mitochondria, which impacted on linear motility pattern through52 | P a g e

mitochondrial ATP production. However, LM contains both fatty acids and cholesterol,
hypothesizing that cholesterol would be incorporated to frozen-thawed sperm to recover the sperm
motility. To clarify the hypothesis firstly the sperm motility parameters were examined by CASA
using cholesterol in thawing media. After that, the localization of incorporated cholesterol specificsignals on sperm were analyzed by fluorescence tagged cholesterol. Finally, the mitochondria
metabolic activity was detected using flux analyzer system.
0DWHULDOV DQGPHWKRGV0DWHULDOV
Routine chemicals were obtained from FUJIFILM Wako Pure Chemical Industries, Ltd., (Osaka,
Japan) and Nakarai Chemical Co. (Osaka, Japan). Frozen straws of bovine sperm were kindly
gifted from Japan and Livestock improvement association of Japan. INC (Maebashi, Japan).
6SHUPSUHSDUDWLRQDQGLQFXEDWLRQ
Frozen bull semen was thawed in water at 37 °C and washed by centrifugation twice (600 g, 4min) with 6 mL of Bracket-Oliphant (BO) medium (Table-1) (Brackett et al. 1978) using the heated
and refrigerated centrifuge machine (Kubota 5910, Japan). The sperm pellet was resuspended with
BSA containing BO medium without Cholesterol Lipid Concentrate (250X) (0.1%) (Gibco™
12531018) that were used in thawing media, and then the sperm were incubated for 30 min at
37 °C under a humidified atmosphere of 5% CO2 in the air.
'HWHFWLRQRI6SHUP0RWLOLW\ E\&RPSXWHU$VVLVWHG6SHUP$QDO\VLV &$6$ 6\VWHPSperm motility was evaluated using CASA according to our previous study (Zhu et.al. 2019). A
10μL of the sample was placed in a pre-warmed counting chamber to take the CASA reading after
incubation of sperm at different time intervals. Sperm tracks (0.5s, 45 frames) were captured at 60
Hz according to our previous study (Zhu et.al. 2019), using a CASA system (HT CASA-CerosII;
Hamilton Thorne, MA, United States). Minimum three replications were used for each sample andmore than 200 trajectories were recorded.
0LWRFKRQGULDO $FWLYLW\Mitochondrial activity of sperm was measured using MitoPT® JC-1 Assay Kit (911, Immuno
Chemistry Technologies, LLC.) according to Zhu et.al. 2019. Briefly, sperm samples were53 | P a g e

incubated with 200 μL of 1x working solution at 37ºC for 30 min in dark. The mitochondrial
activity was analyzed by flow cytometry using a filter with a bandwidth of 574/26 nm (Attune®
NxT Acoustic Focusing Cytometer, Invitrogen) and measured as mean fluorescence intensity(MFI) of JC-1 orange aggregates. A total of 20,000 sperm events were analyzed.
0HPEUDQHLQWHJULW\The sperm membrane integrity was measured using a LIVE/DEAD Sperm Viability Kit (L7011;
Thermo Fisher Scientific) according to manufacturer protocol. Briefly, Sperm were stained withSYBR-14/PI and incubated with 200 μL 1x working solution at 37ºC for 10 min in dark. The
staining was analyzed by flow cytometer (Attune® NxT Acoustic Focusing Cytometer,
Invitrogen) with Excitation/ Emission= 515/545 nm for SYBR-14 fluorescence, Excitation/
Emission= 675/715 nm for PI fluorescence. A total of 20,000 sperm events were analyzed. There
were four quadrants on the specific events gated area. Q1 (-/+) indicating the percentage of SYBR14 containing sperm cells, Q2 (+/+) indicating the percentage of both SYBR-14 and PI containing
sperm cells, Q3 (+/-) indicating the percentage of PI containing sperm cells and Q4 (-/-) indicating
the percentage of sperm cells neither containing PI nor SYBR-14.
$QQH[LQYVWDLQLQJThe sperm membrane apoptosis was measured using an Annexin v conjugate: Alexa Fluor®647
Kit (A23204; Invitrogen, Molecular probes) according to manufacturer protocol. Briefly, Spermwere stained with Alexa Fluor®647 /PI and incubated with 200 μL 1x working solution at 37ºC
for 30 min in dark. The staining was analyzed by flow cytometer (Attune® NxT Acoustic Focusing
Cytometer, Invitrogen) with Excitation/ Emission= 663/677 nm for Alexa Fluor®647
fluorescence, Excitation/ Emission= 561/587 nm for PI fluorescence. A total of 20,000 sperm
events were analyzed. There were four quadrants on the specific events gated area. Q1 (-/+)
indicating the percentage of necrotic sperm cells, Q2 (+/+) indicating the percentage of earlynecrotic sperm cells, Q3 (+/-) indicating the percentage of apoptotic sperm cells and Q4 (-/-)
indicating the percentage of live sperm cells.
2[\JHQ&RQVXPSWLRQ$VVD\Cellular oxygen consumption rate (OCR) was monitored in real time using a Seahorse Bioscience
Extracellular Flux Analyzer (XF HS Mini, Agilent, Santa Clara, CA). For flux analyzer, NaHCO 354 | P a g e

Free HTF medium was made according to previous study (Balbach et al., 2020). Frozen bovine
sperm were thawed with 0.1% cholesterol as above. After washing by NaHCO 3 -Free HTF
medium, cell numbers were quantified and diluted to 3,000,000 sperms/180 μL of NaHCO 3 -FreeHTF medium. Assay plates were coated with concanavalin A (0.5 mg/mL, Fujifilm Wako
Chemicals, Osaka, Japan) overnight the day before the assay. Each well was then seeded with 180
μL of NaHCO 3 -Free HTF medium containing 3,000,000 sperm. The assay was performed in 6 mincycles of mix (3 min) and measure (3 min) as per manufacturer’s recommendations. Two inhibitors
were used separately in this study namely oligomycin (04876, Sigma, USA) and FCCP (Carbonyl
cyanide-p-trifluoro methoxy phenylhydrazone) (C2920, Sigma, USA). Final concentration was 5
μM for these inhibitors. ATP production rate was calculated according to previous guideline(Romero et al., 2018).
)ORZF\WRPHWU\IRUTXDQWLI\LQJWKHLQWDNHRIFKROHVWHURO
To detect the fluorescence intensity in each sperm by flow cytometry, frozen-thawed bovine sperm
were incubated with fluorescence labeled cholesterol (TopFluor® Cholesterol 810255, Nanocs,
NY, USA) in a dose dependent manner (1.0%, 5.0% and 10.0%). After washing, the fluorescencelevel was analyzed by flow cytometry using a filter with a bandwidth of 530/30 nm (Attune® NxT
Acoustic Focusing Cytometer, Invitrogen) and measured as the intensity of the average value. A
total of 20,000 sperm events were analyzed. Digital images were captured using a Keyence BZ9000 microscope (Keyence Co., Osaka, Japan). Trypan blue (0.4%) (355-25, Nakarai Chemicals
Ltd. Japan) was used as quenching media to detect the actual region of cholesterol uptake.
6WDWLVWLFDO DQDO\VLV
Statistical analyses of data from three or four replicates for comparison were carried out by either
Student's t-test or one-way ANOVA followed by Student's t-test (Statview; Abacus Concepts, Inc.,
Berkeley, CA).
5HVXOWV6SHUPPRWLOLW\ SDUDPHWHUVZLWKFKROHVWHURO
Sperm motility parameters were calculated by CASA-derived motility tracks after 30-min
incubation. In the sperm treated with 0.1% cholesterol (CHL), BCF, VCL, VSL and TM weresignificantly (P < 0.05) higher than the control (F-) group and other concentrations of CHL treated
55 | P a g e

group (Figure 21). The values of VCL, VSL and TM were highest in 0.1% CHL treated group. In
CASA motility track, stretched zigzag motility pattern was observed in 0.1% CHL treated groupcompared to other concentrations of CHL treated group and control group (Figure 22). Scatter plot
was made considering the parameters of individual sperm data that were significantly increased
by 0.1% CHL treatment, BCF (Y Axis of graph) and VSL (X axis of graph), between BCF (YAxis of graph) and VCL (X axis of graph), and between VSL (Y Axis of graph) and VCL (X axisof graph) in 0.1% CHL (Figure 23 & 24). The lines were made in scatter plot considering the meanvalue of each motility parameter in control group of sperm. The percentage of the number of sperm
in the upper right quadrant of scatter plot was significantly (P<0.05) higher in 0.1% CHL treatmentgroup rather than control group (Figure 23,24).
&KROHVWHURODIIHFWVSHUPPHPEUDQHLQWHJULW\The sperm membrane integrity was analyzed by SYBR-14/PI staining. The intact membrane
integrity of the sperm incubated for 30 min at 37ºC thawed under the presence of 0.1 % (v/v)
cholesterol, stained by SYBR-14 showed significantly (P<0.05) higher percentages than withoutcholesterol (Figure 25-A). Damages of sperm membrane integrity stained by PI was significantly(P<0.05) lower in the cholesterol condition group than in the group without cholesterol condition
thawing (Figure 25-A).
Live, apoptotic, necrotic, and early necrotic cells were analyzed by Annexin V staining. The
number of live sperm cells was significantly (P<0.05) higher in the cholesterol condition groupthan without cholesterol condition thawing (Figure 25-B). The numbers of necrotic and earlynecrotic sperm cells were significantly (P<0.05) lower in the cholesterol group than withoutcholesterol condition thawing (Figure 25-B).
/RFDOL]DWLRQRIFKROHVWHURO VLJQDOVLQVSHUP
When frozen-thawed sperm were incubated with fluorescence labeled cholesterol, the fluorescencesignals were selectively observed in midpiece of sperm. The fluorescence intensity in each sperm
detected by FACS was significantly (P<0.05) increased in dose dependent manner (Figure 26-A,
B). Furthermore, incorporated cholesterol was localized in the midpiece and head of sperm (Figure26-C).

56 | P a g e&KROHVWHURODIIHFWVSHUPPLWRFKRQGULDO DFWLYLW\Mitochondrial membrane activity was analyzed by JC-1 staining. Mean fluorescence intensity(MFI) of the sperm incubated for 30 min at 37ºC thawed in the cholesterol condition significantly(P<0.05) differed from the sperm incubated for the same time and under the same conditions but
underwent thawing without cholesterol condition (Figure 27-E). Sperm showed a significantly
lower MFI in without cholesterol condition than with cholesterol condition.
To quantify the metabolic changes of mitochondrial activity, real-time changes of mitochondria
metabolic activity were monitored using a Seahorse Bioscience Extracellular Flux Analyzer (XF
HS Mini, Agilent, Santa Clara, CA). The significant increase of OCR was induced 0.1% CHL usedin thawing of sperm. Oligomycin, an inhibitor of oxidative phosphorylation significantly (P<0.05)
suppressed the CHL-induced OCR rate (Figure 27-A), whereas FCCP accelerated the OCR rate
(Figure 27-C & D). Most interestingly, the ATP production rate was significantly (P<0.05)
increased in CHL thawed group of sperm compared to the control (Figure 27-B).
'LVFXVVLRQSperm motility is thought to be a strong prognostic marker of male fertility potential along withall the semen parameters and it is controlled by its complex, structural, and molecular signalingmechanisms (Dcunha et al., 2020). In vivo fertilization of mammalian animals is required tomaintain linear motility of sperm during migration process through the uterus to oviduct for
penetrating the oocyte and ATP generation is indispensable for flagellar movement to preserve
this type of motility pattern (Shalgi et al., 1992, Suarez & Pacey, 2006). Most commonly it isbelieved that the sperm motility is controlled by ATP produced either from glycolytic pathway incytoplasm or OXPHOS in mitochondria or both (Storey, 2008; Mukai & Travis, 2012). It was
uncovered in boar sperm study that mitochondrial activity was increased under low glucose
condition, and the ATP produced in mitochondria was correlated with a high-speed linear motility(Zhu et al., 2019), which is described by high straight-line velocity (VSL) and low lateralamplitude (ALH) (Shalgi et al., 1992), suggesting that sperm linear motility is directly related withmitochondrial activity.

57 | P a g e

Kinematic parameters, such as straight-line velocity (VSL) and curvilinear velocity (VCL), haveprognostic value in predicting the fertilization potential of spermatozoa (Tan et al., 2014; Joshi etal., 1996). Sperm motility is supposed to have a strong correlation with assisted reproductive
technology (For example, AI and IVF) success and pregnancy outcome, and it was reported thatvalues for VAP, VSL, and VCL were significantly higher in samples that results more than 50%
fertilization, representing positive correlation between progressive motility and fertilization ability(Donnelly et al., 1998).
In this study with cholesterol, the progressive linear motility was improved in bovine frozen spermwith high straight-line velocity (Figure 21). The high curvilinear velocity (VCL) was also observedin cholesterol treatment compared to control (Figure 21); however, ALH was not increased,suggesting that hyperactivation was not induced by the treatment with cholesterol because both of
ALH and VCL are increased whereas VSL is decreased in hyper-activated sperm (Zhu et al., 2019).
Individual sperm effect of cholesterol on sperm motility parameters also revealed that cholesterol
affected significantly (P< 0.05) individual sperm VSL and VCL (Figure 23, 24), indicating thatlinear motility pattern was induced after the treatment of cholesterol. Therefore, it is suggestingthat cholesterol would be incorporated and utilized in the mitochondria of sperm for the productionof ATP.
It was described that cryo-damage occur at freeze thaw process can be reduced by supplying
cholesterol in freezing diluents (Wessel and Ball, 2004). Moreover, it was stated that membranecholesterol to phospholipid ratio (C:P) influences the sperm sensitivity to cold shock damage(Holt, 2000). Sperm possessing elevated C:P ratios for example, rabbit and human sperm; 0.88
and 0.99 respectively, are more resistant to the “cold shock” damage compared to sperm having
low C:P ratios for instances, boar, ram, and bull sperm; 0.35, 0.37 and 0.45 respectively (Davis,
1981, White, 1993). Prevention of the cryo-capacitation enhances the durability of the sperm cells
after thawing which can be achieved by adding cholesterol in sperm during freezing (Moce et al.,2010). Additionally, it was described that cholesterol loaded methyle β-cyclodextrin in extenderadvanced survivability of stallion (Combes et al., 2000; Graham, 1998) and bull spermatozoa(Amorim et al., 2009) after cryopreservation suggesting that cholesterol can be up taken by sperm
to recover membrane damage and motility.
Interestingly, in this cholesterol study it was observed that the sperm membrane integrity (Figure25) and mitochondrial activity (Figure-27) was significantly (P<0.05) increased in cholesterol
58 | P a g e

treated group compared to control. Additionally, it was discovered in this study that cholesterol
was localized in the mid piece and head region of sperm (Figure-6). As cholesterol was up takenby sperm in the midpiece region so new story raised from this study that up taken cholesterol may
be used as mitochondrial membrane structure recovery to maintain sperm linear motility pattern.
In fact, cholesterol showed good result in real ATP production as well as increasing the potentialof mitochondria ATP production (Figure 7), suggesting that cholesterol in thawing media of frozen
bovine sperm can improve the structure of mitochondria in sperm midpiece to induce and maintain
ATP production for linear motility.
In concluding remarks, it can be said that cholesterol is up taken to the sperm mitochondria where
they acted as components of not only cell membrane but also mitochondria membrane structure to
produce energy for the sperm to keep linear motility. Thus, to increase and keep sperm fertilization
ability, exogenous cholesterol is an advantageous factor to induce and keep sperm linear motility
which is very maiden in the discovery of sperm research. It may possible that thawing sperm withcholesterol containing insemination medium will boost sperm linear motility in the female genitaltracts to improve fertilization as a simple and low-cost approach.

59 | P a g e

$EVWUDFW
Mitochondria are an important source of energy required for sperm motility. Abnormalities of
mitochondrial membrane integrity are vitally related with abnormal sperm motility. For successful
fertilization sperm linear motility is very important and this is induced by mitochondrial ATP
production. Present study was undertaken to clarify whether bovine sperm could uptake cholesterol(CHL) in thawing media and recover the mitochondrial membrane structure which was distorted
in cryopreservation of sperm. Bovine frozen semen was thawed in CHL contained in thawing
media and the sperm motility was analyzed by commuter assisted sperm analysis (CASA). The
cholesterol uptake signal was observed by fluorescence intensity in the mid piece and head regionof sperm after the treatment with fluorescence-tagged CHL. The mitochondrial activity of sperm
treated with CHL was analyzed as fluorescence intensity of JC-1 staining and oxygen consumption
rate (OCR). OCR was higher in CHL treated group due to up taken CHL was used to renovate thealtered mitochondrial structure. Sperm linear motility was significantly (P<0.05) increased by thethawing with CHL. Sperm linear motility is prompted by CHL in thawing media of bovine frozen
sperm might provide a new vision for modernizing the artificial insemination technique of both
livestock animals and human infertility care.













60 | P a g e)LJXUHCASA motility parameters of sperm incubated with different doses of cholesterolfor 30 min
Values are specified (Mean±SD) with three replications. Different superscripts differsignificantly at P < 0.05.












61 | P a g e of cholesterol (CHL))LJXUH CASA motility track at different doses








62 | P a g e)LJXUH  Correlation between ALH and VCL (A.) and BCF and VSL(B.) of each sperm
treated with Cholesterol (CHL) in thawing media compared with control. The lines in scatterplot were made considering the mean value of each motility parameter in control group of
sperm.
In each correlation figure the second quadrant (ii) region, indicating the lower value of bothparameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
different in CHL treated group compared to control.

63 | P a g e)LJXUH  Correlation between BCF and VCL (A.) and VCL and VSL(B.) of each sperm
treated with Cholesterol (CHL) in thawing media compared with control. The lines in scatterplot were made considering the mean value of each motility parameter in control group of
sperm.
In each correlation figure the second quadrant (ii) region, indicating the lower value of bothparameters compared to mean value of control and third quadrant (iii) region, indicating thehigher value of both parameters compared to mean value of control are significantly (P<0.05)
different in CHL treated group compared to control.

64 | P a g e)LJXUHCholesterol improves sperm survivability. 
Values are specified (Mean±SD) with three replications. Different superscripts with various
parameters differ significantly at P < 0.05 in One way ANOVA with t-test.
(A) Membrane integrity (B) Annexin v Staining
Here, CHL = Cholesterol (0.1%) and control = Without cholesterol.

65 | P a g e)LJXUHFluorescence intensity of sperm and localization of cholesterol uptakeValues are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
A. Overlay of fluorescence intensity at different doses of fluorescence tagged cholesterol
B. Mean fluorescence intensity at different doses of fluorescence tagged cholesterol
C. Localization of cholesterol uptake signal in sperm

66 | P a g e)LJXUHEffect of Cholesterol on oxygen consumption rate (OCR) of sperm. Values
are specified (Mean±SD) with three replications. Different superscripts differ
significantly at P < 0.05.
A indicating the effect of oligomycin injection
B indicating the ATP production rate of sperm
C &D indicating the effect of FCCP injection
E indicating the mitochondrial activity of sperm

67 | P a g e

&KDSWHU






*HQHUDO'LVFXVVLRQ
3DJH1R 
Frozen sperm are being used highly in Artificial Insemination (AI), vital reproductive technologythat has been utilized to advance livestock farming and have an important, positive impact on cattle
production especially, and product quality allowing for accelerated genetic progress and selection(Medeiros et al., 2002). Sperm cryopreservation procedures are not always effective because ahuge number of sperm undergo damage which leads to the loss of fertility following freezing andthawing processes (Bailey et al., 2000). The main cause of cellular injury in cryopreservation isthe damage endured by the plasma membrane due to cold shock that is associated with the lipid
composition of the membrane bilayer (Watson, 1995). Mammalian sperm maintain linear motility
during fertilization, to migrate through the uterus and reach the oviduct for penetrating the oocyte(Shalgi et al., 1992). During the journey ATP production is crucial for flagellar movement (Suarez& Pacey, 2006). In this critical situation of cryopreserved sperm to recover membrane integrity
and supply adequate energy production for migration from insemination site to fertilization site
aiming successful fertilization, thawing of sperm is utmost important with an optimum media from
where sperm can get both energy substrates and membrane components is really a challenge.
In this study underlying mechanism of LM on keeping linear motility and sperm quality of bovine
frozen sperm (Experiment 1) have become clear. The molecular mechanism of fatty acids to
produce energy through mitochondrial β oxidation and localization of fatty acid transporter to themitochondria of sperm (Experiment 2) is the maiden report. Although there are some studies
regarding on effect of fatty acids on sperm motility and quality parameters, but nobody described
the process of fatty acid transport to the sperm mitochondria and energy production mechanism.
Moreover, the actual cholesterol uptake and localization on sperm mid piece and head region isentirely a new story regarding sperm membrane potential and mitochondrial membranepotentiality for ATP production (Experiment 3).
It was reported that the cold shock during freeze-thaw process injures mitochondria (Pena et al.,2009), plasma and acrosome membranes (Meyers, 2005) of spermatozoa which may lead todecreasing the spermatozoa functional integrity and declining its fertilizing ability (Gillan &Maxwell, 1999). Moreover, it was observed that after freezing of the spermatozoa acrosome
reaction was reduced, distorted mitochondria increased, chromatin damage and DNA breaks wasenhanced (Khalil et al., 2017). In this study, it was observed that LM not only improved the
motility parameters of sperm but also the membrane potentials of bovine frozen sperm. Therefore,
68 | P a g e

this study might be effectively applied in improving the success rate of AI in livestock industry by
diluting frozen sperm in LM containing thawing media as low- cost approach.
Research on bovine species (Neill & Masters,1972) and ovine species (Neill & Masters,1973)
reported that sperm cells can take up lipid components or fatty acids from the surrounding
environment during incubation (Cerolini et al., 2001). It has been described that fatty acids wereincorporated into the lipids of sperm cell membranes within two hours of addition to cell cultures(Neill & Masters,1971). Present study revealed the molecular mechanism of exogenous fatty aciduptake and effect on the sperm quality in cryopreserved semen technology (Experiment 2).
LM is composed of both saturated and unsaturated fatty acids with cholesterol. The effect of fatty
acids already discussed so far. In this present study it was revealed that cholesterol in thawing
media of bovine frozen sperm was beneficial for improving the linear motility pattern andmembrane potentials of sperm mitochondria. Amusingly, the cholesterol was up taken with thetreatment of fluorescence tagged cholesterol and located in the mid piece and head region of sperm(Experiment 3). Additionally, these up taken cholesterol was used to recover the mitochondrial
modified structure due to cryopreservation of sperm and thereby ATP production rate wassignificantly increased in cholesterol treated group compared to control (Experiment 3).
Therefore, this study is beneficial to frozen sperm technique to recover sperm membrane integrity
and mitochondrial potentiality to maintain linear motility of cryopreserved sperm.
Artificial insemination (AI) is the most regularly used biotechnology in livestock industry invarious developed and developing countries. In AI industry, the main constraint to the genetic
impact of sires is decreased sperm motility and functions during cryopreservation (Woelders et al.,
1997; Celeghini et al., 2008). It was reported that 20-30 million sperm per straw have nowadays
been used as a standard for dairy industry and 10-15 million progressive motile sperm per dosewere necessary for achieving desirable conception rate in French midi straws of cattle (Sullivan,
1970; Allen and Seidel,1996). The artificial insemination technique is commercially applied
worldwide to breed cattle, however, the technique is still not efficient, as the large sperm numbers
per cow in estrus is required for getting high reproductive performance. The substantial sperm
numbers are a vital limitation for artificial insemination application not only in cattle but also inhuman infertility treatment. Because in human artificial insemination, 1 ×107 or more motile sperm
are required (Van Voorhis et al., 2001), in vitro fertilization or intracytoplasmic sperm injection(ICSI) are usually selected in the case of oligospermia. From the above-mentioned references, it is
69 | P a g every clear that in any type of reproductive technology sperm quality with good motility is theprerequisite to get optimum success rate. The novel findings of the present study are directly
related to sperm quality specifically linear motility which is very crucial for desired fertilization
to get optimum conception rate. In another view, it can be said that the new information of thisstudy can minimize the sperm content of frozen straws as LM treatment in thawing media canrecover the cryodamage after thawing and washing. When sperm content can be reduced in single
straw then the number of straws can be increased from single collection and ultimately it will bebeneficial to make profitable farming. In a single sentence, the inventory of the current study mightprovide a new strategy for improving the artificial insemination technique of both livestock
animals and human infertility care.
The human connection to livestock is also absolutely intimate. Domestic animals are crucial to thesurvival of populations living in variable environments (FAO, 2009), and were historically central
to the formation of societies and cultures (Smil, 2009). According to the most recent (around 2000)
World Census of Agriculture, of the approximately 308 million farmers asked in 83 countries
across the world, 23.4% reported that they own at least one cow or head of cattle (FAO, 2013).
While the number of livestock owned typically increases with farm size, livestock are also
important for smaller farms with lower income levels (FAO, 2009). AI using cryopreserved sperm
improves desired characteristics more rapidly through intensive genetic selection and has
significant advantages, such as enabling the use of superior quality sires even the animal is deador injured, and greatly reducing the risk of introducing venereal diseases into the herd (Fontes etal., 2020; Vishwanath, 2003). It is a tool for improving reproductive performance and genetic
quality of livestock.
It was investigated that the livestock sector affects at least 58% (10 out of the 17) of the SDGs
(Sustainable Development Goals) and 16% (28 out of the 169) of the SDG targets (Mehrabi et al.,2020). Globally, livestock products contribute 34 percent of protein and 17 percent of calorie
intake of diets and animal-source foods are energy and protein-dense and sources of many otheressential nutrients, (FAO/ WHO,2019). Animal food products provide livelihoods for millions ofpeople (Pica-Ciamarra et al., 2011), deliver essential protein and micronutrients for the world’s
poor (Frelat et al., 2016; Cordain et al., 2002) and provide a vital mechanism for reducing
inhibiting and wasting globally (Grace et al., 2018).
There is an emergency to improve the efficiency and sustainability of producing animals for food70 | P a g e

to face the demand of ever-increasing world population. Increasing the fertility of livestock,
especially cattle, around the world is important for overcoming this problem. Improved
understanding of mechanisms and challenges of reproductive technologies are vital for improving
the viability of the livestock industry. Among such reproductive technologies, artificial
insemination (AI) is a significant technology that has been utilized to advance livestock farming,allowing for promoting genetic progress and selection (Medeiros et al., 2002) where successful
semen cryopreservation improves the efficiency and success rate of AI. It was reported thatconception rate after artificial insemination of cattle were in the range of 60–73% with an average
71% (Ricardo et al., 2004). Sperm cryopreservation procedures are not always efficient because alarge number of sperm suffer physiological damage which leads to the loss of fertility following
freezing and thawing (Bailey et al., 2000). Techniques and novel findings of the present study can
improve the cryodamage of sperm during thawing and ultimately the success rate of AI.
Considering above all discussion it is very clear that LM in thawing media is very helpful torecover the sperm linear motility pattern with other parameters. Furthermore, LM is beneficial torescue the sperm damage after thawing and washing of bovine frozen sperm. However, an
increased number of sperm linear motility for IVF or AI is the aim of animal reproduction. In thisstudy, LM in thawing media of bovine frozen sperm did neither any abnormalities in sperm rather
improved their quality with increased linear motility pattern. The findings of this study will be
useful for in vitro fertilization and AI decision in both human and animal fertility improvement.
Additionally, exogenous LM treatment in thawing of frozen sperm could be successfully appliedin livestock production program, which will improve the non -return rate and pregnancy with anaugment in ensuring profitable farming.
In conclusion, this study unveiled the mechanism of LM to improve the linear motility pattern andmembrane integrity of bovine frozen sperm. Two parts in LM fatty acids and cholesterol contributeseparately to rescue the quality of bovine frozen sperm after thawing in a LM containing media.
Interestingly, this study uncovers the energy metabolism of bovine frozen sperm thawed in LM or
fatty acid containing thawing media with the molecular strategy of fatty acid uptake to the sperm
mitochondria from the membrane. Moreover, cholesterol helps to recover the membrane integrity
and mitochondrial membrane potentials declined during cryopreservation process and contributeto regain the linear motility pattern through accelerate ATP production rate. This study invented
new information for reproductive science to maintain linear motility of sperm after thawing and
71 | P a g ewashing which is prerequisite for in vivo fertilization. Therefore, the new story developed fromthe present study is strongly believed to be fruitful to the future research in AI technology forlivestock and human infertility care as inexpensive technique. New approach of thawing frozen
sperm with LM containing media from the present study will increase the conception rate
definitely and ultimately the production of livestock will increase which may be beneficial to
overcome poverty, increase income and attain the SDGs of the United Nations (UN).













72 | P a g e











&KDSWHU







6XPPDU\
3DJH1R Mammalian sperm migrate through uterus to oviduct by maintaining linear motility pattern duringin vivo fertilization process. For the migration process sperm require adequate energy to reach theoocyte for fertilization. Sperm gets maximum energy from mitochondria ATP production. The
quality of sperm deteriorates in the process of cryopreservation. Sperm membrane integrity,
acrosomal integrity and mitochondrial abnormality are the main physical negative effect of sperm
cryopreservation. Therefore, the present study was aimed to investigate the recovery of bovine
frozen sperm quality after thawing and washing. To achieve the aim, firstly effect of LM in thawing
media of bovine frozen sperm were studied. After that, molecular mechanism of mitochondrial
ATP production by using fatty acids were investigated. Finally, the sperm membrane potentiality
with mitochondrial membrane potentials of sperm with cholesterol were studied.
 /0 /0  LQ WKDZLQJ PHGLD LV EHQHILFLDO WR NHHS VSHUP PRWLOLW\ ZLWK RWKHU TXDOLW\SDUDPHWHUVLQKHDOWK\VWDWXVRIERYLQHIUR]HQVSHUP
Cryodamage affect sperm motility and membrane structure highly. In Experiment 1, it was
hypothesized that LM in thawing media maybe beneficial for bovine frozen sperm to keep good
motility with sound membrane structure by maintaining their membrane lipid composition healthy
and there may be a correlation between lipid components and sperm motility. To clear the
hypothesis CASA, GC-MS and FCM with different stains were performed using bovine frozen
sperm. It was observed that fatty acids from LM was up taken by LM thawed sperm compared tocontrol and there was a positive correlation between fatty acids amount and total sperm motility.
It was also observed that LM thawed bovine frozen sperm showed significantly good motility
parameters compared to control group. Moreover, it was also uncovered that membrane integrity,
mitochondrial activity and live sperm percentage was significantly higher in LM thawed spermcompared to without LM thawed sperm used as control. Although, it is known that lipid
components can improve sperm motility, but this is the maiden report considering the variability
in the amount of lipid components in sperm membrane of bovine frozen sperm and their correlation
to sperm motility according to author best of knowledge.
73 | P a g e6SHUPOLQHDU PRWLOLW\ LVLQGXFHGE\VDWXUDWHGIDWW\DFLGEHLQJXVHGDVHQHUJ\VXEVWUDWHLQ
WKHPLWRFKRQGULD RIERYLQHIUR]HQVSHUP
Successful fertilization depends on sperm linear motility and motility patterns are dependent onenergy substrate and their effective utilization in energy producing segments of sperm. In
Experiment 2, it was hypothesized that bovine sperm could uptake fatty acids (FAs) in thawing
media and produce ATP in mitochondria for maintaining linear motility. To clear this hypothesis;
bovine frozen sperm was thawed in FAs containing thawing media and the sperm motility was
analyzed by commuter assisted sperm analysis (CASA). The kinetic changes of FAs level in sperm
were detected by GC-MS. The mitochondrial activity of sperm treated with FAs was analyzed as
fluorescence intensity of JC-1 staining and oxygen consumption rate (OCR). FA transporter (CD36
and GOT2) was observed by whole-mounted immunofluorescence. It was observed that sperm
linear motility was significantly increased by the thawing with FA. Interestingly, it was revealed
that long chain saturated fatty acids (LCSFA) (C14, C16, C18) were decreased during 30 min
incubation, except very long chain saturated fatty acid (C22). Moreover, another crucial factor wasobserved that the bovine frozen sperm possessed FA transporter in midpiece where thefluorescence signals were detected after the treatment with fluorescence-tagged FA. Furthermore,
it was discovered that the treatment with FA activated electron transportation in mitochondria
through β-oxidation. These findings highlight the molecular mechanism of fatty acid uptake from
sperm membrane to mitochondria and ATP production through β-oxidation process.
 &KROHVWHURO LQ WKDZLQJ PHGLD UHJDLQ PLWRFKRQGULDO SRWHQWLDO ZKLFK DFFHOHUDWHV $73SURGXFWLRQLQPLWRFKRQGULDWKDWLQGXFHVOLQHDUPRWLOLW\ LQERYLQHIUR]HQVSHUPFor successful fertilization sperm linear motility is very important and this is induced by
mitochondrial ATP production. In Experiment 3 it was supposed that cholesterol may be up takenby sperm and used to remake the mitochondrial membrane potential along with sperm membranepotential which was damaged during cryopreservation process. To clarify the hypothesis bovine
frozen sperm was thawed in CHL contained thawing media and the sperm motility was analyzed
by commuter assisted sperm analysis (CASA). The cholesterol uptake signal was observed byfluorescence intensity in the mid piece and head region of sperm after the treatment withfluorescence-tagged CHL. The mitochondrial activity of sperm treated with CHL was analyzed as
fluorescence intensity of JC-1 staining and oxygen consumption rate (OCR). OCR was higher in
74 | P a g e

CHL treated group due to up taken CHL was used to renovate the altered mitochondrial structure.
ATP production rate was also significantly higher in CHL thawed group compared to control.
Sperm linear motility was significantly (P<0.05) increased by the thawing with CHL. Sperm linear
motility is prompted by CHL in thawing media of bovine frozen sperm might provide a new vision
for modernizing the artificial insemination technique of both livestock animals and humaninfertility care.
In conclusion, this study unveiled the mechanism of LM to improve the linear motility pattern andmembrane integrity of bovine frozen sperm. Two parts in LM fatty acids and cholesterol contributeseparately to rescue the quality of bovine frozen sperm after thawing in a LM containing media.
Interestingly, this study uncovers the energy metabolism of bovine frozen sperm thawed in LM or
fatty acid containing thawing media with the molecular strategy of fatty acid uptake to the sperm
mitochondria from the membrane. Moreover, cholesterol helps to recover the membrane integrity
and mitochondrial membrane potentials declined during cryopreservation process and contributeto regain the linear motility pattern through accelerate ATP production rate. This study invented
new information for reproductive science to maintain linear motility of sperm after thawing andwashing which is prerequisite for fertilization. Therefore, the new story developed from the present
study is strongly believed to be fruitful to the future research in AI technology for livestock andhuman infertility care as inexpensive technique. New approach of thawing frozen sperm from thepresent study will increase the conception rate definitely and ultimately the production of livestock
will increase which may be beneficial to overcome poverty, increase income and attain the SDGsof the United Nations (UN).



75 | P a g e









5HIHUHQFHV
3DJH1R 













Abavisani, A., Arshami, J., Naserian, A. A., Sheikholeslami, K. M. A., and Azizzadeh, M. (2013).
Quality of bovine chilled or frozen-thawed semen after addition of omega-3 fatty acids
supplementation to extender. International Journal of Fertility and Sterility, 7(3): 161-168.
Ahmad, M., Nasrullah, R., Riaz, H., Sattar, A., Ahmad, N. (2014). Changes in motility,
morphology, plasma membrane and acrosome integrity during stages of cryopreservationof buck sperm. Journal of the South African Veterinary Association, 85:1–5.
Aitken, R. J. (1995). Free radicals, lipid peroxidation and sperm function. Reproduction, Fertility
and Development, 7(4): 659-668.
Albarracın, J. L., Mogas, T., Palomo, M. J., Pena, A., Rigau, T., and Rodrıguez-Gil, J. E., (2004).
“In vitro capacitation and acrosome reaction of dog spermatozoa can be feasibly attainedin a defined medium without glucose.” Reproduction in Domestic Animals, 39(3):129–135.
Allen, C. H., Seidel, G. E. (1996). Atlantic’s experience with non-frozen sperm cells. Proceedingsof the National Association of Animal Breeders. Wisconsin, USA, 55-56.
Alvarez, J. G., Touchstone, J. C., Blasco, L. and Story, B. T. (1987). Spontaneous lipid
peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa.
Journal of Andrology, 8: 338–348.
Alvarez, J. G., and Storey, B. T. (1992). Evidence for increased lipid per oxidative damage andloss of superoxide dismutase activity as a mode of sublethal cryodamage to human sperm
during cryopreservation. Journal of Andrology, 13(3): 232–241.
Amann, R. P., Pickett, B.W. (1987). Principles of cryopreservation and a review ofcryopreservation of stallion spermatozoa. Journal of EquineVeterinary Science, 7:145-173.
Amaral, A., Lourenco, B., Marques, M., Ramalho-Santos, J. (2013). Mitochondria functionality
and sperm quality. Reproduction, 146 (5): R163–R174.
Amorim, E. A. M., Graham, J. K., Spizziri, B., Meyers M., and Torres, C. A. A. (2009). Effect ofcholesterol or cholesteryl conjugates on the cryosurvival of bull sperm. Cryobiology, 58:
210-214.
Aon, M.A., Bhatt, N., Cortassa, S.C. (2014). Mitochondrial and cellular mechanisms for managing
lipid excess. Frontiers in Physiology, 5(282).
Bailey, J. L., Bilodeau, J. F., Cormier, N. (2000). Semen cryopreservation in domestic animals: A
damaging and capacitating phenomenon. Journal of Andrology, 21: 1–7.

76 | P a g e

Bailey, J. L., Lessard, C., Jacques, J., Breque, C., Dobrinski, I., Zeng, W., and Galantino-Homer
H. L. (2008). Cryopreservation of boar semen and its future importance to the industry.
Theriogenology,70: 1251-1259. Oberoi B., Kumar S., & Talwar P. Study of human sperm
motility post cryopreservation. Med. J. Armed Forces India. 2014;70(4):349-53.
Balbach, M., Gervasi, M. G., Hidalgo, D. M., Visconti, P. E., Levin, L. R., and Buck, J. (2020).
Metabolic changes in mouse sperm during capacitation. Biology of Reproduction, 1–11.
Barth, A. D. and Oko, R. J. (1989). Normal bovine spermatogenesis and sperm maturation. In:
Barth, A. D., Oko, R. J. (Eds.). Abnormal morphology of bovine spermatozoa. Ames: Iowa
State University Press, 19-88.
Barbonetti, A., Vassallo, M. R., Fortunato, D., Francavilla, S., Maccarrone, M., and Francavilla,
F. (2010). Energetic metabolism and human sperm motility: impact of CB (1) receptor
activation. Endocrinology, 151: 5882–5892.
Barthelemy, C., Royere, D., Hammahah, S., Lebos, C., Tharanne, M.-J., and Lansac, J. (1990).
Ultrastructural changes in membranes and acrosome of human sperm during
cryopreservation. Systems Biology in Reproductive Medicine, 25: 29–40.
Belleannee, C., Calvo, E., Caballero, J., Sullivan, R. (2013). Epididymosomes convey different
repertoires of microRNAs throughout the bovine epididymis. Biology of Reproduction,
89:30.
Brackett, B. G., Oh, Y. K., Evans, J. F., Donawick, W. J. (1978). In vitro fertilization of cow ova.
Theriogenology, 9:89.
Bratic, I., Trifunovic, A. (2010). Mitochondrial energy metabolism and ageing, Biochimica etBiophysica Acta, 1797 (6–7) 961–967.
Breton, S., Ruan, Y. C., Park, Y. J, and Kim, B. (2016). Regulation of epithelial function,differentiation, and remodeling in the epididymis. Asian Journal of Andrology, 18:3–9.
Brokaw, C. J. (1972). Flagellar movement: a sliding filament model. Science,178: 455–462.
Braun, J., Sakai, M., Hochi, S., and Oguri, N., (1994). Preservation of ejaculated and epididymal
stallion spermatozoa by cooling and freezing. Theriogenology, 41: 809–818.
Caballero, J., Frenette, G., and Sullivan, R., (2011). Post Testicular Sperm Maturational Changes
in the Bull: Important Role of the Epididymosomes and Prostasomes. Veterinary Medicine
International, 2011: 1-13.

77 | P a g e

Calvete, J. J., Mann, K., Sanz, L., Raida, M., and Topfer- Petersen, E. (1996). “The primarystructure of BSP-30K, a major lipid-, gelatin-, and heparin-binding glycoprotein of bovine
seminal plasma,” FEBS Letters, 399(1-2): 147–152.
Celeghini, E. C. C., Arruda, R. P., Andrade, A. F. C., Nascimento, J., Raphael, C. F., Rodrigues,
P. H. M. (2008). Effects that bovine sperm cryopreservation using two different extenders
has on sperm membranes and chromatin. Animal Reproduction Science, 104: 119-131.
Cerolini, S., Maldjian, A., Pizzi, F., and Gliozzi, T. M. (2001). Changes in sperm quality and lipid
composition during cryopreservation of boar semen. Reproduction, 121(3):395–401.
Chakrabarty, J., Banerjee, D., Pal, D., De, J., Ghosh A. and Majumder G. C. (2007). Shedding off
specific lipid constituents from sperm cell membrane during cryopreservation.
Cryobiology, 54(1): 27-35.
Chauhan, M.S., Kapila, R., Gandhi, K. K., Anand, S. R. (1994). Acrosome damage and enzyme
leakage of goat spermatozoa during dilution, cooling and freezing. Andrologia, 26: 21–26.
Cincik, M., Ergur, A. R., Tutuncu, L., Muhcu, M., Kilic, M., Balaban, B. & Urman, B. (2007).
Combination of hypoosmotic swelling/eosin Y test for sperm membrane integrity
evaluation: correlations with other sperm parameters to predict ICSI cycles. Archives ofAndrology,53: 25–28.
Clermont, Y. (1966). Renewal of spermatogonia in man. American Journal of Anatomy, 118:509529.
Combes, G. B., Varner, D. D., Schroeder, F., Burghardt, R. C. and Blanchard, T. L. (2000). Effectof cholesterol on the motility and plasma membrane integrity of frozen equine sperm afterthawing. Journal of Reproduction and Fertility, 56: 127-132.
Cooper T. G. (1998). “Interactions between epididymal secretions and spermatozoa,” Journal of
Reproduction and Fertility, 53: 119–136.
Cooper, T. G. and Yeung, C. H., (2006). “Sperm maturation in the human epididymis,” in The
Sperm Cell. Production, Maturation, Fertilization, Regeneration. Cambridge University
Press, Cambridge, UK, 72–107.
Cordain, L., Eaton, S., Miller, J. B., Mann, N. & Hill, K. (2002). The paradoxical nature of huntergatherer diets: meat-based, yet non-atherogenic. European Journal of Clinical Nutrition,
56, 42–52.

78 | P a g e

Cross N. L. (2004). “Reorganization of lipid rafts during capacitation of human sperm.” Biology
of Reproduction, 71(4): 1367–1373.
Cuasnicu, P. D., Ellerman, D., Busso, D., DaRos, V., and Morgenfeld, M. (2002). “Changes insperm proteins during epididymal maturation,” in The Epididymis. From Molecules to
Clinical Practice, Plenum Press, New York, USA, 389–404.
Cunha, A. T. M., Carvalho , J. O., Kussano , N. R., Martins , C. F., Mourao , G. B., Dode M. A.
N. (2016). Bovine epididymal spermatozoa: Resistance to cryopreservation and binding
ability to oviductal cells. Cryobiology, 73, 348–355
Curtis, M. P., Kirkman-Brown J. C., Connolly, T. J., and Gaffney, E. A. (2012). Modelling atethered mammalian sperm cell undergoing hyperactivation. Journal of Theoretical
Biology,309: 1–10.
Dacheux, J. L. and Dacheux, F. (2002). “Protein secretion in the epididymis,” in The Epididymis.
From Molecules to Clinical Practice. Kluwer Academic/Plenum Publishers, New York,USA, 151–168.
Dacheux, J. L., Belghazi, M. Y., Lanson, and Dacheux, F. (2006). “Human epididymal secretome
and proteome,” Molecular and Cellular Endocrinology, 250(1-2): 36–42.
Darin-Bennett, A., White, I. G. (1977). Influence of the cholesterol content of mammalianspermatozoa on susceptibility to cold shock. Cryobiology, 14: 466–470.
Davis, B. K. (1981). Timing of fertilization in mammals: Sperm cholesterol/phospholipid ratio asa determinant of the capacitation interval. Cell Biology, 78: 7560-7564.
Dcunha, R., Hussein, R. S., Ananda, H., Kumari, S., Adiga, S. K., Kannan, N., Zhao, Y., Kalthur,G. (2020). Current Insights and Latest Updates in Sperm Motility and AssociatedApplications in Assisted Reproduction. Reproductive Sciences, 1-19.
De Kretser, D. M. and Kerr, J. B. (1994). The cytology of the testis. In Knobil, E. and Neill, J. D.
(eds), Physiology & Reproduction. Raven Press, New York, 1177-1290.
De Lamirande, E., Leclerc, P., and Gagnon, C. (1997). Capacitation as a regulatory event thatprimes spermatozoa for the acrosome reaction and fertilization. Molecular Human
Reproduction, 3: 175–194.
Demott, R. P., and Suarez, S. S. (1992). Hyperactivated sperm progress in the mouse oviduct.
Biology of Reproduction, 46: 779–785.

79 | P a g e

Depuydt C. E., Zalata, A. and de Potter C. R. (1996). The receptor encoded by the human C-met
oncogene is expressed in testicular tissue and on human spermatozoa. Molecular Human
Reproduction, 2 :2–8.
Desnoyers, L. and Manjunath, P. (1992). “Major proteins of bovine seminal plasma exhibit novel
interactions with phospholipid.” Journal of Biological Chemistry,267(14): 10149–10155.
Donnelly, E. T., Lewis, S. E., McNally, J. A., Thompson, W. (1998). In vitro fertilization andpregnancy rates: the influence of sperm motility and morphology on IVF outcome. Fertility
and Sterility, 70(2):305–14.
Drobnis, E. Z., Crowe, L.M., Berger, T., Anchordoguy, T.J., Overstreet J.W., and Crowe J. H.
(1993). Cold shock damage is due to lipid phase transitions in cell membranes: ademonstration using sperm as a model. Journal of Experimental Zoology, 265: 432-437.
Dravland, E., and Meizel, S. (1981). “Stimulation of hamster sperm capacitation and acrosome
reaction in vitro by glucose and lactate and inhibition by the glycolytic inhibitor ߙchlorohydrin.” Gamete Research, 4(6): 515–523.
Du Plessis, S. S., Agarwal, A., Mohanty, G., van der Linde, M., (2015). Oxidative phosphorylation
versus glycolysis: what fuel do spermatozoa use. Asian Journal of Andrology, 17 (2) ,230–
235.
Eddy, E. M., O Brien, D. A., (1994). in: E. Knobil, J.D Neill (Eds.), The Physiology of
Reproduction, Raven Press, New York, pp. 29-77.
Ehehalt, R., Füllekrug, J., Pohl, J., Ring, A., Herrmann, T., Stremmel, W. (2006). Translocationof long chain fatty acids across the plasma membrane lipid rafts and fatty acid transport
proteins. Molecular and Cellular Biochemistry, 284: 135–140.
Eickhoff, R., Wilhelm, B., Renneberg, H., Wennemuth, G., Bacher, M., Linder, D., Bucala, R.,
Seitz, J., Meinhardt, A. (2001). Purification and characterization of macrophage migration
inhibitory factor as a secretory protein from rat epididymis: evidence for alternative releaseand transfer to spermatozoa. Molecular Medicine.7:27–35.
Esch, F. S., Ling, N. C., and Bohlen, P. (1983). “Primary structure of PDC-109, a major protein
constituent of bovine seminal plasma.” Biochemical and Biophysical ResearchCommunications, 113(3): 861–867.
FAO, (2009). The State of Food and Agriculture: Livestock in Balance.
FAO, (2013). 2000 World Census of Agriculture.
80 | P a g e

FAO/WHO. (2019). Guiding Principles Sustainable Healthy Diet.
Farkas, R. (2015). Apocrine secretion: New insights into an old phenomenon. Biochimica etBiophysica Acta.1850(9):1740-1750.
Fernandez-Santos, M.R., Esteso, M. C., Montoro, V., Soler, A. J., Garde, J. J. (2006).
Cryopreservation of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa:
Effects of egg yolk, glycerol, and cooling rate. Theriogenology, 66:1931–1942.
Ferreira, G., Costa, C., Bassaizteguy, V., Santos, M., Cardozo, R., Montes, J., Settineri, R.,
Nicolson, G. L.

(2018). Incubation of human sperm with micelles made fromglycerophosphoLMs increases sperm motility and resistance to oxidative stress. PLoS
ONE, 13(6): e0197897.
Flesch, F. M., Gadella, B. M. (2000). Dynamics of the mammalian sperm plasma membrane in theprocess of fertilization. Biochimica et Biophysica Acta, 1469:197-235.
Fontes, P. L. P., Oosthuizen, N., & Cliff Lamb, G. (2020). Reproductive management of beef
cattle Animal Agriculture, 57–73.
Ford, W. C. (2006). Glycolysis and sperm motility: does a spoonful of sugar help the flagellum goround? Human Reproduction Update; 12: 269–74.
Fouchecourt, S., Metayer, S., Locatelli, A., Dacheux, F., and Dacheux, J. L. (2000). “Stallion
epididymal fluid proteome: qualitative and quantitative characterization; secretion anddynamic changes of major proteins.” Biology of Reproduction, 62(6): 1790–1803.
Fraser L. (1977). Motility patterns in mouse spermatozoa before and after capacitation. Journal of
Experimental Zoology, 202:439–444.
Fraser, L. R., Harrison, R. A., and Herod, J. E. (1990). Characterization of a decapacitation factor
associated with epididymal mouse spermatozoa. Journal of Reproduction and Fertility, 89:
135–148.
Frelat, R., Lopez-ridaura, S., Giller, K. E., Herrero, M. & Douxchamps, S. (2016). Drivers ofhousehold food availability in sub-Saharan Africa based on big data from small farms.
Proceedings of the National Academy of Sciences, USA ,113: 458–463.
Frenette, G., Lessard, C., and Sullivan, R., (2004). “Polyol pathway along the bovine epididymis.”
Molecular Reproduction and Development, 69(4):448–456.
Frenette, G., Legare, C., Saez, F., Sullivan, R. (2005). Macrophage migration inhibitory factor in
the human epididymis and semen. Molecular Human Reproduction, 11:575–582.
81 | P a g e

Frits, M. F. and Gadella, B. M. (2000). Dynamics of the mammalian sperm plasma membrane in
the process of fertilization. Biochimica et Biophysica Acta, 1469: 197-235.
Garcia-Alvarez, O., Maroto-Morales, A., Martinez-Pastor, F., Garde, J. J., Ramon, M., FernandezSantos, M. R., Esteso, M. C., Perez-Guzman, M. D., Soler, A. J. (2009). Spermcharacteristics and in vitro fertilization ability of thawed spermatozoa from black
manchega ram: Electroejaculation and postmortem collection. Theriogenology, 72: 160–
168.
Gillan, L., Maxwell, W. M. (1999). The functional integrity and fate of cryopreserved ram
spermatozoa in the female tract. Journal of Reproduction and Fertility, 54: 271-283.
Gimeno, R. E., Ortegon, A. M., Patel, S., Punreddy, S., Ge, P., Sun, Y., Lodish, H. F., and Stahl,
A. (2003). Characterization of a heart-specific fatty acid transport protein. Journal of
Biological Chemistry, 278 (18): 16039–16044.
Girouard, J., Frenette, G., and Sullivan, R., (2008). “Seminal plasma proteins regulate theassociation of lipids and proteins within detergent-resistant membrane domains of bovine
spermatozoa,” Biology of Reproduction, 78(5): 921– 931.
Gohil, V. M., Sheth, S. A., Nilsson, R., Wojtovich, A. P., Lee, J. H., Perocchi, F. (2010). Nutrientsensitized screening for drugs that shift energy metabolism from mitochondrial respiration
to glycolysis. Nature Biotechnology, 28: 249–255.
Grace, D., Dominguez-Salas, P., Alonso, S., Lannerstad, M., Muunda, E., Ngwili, N., Omar, A.,
Khan, M. and Otobo, E. (2018). The influence of livestock-derived foods on nutrition
during the first 1,000 days of life. ILRI Research Report 44. Nairobi, Kenya: ILRI.
Graham, J.K. (1996). Cryoservetion of stallion spermatozoa. Veterinary Clinics of North
America: Equine Practice.; 12:131-147.
Graham, J. K. (1998). An update on semen extenders and cryoprotectants, In Proceedings of the
17th Technical Conference on A.I. and Reproduction, Madison, WI.
Grotter, L. G., Cattaneo, L., Estela, P., Kjelland, M. E., Ferre, L. B. (2019). Recent advances inbovine sperm cryopreservation techniques with a focus on sperm post—Thaw quality
optimization. Reproduction in Domestic Animals, 54: 655–665.
Gruning, N. M., Du, D., Keller, M. A., Luisi, B. F., Ralser, M. (2014). Inhibition of triosephosphate
isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis. Open Biology,
4:130232.
82 | P a g e

Hall, J. C., Hadley, J., & Doman, T. (1991). Correlation Between Changes in Rat Sperm
Membrane Lipids, Protein, and the Membrane Physical State During Epididymal
Maturation. Journal of Andrology, 12:76-87.
Hajri, T. & Abumrad, N. A. (2002). Fatty acid transport across membranes: relevance to nutrition
and metabolic pathology. Annual Review of Nutrition, 22: 383–415.
Hereng, T. H., Elgstoen, K. B. P., Cederkvist F. H. (2011). “Exogenous pyruvate accelerates
glycolysis and promotes capacitation in human spermatozoa.” Human Reproduction,
26(12): 3249–3263.
Hermo, L., Robaire, B. (2002). Epidydimal cell types and their functions. In: Robaire, B., Hinton,BT.,

editors. The Epidydims: from molecules

to clinicalpractice.: Kluwer

Academic/Plenum Publishers, New York, 81-102.
Ho, H.C., Granish, K. A., and Suarez S. S. (2002). Hyperactivated motility of bull sperm istriggered at the axoneme by Ca2C and not cAMP. Developmental Biology, 250: 208–217.
Holstein, A. F., Roosen-Runge, E. C. (1981). Atlas of Human Spermatogenesis. Grosse Verlag,
Berlin, 1-224.
Holt, W.V. (2000). Basic aspects of frozen storage of semen. Animal Reproduction Science, 62: 322.
Hossain, M.S., Tareq, K., Hammano, K. I., Tsujii, H. (2007). Effect of fatty acids on boar sperm
motility, viability, and acrosome reaction. Reproductive Medicine and Biology, 6:235–239.
Hyne, R. V. and Edwards, K. P. (1985). “Influence of 2-deoxy-D-glucose and energy substrates
on guinea-pig sperm capacitation and acrosome reaction.” Journal of Reproduction andFertility, 73(1):59–69.
Isachenko, E., Isachenko, V., Katkov, Ii, Dessole, S., and Nawroth, F. (2003). Vitrification of
mammalian spermatozoa in the absence of cryoprotectants: From past practical difficulties
to present success. Reproductive Biomedicine Online, 6: 191–200.
Ishijima S. (2015). Ca2+ and cAMP regulations of microtubule sliding in hyperactivated motilityof bull spermatozoa. Proceedings of Japan. Academy, Series. B, 91: 99-108.
Jones, R. C., and Murdoch, R. N., (1996). “Regulation of the motility and metabolism ofspermatozoa for storage in the epididymis of eutherian and marsupial mammals.”
Reproduction, Fertility and Development, 8(4): 553–568.

83 | P a g e

John Morris, G., Acton, E., Murray, B. J., and Fonseca, F. (2012). Freezing injury: The special
case of the sperm cell. Cryobiology, 64:71–80.
Jose, C., Bellance, N., Rossignol, R., (2011). Choosing between glycolysis and oxidative
phosphorylation: a tumor's dilemma? Bba-Bioenergetics, (6) (1807): 552–561.
Joshi, N., Kodwany, G., Balaiah, D., Parikh, M., Parikh, F. (1996). The importance of computerassisted semen analysis and sperm function testing in an IVF program. International
Journal of Fertility and Menopausal Studies, 41(1):46–52.
Kaka, A., Wahid, H., Rosnina, Y., Yimer, N., Khumran, A. M., Behan, A. A. and Ebrahimi A.
(2015). Alpha-Linolenic Acid Supplementation in Tris Extender Can Improve Frozen–
Thawed Bull Semen Quality. Reproduction in Domestic Animals, 50: 29–33.
Kandelousi, M. S., Arshami, J., Naserian, A., and Abavisani, A. (2013). The effects of addition ofomega 3, 6, 9 fatty acids on the quality of bovine chilled and frozen thawed sperm. Open
Veterinary, 3 (1):47-52.
Kelso, K., Redpath, A., Noble, R., Speake, B. (1997). Lipid and antioxidant changes inspermatozoa and seminal plasma throughout the reproductive period of bulls.
Reproduction, 109: 1–6.
Khalil, W. A., Mostafa, A., El-Harairy, Alaa, E. B., Zeidan, Mahmoud, A. E. Hassan, MoheyElsaeed, O. (2017). Evaluation of bull spermatozoa during and after cryopreservation withstructural and ultrastructural insights. International Journal of Veterinary Science andMedicine, 6:49-56.
Khalil, W.A., El-harairy, M. A.; Zeidan, A. E. B., Hassan, M. A. E.; Mohey-Elsaeed, O. (2018).
Evaluation of bull spermatozoa during and after cryopreservation: Structural andultrastructural insights. International Journal of Veterinary Science and Medicine, 6: S49–
S56.
Kiernan, M., Fahey, A.G., Fair, S. (2013). The effect of the in vitro supplementation of exogenous
long-chain fatty acids on bovine sperm cell function. Reproduction Fertility andDevelopment, 25: 947–954.
Kirchhoff, C., Hale, G. (1996). Cell-to-cell transfer of glycosylphosphatidylinositol-anchoredmembrane proteins during sperm maturation. Molecular Human Reproduction, 2:177–184.
Kirchhoff, C. (1999). “Gene expression in the epididymis,” International Review of Cytology, 188:
133–202.
84 | P a g e

Kopeika, J., Thornhill, A., and Khalaf, Y. (2015). The effect of cryopreservation on the genomeof gametes and embryos: Principles of cryobiology and critical appraisal of the evidence.
Human Reproduction, 21: 209–227.
Kumar, D., Kumar, P., Singh, P., Yadav, S.P., Yadav, P.S. (2016). Assessment of sperm damages
during different stages of cryopreservation in water buffalo by fluorescent probes.
Cytotechnology, 68: 451–458.
Leahy, T., and Gadella, M. (2015). New insights into the regulation of cholesterol efflux from thesperm membrane. Asian Journal of Andrology, 17: 561–567.
Lemma, A. (2011). Effect of Cryopreservation on sperm quality and fertility. Artificial
Insemination in Farm Animals, 191–216.
Lenzi, A., Picardo, M., Gandini, L., and Dondero, F. (1996). Lipids of the sperm plasma
membrane: from polyunsaturated fatty acids considered as markers of sperm function topossible scavenger therapy. Human Reproduction Update, 2: 246–256. 
Lewis, S. E and Maccarrone, M. (2009). Endocannabinoids, sperm biology and human fertility.
Pharmacological Research, 60:126–131.
Mannowetz, N., Wandernoth, P. M, Wennemuth, G. (2012). Glucose is a pH‑dependent motor for
sperm beat frequency during early activation. PLoS One; 7: e41030.
Mann, T., and Mann, C. L. (1981). Male Reproductive Function and Semen. Springer, Berlin.;137.
Martin-DeLeon, P. A. (2015). Epididymosomes: transfer of fertility-modulating proteins to thesperm surface. Asian Journal of Andrology,17:720–725.
Martinez -Soto, J. C., Landeras, J., & Gadea, J. (2012). Spermatozoa and seminal plasma fatty
acids as predictors of cryopreservation success. American Society of Andrology andEuropean Academy of Andrology, 1-11.
Martinez -Soto, J. C., Landeras, J., & Gadea, J. (2013). Spermatozoa and seminal plasma fatty
acids as predictors of cryopreservation success. Andrology, 1(3): 365–375.
McArthur, M. J.,

Atshaves, B. P., Frolov, A., Foxworth, W. D., Kier, A. B., Schroeder, F.

(1999). Cellular uptake and intracellular trafficking of long chain fatty acids. Journal of
Lipid Research, 40: 1371–1383.
Medeiros, C. M. O., Forell, F., Oliveira, A. T.D., Rodrigues, J. L. (2002). Current status of sperm
cryopreservation: why isn’t it better? Theriogenology, 57:327–44.
85 | P a g e

Mehrabi Z., Gill M., Wijk M., Herrero M.,

and Ramankutty N. (2020) Livestock policy forsustainable development. Nature Food,1: 160–165.
Meyers, S. A. (2005). Spermatozoal response to osmotic stress. Animal Reproduction Science, 89:
57-64.
Mishro, M. M, Ramya, T. (2012). Fuel/energy sources of spermatozoa. In: Parekattil JS, Agarwal
A, editors. Male Infertility. New York: Springer‑Verlag; p. 209–23.
Moce, E., Purdy P. H., and Graham J. K. (2010). Treating ram sperm with cholesterol-loaded
cyclodextrins improves cryosurvival. Animal Reproduction Science, 118: 236-247.
Moore, A. I., Squires, E. L. and Graham, J. K. (2005). Adding cholesterol to the stallion sperm
plasma membrane improves cryosurvival. Cryobiology,51: 241-249.
Morris, G. (2006). Rapidly cooled human sperm: No evidence of intracellular ice formation.
Human Reproduction, 21: 2075–2083.
Mujica, A., Navarro-Garci, F., Hernandez-Gonzalez, E. O., and Juarez-Mosqueda, M. D. L.
(2003). “Perinuclear theca during spermatozoa maturation leading to fertilization,”
Microscopy Research and Technique, 61(1):76–87.
Mukai, C., Travis, A. J. (2012). What sperm can teach us about energy production. Reproduction
in Domestic Animals; 47(4): 164–9.
Nakada, K., Sato, A., Yoshida, K., Morita, T., Tanaka, H., (2006). Mitochondria‑related maleinfertility. Proceedings of the National Academy of Science U S A; 103: 15148–53.
Neill, A. R., and Masters, C. J. (1971). Incorporation of (U-14C) palmitic acid into thephospholipids of bovine semen. Journal of Reproduction and Fertility, 24(2): 295–297.
Neill, A. R. and Masters, C. J. (1972). Metabolism of fatty acids by bovine spermatozoa.
Biochemical Journal, 34: 279–287.
Neill, A. R. and Masters, C. J. (1973). Metabolism of fatty acids by ovine spermatozoa. Journalof Reproduction and Fertility, 34(2): 279–287.
Nur, Z., Zik, B., Ustuner, B., Sagirkaya, H., Ozguden, C. G. (2010). Effects of different
cryoprotective agents on ram sperm morphology and DNA integrity. Theriogenology,
73:1267–1275.
Oberoi, B., Kumar, S., and Talwar, P. (2014). Study of human sperm motility post
cryopreservation. Medical Journal Armed Forces India,70(4):349-53.

86 | P a g e

Odet, F., Duan, C., Willis, W. D., Goulding, E. H., Kung, A., Eddy, E. M. (2008). Expression of
the gene for mouse lactate dehydrogenase C (Ldhc) is required for male fertility. Biology
of Reproduction, 79: 26–34.
Oh, J. S, Han, C., and Cho, C. (2009). ADAM7 is associated with epididymosomes and integrated
into sperm plasma membrane. Molecules and Cells, 28: 441–446.
Okamura, N., Kiuchi, S., Tamba, M. (1999). “A porcine homolog of the major secretory proteinof human epididymis, HE1, specifically binds cholesterol.” Biochimica et Biophysica Acta,
1438 (3): 377–387.
Ozkavukcu, S., ErdemLi, E., Isik, A., Oztuna, D., and Karahuseyinoglu, S. (2008). Effects ofcryopreservation on sperm parameters and ultrastructural morphology of humanspermatozoa. Journal of Assisted Reproduction and Genetics, 25: 403–411.
Parrish, J. J., Susko-Parrish, J. L., and First, N. L., (1989). “Capacitation of bovine sperm byheparin: inhibitory effect of glucose and role of intracellular pH,” Biology of Reproduction,
41(4) :683–699.
Pena, F. J., Rodriguez Martinez, H., Tapia, J. A., Ortega Ferrusola, C., Gonzalez Fernandez, L.,
Macias Garcia, B. (2009). Mitochondria in mammalian sperm physiology and pathology:
a review. Reproduction in Domestic Animals, 44: 345-349.
Pepino, M. Y., Kuda, O., Samovski, D., Abumrad, N. A. (2014). Structure-function of CD36 andimportance of fatty acid signal transduction in fat metabolism. Annual Review of Nutrition,
34: 281–303.
Perumal, P. (2018). Low density lipoprotein in cryopreservation of semen. Asian Pacific Journalof Reproduction, 7(3): 103-116.
Perez, C. Parrilla , I., Li , J., Barranco , I., Martinez , E. A., Rodriguez-Martinez , H., Roca J.,(2018). The proteome of pig spermatozoa is remodeled during ejaculation. Molecular andCellular Proteomics, 18: 41–50.
Perez, C., Barranco , I., Li , J.,

Martinez , E. A., Padilla, L.,

Roca J.,

Parrilla ,I., (2019). Cryopreservation differentially alters the proteome of epididymal andejaculated pig spermatozoa. International Journal of Molecular Science, 20: 1791.

87 | P a g e

Pica-Ciamarra, U., Tasciotti, L., Otte, J. & Zezza, A. (2011). Livestock Assets, Livestock Income
and Rural Households: Cross-Country Evidence from Household Surveys 18 (World Bank,
2011).
Pini, T., Leahy , T., Soleilhavoup , C., Tsikis , G., Labas , V., Combes-Soia , L., Harichaux , G.,
Rickard , J. P., Druart , X., de Graaf S. P. (2016). Proteomic investigation of ram
spermatozoa and the proteins conferred by seminal plasma. Journal of Proteome Research,
15: 3700–3711.
Plaza Davila M., Bucci, D., Galeati, G., Pena, F. J., Mari, G., Giaretta, E., Tamanini, C., andSpinaci, M. (2015). Epigallocatechin-3-Gallate (EGCG) Reduces Rotenone Effect on
Stallion Sperm–Zona Pellucida Heterologous Binding. Reproduction in Domestic
Animals,50: 1011–1016.
Potter, M., Newport, E., and Morten, K. J. (2016). The Warburg effect: 80 years on. Biochemical
Society Transactions, 44: 1499–1505.
Purdy, P. H., Graham, J. K. (2004). Effect of cholesterol-loaded cyclodextrin on the cryosurvivalof bull sperm. Cryobiology, 48:36–45.
Purdy, P. H., Fox, M. H., and Graham J. K. (2005). The fluidity of Chinese hamster ovary cell andbull sperm membranes after cholesterol addition. Cryobiology, 51: 102-112.
Purdy P.H. (2006). A review on goat sperm cryopreservation. Small Ruminant Research, 63: 215225.
Quinn, P.J. (1989). Principles of membrane stability and phase behavior under extreme conditions.
Journal of Bioenergetics and Biomembranes, 21: 3-19.
Rasul, Z., Ahmad, N., Anzar, M. (2001). Changes in motion characteristics, plasma membrane
integrity, and acrosome morphology during cryopreservation of buffalo spermatozoa.
Journal of Andrology, 22: 278–283.
Ricardo, C., Chebel., J. E. P. Santos., J. P. Reynolds., R.L.A. Cerri., S. O. Juchem and O. Michael.
(2004). Factors affecting conception rate after artificial insemination and pregnancy loss
in lactating dairy cows. Animal Reproduction Science; 239-255.
Rodriguez, C. M., Kirby, J. L., and Hinton, B. T., (2001). “Regulation of gene transcription in theepididymis.” Reproduction, 122(1): 41–48.

88 | P a g e

Rogers, B. J., Ueno, M., Yanagimachi, R. (1977). Inhibition of hamster sperm acrosome reaction
and fertilization by oligomycin, antimycin A, and rotenone. Journal of Experimental
Zoology. 199(1):129-36.
Romero, N., Rogers, G., Neilson, A., and Dranka B. P. (2018). White paper: Quantifying Cellular
ATP Production Rate Using Agilent Seahorse XF Technology. Agilent Technologies, Inc.
Lexington, MA, USA ,5991-9303EN.
Royere, D., Barthelemy, C., Hamamah, S., and Lansac, J. (1996). Cryopreservation of
spermatozoa: A review. Human Reproduction, 2: 553–559.
Sa-Ardrit, M., Saikhun, J., Thongtip, N., Damyang, M., Mahasawangkul, S., Angkawanish, T.,
Jansittiwate, S., Faisaikarm, T., Kitiyanant, Y., Pavasuthipaisit, K., and Pinyopummin, A.
(2006). Ultrastructural alterations of frozenthawed asian elephant (elephas maximus)
spermatozoa. International Journal of Andrology, 29: 346–352.
Sahin, E., Petrunkina, A. M., Waberski, D., Harrison, R. A. P., and Topfer-Petersen, E. (2009).
“Control of bull sperm cell volume during epididymal maturation.” Reproduction, Fertility
and Development, 21(3): 469–478.
Schoff, P. K., Cheetham, J., and Lardy, H. A., (1989). “Adenylate kinase activity in ejaculated
bovine sperm flagella.” Journal of Biological Chemistry, 264(11): 6086–6091.
Schwieterman, W., Sorrentino, D., Potter, B. J., Rand, J., Kiang, C. L., Stump, D., and Berk, P. D.
(1988). Uptake of oleate by isolated rat adipocytes is mediated by a 40-kDa plasma
membrane fatty acid binding protein closely related to that in liver and gut. Proceedings of
the National Academy of Sciences, USA, 85: 359–363.
Scott, T. W., Voglmayr, J. K. & Setchell, B. P. (1967). Lipid composition and metabolism intesticular and ejaculated ram spermatozoa. Biochemical Journal, 102: 456–461.
Seidah, N. G., Manjunath, P., and Rochemont, J., (1987). “Complete amino acid sequence of BSPA3 from bovine seminal plasma. Homology to PDC-109 and to the collagen-binding
domain of fibronectin.” Biochemical Journal, 243(1): 195– 203.
Shalgi, R., Smith, T.T., Yanagimachi, R., (1992). A quantitative comparison of the passage ofcapacitated and uncapacitated hamster spermatozoa through the uterotubal junction.
Biology of Reproduction, 46 (3): 419–424.
Shum, W.W, Ruan Y. C, Da Silva N, Breton, S. (2011). Establishment of cell-cell cross talk in theepididymis: control of luminal acidification. Journal of Andrology, 32:576–586.
89 | P a g e

Sleight, S. B., Miranda, P. V., Plaskett, N. W. (2005). “Isolation and proteomic analysis of mouse
sperm detergent-resistant membrane fractions: evidence for dissociation of lipid rafts
during capacitation.” Biology of Reproduction, 73(4): 721–729.
Smil, V. (2009). Eating meat. Population and Development Review, 28: 299–639.
Soler, C., Yeung, C. H., and Cooper, T. G. (1994). Development of sperm motility patterns in themurine Epididymis. International journal of andrology,17:271-278.
Spiropoulos, J., Turnbull, D. M., Chinnery, P. F. (2002). Can mitochondrial DNA mutations cause
sperm dysfunction? Molecular Human Reproduction, 8: 719–21.
Stauss, C. R, Votta, T. J., and Suarez, S. S. (1995). Sperm motility hyperactivation facilitates
penetration of the hamster zona pellucida. Biology of Reproduction, 53: 1280–1285.
Stincone, A., Prigione, A., Cramer, T., Wamelink, M. M., Campbell, K., Cheung, E., OlinSandoval, V., Gruning, N. M., Kruger, A., Tauqeer, A. M., Keller, M. A., Breitenbach, M.,
Brindle, K. M., Rabinowitz, J. D., Ralser, M. (2015). The return of metabolism:
biochemistry and physiology of the pentose phosphate pathway. Biological reviews of theCambridge Philosophical Society, 90 (3): 927–963.
Storey, B. T. (2008). Mammalian sperm metabolism: oxygen and sugar, friend , and foe.
International Journal of Developmental Biology, 52: 427–37.
Su, X., and Abumrad, N. A. (2009). Cellular fatty acid uptake: a pathway under construction.
Trends in Endocrinology and Metabolism, 20:72–77.
Suarez, S. S., Osman, R. A. (1987). Initiation of hyperactivated flagellar bending in mouse sperm
within the female reproductive tract. Biology of Reproduction, 36:1191–1198.
Suarez, S. S., Katz, D. F., Owen, D. H., Andrew, J. B., Powell, R. L. (1991). Evidence for thefunction of hyperactivated motility in sperm. Biology of Reproduction, 44 (2): 375–381.
Suarez, S. S., Pacey A. A., (2006). Sperm transport in the female reproductive tract. Human
Reproduction Update,12 (1): 23–37.
Suarez S. S., (2008). Control of hyperactivation in sperm. Human Reproduction Update,14: 647–
657.
Sullivan, J.J. (1970). Sperm numbers required for optimum breeding efficiency in cattle.
Proceedings of the 3rd Technical Conference on Artificial Insemination and Reproduction.
Chicago, USA: National Association of Animal Breeder, 36-43.

90 | P a g e

Sullivan, R. (1999). “Interaction between sperm and epididymal secretory proteins,” in The Male
Gamete: From Basic to Clinical Applications, Cache River Press, Vienna,Ill, USA,130–136.
Sullivan, R., Frenette, G., Girouard, J. (2007). Epididymosomes are involved in the acquisition of
new sperm proteins during epididymal transit. Asian Journal of Andrology, 9:483–491.
Tan, O., Ha, T., Carr, B. R., Nakonezny, P., Doody, K. M., Doody, K. J. (2014). Predictive valueof postwashed total progressively motile sperm count using CASA estimates in 6871 nondonor intrauterine insemination cycles. Journal of Assisted Reproduction and Genetics,
31(9):1147–53.
Therien, I., Moreau, R., and Manjunath, P. (1998). “Major proteins of bovine seminal plasma andhigh-density lipoprotein induce cholesterol efflux from epididymal sperm.” Biology of
Reproduction, 59(4): 768–776.
Towhidi, A. and Parks, J. E. (2012). Effect of n-3 fatty acids and α-tocopherol on post-thawparameters and fatty acid composition of bovine sperm. Journal of Assisted Reproduction
and Genetics, 29:1051–1056.
Tsikis, G., Reynaud, K., Ferchaud, S. & Druart, X. (2018). Seminal plasma differentially alters theresistance of dog, ram, and boar spermatozoa to hypotonic stress. Animal Reproduction
Science, 193:1–8.
Van Voorhis, B. J., Barnett, M., Sparks, A. E., Syrop, C. H., Rosenthal, G., and Dawson, J. (2001).
Effect of the total motile sperm count on the efficacy and cost-effectiveness of intrauterine
insemination and in vitro fertilization. Fertility and Sterility, 75, 661–668.
VandeVoort, C. A. and Overstreet, J. W. (1995). “Effects of glucose and other energy substrates
on the hyperactivated motility of macaque sperm and the zona pellucida-induced acrosome
reaction.” Journal of Andrology, 16(4): 327–333.
Varisli, O., Uguz, C., Agca, C. and Agca, Y. (2009). Motility and acrosomal integrity comparisons
between electro-ejaculated and epididymal ram sperm after exposure to a range ofanisosmotic solutions, cryoprotective agents and low temperatures. Animal Reproduction
Science, 110, 256–268
Verma, R. J. (2001). Sperm quiescence in cauda epididymis. A mini review. Asian Journal ofAndrology, 3: 181–183.

91 | P a g e

Vishwanath, R. (2003). Artificial insemination: The state of the art. Theriogenology, 59(2), 571–
584.
Watson, P. F. (1995). Cooling of spermatozoa and fertilizing capacity. Reproduction in Domestic
Animals, 31:135–40.
Watson P.F. (2000). The causes of reduced fertility with cryopreserved semen. Animal
Reproduction Science, 60-61: 481-492.
Wessel, M. T. and Ball, B. A. (2004). Step-wise dilution for removal of glycerol from fresh andcryopreserved equine spermatozoa. Animal Reproduction Science, 84: 147-156.
White, I. G. (1993). Lipids and calcium uptake of sperm in relation to cold shock and preservation:a review. Reproduction Fertility and Development, 5:639–658.
Woelders, H., Matthijs, A., and Engel, B. (1997). Effects of trehalose and sucrose, osmolality of
the freezing medium and cooling rate on viability and intactness of bull sperm after
freezing and thawing. Cryobiology, 35: 93-105.
Yanagimachi, R. (1970). The movement of golden hamster spermatozoa before and after
capacitation. Journal of Reproduction and Fertility, 23: 193–196.
Yeste, M. (2016). Sperm cryopreservation update: Cryodamage, markers, and factors affecting thesperm freezability in pigs. Theriogenology, 85: 47–64.
Yoon, S. J., Kwon, W. S., Rahman, M.S., Lee, J.S., Pang, M.G. (2015). A novel approach toidentifying physical markers of cryo-damage in bull spermatozoa. PLoS ONE, 10,e0126232.
Zalata, A. A., Christophe, A. B., Depuydt, C. E, Schoonjans, F., Comhaire, F. H. (1998). The fatty
acid composition of phospholipidsof spermatozoa from infertile patients. Molecular
Human Reproduction, 4(2): 111-118.
Zheng J., (2012). Energy metabolism of cancer: glycolysis versus oxidative phosphorylation(Review), Oncology Letters, 4 (6):1151–1157.
Zhu, Z., Umehara, T., Okazaki, T., Goto, M., Fujita, Y., Hoque, S.A.M., Kawai, T., Zeng W.,
Shimada, M. (2019). Gene expression and protein synthesis in mitochondria enhance theduration of high-speed linear motility in boar sperm, Frontiers in Physiology, 10 (252):113.

92 | P a g e

Zhu, Z., Li, R., Feng, C., Liu, R., Zheng, Y., Hoque, S. A. M., De W., Lu, H., Zhang, T., and Zeng,
W. (2020). Exogenous Oleic Acid and Palmitic Acid Improve Boar Sperm Motility via
Enhancing Mitochondrial B-Oxidation for ATP Generation. Animals, 10: 591.

93 | P a g e