V K Gandotra

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.

Characterization of Mongrel Dog Seminal Plasma Proteins and Their Correlation with Semen Characteristics

The objective of this study was to characterize mongrel dog seminal plasma proteins with SDS-PAGE and to determine the correlation between different proteins and semen characteristics. Ejaculate volume, sperm motility, total sperm count, live sperm percent, percentage abnormalities and membrane integrity (hypo osmotic swelling test) were assessed in 5 ejaculates of seven dogs. For each dog, seminal plasma was also pooled from five ejaculates and proteins were separated by SDS-PAGE using polyacrylamide concentration of 13% and 15% in separating gels. There was considerable variation in the semen characteristics among the ejaculates of different dogs. Total protein content of seminal plasma varied from 12.06 to 21.3 mg/ml in different dogs. The number of protein bands ranged from 10-12 in different dogs. The proteins with mol wt of 42, 33, 29, 24 and 14.0 kDa were major proteins in seminal plasma of mongrel dog and ranged from 61.3% to 74.3% in different dogs. There were differences in live sperm percent (CD 5% =21.7) and HOS positive spermatozoa (CD 5% =21.01) among different dogs. There was strong correlation between volume of semen and total sperm concentration (r= +0.87), whereas the correlation between motility vs HOST (r=+0.58) and motility vs live percent (r=+0.60) was moderate. A positive correlation was observed between concentrations of 82, 70, 24, 14 kDa proteins vs percent motility, live sperm percent and percent HOS positive spermatozoa. Our study confirmed the previous conclusion that HOS-test could also be included in routine evaluation of semen. The positive correlation of 14, 24, 70 and 82 kDa proteins with semen characteristics also inferred the role of these proteins in the fertility of mongrel dog semen, which needs to be worked out further for their role in the process of fertilization. The author(s) have nothing to declare. Supported by Dept of Biotechnology, Ministry of Science & Technology, India, Ref: : BT/PR10394/AAQ/01/360/2008 J Reprod Stem Cell Biotechnol (Suppl) 2(1):55-63,2011 Correspondence: Name of Corresponding Author, PhD/MD, Dept of Veterinary Gynaecology and Obstetrics, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India. Email addrss: ranjna_cheema@hotmail.com T: 91-161-2414003 F: 91-161-2400822

Mutual interaction of dog sperm LDHC4, PH-20, actin and tubulin proteins and their immunocontraceptive potential in bitches

In this paper, interaction of LDHC4, PH-20, a-actin and b-tubulin on dog’s sperm surface and their immunocontraceptive effect in bitches has been elucidate. Anti spam -1 and anti LDHC4 recognized 46/32 and 36/30/28 kDa proteins in dog sperm extracts on immunoblots. Whereas, proteins of 43, 36 kDa and 62, 46, 43, 30 and 28 kDa were detected on immunoblots with anti-a-actin and anti-b-tubulin. In MALDI-TOF analysis, sequence of 46, 30, 28 and 43, 36 kDa antigens matched twice to b-tubulin and a-actin respectively. Therefore, one sub unit each of LDHC4 and a-actin had same mol wt of 36 kDa and two other sub units each of LDHC4 and b-tubulin shared the same mol wt of 30/28 kDa. Reaction of 46 kDa protein both with anti-spam-1 and b-tubulin indicated similarity in their mol wts. Similarly the reaction of 43 kDa protein both with anti-a-actin and anti- b-tubulin revealed some similarity between both. Sequence analysis revealed 19.49%, 24.28%, 21.42% and 18.75% similarity of LDHC4/ a-actin, PH-20/ a-actin, LDHC4/ b-tubulin and LDHC4/tubulin respectively. Immunofluoresence staining of dog sperm smears also indicated similarity in localization of these four proteins. Localization of PH-20 and b-tubulin was mainly on the entire head region. Whereas, LDHC4 is uniformly distributed on the whole sperm and actin is more concentrated on the post acrosomal cap. Purified native proteins of 46/32 and 36/30/28 kDa were injected to a group of two bitches respectively through i/m route to elucidate their immunocontraceptive potential. Immunization of bitches in both the groups resulted in suppressed heat, and inhibited natural mating. On being subjected to AI, ultrasonography of four immunized and two unimmunized (control) bitches 35 days after insemination confirmed the presence of 0 and 3-4 fetuses respectively. Therefore, it seems that LDHC4/PH-20 and a-actin/ tubulin share their molecular weights or interact with each other or actin/ b-tubulin superimpose LDHC4 /PH-20 on sperm surface. In view of observed contraceptive effect, it can be concluded that LDHC4 and PH-20 collectively with a-actin and b-tubulin affect estrus, process of natural mating and ultimately the fertility in bitches.