Molecular weight-based profiling of sperm proteins and their association with semen quality in Kokok Balenggek roosters

The study aimed to assess sperm motility characteristics, kinematic parameters, and sperm protein molecular weight (MW) in Indonesian buffalo to predict fertility. Frozen semen from Silangit (4 bulls), Murrah (4 bulls), and Toraya (2 bulls)-aged 7-10 years, was analyzed. Sperm motility was assessed using Computer Assisted Semen Analysis, viability and abnormality were evaluated using eosin-nigrosin staining, plasma membrane integrity was evaluated using the hypoosmotic swelling test, acrosomal status was evaluated using lectin peanut agglutinin, protamine deficiency was evaluated using chromomycin A3, and deoxyribonucleic acid (DNA) integrity was evaluated using Halomax. Protein concentration was determined using the bicinchoninic acid method and characterized with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The study revealed breed-specific variations in semen quality. Silangit buffaloes exhibited lower DNA integrity, while Murrah buffaloes showed elevated motility and membrane integrity. Toraya buffaloes displayed higher normal morphology and protamine status, though they had lower viability. Notable differences in protein expression included the presence of SPAG9 and the absence of IZUMO1 in Toraya buffaloes. Protein MW analysis further showed correlations with sperm characteristics. In Murrah buffaloes, proteins within the 130-125 kilodalton (kDa) range were negatively correlated with acrosome integrity, whereas in Toraya buffaloes, proteins within the 55-50 kDa range were negatively correlated with sperm abnormalities. Silangit buffaloes showed a positive correlation between proteins at 32 kDa and sperm abnormalities. Analyzing protein MW through SDS-PAGE provides a promising approach for assessing semen quality in indigenous Indonesian buffalo bulls. Although the semen quality of the buffaloes in this study was variable, all bulls met the established Indonesian standards for semen quality and exhibited adequate fertilization potential. These results provide valuable insights into the reproductive biology of Indonesian buffalo bulls and form the basis for predicting fertility capacity through a comprehensive analysis of sperm characteristics and molecular profiles of sperm proteins.

Background:The Kokok Balenggek rooster (KBR), with its distinct phenotypes—Biriang, Balang, Kinantan, and Kuriak—offers a unique opportunity to study variations in semen characteristics and protein profiles. Understanding these variations can aid in the development of better management strategies for poultry breeding programs. Aim:This study aimed to characterize the spermatozoa and seminal plasma protein profiles based on molecular weight (MW) across four phenotypes of KBRs, focusing on semen parameters, such as motility, viability, and protein concentration. Methods:Semen samples were collected from the KBR of four phenotypes: Biriang, Balang, Kinantan, and Kuriak. The parameters analyzed included semen volume, color, smell, consistency, sperm concentration, motility, viability, abnormality, plasma membrane integrity, and protein concentration. Protein profiles of spermatozoa and seminal plasma were analyzed using MW markers via gel electrophoresis. Results:The results revealed significant variations in semen volume (p < 0.05) and protein concentration (p < 0.01), with the Kinantan phenotype exhibiting the highest protein concentration (2.23 mg/ml). Sperm motility (p < 0.05) and viability (p > 0.05) were highest in the Biriang, Balang, and Kinantan phenotypes, whereas the Kuriak phenotype showed lower motility (64%, p < 0.01). Protein profile analysis indicated the presence of proteins in sperm with MWs of 10, 25–35, 35–45, 45–65, and 100 kDa and in seminal plasma with MWs of 10, 20–25, 25–35, 45, 65, 75, 140, and 180–245 kDa, respectively, across all phenotypes.Conclusion:This study highlighted variations in sperm characteristics and protein profiles among KBR phenotypes, with the Kuriak phenotype showing lower motility, providing insights for improving genetic resource management and semen preservation.

Background and Aim:Holstein cows and heifers are widely bred in Indonesia by artificial insemination (AI) to increase population and milk production. Sperm fertility is modulated by genetic factors, but the analysis of sperm quality is still based on macro- and microscopic characteristics. This study aimed to analyze both sperm quality and proteins of Holstein bulls at different fertility levels.Materials and Methods:The frozen semen samples were collected from the Indonesia National AI Center. They were classified based on the reproductive efficiency data and were grouped into high fertile (HF) and low fertile (LF). Sperm qualities were evaluated by microscopic evaluation. The Holstein sperm proteins were extracted using phenylmethanesulfonyl fluoride as a protease inhibitor and the benzidine detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted to analyze the molecular weights (MWs) of the sperm proteins. The data obtained were analyzed by a t-test using the one-factor bull fertility level, and Spearman’s correlation analysis was used to identify the correlation between the sperm microscopic evaluation parameters and protein bands.Results:The sperm motility post-freeze thawing was not significantly different between the HF and LF (p>0.05). The HF level had a higher percentage of viability, intact plasma membrane integrity, and intact acrosomes than the LF (p<0.05). Five protein bands were found in the SDS-PAGE of sperm proteins of Holstein bulls with different concentrations. Sperm proteins with MWs of 17.51 kDa, 14.87 kDa, 33.71 kDa, and 41.97 kDa were abundant in the Holstein bulls with an HF level, while 55 kDa proteins were abundant in the LF level of Holstein bulls. The sperm of Holstein bulls in the HF level contained proteins of about 33.71 kDa that were not detected in the LF.Conclusion:The sperm protein with a molecular weight of 33.71 kDa was predicted to be a specific protein biomarker that influences bull fertility. Sperm fertilization abilities were also determined by the sperm proteins, the morphology of sperm acrosomes, and the quality of plasma membranes. This method can be used to select bulls with high fertility to increase the population of Holstein bulls.

ABSTRACTBackground and Aim:The reproductive efficiency of livestock, especially indigenous breeds such as Donggala bulls, is pivotal to successful breeding programs. While conventional semen parameters are widely used, molecular markers, such as sperm protein profiles and DNA integrity, are emerging as reliable indicators of fertility. This study aimed to characterize the sperm protein profiles of Donggala bulls and examine their correlation with sperm DNA integrity and protamine deficiency.Materials and Methods:Frozen semen samples were collected from six Donggala bulls (aged 5–7 years). Pre-freezing evaluations included progressive motility (via computer-assisted sperm analysis), sperm morphology (using Diff-Quik staining), DNA integrity (assessed by the acridine orange assay), and protamine deficiency (assessed by the chromomycin A3 assay). Protein concentrations were determined using the bicinchoninic acid assay, and protein profiling was performed using 1D sodium dodecyl sulfate polyacrylamide gel electrophoresis. Band intensities and distributions were analyzed using ImageJ. Statistical correlations were analyzed using a one-way analysis of variance and Pearson’s correlation coefficients.Results:Significant individual variation was observed in semen quality among bulls. Progressive motility ranged from 38.3% to 46.1%, DNA integrity from 79.5% to 96.8%, and protamine deficiency from 96.0% to 98.7%. The number of protein bands per sample varied between 8 and 11, with molecular weights ranging from 5 to 175 kilodaltons (kDa). Protein concentration ranged from 8.32 to 20.70 μg/mL. A 35 kDa protein band was notably absent in one bull, which may be linked to lower motility. Strong correlations were observed between sperm motility and DNA fragmentation (r = 0.628), protamine deficiency (r = 0.539), protein concentration (r = 0.658), and protein band expression (r = 0.788).Conclusion:Sperm protein profiles in Donggala bulls are significantly correlated with DNA integrity and protamine deficiency, indicating their potential as molecular biomarkers for fertility prediction. These findings provide a foundation for integrating protein profiling into breeding soundness evaluations, suggesting that targeted proteomic analysis may enhance reproductive management strategies.

The concentration of several protein (β2-microglobulin, orosomucoid, albumin, transferrin) and of total amino acids was determined in the urine of 18 cadmium-exposed workers and in a group of matched nonexposed workers. The results were compared with the electrophoretic pattern of urinary proteins on agarose gel. Ten of the cadmium-exposed workers had an abnormal electrophoretic pattern, eight of them excreted larger quantities of high and low molecular weight proteins, and the other two showed only an increased excretion of high molecular weight proteins. An increased β2-microglobulin excretion was found in five workers with a normal urinary protein electrophoresis whereas only the urine of three exposed workers were found to be normal. We have confirmed a previous observation that in the majority of the cadmium-exposed workers with an abnormal electrophoretic pattern or an increased total proteinuria, not only low molecular weight proteins (β2-microglobulin) are excreted in greater amount but also high molecular weight proteins such as albumin and transferrin. Furthermore, in cadmium-exposed workers proteinuria is more closely related to the concentrations of albumin and orosomucoid in urine than that of β2-microglobulin. The change in urinary concentration of total amino acids was less marked than that of protein. The determination of both low and high molecular weight proteins ought to be recommended for detecting renal damage due to cadmium.

The present study determined the effects of conventional, and controlled freezing method adopting three freezing rates, viz. 20°C, 40°C and 60°C/min on quality of sperm (motility, viability and plasma membrane integrity), DNA integrity and plasma membrane protein profile of cryopreserved boar semen. Sixty sperm-rich fractions of ejaculates from six boars were utilized for freezing of semen with different freezing methods in lactose-egg yolk glycerol extender. Semen samples were evaluated for sperm motility, viability (Propidium Iodide assay), functional integrity of plasma membrane (HOST), DNA integrity (Acridine Orange stain) and plasma membrane protein profile (SDSPAGE) after equilibration and after freezing. The results revealed that the post thaw sperm motility, sperm viability, and plasma membrane integrity (HOST-reacted) were significantly higher in all the three controlled freezing methods (20°C, 40°C and 60°C/min) as compared to that in conventional method. In addition, the number of sperm plasma membrane protein loss was less in controlled freezing methods as compared to that in conventional freezing. However, the post thaw sperm DNA integrity did not influence by difference in freezing methods. No significant difference on the post thaw sperm characteristics was recorded among the three controlled freezing rates. All the sperm parameters assessed declined significantly after freezing as compared to that after equilibration irrespective of freezing method employed. In conclusion, controlled freezing methods conferred better post thaw sperm quality as compared to conventional method, and thus the freezing rates of either 20, 40 or 60°C/min could provide better freezability of boar semen.

Significant deterioration in semen quality is associated with increased male infertility. Alterations in the sperm proteome resulting from infertility are not well understood. The aim of this study was to examine the connection between the proteome and the quality of spermatozoa in fertile and infertile dogs. The research was conducted on 11 male dogs, including 3 German shepherds, 3 Turkish Kangal, 2 Cane Corso, and 3 Golden Retrievers, aged 4 and 6 years (4.81 ± 0.75). Four of the 11 dogs constituted the infertile study group because of conception failure observed in at least 3 (3-5) matings with different fertile females within 1 year, and seven dogs from different owners constituted the fertile control group. Semen was manually manipulated and collected from all dogs in the presence of a female dog in heat. Ejaculates were used for spermatological examination and protein analysis. Computer-assisted semen analysis (CASA) was used to evaluate semen samples. In spermatological parameters, motility, kinematic parameters, morphology and plasma membrane integrity were evaluated. Gel electrophoresis (SDS-PAGE) was used to analyze proteins in sperm. The semen quality of infertile dogs was significantly lower. Motility, progressive motility, viability and plasma membrane integrity were statistically significantly lower in the infertile group than in the fertile group (P &lt; 0.05). In morphological parameters, acrosome, head, middle part, tail and total defects were found to be statistically significantly higher in sterile dogs than in fertile dogs (P &lt; 0.05). In the study, a total of 8 protein bands with molecular weights (MW) ranging from 11.0 to 245.0 kDa were identified in the protein analysis of semen. Variations in the sperm proteome composition were shown to be dependent on fertility. These proteins were associated with important metabolite pathways. Additionally, correlations of these bands with various spermatological parameters were revealed. The result of the study suggests that seminal plasma proteins play a role in semen quality and may be potential fertility biomarkers.

Protein profile and functionality of spermatozoa from two semen collection methods in Bali bulls

Aim:This study aimed to study the electrophoretic properties of seminal plasma and sperm proteins of Black Bengal buck semen and their correlation with in vitro sperm characters and freezability.Materials and Methods:Semen ejaculates from nine Black Bengal bucks were collected by artificial vagina (n=20/buck). Ejaculates were evaluated for in vitro sperm characters and electrophoretic profile of seminal protein. In vitro sperm characters were evaluated immediately after collection, after completion of equilibration period, and after freeze-thawing. For seminal protein studies, seminal plasma proteins were precipitated by ice-cold ethanol method, and sperm proteins were extracted by Triton X detergent extraction method. Discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to assess the molecular weight of seminal proteins. Correlation between in vitro sperm characters and protein bands was determined by Pearson’s correlation coefficient, and two-way ANOVA was applied to find the individual buck differences.Results:Significant difference (p<0.01) among the bucks was noticed in the in vitro sperm characters evaluated at all the three stages of semen evaluation such as immediately after collection, after completion of equilibration period, and post-freeze thawing. Progressive loss of sperm motility, membrane integrity, and other in vitro sperm characters were noticed during cryopreservation. A total of ten protein bands in the molecular weight ranging from 17 to 180 kDa were found in the SDS-PAGE of seminal plasma proteins, while nine bands of 17-134 kDa were observed in sperm proteins. Seminal plasma proteins of molecular weight 75, 62-49, 20, and 17 kDa and sperm proteins of 75, 20, and 17 kDa were present in all the nine bucks (100%) screened, and variation among the bucks was noticed for the presence of other proteins. Seminal plasma protein of 180-134 kDa showed a negative correlation with individual motility (−0.716) and functional membrane integrity of sperm cells (−0.724) in post-freeze–thaw analysis and 48 kDa protein had a positive correlation with individual motility (0.649) and functional membrane integrity of sperm cells (0.664) in post-thaw analysis. Sperm proteins of 63 kDa had a negative correlation (−0.616) with sperm concentration in neat semen.Conclusion:Variation among the bucks was noticed in the in vitro sperm characters and semen freezability. Correlation between seminal proteins and in vitro sperm characters and semen freezability had been found which might be useful as a tool to select breeding bucks.

Significant deterioration in semen quality is associated with increased male infertility. Alterations in the sperm proteome resulting from infertility are not well understood. The aim of this study was to examine the connection between the proteome and the quality of spermatozoa in fertile and infertile dogs. The research was conducted on 11 male dogs, including 3 German shepherds, 3 Turkish Kangal, 2 Cane Corso, and 3 Golden Retrievers, aged 4 and 6 years (4.81 ± 0.75). Four of the 11 dogs constituted the infertile study group because of conception failure observed in at least 3 (3-5) matings with different fertile females within 1 year, and seven dogs from different owners constituted the fertile control group. Semen was manually manipulated and collected from all dogs in the presence of a female dog in heat. Ejaculates were used for spermatological examination and protein analysis. Computer-assisted semen analysis (CASA) was used to evaluate semen samples. In spermatological parameters, motility, kinematic parameters, morphology and plasma membrane integrity were evaluated. Gel electrophoresis (SDS-PAGE) was used to analyze proteins in sperm. The semen quality of infertile dogs was significantly lower. Motility, progressive motility, viability and plasma membrane integrity were statistically significantly lower in the infertile group than in the fertile group (P &amp;lt; 0.05). In morphological parameters, acrosome, head, middle part, tail and total defects were found to be statistically significantly higher in sterile dogs than in fertile dogs (P &amp;lt; 0.05). In the study, a total of 8 protein bands with molecular weights (MW) ranging from 11.0 to 245.0 kDa were identified in the protein analysis of semen. Variations in the sperm proteome composition were shown to be dependent on fertility. These proteins were associated with important metabolite pathways. Additionally, correlations of these bands with various spermatological parameters were revealed. The result of the study suggests that seminal plasma proteins play a role in semen quality and may be potential fertility biomarkers.

Seminal plasma proteins (SPPs) significantly influence sperm quality, playing a critical role in fertility. This study aims to investigate the molecular weight (MW) profiles of SPPs in horned and polled Bali bulls and their correlation with sperm quality parameters. Semen samples were collected from six Bali bulls (3 horned, 3 polled). Sperm quality was evaluated based on motility, viability, abnormalities, intact membranes, and acrosomes. SPPs were extracted and analyzed using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis to determine protein MWs. Pearson's correlation was used to analyze relationships between MW profiles and sperm quality metrics. SPPs were identified across a MW range of 15-165 kDa, with specific proteins showing strong correlations with sperm quality. Proteins at 50 and 46 kDa positively correlated with motility (r = -0.96), viability (r = -0.99), and intact membranes (r = -0.86). Conversely, proteins at 40 kDa negatively correlated with these parameters. A 25 kDa protein displayed a positive correlation with intact acrosomes (r = -0.93) and a negative correlation with abnormalities (r = -0.99). Differences in sperm quality metrics between horned and polled bulls were observed, with polled bulls exhibiting fewer abnormalities. This study highlights the potential of SPP MW profiles as biomarkers of sperm quality in Bali bulls. Proteins at 50, 46, and 25 kDa are promising markers for sperm motility, viability, and intact acrosomes, respectively. These findings could inform bull selection and reproductive management strategies. Further research is recommended to validate these biomarkers using advanced proteomic approaches.

Boar sperm protein tyrosine phosphorylation in the presence of egg yolk soluble and low density lipoprotein fractions during cooling

In order to estimate the degree of tubular lesions and to detect a possible glomerular involvement, proteinuria was studied in tubular and/or interstitial nephropathies by determination of total protein and beta-2 microglobulin and by micro-gradientgel electrophoresis. The excretion of albumin, low molecular weight proteins (LMW) and high molecular weight (HMW) proteins was measured in addition. Other proximal tubulus functions such as glucose and phosphate reabsorption were also measured. The clinical entities studied were: chronic postinfectious nephritis, patients with nephropathy following analgesic abuse, tubular glucosuria in pregnancy and cis-platinum treated patients suffering from testicular tumor. The results were interpreted basing on the “selectivity” or urinary protein patterns, which means the relation between LMW and HMW proteins. Micro-gradientgel electrophoresis proved to be a fast and sensitive tool for quantitative as well as qualitative analysis of albumin, LMW and HMW proteins.

In situ immunocytochemical detection of cells containing antibodies specific for Eimeria tenella antigens

Experimental confirmation in rats of the mixed type proteinuria observed in workers exposed to cadmium