Studies on minimization of cryocapacitation of buffalo (Bubalus bubalis) sperm during cryopreservation
Abstract
Keeping fact in mind that cryocapacitation is detrimental to sperm, the present study was designed to
minimize the cryocapacitation in buffalo bull sperm by using low density glycoprotein (LDL) in place of yolk,
mifepristone (RU 486), NNC 55-0396 (NNC), H-89 and EC Oxyrase in order to protect sperm membrane,
antagonising progesterone of egg yolk, minimize Ca2+ influx, inhibit signalling molecule protein kinase A (PKA)
and oxidative stress by digesting O2 during cryopreservation, respectively. The LDL (10, 12 and 14%) was used to
replace 20% of egg yolk (EY) in the semen extender. The semen ejaculates having mass motility (MM) ≥ 3.0 and
Individual motility ≥ 75% were cryopreserved in EY and LDL extenders. Further, LDL 12 % was compared with
egg yolk to cryopreserve the poor and marginal quality ejaculates (MM ≤2.5; TM ≤ 65%). We also measured the
progesterone and Ca2+ in LDL and EY. The ejaculate was divided into four equal aliquots and extended using egg
yolk- based extender fortified with different concentration of RU 486 (0, 5, 10 and 20 μM), NNC (0, 2, 5 and 10
μM), H-89 (0, 2, 10, 30 μM) and Oxyrase (0, 0.3, 0.6, 0.9, 1.2 u/mL) separately and cryopreserved. The semen
samples were analysed for sperm kinetics and motility, incubation test, integrity of sperm membrane (HOST),
sperm cholesterol, expression of CatSper-2 proteins (LDL and mifepristone), Ca2+ influx in sperm, expression of
tyrosine phosphorylated proteins (TPP), cryocapacitation of sperm (CTC assay), lipid peroxidation (MDA
concentration), and antioxidative capacities of LDL and mifepristone through FRAP assay. Differential expression
of CatSper-1, CatSper-2, e-NOS, n-Nos and c-Myc was also assessed in sperm cryopreserved in LDL and EY
extender. Further mifepristone, NNC and H-89 treated samples were analysed for in vitro capacitation and zona
binding ability. Moreover, mifepristone treated sperm were also analysed for in vitro fertilizing potential. We
found that progesterone and Ca2+ were extremely low in LDL as compared to EY (P<0.05). Sperm motility and
kinetic parameters were better in LDL as compared to EY. The RU 486, NNC and H-89 and NNC did not affect
the sperm motility (P>0.05). But, LDL and Oxyrase prolonged the sperm longevity (P<0.05). The LDL and RU
486 protected the sperm membrane integrity (P<0.05). The LDL, RU 486, NNC, H-89 and Oxyrase prevented the
efflux of cholesterol, influx of Ca2+ (P<0.05). Lipid peroxidation of sperm membrane was lower as indicated by
lower MDA concentration in sperm cryopreserved in LDL extender and in sperm treated RU 486, NNC and
Oxyrase as compared to control (P<0.05). The expression of CatSper-2 proteins was higher in sperm
cryopreserved in LDL extender and sperm treated with mifepristone as compared to EY (P<0.05). The expression
of TPP was lowered in sperm cryopreserved in LDL as compared to EY (P<0.05). Also, expression of TPP was
lowered in sperm treated with RU 486, NNC, H-89 and Oxyrase (P<0.05). The RU 486, NNC and H-89 treated
sperm were shown normal in vitro capacitation and attached tightly to zona pellucida (P<0.05) as compared to
control. Also, RU 486 treated sperm had better in vitro fertilization rate as compared to control (P<0.05). LDL and
RU 486 fortified extender showed more antioxidant power based on DDPH and FRAP assays. The expression of
CatSper-1, CatSper-2, n-NOS and c-Myc were upregulated where as that of e-NOS was downregulated in sperm
cryopreserved in LDL extender as compared to EY. The LDL 12 % extender was more efficient in successful
cryopreservation of poor and marginal ejaculates as compared to EY (P<0.05). Taken all together, LDL, RU 486,
NNC, H-89 and Oxyrase in the present study found to beneficial in preventing the cryocapacitation in buffalo bull
sperm.
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