Ꮫ ఩ ㄽ ᩥ ࡢ せ ᪨  ㄽᩥ㢟┠   Study of Lipid Components in Bovine Frozen Sperm㸦࢘ࢩ෾⤖⢭Ꮚ࡟ྵ᭷ࡍࡿ⬡㉁ᡂศ࡟㛵ࡍࡿ◊✲㸧                   ᗈᓥ኱Ꮫ኱Ꮫ㝔⏕≀ᅪ⛉Ꮫ◊✲⛉
Bioresource Science ᑓᨷ                   Ꮫ⏕␒ྕ  D180963
                   Ặ  ྡ Md. Mazharul Islam

Mammalian sperm migrate through uterus to oviduct by maintaining linear motility patternduring in vivo fertilization process. For the migration process sperm require adequate energy toreach the oocyte for fertilization. Sperm gets maximum energy from mitochondria ATPproduction. The quality of sperm deteriorates in the process of cryopreservation. Spermmembrane integrity, acrosomal integrity and mitochondrial abnormality are the main physicalnegative 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 lipid mixture 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 spermwith cholesterol were studied.
1. Lipid Mixture (LM) in thawing media is beneficial to keep sperm motility with otherquality parameters in healthy status of bovine frozen sperm
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 compositionhealthy 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 lipid mixture was up taken by LM thawedsperm compare to control and there was a positive correlation between fatty acids amount andtotal sperm motility. It was also observed that LM thawed bovine frozen sperm showedsignificantly good motility parameters compare to control group. Moreover, it was also
uncovered that membrane integrity, mitochondrial activity and live sperm percentage wassignificantly higher in LM thawed sperm compare 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 ofbovine frozen sperm and their correlation to sperm motility according to author best ofknowledge.
2. Sperm linear motility is induced by saturated fatty acid being used as energy substratein the mitochondria of bovine frozen sperm
Successful fertilization depends on sperm linear motility and motility patterns are dependent onenergy substrate and their effective utilization in energy producing segments of sperm. InExperiment 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 thishypothesis; 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 ofFAs 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).

FAtransporter(CD36andGOT2)

wasobservedbywhole-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 was observed that the bovine frozen sperm
possessed FA transporter in midpiece where the fluorescence signals were detected after thetreatment 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.
3. Cholesterol in thawing media regain mitochondrial potential which accelerates ATP
production in mitochondria that induces linear motility in bovine frozen sperm
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
taken by sperm and used to remake the mitochondrial membrane potential along with sperm
membrane potential which was damaged during cryopreservation process. To clarify thehypothesis 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 by fluorescence intensity in the mid piece and head region of sperm afterthe treatment with fluorescence-tagged CHL. The mitochondrial activity of sperm treated withCHL 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. ATP production rate was also significantly higher in CHL
thawed group compare to control. Sperm linear motility was significantly (P<0.05) increasedby the thawing with CHL. Sperm linear motility is prompted by CHL in thawing media ofbovine frozen sperm might provide a new vision for modernizing the artificial insemination
technique of both livestock animals and human infertility care.
In conclusion, this study unveiled the mechanism of lipid mixture to improve the linear motility
pattern and membrane integrity of bovine frozen sperm. Two part in lipid mixture fatty acids
and cholesterol contribute separately to rescue the quality of bovine frozen sperm after thawing
in a lipid mixture containing media. Interestingly, this study uncovers the energy metabolism ofbovine frozen sperm thawed in lipid mixture or fatty acid containing thawing media with themolecular strategy of fatty acid uptake to the sperm mitochondria from the membrane.

Moreover, cholesterol helps to recover the membrane integrity and mitochondrial membranepotentials declined during cryopreservation process and contribute to regain the linear motility
pattern through accelerate ATP production rate. This study invented new information forreproductive science to maintain linear motility of sperm after thawing and washing which isprerequisite for fertilization. Therefore, the new story developed from the present study isstrongly believed to be fruitful to the future research in AI technology for livestock and humaninfertility care as inexpensive technique. New approach of thawing frozen sperm from thepresent study will increase the conception rate definitely and ultimately the production oflivestock will increase which may be beneficial to overcome poverty, increase income and attainthe SDGs of the united nations (UN).

Keywords: Frozen sperm, Linear motility, ATP Production, Mitochondrial Potential.