Ranjna S. Cheema

Characterization and immunolocalization of HBP, FA-1 and TIMP-2 like proteins in cattle bull semen: HBP modification in in vitro capacitated spermatozoa

Characterization and localization of HBP, FA-1 and TIMP-2 like proteins in spermatozoa and seminal plasma of cattle bulls was carried out by immunoblotting and immunofluoresence. Anti-HBP, anti-TIMP-2 and anti-FA-1 reacted with 55, 48, 45, 42, 35, 30, 24, 18, 16 kDa; 65, 45, 24, 16 kDa and 55, 48, 16 kDa sperm proteins on immunoblots. Immunofluoresence indicated that HBP/TIMP-2 are localized mainly on acrosomal cap, whereas, FA-1 predominantly on post acrosomal cap. Among the bulls, positive for 60, 45, 16 kDa FA-1 like proteins in sperm extracts and 11/16 kDa in SP; 7, 8, 6 and 7 bulls also showed higher rate of in vitro AR. Number of bulls positive for 65, 24 kDa-TIMP-2 and with higher rate of AR was more as compared to other anti-TIMP-2 reactive sperm/SP proteins. Therefore, 60, 45 and 16 kDA-FA-1, 65 and 24 kDa-TIMP-2 like proteins may serve as indicators of higher rate of in vitro AR vis a vis fertility of cattle bulls. Immunoblotting of cpapacitatd and un-capacitated spermatozoa with anti-HBP suggested that removal of 55, 48, 45, 40, 37 and 30 kDa HBP from in vitro acrosome reacted cattle bull spermatozoa allowed heparin to mediate in vitro AR and an increase in intensity of 110, 90 kDa and exposure of 65, 60, 26 and 16 kDa HBP after AR may be important for binding of sperm to ovum, penetration and fertilization.

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

Homology between cattle bull sperm and bacterial antigenic proteins viz a viz possible role in immunological infertility

This study was carried out to investigate the possible presence of identical sperm and bacterial antigens which may cause similar antisperm antibody production leading to lower fertility. Cross-reactive antigens of cattle bull spermatozoa and different bacteria including Escherichia coli (E.xa0coli), Bacillus sp., and Staphylococcus sp. were characterized by immunoblotting and mass fingerprinting. Significant cross-reactivity was obtained for 75, 72, 44, 40, 33, 30, 25, 18, 14xa0kDa proteins with purified IgG of calves, heifers and cows between spermatozoa and the studied bacteria. Significantly (pxa0<xa00.05) matched cross-reactive 40/33/30xa0kDa sperm, 33xa0kDa Staphylococcus sp/Bacillus sp and 40/25xa0kDa E.xa0coli proteins were analyzed. Mass fingerprinting of 40/33/30xa0kDa (spermatozoa); 40/25xa0kDa (E.xa0coli) and 33xa0kDa (Bacillus/Staphylococcus) proteins revealed their matching with vitellogenin-1-like/mitochondrial malate dehydrogenase 2, NAD/acrosin-binding protein isoform XI; outer membrane insertion signal domain/spore coat protein and glyceraldehyde-3-phosphate dehydrogenase, respectively. Acrosin-binding protein isoform X1 and mitochondrial malate dehydrogenase 2, NAD contributes to the capacitation of spermatozoa. Spore coat protein; glyceraldehyde-3-phosphate dehydrogenase of E.xa0coli; Bacillus/Staphylococcus are 37.6% and 39.01% identical to acrosin-binding protein isoform X1; mitochondrial malate dehydrogenase 2, NAD of cattle bull spermatozoa. It can be interpreted from these observations that cross-reacting antibodies developed against 33/30xa0kDa sperm proteins and 25, 33xa0kDa bacterial proteins in cows may affect the functional activity of spermatozoa leading to delayed fertility in heifers and cows.

Bacterial Pathogens and Antibiotic Susceptibility Patterns of Cervico-Vaginal Discharges in Cross Bred Repeat Breeding Heifer Cows

Well managed and profitable dairy farming requires animal with good reproductive performance (Nebel and Jobst, 1998). However, various reports of decreasing profit in dairy farming have been identified where improvements can be made (Bishop, 1964). Of them, postpartum uterine infection is the majorcause of economic loss in dairy cows (Fox et al., 2002; Sriskandan et al., 2000). It may be due to a longer calving interval, costs of extra services, treatment and increased culling rate (Drillich et al., 2005; Gilbert et al., 2005). Fertility of cow is affected by many nonspecific and specific pathogens of the genital tract. Uterine environment favors the growth of anaerobic organisms which in turn promotes the growth of various pathogenic aerobes that act synergistically to cause bacterial infection (Huszenicza et al., 1999). A higher percentage of cows (80 to 100%) found positive for bacterial contamination of the uterus in the first 2 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 6 (2017) pp. 1769-1775 Journal homepage: http://www.ijcmas.com

Purification and Characterization of Heparin Binding Proteins from Seminal Plasma of Cross- bred Cattle Bulls by Affinity Chromatography, SDS-PAGE and Mass Spectrometry

Heparin binding proteins (HBP) play a crucial role in the fertility of bovine semen. In this study HBPs purified from cross-bred cattle bull seminal plasma (SP) by sepharose-affinity chromatography were characterized by SDS-PAGE, and mass- spetrometery. Affinity chromatographic analysis indicated two peaks of unbound (non-HBP) and bound proteins (HBP). On average, seminal plasma of cross-bred bulls contained 39.36 ± 4.41% HBP with a peak area of 2.74 ± 0.82 cm2. Sixteen bands with molecular weights ranging from 14 kDa to 150 kDa could be separated by SDS-PAGE from seminal plasma of 11 bulls. SDS-PAGE analysis of the eluted HBP peaks identified 14 bands, with molecular weights ranging from 14 kDa to 150 kDa. However, variation in number of bands, separated in SP and SP-HBP was observed among the bulls. The matched peptides of 60; 40, 35; 31, 28; 25 and 20 kDa proteins with highest score (>61-67) were identified to have significant matching with the peptides of Platelet activatin factor AH; Clusterin preprotein; DNase1-L3 and TIMP-2, respectively. This study envisaged that four characterized SP-BHPs have important functions in reproduction. Moreover, role of DNASE-1L-3 and TIMP-2 kDa proteins is related to higher conception rate of bovine. Therefore, this study opens a further scope to analyze these SP – HBP as potential biomarkers of fertility in cross – bred bulls.

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.