Juvenile in vitro embryo transfer can shorten generation intervals, but its application in goats is constrained by the quantity-quality trade-off in oocytes from prepubertal donors. We established an age-specific workflow for Yudong black goats by separately optimizing ovarian superstimulation in juvenile and adult donors, evaluating anti-Müllerian hormone (AMH) as an exploratory indicator of ovarian response, improving in vitro maturation (IVM), and optimizing somatic cell nuclear transfer (SCNT). Under the selected juvenile protocol (1-2 months, 220 IU FSH plus 330 IU PMSG, collection 12 h after the final FSH injection), donors yielded 113.4 ± 10.9 follicles and 73.5 ± 6.3 cumulus-oocyte complexes, with 35.9 ± 0.9% Grade A/B oocytes. Pre-stimulation serum AMH was positively associated with follicle number; an exploratory cutoff of 4.54 ng/mL predicted ≥ 90 follicles (AUC 0.906; sensitivity 87.5%; specificity 80.0%). Supplementing IVM medium with 20% adult follicular fluid increased juvenile oocyte maturation from 53.7 ± 0.8% to 62.2 ± 0.8% and reduced intracellular reactive oxygen species. Optimal electrofusion voltages were 18 V for juvenile and 21 V for adult cytoplasts. Adult-derived parthenogenetic embryos had higher blastocyst rates than juvenile-derived embryos. SCNT embryos were transferred at the 2-4-cell stage; therefore, no in vitro SCNT blastocyst endpoint was generated. Preliminary Day-45 pregnancy establishment was 1/6 recipients for each cytoplast source. These findings support an age-specific, conservation-oriented workflow while emphasizing the need for independent AMH validation, minimally invasive oocyte recovery, and larger full-term SCNT studies.
Efficient superovulation protocols are essential to improve efficiency and facilitate scheduling in porcine embryo transfer (ET) programs. Although hCG is widely used to synchronize ovulation following eCG-based superovulation, constraints in its availability have highlighted the need for hCG-free strategies. GnRH agonists could expand ovulation induction options within eCG-based superovulation protocols. In this study, multiparous weaned donor sows received eCG alone (1000 IU, 24 h post-weaning), eCG plus buserelin, a GnRH agonist (10 µg at estrus onset), or no hormonal treatment (control), and embryos were recovered on Day 6 after estrus to evaluate ovulatory response, embryo production efficiency, developmental synchrony, and blastocyst transcriptomic profiles. Both hormonal protocols independently increased ovulatory response by approximately 48% (~37 vs 25 corpora lutea; P < 0.001) and doubled transferable embryo production efficiency compared with controls (P < 0.001), without affecting pregnancy and fertilization rates, embryonic viability, total cell number, or apoptosis. Untreated sows showed the greatest developmental dispersion and a higher proportion of hatched blastocysts, whereas eCG-treated sows showed intermediate variability. The addition of buserelin further improved ovulation-embryo synchrony, with most embryos at the blastocyst stage and all viable embryos classified as transferable. Transcriptomic profiling identified a limited number of differentially expressed genes between treated and control embryos. In conclusion, both hCG-free treatments increased embryo production in weaned sows without adversely affecting the Day-6 blastocyst quality parameters assessed, whereas the addition of buserelin further improved embryo-stage synchrony at collection. Overall, both protocols represent practical hCG-free strategies for in vivo porcine embryo production under commercial conditions.
This study examined interactions between cryoprotectant (CPA) composition and processing factors (i.e., pre-freeze cooling rate, egg yolk concentration, cryopreservation step, final sperm concentration) that may influence post-thaw quality of stallion sperm. In Experiment 1, 24 ejaculates were cryopreserved in extenders with either 20% egg yolk (EY) or 2% egg yolk with added milk proteins (SMEY) and containing either glycerol (Gly) and methyl formamide (MF; Gly + MF), methyl formamide (MF), or dimethylformamide (DF). Post-thaw sperm quality was assessed via computer-assisted motion analysis and flow cytometry. An interaction between egg yolk concentration (EY vs. SMEY) and CPA (Gly+MF vs. MF vs. DF) was observed, whereas an effect of pre-freeze cooling rate (-0.1 vs. -1 °C/min) was not detected. Sperm cryopreserved in EY-Gly + MF exhibited the highest sperm motility parameters and plasma membrane integrity values, as well as the lowest levels of apoptotic and oxidative stress-associated changes. In Experiment 2 (n = 24 ejaculates), sperm quality was not affected immediately after CPA addition (Gly vs. MF vs. Gly+MF) or pre-freeze cooling but decreased significantly after freezing and thawing. Post-thaw motility and viability were similar between Gly and Gly + MF, both higher than for MF. Exposure to hyperosmotic (CPA-containing) or isosmotic (CPA-free) media during analysis at every step of the cryopreservation process had no impact on sperm quality. In Experiment 3 (n = 24 ejaculates), sperm cryopreserved at 200, 400, or 800 × 106 sperm/mL exhibited higher post-thaw motility and viability than sperm cryopreserved at 50 or 100 × 106 sperm/mL, regardless of CPA.
Sex identification in teleosts with limited external sexual dimorphism remains a major challenge for aquaculture and reproductive management. To address this limitation, Pimelodus maculatus was used as a model to evaluate the potential of circulating sex steroids, including 17β-estradiol (E2), testosterone (T), and 11-ketotestosterone (11-KT), and the hormonal ratios T/E2 and 11-KT/E2 as minimally invasive biomarkers for sex identification. Females and males maintained in a recirculating aquaculture system (RAS) were evaluated at two sampling time points, in July and December 2024. Individual hormones showed limited or variable discriminatory performance between sampling time points, whereas the T/E2 ratio yielded the highest numerical classification metrics, with substantial to almost perfect agreement with ultrasonographic sex determination. The 11-KT/E2 ratio also showed discriminatory potential but exhibited greater variation between sampling time points. Histological analysis of a complementary group showed gonadal characteristics consistent with sexually mature adults that had not reached advanced stages of gonadal development, providing physiological context for interpreting the endocrine profiles under the experimental conditions. Overall, these findings demonstrate the potential of circulating endocrine indicators for minimally invasive sex identification in P. maculatus and support further investigation of the T/E2 ratio as a candidate composite endocrine indicator.
Semen cryopreservation is an invaluable tool for facilitating artificial insemination and preserving genetic resources in many animal species. However, semen cryopreservation is often less successful in poultry than in mammals, due to unique biological and physiological features of avian semen. Sperm cryopreservation promotes ice formation and osmotic stress, producing reactive oxygen species (ROS); furthermore, the high content of polyunsaturated fatty acids in poultry sperm cell membranes renders these cells particularly susceptible to lipid peroxidation. To ensure proper functioning of cells, it is necessary to have an appropriate balance between ROS formation and protective actions of the antioxidant system. The objective of this review is to discuss molecular and cellular aspects of rooster sperm, the antioxidant system in rooster semen, and adding antioxidants to extenders to improve rooster semen quality and fertility and to delay or prevent age-related declines in broiler breeder roosters, with benefits for poultry production.
Steroid hormones regulate ovarian development, but hsd3b7 function in fish remains unclear. In this study, we cloned full-length hsd3b7 cDNA from Qihe gibel carp (Carassius gibelio var. Qihe) and generated a polyclonal antibody. Real-time PCR, Western blotting, and immunohistochemistry revealed widespread hsd3b7 expression in brain, heart, spleen, muscle and ovary. During ovarian development and maturation, hsd3b7 mRNA expression peaked at 150 days after hatching (dah) and remained at a relatively high level until 720 dah. Immunohistochemical analysis further revealed that Hsd3b7 protein was localized in both gonadal germ cells and somatic cells, suggesting its involvement in ovarian development and maturation. To further investigate its function, hsd3b7 was knocked down for 30 days by ovarian injection of double-stranded RNA (dsRNA) during the previtellogenic stage. Silencing of hsd3b7 markedly inhibited ovarian development, with most oocytes remaining at phase II. In addition, the expression levels of the germ cell marker genes vasa and piwil, as well as the cell proliferation-related genes pcna and ki-67, were significantly reduced. Protein interaction analysis suggested that Hsd3b7 is associated with several steroidogenic enzymes, including Cyp19a1a, Cyp11c1, Hsd17b3, and Hsd17b7. Moreover, hsd3b7 knockdown altered the expression of genes involved in steroidogenesis and lipid accumulation, and significantly affected serum E2 and total cholesterol levels. Collectively, these findings indicate hsd3b7 regulates ovarian development and maturation via steroid hormone synthesis and lipid metabolism, providing insights for addressing precocious maturation in Qihe gibel carp.
The present study investigated the dose- and time-dependent effects of synthesized calcium oxide (CaO) and silver (Ag) nanoparticles (NPs) on sperm quality parameters and their antimicrobial efficacy during liquid preservation of boar semen. The synthesized NPs were characterised using standard analytical techniques, which confirmed their nanoscale size and stability. Preliminary cytotoxicity screening revealed that CaO-NPs at 10-1000 µM were non-cytotoxic to spermatozoa, thereby maintaining progressive motility, plasma membrane integrity (PMI), and mitochondrial membrane potential (MMP) during incubation at 37°C, while higher concentrations (≥5000 µM) were cytotoxic. Supplementation of CaO-NPs (50 and 100 µM) in Modena extender during liquid preservation at 16°C significantly (P ≤ 0.05) improved sperm motility, PMI and MMP for up to 10 days in comparison to control (CON). Similarly, Ag-NPs tested at 0.01-10 µM showed dose- and time-dependent effects; 10 µM concentration exhibited cytotoxicity, whereas 0.01-1 µM were non-toxic. Supplementation of 0.01 µM Ag-NPs significantly improved (P ≤ 0.05) sperm motility and PMI up to 10 days of preservation, maintaining MMP until day 7, while 0.1 µM preserved sperm quality parameters up to day 5 of preservation. Both CaO-NP and Ag-NP treatments markedly reduced colony-forming units (CFUs) by more than 1000-fold compared with the negative CON. However, neither NP treatment achieved antibacterial efficacy comparable to the antibiotic-treated positive CON. Overall, the study highlights the dual role of CaO and Ag NPs, as biocompatible and effective additives for enhancing boar semen preservation. By improving sperm quality and reducing microbial growth, these NPs may point towards decreased reliance on antibiotics in semen extenders. These findings provide new insights into the safe and functional application of nanomaterials in reproductive biotechnology.
The success of spermatogenesis depends on the interplay of various biomolecules that ultimately determine sperm quality. In this study, RNA-seq analysis of frozen-thawed buffalo sperm (n = 8) revealed the presence of 263 mineral-associated genes (>1 FPKM) in high (n = 4) and 181 in low motile (n = 4) sperm groups. Among these, 177 mineral-associated genes were commonly expressed between them, and the majority were upregulated (>1 fold), LOC102391588 (ncRNA; 37-fold), ZNF699 (26.5-fold), MYZAP (13-fold), etc., in the high motile group. The expression of selected mineral-associated genes was validated. The top enriched functions in commonly expressed genes were regulation of transcription by RNA polymerase II (FDR: 4.7 × 10⁻2; ZNF331, ZNF692, ZNF180, etc.), followed by spermatogenesis (FDR: 2.9 × 10⁻2; CALR3, ADAM18, ADAM29, etc.), proton transmembrane transport (FDR: 4.0 × 10⁻2; ATP6V0E1, ATP1A4, ATP6V1B2, etc.) and flagellated sperm motility (FDR: 2.3 × 10⁻1; CATSPERD, EFCAB6, CABS1, etc.). Additionally, the chromatin remodeling pathway (FDR: 3 × 10⁻2; PTP4A1, PPM1A, DUSP1, etc.) emerged as the most significant and may suggest that these minerals influence genome packaging and sperm functionality. Mineral-associated genes were predominantly associated with zinc (49%), followed by calcium (20%), phosphorus (10%), iron (5%), sodium (2%), potassium (2%), copper (1%) and other trace elements (11%). Although the current study uses frozen-thawed sperm, the findings indicate that mineral-associated genes are crucial for promoting membrane stability, energy production, motility and chromatin integrity, which may contribute to the superior fertilizing ability of sperm.
Sperm morphology is a critical predictor of fertility across a range of species; however, its assessment remains inherently subjective and thus susceptible to variability and inaccuracy. Morphological abnormalities can present in diverse forms, with over 25 distinct categories recognised in the current literature. These abnormalities may arise during spermatogenesis due to stressors, as well as post-maturation during epididymal storage, ejaculation, or as a consequence of suboptimal handling during processing. Researchers have long sought to characterise these abnormalities through classification systems. Numerous classification frameworks have been developed to categorise observed defects. However, inconsistencies in the classification of specific abnormalities have led to disputes among experts. Preferences for particular classification schemes vary among laboratories, species, industries, and geographic regions, resulting in a lack of standardisation that hampers comparability and contributes to considerable inter- and intra-observer variability. While advancements in automation have begun to emerge, subjective visual assessment remains the predominant method of evaluation. This reliance, combined with inconsistent classification standards, absence of formal training, and inherent human bias, continues to drive substantial variability. In response, recent research has turned to novel technologies such as machine learning to achieve objective, scalable assessment. However, training these models necessitates high-quality training data -something difficult to obtain given the challenges of standardising subjective assessments. To preserve the utility of sperm morphology as a useful indicator of fertility, it is essential that future efforts focus on improving assessor training and standardisation protocols, thereby reducing variability while the development of automated, objective technologies continues to progress.
Apoptosis of mammalian granulosa cells (GCs) is considered to be the main cause of follicular atresia. MiRNA-mediated post-transcriptional gene regulation plays a pivotal role in cell apoptosis. Our previous studies demonstrated that miR-424 is highly expressed in atretic follicles and may promoter of buffalo follicular atresia. In this study, we investigated the role and molecular mechanisms of miR-424 in buffalo follicular atresia. GCs apoptosis, proliferation and estrogen secretion were assessed by flow cytometry, EdU and ELISA, respectively. The expression levels of relevant genes were measured by qRT-PCR. Last, miR-424 agomir and antagomir was administered to mice via tail vein injection. The results showed that miR-424 promotes follicular atresia by inducing cell cycle arrest, inhibiting GC proliferation, suppressing the secretion of estradiol, and enhancing apoptosis. SERPINB2, as a target gene of miR-424, inhibits follicular atresia by promoting GCs proliferation, hormone secretion and inhibiting GCs apoptosis. Overexpression of miR-424 or silencing of SERPINB2 significantly reduced the number of follicles at various developmental stages in mice, whereas inhibition of miR-424 yielded opposite effects. In conclusion, our findings establish miR-424 as a key regulator of follicular atresia in buffalo, through direct targeting of SERPINB2 to modulate granulosa cell functions.
Erythropoietin (EPO) is primarily known for its role in erythropoiesis. However, recent data confirm the presence and function (autocrine or paracrine) of EPO in human and rat testicular tissue and seminal plasma, suggesting a role in reproductive physiology. Moreover, a cytoprotective and antioxidant role of EPO has been documented in thawed bovine semen. Based on these, the present study aims to explore the possible effect of EPO on various parameters of fresh ram spermatozoa during prolonged cold storage. Semen from five Chios rams was supplemented with EPO at two concentrations [10 (EPO 10) and 100 mIU/μl (EPO 100)] or left untreated (control) and stored at 5°C for up to 3 days. Sperm motility, viability, oxidative status, apoptotic markers, and heat shock response were evaluated. EPO, and more specifically the 100 mIU/μl concentration, significantly mitigated the decline in motility parameters from Day 1 to Day 3 compared to the control. Computer Assisted Sperm Analyzer (CASA) parameters and glutathione (GSH) levels were better preserved, indicating maintenance of flagellar activity, membrane integrity, and antioxidant capacity. In addition, apoptotic signaling, as witnessed by a Bax/Bcl-2 ratio decrease, and Heat Shock Protein (Hsp70) expression were modulated by EPO, consistent with the potential involvement of cytoprotective pathways. These findings offer new insights into the role of EPO in the field of reproductive physiology, indicating that EPO helps ram spermatozoa to maintain their homeostasis.
Timely and efficient degradation of maternal mRNA is essential for early embryonic development, which occurs from fertilization through the initiation of zygotic genome activation (ZGA). Yet, the regulatory mechanisms governing this process remain poorly characterized. In the present study, we investigated the function of CCR4-NOT transcription complex subunit 1 (CNOT1) during goat embryogenesis. We found that CNOT1 was upregulated during mammalian ZGA, and that its knockdown led to developmental arrest and a marked reduction in blastocyst formation. Moreover, CNOT1 knockdown impaired nascent RNA activity, resulting in 814 upregulated and 1014 downregulated genes, which were enriched for RNA splicing, regulation of chromosome organization, and RNA localization. RNA splicing analysis revealed differential splicing events in 2959 genes, of which 259 were downregulated following CNOT1 knockdown. Notably, CNOT1 was predicted to crosstalk with the m6A reader YTHDF2. Knockdown of YTHDF2 resulted in CNOT1 downregulation at the 8-cell stage in goats and increased transcription levels around polyadenylation sites during ZGA in mice. Together, these findings indicate that CNOT1 is a potential YTHDF2 target that orchestrates maternal mRNA decay and ZGA during goat embryogenesis. Our work provides new insight into the complex regulatory landscape underlying ZGA and may inform strategies to improve the efficiency of goat embryogenesis.
Sperm cryodamage is a key factor affecting its post-thaw quality. This study aims to systematically elucidate the molecular response mechanisms of bovine sperm cryodamage by integrating transcriptomic and proteomic analyses. By utilizing the swim-up technique to isolate sperm subpopulations with mild and severe cryodamage, thereby minimizing interference from individual genetic variation. Results demonstrated that mildly damaged sperm exhibited superior motility, structural integrity, and antioxidant capacity, along with less reactive oxygen species accumulation and more active Wnt, mTOR, and Hippo signaling pathways at the transcriptional level. Severely damaged sperm exhibited activation of pathways related to oxidative phosphorylation, thermogenesis, peroxisome, glutathione metabolism, phagocytosis, and DNA repair at the protein level, with ATP5MG, GPX1, XRCC1, and AP1M2 serving as core regulatory factors in these pathways. Additionally, integrated analysis identified eight potential biomarker proteins, including apoptosis-related protein ARL6IP5, anti-apoptotic protein GRINA, adhesion factors CDHR1/LGALS1, DNA repair proteins CUL4B/POLR2E, and core factors RPL14/A1BG in the protein-protein interaction network. Notably, the protein levels of ARL6IP5, CDHR1, and LGALS1 were inconsistent with their gene expression levels. These candidate molecules showed significant correlations with post-thaw sperm quality and were quantitatively validated. The results indicate that mitochondrial energy metabolism, antioxidant and anti-apoptotic defense systems, as well as self-repair and clearance responses, including nucleic acid repair and phagocytosis, represent the molecular response characteristics of sperm cryodamage. These findings complement the molecular mechanisms and regulatory networks of sperm cryodamage, and provide potential targets for evaluating the cryopreservation potential of bull semen and optimizing cryoprotectants.
This review critically reflects on the use of immunocrit in neonatal piglets as an indicator of passive immunity status. Under intensive production conditions and with the development of hyperprolific sow lines, reliable techniques are required to objectively assess the success of passive immunity transfer to the piglets. The immunocrit is a cost-effective indirect method for assessing immunoglobulin concentration in serum or plasma samples obtained 12-48 h after the birth of the first piglet, by using the principle of protein precipitation. Reduced growth performance, delayed development, greater vulnerability to illness, and higher pre-weaning mortality are all consistently associated with low immunocrit readings. Higher numbers, on the other hand, indicate effective immunoglobulin absorption and better long-term productivity. Various management strategies, including split suckling, cross-fostering, and farrowing system design, directly influence immunocrit values by affecting colostrum intake and competition among piglets. Certain interventions, such as oxytocin administration or anti-inflammatory treatments, may influence colostrum composition or farrowing conditions, but they do not always result in appreciable changes in immunocrit levels. Its usefulness, quickness, and field application make it an important tool in contemporary swine production, even though it lacks the analytical precision of enzyme-linked immunosorbent assay and radial immunodiffusion that quantify individual immunoglobulin isotypes, such immunoglobulin G. For herd level interpretations the immunocrit results should be complementary to the evaluation of the farrowing and colostrum management practices, the sow diet and the handling of the piglets in supporting corrective measures prenatally.
