Assessing lameness prevalence and associated risk factors in crossbred dairy cows across diverse management environments.

BACKGROUND: A thorough understanding of lameness prevalence is essential for evaluating the impact of this condition on the dairy industry and assessing the effectiveness of preventive strategies designed to minimize its occurrence. Therefore, this cross-sectional study aimed to ascertain the prevalence of lameness and identify potential risk factors associated with lameness in Holstein Friesian crossbred cows across both commercial and smallholder dairy production systems in Bengaluru Rural District of Karnataka, India. METHODS: The research encompassed six commercial dairy farms and 139 smallholder dairy farms, involving a total of 617 Holstein Friesian crossbred cattle. On-site surveys were conducted at the farms, employing a meticulously designed questionnaire. Lameness in dairy cattle was assessed subjectively using a locomotion scoring system. Both bivariate and binary logistic regression models were employed for risk assessment, while principal components analysis (PCA) was conducted to address the high dimensionality of the data and capture the underlying structure of the explanatory variables. RESULTS: The overall lameness prevalence of 21.9% in commercial dairy farms and 4.6% in smallholder dairy farms. Various factors such as age, body weight, parity, body condition score (BCS), floor type, hock and knee injuries, animal hygiene, provision of hoof trimming, and the presence of hoof lesions were found to be significantly associated with lameness. Binary logistic regression analysis indicated that the odds of lameness in crossbred cows increased with higher parity, decreased BCS, presence of hard flooring, poor animal hygiene, and the existence of hoof lesions. These factors were identified as potential risk factors for lameness in dairy cows. Principal component analysis unveiled five components explaining 71.32% of the total variance in commercial farms and 61.21% in smallholder dairy farms. The extracted components demonstrated higher loadings of housing and management factors (such as hoof trimming and provision of footbath) and animal-level factors (including parity, age, and BCS) in relation to lameness in dairy cows. CONCLUSIONS: The findings suggest that principal component analysis effectively reduces the dimensionality of risk factors. Addressing these identified risk factors for lameness is crucial for the strategic management of lameness in dairy cows. Future research in India should investigate the effectiveness of management interventions targeted at the identified risk factors in preventing lameness in dairy cattle across diverse environments.

Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

Sperm proteomic landscape is altered in breeding bulls with greater sperm DNA fragmentation index.

Although, it is well understood that sperm DNA damage is associated with infertility, the molecular details of how damaged sperm DNA affects fertility are not fully elucidated. Since sperm proteins play an important role in fertilization and post-fertilization events, the present study aimed to identify the sperm proteomic alterations in bulls with high sperm DNA Fragmentation Index (DFI%). Semen from Holstein-Friesian crossbred breeding bulls (n = 50) was subjected to Sperm Chromatin Structure Assay. Based on DFI%, bulls were classified into either high- (HDFI; n = 6), or low-DFI (LDFI; n = 6) and their spermatozoa were subjected to high throughput proteomic analysis. Liquid chromatography and mass spectrometry analysis identified 4567 proteins in bull spermatozoa. A total of 2660 proteins were found common to both the groups, while 1193 and 714 proteins were unique to HDFI and LDFI group, respectively. A total of 265 proteins were up regulated and 262 proteins were down regulated in HDFI group. It was found that proteins involved in capacitation [heparin binding (molecular function), ERK1 and ERK2 cascade (biological process), PI3K-Akt signalling (pathway), Jak-STAT signalling (pathway)], spermatogenesis [TLR signalling (pathway), gamete generation (biological process)] and DNA repair mechanism (biological process) were significantly altered in the bulls with high DFI%.

High-throughput proteomic characterization of seminal plasma from bulls with contrasting semen quality.

Seminal plasma proteins are the major extrinsic factors that can modulate the sperm quality and functions. The present study was carried out to compare the proteomic profiles of seminal plasma from breeding bulls producing good and poor quality semen in an effort to understand the possible proteins associated with semen quality. A total of 910 and 715 proteins were detected in the seminal plasma of poor and good quality semen producing bulls, respectively. A total of 705 proteins were common to both the groups, in which 380 proteins were upregulated and 89 proteins were downregulated in the seminal plasma of poor quality semen, while 236 proteins were co-expressed. The proteins negatively influencing sperm functions such as CCL2, UQCRC2, and SAA1 were among the top ten upregulated proteins in the seminal plasma of poor quality semen. Proteins having a positive role in sperm functions (NGF, EEF1A2, COL1A2, IZUMO4, PRSS1, COL1A1, WFDC2) were among the top ten downregulated proteins in the seminal plasma of poor quality semen. The upregulation of oxidation-reduction process-related proteins, histone proteins (HIST3H2A, H2AFJ, H2AFZ, H2AFX, HIST2H2AB, H2AFV, HIST1H2AC, HIST2H2AC, LOC104975684, LOC524236, LOC614970, LOC529277), and ubiquinol-cytochrome-c reductase proteins (UQCRB, UQCRFS1, UQCRQ, UQCRC1, UQCRC2) indicate deranged oxidation-reduction equilibrium, chromatin condensation and spermatogenesis in poor quality semen producing bulls. The expression of proteins essential for motile cilium (CCDC114, CFAP206, TEKT4), chromatin integrity (PRM2), gamete fusion (IZUMO4, EQTN), hyperactivation, tyrosine phosphorylation, and capacitation [PI3K-Akt signalling pathway-related proteins (COL1A1, COL2A1, COL1A2, SPP1, PDGFA, NGF)] were down regulated in poor quality semen producing bulls. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03474-6.

Optimized addition of nitric oxide compounds in semen extender improves post-thaw seminal attributes of Murrah buffaloes.

Semen dilution and cryopreservation alter the homogeneity of seminal plasma, resulting in a non-physiological redox milieu and consequently poor sperm functionality. Considering the concentration-specific bimodal action of nitric oxide (NO) in the regulation of sperm functions, cryopreservation media supplemented with optimized concentrations can improve the semen attributes. The present study aimed to evaluate the effect of adding an optimized concentration of sodium nitroprusside (SNP) and N-nitro-L-arginine methyl ester (L-NAME) in an extender on in vitro semen quality. An aliquot of semen samples (n = 32) from Murrah buffalo bulls (n = 8) was divided into control (C) and treatment (T-I: SNP in extender at 1 µmol/L; T-II: L-NAME in extender at 10 µmol/L). Fresh semen quality parameters showed no significant difference at 0 h except for the structural integrity in the T-II group. Post-thaw semen quality parameters and sperm kinematics using computer-aided sperm analysis (CASA) revealed significantly higher (p < 0.05) cryoresistance in the treatment groups. Viability, acrosome integrity, and membrane integrity were significantly higher (p < 0.05) in both treatment groups; however, the results were pervasive in T-II. Lower abnormal spermatozoa were observed in both T-I and T-II. SNP supplementation led to a significant rise (p < 0.05) in NO, whereas L-NAME reduced the NO concentration in post-thawed samples, which was directly correlated with different sperm functionality and associated biomarkers viz. total antioxidant capacity (TAC) and thiobarbituric acid reactive substance (TBARS). It was concluded that the cryopreservation media supplemented with SNP and L-NAME at 1 µmol/L and 10 µmol/L, respectively, lower the cryo-damage and improve post-thaw seminal attributes.

Filtration techniques are advantageous over colloidal centrifugation in improving freezability of low-quality buffalo bull (Bubalus bubalis) ejaculates.

The study compared efficacy of three sperm selection techniques in improving freezability of low-quality Murrah buffalo bull ejaculates. Sephadex (SEP), Sephadex ion-exchange filtration (SIE), and 40/80% BoviPure™ (BP) gradient centrifugation protocols were standardized (ejaculates, n = 24). In Experiment-I, Sephadex G-75, G-100, and combined Sephadex G (75-100) column filtrates were compared. In Experiment-II, BP protocols: 200 g-10 min, 250 g-5, and 10 min, 300 g-10, and 15 min were compared. In fresh semen, Sephadex G (75-100) filtration and 250 g-5 min BP protocol improved sperm functions and were used in Experiment-III, where SEP G (75-100), SIE G (75-100), and 250 g-5 min BP processed ejaculates (n = 48) were cryopreserved and compared at post-thaw stage. The mean recovery rate differed in order: SEP > SIE > BP. SIE filtration significantly improved progressive motility, livability, membrane integrity, bovine cervical mucus penetration and live non-apoptotic sperm. Compared with control, all three techniques equally reduced post-dilution and post-thaw lipid peroxidation (LPO) rate. SEP post-thaw filtrates observed lower cryocapacitation-like changes, LPO (C11-BODIPY581/591), and higher active mitochondria than other treatments. SIE and SEP equally improved post-thaw acrosome-intact sperm over BP. Filtration techniques, preferably, Sephadex ion-exchange filtration can most efficiently process low-quality buffalo bull ejaculates for cryopreservation and improve freezability.

Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus × Bos indicus) bull spermatozoa.

Spermatozoa from high-fertile (HF) and low-fertile (LF) breeding bulls were subjected to high-throughput next-generation sequencing to identify important Single nucleotide polymorphisms (SNPs) and novel variants associated with fertility. A total of 77,038 genome-wide SNPs were identified, among which, 10,788 were novel variants. A total of 42,290 and 34,748 variants were recorded with 6115 and 4673 novel variants in in HF and LF bulls, respectively. Higher number of SNPs were identified in HF compared to LF bulls. GO analysis of filtered genes with significant variations in HF bulls indicated their involvement in oxidative phosphorylation and metabolic pathways. GO analysis of filtered genes with significant variation in LF bulls revealed their involvement in Ca2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly. The study identified SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13, and RPS27A, which are linked to sperm functionality, survival, oxidative stress, and bull fertility. The identified SNPs could be used in selection of bulls for high fertility and the variation in these genes could be established as an explanation for the fertility differences in bulls upon validation in large number of bulls.

Genom-wide analysis identifies single nucleotide polymorphism variations and altered pathways associated with poor semen quality in breeding bulls.

The reason for poor semen quality among the breeding bulls is not well understood. In the present study, we performed high-throughput RNAseq analysis of spermatozoa to identify the SNPs present in good and poor-quality semen-producing Holstein Friesian breeding bulls. A total of 21,360 and 44,650 SNPs were identified in good and poor-quality semen with a minimum read depth of 20, among which 4780 and 8710 novel variants were observed in good and poor-quality semen, respectively. Greater SNPs and indels variations were observed in poor compared to good-quality semen. In poor-quality semen, SNP variations were observed in ZNF280B, SLC26A2, DMXL1, OR52A1, MACROD2 and REV1 genes, which are associated with regulation of spermatogenesis, post-testicular maturation, Cl- channel activity, V-ATPase-mediated intracellular vesicle acidification, a mono-ADP-ribosyl hydrolase and ATR-Chk1 checkpoint activation. GO analysis of filtered genes with significant variations between good and poor-quality semen showed enrichment in important pathways related to semen quality such as MAPK signalling pathway, Akt signalling pathway, focal adhesion, cAMP signalling pathway, and Rap1 signalling pathway. Network analysis of filtered genes in poor-quality semen showed variations in pathways of purine metabolism, pyrimidine metabolism, prolactin signalling pathway and RNA cap-binding complex. It is inferred that SNP in genes involved in maintaining sperm functions could be the reason for poor-quality semen production in bulls, and the identified SNPs hold potential to be used as biomarkers for semen quality in bulls.

High throughput deep proteomic analysis of seminal plasma from stallions with contrasting semen quality.

Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility (n = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low- motile (LM) sperm of stallions, respectively. A total number of 1436 proteins were co-expressed in both the groups; 191 (11%) and 60 (3.5%) proteins were exclusively detected in HM and LM groups, respectively. A total of 220 proteins were upregulated (>1-fold change) and 386 proteins were downregulated in SP from LM group stallions as compared to HM group stallions, while 830 proteins were neutrally expressed in both the groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed dysregulation of the important proteins related to mitochondrial function, acrosome, and sperm cytoskeleton in the seminal plasma of stallions producing ejaculates with low sperm motility. High abundance of peroxiredoxins and low abundance of seminal Chaperonin Containing TCP1 Complex (CCT) complex and Annexins indicate dysregulated oxidative metabolism, which might be the underlying etiology for poor sperm motility in LM group stallions. In conclusion, the current study identified the seminal plasma proteomic alterations associated with poor sperm motility in stallions; the results indicate that poor sperm motility in stallions could be associated with altered expression of seminal plasma proteins involved in oxidative metabolism.

Implications of cryopreservation on structural and functional attributes of bovine spermatozoa: An overview.

Sperm cryopreservation is an important adjunct to assisted reproduction techniques (ART) for improving the reproductive efficiency of dairy cattle and buffaloes. Improved understanding of mechanisms and challenges of bovine semen cryopreservation is vital for artificial insemination on a commercial basis. Although cryopreservation of bovine spermatozoa is widely practiced and advanced beyond that of other species, there are still major gaps in the knowledge and technology. Upon cryopreservation, disruption of spermatozoal plasma membrane configuration due to alterations in metabolic pathways, enzymes and antioxidants activity add to lower efficiency with loss of sperm longevity and fertilising ability. Therefore, the effective amalgamation of cryo-variables like ambient temperature, cooling and thawing rates, nucleation temperature, type and concentration of the cryoprotectant, seminal plasma composition, free radicals and antioxidant status are required to optimise cryopreservation. Novel strategies like supplementation of cholesterol-loaded cyclodextrins (CLC), nanovesicles, osteopontin, antioxidants, etc., in an extender and recent techniques like nano-purification and modified packaging have to be optimised to ameliorate the cryodamage. This article is intended to describe the basic facts about the sperm cryopreservation process in bovine and the associated biochemical, biophysical, ultra-structural, molecular and functional alterations.