The puerperium represents a critical physiological period during which the bovine reproductive tract transitions from pregnancy to renewed fertility. In the modern high-producing dairy cow, this transition is challenged by profound metabolic, endocrine, immunological, and structural demands that collectively influence uterine health, ovarian function, and subsequent reproductive performance. This review examines current understanding of the physiology of the puerperium in dairy cattle, with particular emphasis on uterine involution, immune clearance of postpartum contamination, endocrine regulation, and resumption of ovarian cyclicity. Further, it contrasts high-yielding Holsteins with fertility selected dairy populations. Normal uterine involution involves coordinated myometrial contraction, tissue remodelling, endometrial regeneration, and tightly regulated inflammatory responses. Failure of these processes predisposes cows to postpartum uterine disorders, including retained fetal membranes, metritis, endometritis (purulent vaginal discharge with cytological confirmation), and pyometra, which remain major contributors to subfertility and economic loss. Central to the pathophysiology of puerperal disease is negative energy balance, which disrupts immune competence, alters hepatic steroid metabolism, impairs ovarian signalling, and compromises oocyte and embryo quality. Emerging evidence highlights the complex interplay between metabolism, immunity, and the uterine microbiome, shifting current perspectives away from pathogen-centric models toward host resilience. Advances in biomarkers, genomic selection, and precision monitoring offer new opportunities for targeted reproductive management. Ultimately, optimisation of transition period management remains the cornerstone of supporting physiological puerperal recovery and sustaining reproductive efficiency in modern dairy systems.
Various uterine dysfunctions in non-pregnant and pregnant dogs and cats exist and can have a significant impact on the animal's health and welfare, as well as the offspring in case of pregnant and postpartum animals. This article aims to provide an overview, focusing on pyometra, endometritis, and uterine inertia, mainly in dogs.
Bovine embryo transfer (ET) has been a consistently growing market worldwide with in vitro produced (IVP) embryos now far outnumbering those produced by traditional superovulation. Because transfer of a viable blastocyst-stage embryo to the uterus bypasses many of the potential biological causes of early pregnancy failure associated with artificial insemination (AI; anovulation, sperm transport, fertilization failure, early embryonic death), pregnancy success should logically be higher following ET than following AI. However, this is not always the case. One of the main factors contributing to this paradox is pregnancy loss following ET, particularly with IVP embryos. This greater embryo mortality presents a significant obstacle to more widespread use of IVP embryos. Improvements in embryo culture media as well as in our understanding of the underlying physiological and molecular regulation of early embryo development and embryo-maternal communication leading to a successful pregnancy will significantly contribute to improved reproductive efficiency and sustainability in agri-food production.
Genetic selection is crucial to increase the reproductive performance of stud donkeys. Testicular development and spermatogenesis involve numerous genes, including circular RNAs (circRNAs). However, circRNAs in the male reproductive system of donkeys remain unexplored. This study comprehensively analysed testicular samples from Dezhou donkeys at three different age stages for circRNA using transcriptome data. A total of nine Dezhou donkeys were divided into three groups based on their developmental stages: juvenile (two-month-old, n = 3), adolescent (12-month-old, n = 3) and adult (24-month-old, n = 3) donkeys. We identified 12,084 candidate circRNAs, with 6409 circRNAs specific to the juvenile stage, 6616 to the adolescent stage and 10,031 to the adult stage, respectively. Among these, 3908 circRNAs were expressed across all three age stages. Differential expression analysis revealed 2419 circRNAs exhibiting age-dependent expression changes. Eight circRNAs showed consistent differential expression across all age stages, while specific differential expression patterns were observed between different age pairs. Five representative differentially expressed circRNAs were validated by RT-qPCR, and their expression patterns were consistent with the RNA-sequencing results. Moreover, enrichment analyses of differentially expressed circRNA target genes demonstrated significant enrichment in pathways related to male reproductive processes, including sperm egg recognition and sperm binding to the zona pellucida. Additionally, a circRNA-miRNA-mRNA network, comprising 350 circRNAs, 29 miRNAs and 39 mRNAs, is providing insights into the regulatory mechanisms of non-coding RNAs in testes. In conclusion, circRNA expression patterns are involved in the reproductive biology of testicular development and spermatogenesis in Dezhou donkeys.
This study evaluated boar semen quality, seminal microbiome composition, and antimicrobial susceptibility of bacteria isolated from seminal plasma in a tropical smallholder pig production system in Indonesia. A total of 10 ejaculates were collected from eight sexually mature boars using the gloved-hand technique from two herds located in East Nusa Tenggara (Herd A) and East Java (Herd B). Semen quality was evaluated using conventional methods in Herd A (five ejaculates from three boars) and a mobile semen analysis unit in Herd B (five ejaculates from five boars). Seminal microbiome composition (three ejaculates from three boars in Herd A and five ejaculates from five boars in Herd B) was assessed using next-generation sequencing, and antimicrobial susceptibility of bacterial isolates recovered from seminal plasma (five ejaculates from three boars in Herd A and five ejaculates from five boars in Herd B) was determined using the disk diffusion method. Boar semen quality from Herd A was assessed only for gross motility using subjective observation, yielding a score of 2 out of 3. In Herd B, semen quality was generally high, with a mean ejaculate volume of 232.0 ± 28.6 mL and sperm concentration of 436.9 ± 109.3 × 106/mL. Total and progressive motility were 89.5% ± 6.9% and 84.2% ± 9.5%, respectively, while the proportion of immotile spermatozoa was 10.5% ± 6.9%. Based on 16S rRNA gene sequencing, Chryseobacterium showed the highest relative abundance in semen samples from Herd A, whereas Streptococcus predominated in Herd B. Principal coordinates analysis based on Bray-Curtis dissimilarity demonstrated clear separation of seminal microbiota between herds, with Herd A samples forming a more compact cluster. LEfSe analysis further identified bacterial taxa differentially enriched between herds. Bacterial culture revealed that all semen samples were contaminated with either single or multiple bacterial species, including both Gram-negative and Gram-positive organisms. Escherichia coli and Klebsiella spp. were the most frequently detected isolates. Antimicrobial susceptibility test showed that Herd B exhibited a higher proportion of non-susceptible outcomes (58.3%) compared to Herd A (24.3%). In addition, the proportion of multidrug resistance in Herd A and Herd B were 42.9% and 83.3%, respectively. In conclusion, bacterial contamination in boar semen is unavoidable and may originate from the animal itself or the surrounding environment. Its impact on semen quality may vary depending on the bacterial load and the specific types of microorganisms present. In addition, variations in AMR and seminal microbiota between herds suggest that farm management practices may influence the microbial ecology and resistance patterns in boar semen.
The determination of sperm oxidative stress provides significant information related to semen quality. Most studies investigate oxidative stress markers only at the level of seminal plasma, but this approach may provide an incomplete overview of the oxidative status of semen. The aim of the study is to determine the relationship between protein-normalized oxidative stress markers and spermatological parameters in ram semen fractions. A total of 38 ejaculates obtained from 24 rams were fractionated into seminal plasma, whole semen (non-centrifuged), and pellet at different concentrations. Total antioxidant status (TAS), total oxidant status (TOS), malondialdehyde (MDA), and oxidative stress index (OSI) were measured and normalized to total protein content using the Lowry method. Fraction-wise differences in oxidative stress parameters were analysed using linear mixed-effects models accounting for ram identity, while relationships between spermatological and oxidative parameters were evaluated using Spearman correlation analysis. Principal component analysis (PCA) and hierarchical clustering were performed as exploratory multivariate visualization tools. Motility and viability were positively correlated (ρ = 0.856, p < 0.001), while both parameters were negatively correlated with abnormal sperm rate (p < 0.05). In addition, TOS and MDA levels were significantly lower in seminal plasma than in sperm pellet fractions, supporting the presence of fraction-dependent oxidative profiles (p < 0.05). Exploratory PCA suggested that overall multivariate variation was mainly associated with spermatological parameters, whereas oxidative markers had a more limited contribution. These findings provide insight into the relationship between oxidative status and different semen fractions. Additionally, fraction-specific oxidative profiling may provide complementary information beyond seminal plasma measurements alone.
Cryopreservation of epididymal stallion spermatozoa is a valuable tool for fertility preservation, particularly after orchiectomy. However, the potential interactions between local anaesthesia and extender composition on post-thaw spermatozoal quality remain poorly understood. This study investigated the effects of intratesticular lidocaine injection and the efficacy of various extender (egg yolk [EY] vs. lecithin [LC]) and cryoprotectant (glycerol [GLY] vs. dimethyl sulfoxide [DMSO]) combinations on stallion epididymal spermatological parameters. Using a paired 2 × 2 × 2 factorial design, epididymal spermatozoa were collected from both lidocaine-injected (right) and non-injected (left) testes of eight stallions. Samples were cryopreserved in Tris-based extenders supplemented with EY + GLY, EY + DMSO, LC + GLY or LC + DMSO. Spermatozoal motility (fresh, post-equilibration and post-thaw), viability, plasma membrane and acrosome integrity (PMAI), mitochondrial reactive oxygen species (MROS), spermatozoa malondialdehyde (MDA) and glutathione (GSH) levels were evaluated. Intratesticular lidocaine administration had no significant effect on spermatozoal motility at any evaluation stage, and no differences were detected in any biochemical or spermatologic parameters assessed post-thaw (p > 0.05). Regarding cryopreservation media, a significant main effect of extender type was observed for spermatozoal viability (p < 0.05) and MROS levels (p < 0.001), with EY-based extenders providing superior protection compared to LC-based formulations. A significant Extender × Cryoprotectant interaction was detected for GSH levels (p < 0.05), with the EY + GLY combination yielding the highest antioxidant capacity. No significant differences were observed in MDA levels among treatment groups (p > 0.05). Intratesticular lidocaine administration during orchiectomy does not compromise epididymal spermatozoal quality after cryopreservation. Among the evaluated cryopreservation strategies, the combination of egg yolk and glycerol in a Tris-based extender provides the most effective protection against oxidative stress and membrane damage in stallion epididymal spermatozoa.
Managing aggressive behaviour in bulls often involves a trade-off between handling ease and the anabolic benefits associated with endogenous testosterone production. This study compared the effects of GnRH-immunocastration and a phytogenic feed additive on reproductive parameters, testicular echotexture and behavioural activity in young Holstein bulls aged 12-15 months. Bulls were allocated to either an immunocastration group (IC, n = 6) or a phytogenic feed additive group (PFA, n = 7). The IC group received two subcutaneous injections of 1 mL Bopriva (400 μg GnRH-protein conjugate) at a three-week interval, whereas the PFA group received 50 g/day of a phytogenic feed additive throughout the 90-day experimental period. Evaluations included scrotal circumference (SC), serum testosterone concentrations, ultrasonographic testicular echotexture assessed by mean grey value (MGV) and behavioural observations. Repeated measurements obtained throughout the study were analysed using linear mixed models. Significant group × time interactions (p < 0.001) were observed for SC, testosterone concentrations and MGV. In the IC group, testosterone concentrations and SC progressively decreased beginning on Day 15, while MGV values declined from Day 30 onward. In contrast, the PFA group maintained relatively stable testosterone concentrations and ultrasonographic echotexture throughout the observation period. Qualitative behavioural observations indicated reduced mounting attempts and restlessness in both groups during the follow-up period. Immunocastration induced marked suppression of reproductive parameters and alterations in testicular echotexture in young bulls. Under the conditions of this study, supplementation with the phytogenic feed additive was associated with relatively stable reproductive parameters, while qualitative reductions in behavioural activity were also observed. Further studies including untreated control groups and quantitative behavioural scoring systems are warranted.
South American camelids (SAC), including llamas (Lama glama) and alpacas (Vicugna pacos), exhibit distinctive reproductive features, such as induced ovulation and highly viscous seminal plasma (SP), that complicate conventional semen collection and objective sperm evaluation. Consequently, spermatozoa recovered from the cauda epididymis or through surgical vas deferens deviation (VDD) have become valuable alternatives for research and germplasm preservation. These sperm sources also provide a unique opportunity to investigate sperm physiology in the absence of SP. This review critically compares the functional characteristics of ejaculated, epididymal and VDD-derived spermatozoa and examines how methodological variables, including collection procedures, post-mortem interval, tissue handling and surgical protocols, contribute to variability among studies. Current evidence indicates that many functional differences observed among sperm sources, particularly in motility and plasma membrane functionality, are largely associated with the presence or absence of SP rather than intrinsic differences in sperm quality. These findings support a reinterpretation of SP as a physiological regulator of sperm quiescence, membrane stability and functional competence rather than merely a technical obstacle. We also review advanced analytical approaches, including flow cytometry and imaging flow cytometry, highlighting their potential to improve the evaluation of camelid sperm physiology and cryobiology, while summarizing current evidence on the developmental competence of sperm from different anatomical sources for in vitro embryo production. Overall, this review provides an updated synthesis of the literature, identifies major methodological limitations and highlights priorities for future research to advance reproductive biotechnologies in SAC.
Gestational success in ruminants depends on the maintenance of a dynamic maternal-foetal microenvironment, resulting from complex interactions among the conceptus, extraembryonic membranes, maternal tissues, and gestational fluids. This study performed a comparative and longitudinal proteomic characterization of allantoic and amniotic fluids throughout pregnancy in sheep, aiming to elucidate the molecular mechanisms involved in maternal-foetal communication and embryonic development. Samples of allantoic and amniotic fluids aseptically collected from extraembryonic compartments after uterine opening following animal slaughter and allocated into three gestational groups according to foetal development: early gestation (0-50 days; n = 6), mid gestation (50-100 days; n = 3) and late gestation (100-150 days; n = 5). After centrifugation and protein quantification using the Bradford method, standardized aliquots were subjected to in-solution protein digestion and analysed by nano-LC-ESI-Q-TOF mass spectrometry. A total of 201 proteins were identified in the allantoic fluid and 159 proteins in the amniotic fluid, with five proteins showing significant differences across gestational stages. In the allantoic fluid, differentially abundant proteins included alpha-2-macroglobulin, gelsolin, basement membrane-specific heparan sulphate proteoglycan core protein and alpha-globin chain. In the amniotic fluid, changes were observed in peroxiredoxin-2, mucin-5 AC, fetuin-B, cystatin domain-containing protein and globin domain-containing protein. Multivariate analyses revealed progressive remodelling of the foetal extraembryonic microenvironment, associated with proteolytic control, tissue remodelling, redox homeostasis, epithelial protection and metabolic processes. Collectively, these findings reinforce the active role of foetal extraembryonic fluids in gestational physiology and highlight their potential as sources of biomarkers associated with reproductive success in sheep.
The aim of this study was to evaluate the effect of anethole addition to the ovine oocyte in vitro maturation (IVM) medium on viability and chromatin configuration, intracellular production of reactive oxygen species (ROS), enzymatic activity of superoxide dismutase (SOD) and catalase (CAT), as well as the levels of reduced thiols in IVM medium, in addition to its antioxidant potential (DPPH and ABTS). Cumulus-oocyte complexes (COCs) were randomly matured in IVM medium in the absence (control group, CTRL) or presence of anethole at concentrations of 30 (AN30), 300 (AN300), and 2000 μg/mL (AN2000). COCs were matured in groups under mineral oil at 39°C in a humidified atmosphere containing 5% CO2 in air for 24 h. At the end of IVM, part of the COCs was used to assess viability and chromatin configuration using Hoechst 33,342. The remaining COCs were used to assess intracellular ROS levels using the fluorescent probe H2DCFDA. Furthermore, the IVM medium was collected to evaluate antioxidant status (SOD, CAT, and reduced thiols). Finally, the antioxidant potential of different anethole concentrations was evaluated using DPPH and ABTS assays. It was observed that AN300 promoted higher meiotic resumption rates compared to the other treatments (p < 0.05) and a lower rate of oocyte degeneration compared to AN30 (p < 0.05). However, regarding the proportion of oocytes at MII, it was observed that only AN2000 was lower than AN300 (p < 0.05). Moreover, AN30 resulted in lower ROS levels compared to AN300 and AN2000 (p < 0.05). SOD activity was lower in AN2000 compared to the other treatments (p < 0.05). AN2000 also showed the highest antioxidant capacity in the DPPH and ABTS assays among the anethole concentrations, although it was lower than the control group. In conclusion, anethole supplementation during IVM positively affected oocyte meiotic progression in sheep.
Testicular ultrasonography is increasingly used in ram breeding soundness evaluations, yet methodological standardization of quantitative grayscale image analysis remains limited. The primary objective of this study was to determine the effects of region-of-interest (ROI) placement and imaging plane on testicular parenchymal numerical pixel values (NPVs) under a fixed ultrasonographic acquisition protocol, in Güney Karaman rams. A secondary objective was to compare NPV among age groups and between two predefined reproductive-period sampling points, August and March. Twenty-nine healthy, fertility-proven rams were assigned to three age groups: 1.5 (n = 8), 3.1 (n = 12), and 4.6 years (n = 9). B-mode ultrasonography was performed with a 7.5-MHz linear transducer during the breeding (August) and non-breeding season (March). From each testis, 10 standardized regions of interest were analysed (six longitudinal and four transversal, with five above and five below the mediastinum testis). ImageJ analysis yielded 1160 spot measurements. A linear mixed model with random intercepts for animal and testis ID accounted for the hierarchical repeated-measures structure. NPVs increased with age, were higher in August than in March (71.65 ± 1.75 vs. 60.89 ± 1.75, p < 0.001), and were markedly higher below than above the mediastinum testis (89.50 ± 1.75 vs. 43.03 ± 1.75, p < 0.001). Transversal images yielded higher NPVs than longitudinal images (68.61 ± 1.77 vs. 63.93 ± 1.74, p < 0.001). Random-effects analysis showed that 31%, 7%, and 62% of total variance were attributable to between-animal, between-testis, and spot-level variation, respectively. These findings demonstrate that ram testicular echogenicity is influenced by biological and methodological factors, highlighting the need for standardized multi-site sampling protocols.
The amount and source of nutrition are important factors that influence an ewe's reproductive ability, but the effects of diet on the reproductive performance of its offspring are unclear. This study aimed to investigate the impact of maternal nutrition (level of nutrition and source) on the reproductive performance of ewes and their female lamb offspring. Seventy-two Dorper × Santa Inês ewes were allocated in a completely randomized block design, in five treatments: CTL (n = 14) with 100% of requirement, and the energy source was corn; RES (n = 14) with 90% of predicted requirement, and the energy source was corn; SUPP (n = 15) with 110% of requirement, and the energy source was corn; CR (n = 15) treatment SUPP plus chromium propionate; and FAT (n = 14) treatment SUPP plus calcium salts of palm oil. Non-parametric data were evaluated using the Fisher exact test, and parametric data were evaluated using the Tukey test, both at a 5% significance level. There was no difference in pregnancy rate (p = 0.1944) or prolificacy (p = 0.5729) among the treatments; however, the CR diet resulted in a faster return to estrus after lambing (p < 0.001). The RES diet decreased lamb survival (p < 0.005) and reduced offspring reproductive potential. Using chromium positively affected the reproductive parameters of female offspring, whereas FAT adversely affected them. In conclusion, the source and level of dietary energy influence fetal programming in ewes. The restriction negatively affected the reproductive performance of the offspring, and supplementation with chromium propionate improved it.
Evaluating the reproductive microbiome in small ruminant livestock, such as goats and sheep, is crucial for improving breeding performance, promoting animal health, and optimizing pregnancy outcomes. A healthy microbiome in the reproductive tract inhibits infections, regulates immune activity, and reduces the risk of microbial imbalances that negatively affect pregnancy. Understanding this microbiome also supports better disease control, informs advanced animal husbandry practices for goats and sheep, and contributes to global food security. This study aimed to characterize the genital microbiome of three local Portuguese goat breeds-Preta de Montesinho, Serrana, and Serpentina-using 16S rRNA metabarcoding. Samples were collected from 39 goats and, for comparison, seven sheep from a local breed-Serra da Estrela. Microbiome differences were obtained when integrating breed and sex factors in goats. The predominant genera in both vaginal and preputial samples included Fusobacterium, Aerococcus, Corynebacterium, Sediminibacterium, Campylobacter, and Ralstonia. Significant compositional differences were observed across breed, sex, and species as indicated by Bray-Curtis dissimilarity. By characterizing the caprine reproductive microbiota through metabarcoding, this study fills an important knowledge gap and establishes a baseline for future research on disease-associated changes in the male and female urogenital tract microbiota of small ruminants.
ABSTRACT Advancing liquid storage and cryopreservation technologies for ruminant semen is fundamental to the sustainability and genetic progress of the global livestock industry. Current preservation strategies typically involve three thermal approaches: ambient temperature storage, chilled (5°C) preservation, and cryopreservation (−196°C). However, the efficacy of these methods is frequently undermined by bacterial contamination, which serves as a primary limiting factor that compromises spermatozoal integrity and reduces the shelf‐life of processed semen. The proliferation of bacteria within semen induces significant structural and functional degradation of the spermatozoa. While traditional antibiotic supplementation in cryopreservation media effectively reduces microbial loads, tightening global regulatory restrictions and the emergence of multi‐drug resistance have prompted a shift in preservation strategies. Consequently, the integration of natural bioactive compounds as antimicrobial alternatives offers a sustainable, cost‐effective, and ecologically sound approach for maintaining semen quality and biosecurity. This review critically evaluates the inherent limitations and challenges associated with traditional antibiotic supplementation in the cryopreservation media of various livestock species. Beyond evaluating current shortcomings, we explore the therapeutic potential of natural bioactive molecules as viable antimicrobial substitutes, with a specific focus on their synergistic effects on sperm functionality, ultra‐structural integrity, and metabolic parameters. Additionally, we examine the dynamics of the seminal microbiome, analysing how species‐specific traits, seasonal fluctuations, and environmental variables influence microbial profiles. Ultimately, this review synthesizes the application of natural alternatives as a sustainable and environmentally innovative paradigm for optimizing assisted reproductive technologies and enhancing the global biosecurity of germplasm preservation.
The ZyMōt system is one of the commercially available sperm selection tools based on the microfluidic method. The aim of this study was to evaluate the potential of the ZyMōt system for the selection of canine sperm. Semen samples from six dogs were subjected to selection. Spermatozoa were assessed using basic and advanced methods of semen analysis, including computer-assisted sperm analysis and flow cytometry. Sperm viability, morphology, motility and morphostructural features, such as cell membrane integrity, acrosome integrity and mitochondrial membrane potential, were assessed. The results revealed that the ZyMōt membrane is able to retain dead, slow-moving, immotile and morphologically abnormal spermatozoa. However, no significant improvement was observed in any of the evaluated parameters in the selected sperm population. Moreover, the selection process resulted in substantial losses in the total number of sperm. For this reason, the ZyMōt system is not applicable for dog sperm selection under the currently developed protocol. Further research is necessary to adapt this method for use in this species.
ABSTRACT Cryopreservation of spermatozoa has long been conceptualised primarily as a biophysical preservation challenge. Accumulating evidence, however, reveals a profoundly more complex biological reality: the freeze–thaw cycle constitutes a systems‐level molecular reprogramming event. This review synthesises findings from transcriptomics, proteomics, metabolomics and epigenomics to demonstrate that cryopreservation reshapes the sperm transcriptome and small RNA landscape, induces broad post‐translational modifications, rewires mitochondrial bioenergetics and alters the epigenetic memory encoded in DNA methylation and chromatin architecture. These molecular perturbations converge to compromise not only immediate fertilisation competence but also the developmental trajectories of resulting embryos, with potential intergenerational consequences relevant to domestic animal and human assisted reproduction. Emerging cryoprotective strategies—including mitochondria‐targeted antioxidants, membrane engineering, vitrification and epigenetic priming—are critically evaluated. A unified molecular framework is proposed to guide next‐generation cryoprotection protocols, improve post‐thaw quality assessment beyond motility parameters and inform veterinary and clinical practice in assisted reproductive technologies across domestic animal species.
This study investigated the effects of Spirulina extract, a rich source of bioactive antioxidants, on ram sperm quality during liquid storage. Semen from four mature Lori rams was collected, diluted with a Tris-based extender supplemented with Spirulina extract at concentrations of 0, 10, 20, 30, 40, 50, or 60 μg/mL, and stored at 4°C-5°C. Sperm quality parameters, including motility, viability, plasma membrane integrity, morphology, malondialdehyde (MDA) levels, and antioxidant enzyme activities, were evaluated at 1, 24, 48, 72, and 96 h. A completely randomized design with repeated measures was used, and treatment means were compared using Tukey's HSD test. The 30 μg/mL Spirulina extract (Spir-30) significantly improved total motility (74.17% vs. 63.35% in control, p < 0.05), progressive motility, linearity (28.49%, p < 0.05), straight-line velocity (VSL), and average path velocity (VAP) compared to the control and other concentrations. Plasma membrane integrity was significantly enhanced in Spir-30 (78.77% vs. 58.34% in control, p < 0.05). Additionally, Spir-30 reduced MDA levels (5.32 nmol/mL vs. 7.87 nmol/mL in control, p < 0.05) and increased glutathione peroxidase (GPX) activity (21.35 U/mg vs. 9.28 U/mg in control, p < 0.05) and total antioxidant capacity (TAC; 4.5 mmol/L vs. 3.14 mmol/L in control, p < 0.05). These findings indicate that 30 μg/mL Spirulina extract optimally preserves ram sperm quality during refrigerated storage by enhancing motility, membrane integrity, and antioxidant defences while reducing lipid peroxidation, offering a promising supplement for improving semen longevity in Lori breed reproductive programs.
Sex-specific proteins are unique markers found on either X- or Y-chromosome bearing spermatozoa, acting like molecular signatures, and can be used as a key target to distinguish X- or Y-bearing cells. Towards this goal, by reviewing the literature, a total of 6 proteins were selected from the selected, of which 2, CLRN3 and SCAMP1, were finally chosen for the immunoprecipitation study. For the finally selected proteins, predictions of subcellular localisation and transmembrane topology were generated using online tools such as BUSCA, DeepLoc 2.0, DeepTMHMM, and TMHMM. Additionally, several online platforms like SEMMA 2.0 and IEDB were employed to identify potential immunogenic targets. Following this, commercially available antibodies against the 2 identified proteins were procured for subsequent use in multiple immunoprecipitation-based techniques, such as Western blotting, immunocytochemistry, and ELISA (Enzyme-linked immunosorbent assay). Targeting these proteins revealed their binding intensity on spermatozoa. In this study, the CLRN3 antibody targeting X-spermatozoa showed stronger interaction with X-sorted samples than the Y-specific SCAMP1 antibody in the cell-based ELISA. Results from the study suggest targeting sex-specific surface proteins could enable the development of an immune-based sperm-sorting technique in bovines. Overall, the results support the potential of CLRN3 as an X-specific surface marker that needs further validation for the development of a novel immune-based sperm sorting technique.
Microinjection is a common method for generating gene-edited animals; however, persistent Cas9 activity post-cleavage often results in mosaic embryos due to editing occurring in different blastomeres. This study investigated whether co-injecting the CRISPR/Cas9 system with the small-molecule Cas9 inhibitor BRD0539, or supplementing it in the culture medium, could reduce mosaicism while maintaining editing efficiency in porcine parthenogenetic activation embryos targeting the myostatin (MSTN) gene. The findings are as follows: Co-injection of 10 or 100 μM BRD0539 with Cas9 mRNA: sgRNA significantly reduced gene editing efficiency (28.5% ± 11.6% and 33.8% ± 4.1%, respectively, vs. 86.9% ± 4.5% in control, p < 0.05). Supplementing the culture medium with 10 or 50 μM BRD0539 also reduced both editing efficiency (20.8% ± 12.4% and 47.7% ± 14.6%, respectively, vs. 85.6% ± 4.8%) and mosaicism rate (25.0% ± 15.9% and 12.5% ± 12.5%, respectively, vs. 87.1% ± 7.8%, p < 0.05). Immunofluorescence revealed sustained Cas9 protein expression up to 48 h post-injection. Crucially, short-term addition of 10 μM BRD0539 to the culture medium between 24 and 48 h post-activation significantly reduced mosaicism (32.6% ± 7.5% vs. 78.7% ± 9.6%, p < 0.05) without compromising editing efficiency. Furthermore, this treatment did not adversely affect cleavage rates, blastocyst development, total cell number. These results demonstrate that transient inhibition of Cas9 activity with 10 μM BRD0539 during a critical window effectively reduces mosaicism in microinjected porcine embryos, offering a promising strategy to enhance the efficiency of generating non-mosaic gene-edited livestock.
