Characterization of sperm heparin binding proteins (HBPs) using polyclonal antibodies raised against seminal plasma HBPs: Application in buffalo bull fertility

26-33This study aimed to evaluate rabbit polyclonal antibodies raised against purified seminal plasma sperm membrance extracts (SP) heparin binding protein (HBP) for identifying HBPs in buffalo bull spermatozoa by western blotting. Anti-SP-HBP recognized 11 polypeptides in SDS-sperm membrance extracts (SME) of 31 tested bulls. Thirty one bulls were divided into G-1 (>40%) and G-II (≤40%) based on acrosome reaction. Immunoblotting revealed that HBPs of 24, 30, 38 and 43 kDa were present in 3%, 7.02%, 1.16% and 4.83% more bulls of G-I, whereas, 20 and 46 kDa HBPs were present in 13.2 and 9.65% more bulls of G-II. Immunoblotting of anti-HBP with sperm extracts of 10 bulls (22-31) indicated that 31 kDa positive bulls had 10.9% higher conception rate than 31 kDa negative bulls. Although 24 kDa HBP was detected in 10 bulls, but its expression was very weak in bull number 22, 23 and 26, which had 10.7% lower conception rate than the bulls with strong expression of 24 kDa HBP. In the present study, 17/20 kDa positive bulls exhibited 4.46% and 8.67% low conception rate than 17/20 kDa negative bulls. Mass spectrometry analysis revealed matching of 24, 31, 33 and 38 kDa proteins with MHC class 1 antigen, tRNA methyl transferase 11 homolog partial, parvalbumin alpha-like and cilia- and flagella-associated protein 99. This study suggests that buffalo bull fertility can be predicted from sperm HBP

Manganese provides antioxidant protection for sperm cryopreservation that may offer new consideration for clinical fertility

Reactive oxygen species (ROS) are generated by sperm metabolism. While, ROS are required for maturation, capacitation and acrosome reaction, they also modify many peroxidable cellular compounds. There is production of ROS during cryopreservation and frozen spermatozoa are highly sensitive to lipid peroxidation (LPO). Antioxidants exert a protective effect on the plasma membrane of frozen bovine sperm preserving both metabolic activity and cellular viability. Manganese (Mn++) is proved to be a chain breaking antioxidant in biological system. Therefore, we examined the role of (Mn++) during cryopreservation of cattle bull semen. Semen was divided into four parts and cryopreserved in egg-yolk-citrate extender + glycerol (EYC-G), EYC-G + 100 µM of Mn++, EYC-G + 150 µM of Mn++ and EYC-G + 200 µM of Mn++. After four hours of cooling and 24 hrs of freezing, the spermatozoa were examined for percentage motility, Hypo-osmotic swelling (HOS), LPO and protein leakage. Addition of manganese to the semen during cryopreservation showed a protective effect and accounted for an increase in semen quality parameters [percentage motility, HOS percent and decrease in malondialdehyde (MDA) production and protein leakage]. The effect of manganese on motility and HOS was non-significant (p < 0.05) in cooled spermatozoa but significant with 150 µM of Mn++ in frozen-thawed spermatozoa. MDA production and protein leakage decreased to a significant and maximum level (p < 0.05) on addition of 200 µM of manganese. The addition of manganese to EYC-G dilutor will improve the quality/fertility of semen, which will result in improvement of in vitro fertilization and artificial insemination success rate

Characterization of tissue inhibitor metalloproteinases in semen and their relationship with vital sperm function tests vis-à-vis fertility of breeding buffalo bulls

The present study was undertaken to separate and compare the tissue inhibitor metalloproteinases (TIMP) of seminal plasma and frozen-thawed sperm extracts from 30 buffalo bulls by immunoblotting and determine a relationship between various TIMP with post-thaw sperm function tests vis-à-vis bull fertility. Seven immunoreactive bands in seminal plasma (65, 55, 48, 33, 31, 24 and 11 kDa) and 5 in frozen-thawed spermatozoa (75, 65, 55, 24 and 16 kDa) were detected in Western blots following incubation (TIMP–140) and subsequent washing in vitro, indicating that TIMP is bound to sperm membranes. The frozen-thawed semen was evaluated for first service conception rate (FSCR), per cent HOST, acrosome reaction, viability, DNA integrity and total motility and linked to TIMP. In seminal plasma, the bulls positive for 48, 33 and 24 kDa TIMP had significantly higher FSCR (57.0 ± 2.6 vs 27.0 ± 2.4%, 55.7 ± 3.0 vs 31.3 ± 3.2% and 45.0 ± 3.8 vs 32.8 ± 4.7%, respectively) as compared to their negative counterparts. Except per cent viability, almost all seminal parameters (acrosome reaction, per cent HOST, DNA integrity and total motility) were significantly higher in bulls positive for TIMP of 48, 33, 31 and 24 kDa than in their negative contemporary mates. In frozen-thawed sperm extracts, the bulls positive for TIMP–24 had significantly higher FSCR (51.7 ± 3.7 vs 27.2 ± 3.0%), higher percentage of acrosome-reacted (55.9 ± 2.8 vs 48.9 ± 2.2%) and HOS-positive (69.2 ± 1.5 vs 65.3 ± 1.9%) spermatozoa in comparison to their negative herd mates. These results suggested that TIMP influences semen quality and subsequent fertility of buffalo bulls through inhibition of metalloprotease activity in semen

Assessment and comparison of four lab made tris-base extenders for preservation of Labrador retriever dog semen at 4ºC

131-139The present study was aimed to develop a most effective extender for long term storage of Labrador dog semen at 4oC. Tris-citric acid-fructose was supplemented with egg yolk (TEY), egg yolk plasma (TEYP), low density lipoproteins (TLDL) and coconut water (TCW) @10, 15 and 20; 10, 15 and 20; 11-15; and 25, 50 and 75 percent, respectively. Extended semen was stored at 4oC and analyzed for sperm attributes and lipid peroxidation. Values for motility, viability and plasma membrane integrity remained significantly (p &lt; 0.05) higher in 15% TEY, 15% TEYP, 13% TLDL and 25%/50% TCW from 0 hr to 72 hrs of storage. It indicated superiority of 15% TEY, 15% TEYP, 13% TLDL and 25% /50% TCW over other concentrations for storage of Labrador dog semen at 4oC. A significant (p &lt; 0.05) decline in motility, viability and membrane integrity was observed from 0-72 hrs of preservation in all extenders, but sperm attributes were still &gt;50% at 72 hrs of preservation. Decline was comparatively less in 15% TEY, 15% TEYP, 25%/50% TCW and 13% TLDL compared to other concentrations. Values for acrosome integrity also differ significantly (p &gt; 0.05) among different concentrations of extenders except TEY. Lipid peroxidation did not vary among different extenders except TEY. In conclusion, both 15% TEY and 15% TEYP were better than 13% TLDL and 25% TCW. TEY extender may be substituted with TEYP for preservation of dog semen at 4&deg;C and further interventions may improve TLDL and TCW extenders

Response related enzymatic changes in ovaries of superovulated mice

171-173<span style="font-size:14.0pt;line-height: 115%;font-family:" times="" new="" roman";mso-fareast-font-family:"times="" roman";="" color:black;mso-ansi-language:en-in;mso-fareast-language:en-in;mso-bidi-language:="" hi"="" lang="EN-IN">Adult female mice were superovulated with PMSG followed by HCG and 140 blastocysts and 69 morulae were recovered from 24 mice. On the basis of the response, mice were divided into six groups; non responders, 1-5, 6- 10, 11-20, 21-30 and >30 embryos. The ovaries of the animals were pockd group wise, homogenized in PBS (pH 7.4) and after centrifugation for 10-15 minutes, the supernatant was analyzed for the enzymes, guanine oxaloacetate transaminase (GOT), guanine pyruvate transaminase (GPT), acid phosphatases (ACP) and alkaline phosphatases (AKP). Acid and alkaline phosphatise activities did not show any variation in relation to response to superovulation but GOT and GPT showed significantly increased activity in response to induction of superovulation. A statistically significant positive correlation was found between GOT and GPT activities and the superovulatory response in mice.</span

Immuno-contraceptive potential of sperm specific LDHC&lt;sub&gt;4&lt;/sub&gt; and SPAM-1 (PH-20) sub units in dog

International audienc

Seminal Plasma Heparin Binding Proteins Improve Semen Quality by Reducing Oxidative Stress during Cryopreservation of Cattle Bull Semen

Heparin binding proteins (HBPs) are produced by accessory glands. These are secreted into the seminal fluid, bind to the spermatozoa at the time of ejaculation, favour capacitation, acrosome reaction, and alter the immune system response toward the sperm. The present study was conducted with an objective to assess the effect of purified seminal plasma-HBPs (SP-HBPs) on cross bred cattle bull sperm attributes during two phases of cryopreservation: Pre freezing and freezing-thawing. SP-HBPs were purified from pooled seminal plasma by heparin affinity chromatography. Three doses of SP-HBPs i.e. 10, 20, 40 μg/mL semen were standardized to find out the optimum dose and 20 μg/mL was found to be an optimum dose. Semen as such and treated with SP-HBPs was diluted with sodium citrate-egg yolk diluter and cryopreserved as per the standard protocol. Sperm parameters i.e. motility, viability, Hypo-osmotic swelling test (HOST), acrosome damage, in vitro capacitation and lipid peroxidation were evaluated in SP-HBP treated and untreated (control) semen at both phases of cryopreservation. A considerable variation in percent sperm motility, viability, membrane integrity (HOST), acrosome damage, acrosome reaction and lipid peroxidation was observed at both phases among the bulls irrespective of the treatment. Incubation of neat semen with 20 μg/mL SP-HBP before processing for cryopreservation enhanced the average motility, viability, membrane integrity by 7.2%, 1.5%, 7.9%, and 5.6%, 6.6%, 7.4% in pre-frozen and frozen-thawed semen in comparison to control. There was also an average increase of 4.1%/3.9% in in vitro capacitation and acrosome reaction in SP-HBPs-treated frozen-thawed semen as compared to control. However, binding of SP-HBPs to the sperm declined acrosome damage and lipid peroxidation by 1.3%/4.1% and 22.1/32.7 μM/109 spermatozoa in SP-HBP treated pre-frozen/frozen-thawed semen as compared to control, respectively. Significant (p<0.05) effects were observed only in motility, HOST and in vitro acrosome reaction. It can be concluded that treatment of neat semen with SP-HBPs before cryopreservation minimized the cryoinjury by decreasing the generation of reactive oxygen species