Until recently, no repeatable protocol for equine in vitro fertilization (IVF) had been established, despite decades of trying. Recent studies have demonstrated reproducible IVF outcomes under specific experimental conditions, bringing equine IVF into a new perspective. Prolonged incubation of stallion spermatozoa in a TALP based medium supplemented with penicillamine, hypotaurine and epinephrine (PHE) appears to be important factors of protocols that support sperm capacitation and markedly improved IVF outcomes. The sperm capacitation procedure has now been applied successfully using both fresh and frozen-thawed stallion semen, with the latter requiring a shorter pre-incubation period. Here, we review the molecular, structural, and metabolic processes that drive capacitation in stallion spermatozoa, with comparisons to better-characterized mechanisms in other mammals, focusing on changes in sperm membrane composition, energy metabolism, cytoplasmic content, and on the influences of seminal plasma and the female reproductive tract. Because the newly developed IVF protocol appears to be shorter with frozen-thawed semen, special attention is given to capacitation-like changes triggered by cryopreservation, and their consequences for sperm function and fertilizing capacity. Importantly, although successful fertilization can be achieved under these conditions, this does not constitute direct evidence that the molecular mechanisms governing stallion sperm capacitation have been fully elucidated. Rather, these protocols provide a robust experimental platform for investigating these mechanisms in the horse.

Phosphatidylinositol transfer proteins (PITPs) transfer phosphatidylinositol (PI) between the ER and target membranes to regulate phosphoinositide metabolism. However, the expression patterns of class I PITPs in C. elegans remain largely uncharacterized. Here, we generated an endogenous GFP knock-in reporter for PPIT-2 (formerly Y71G12B.17), a conserved homolog of human PITPNA and PITPNB. GFP::PPIT-2 was prominently expressed in the excretory system and canals, with additional expression in the hypodermis, sperm, developing vulva, and germline-adjacent pseudocoelom. PPIT-2 expression notably overlaps with fatty acid synthase FASN-1, establishing candidate tissues to investigate its roles in PI transfer, membrane trafficking, and lipid homeostasis.

Spermiogenesis, the transformation of round spermatids into streamlined, fertilization-competent sperm, represents cellular morphogenesis at its most extreme [1,2] . Despite decades of effort, spermiogenesis remains difficult to recapitulate outside the testis [2,3] , highlighting the importance of cues within the native tissue environment. The nature of these cues, however, remains poorly understood. Here, we discover that mechanical stimulation elicits Ca [2+] entry in spermatids and identify transmembrane channel-like protein 5 (TMC5) as a principal mediator of this response. In both mouse and human testes, TMC5 is selectively expressed in spermatids and localized to the plasma membrane. TMC5 associates with TMC7 and calcium- and integrin-binding (CIB) proteins, mirroring TMC-CIB mechanotransduction complexes in sensory organs [4,5] . Mechanistically, loss of Tmc5 blocks mechanically evoked Ca [2+] entry, leading to disruptions in cytoskeletal remodeling and failure of CREMτ-dependent transcriptional programs required for spermiogenesis [6] . Thus, TMC5 is required to couple mechanical cues to sperm development, with direct implications for male infertility.

Capacitation is a post-ejaculatory maturation process by which sperm become hyperactive and undergo the acrosome reaction, both of which are required to fertilize an oocyte. Two key features of capacitation are increased intracellular calcium via the calcium channel CatSper and changes in mitochondrial function. In mouse spermatozoa, CatSper-dependent calcium entry promotes mitochondrial calcium uptake and mitochondrial activation, contributing to hyperactivated motility and fertilization ability. Whether this mechanism is conserved in human spermatozoa was unknown. Here, we assessed mitochondrial function and mitochondrial calcium in paired non-capacitated and capacitated human spermatozoa from normozoospermic donors. Capacitation increased mitochondrial respiratory control ratio, coupling efficiency, mitochondrial membrane potential, and mitochondrial calcium. The increase in mitochondrial calcium required extracellular calcium and was prevented by pharmacological inhibition of CatSper or the mitochondrial calcium uniporter (MCU). Inhibition of CatSper or mitochondrial calcium uniporter also attenuated the capacitation-associated increase in mitochondrial membrane potential and reduced hyperactivated motility without affecting progressive or total motility. Pharmacological disruption of mitochondrial function reduced capacitation-associated hyperactivated motility without affecting the acrosome reaction. Together, these findings support a conserved pathway in human and mouse spermatozoa in which CatSper-mediated calcium entry promotes MCU-dependent mitochondrial calcium uptake and mitochondrial activation, contributing to the acquisition of hyperactivated motility during capacitation. These findings identify mitochondrial calcium handling as a potential target for modulating human spermatozoa function.

Male fertility is a key determinant of reproductive efficiency in dairy cattle; however, some sires with normal semen parameters exhibit reduced fertility due to intrinsic sperm defects, including those arising from genetic mutations. Previous research has identified 2 genomic regions, located on BTA11 (2.6 Mb) and BTA25 (19.1 Mb), that severely affect service sire fertility in US Jersey cattle. However, the causal mutations and their impact on male fertility remain unknown. The first goal of this study was to identify the causal mutations using targeted sequencing. For each genomic region, 2 DNA pools were constructed with high- and low-fertility Jersey sires. The DNA-sequencing analysis included genome alignment, variant calling, and variant annotation. We identified a deleterious missense mutation on FER1L5 (BTA11) and a deleterious frameshift variant on ABCA16 (BTA25) carried by low-fertility sires. The second goal of this study was to determine whether these mutations affect sperm function, early embryo, and post-hatching development using frozen semen from Jersey sires homozygous for the FER1L5 and ABCA16 variants or wild-type sperm (WT). Both variants affected sperm acrosomal reaction, with lower proportions of reacted sperm in the FER1L5 and ABCA16 variants compared with WT. As a consequence, oocytes fertilized with sires carrying these variants had reduced fertilization rates (P = 0.0001), with 60.2 ± 8.8%, 76.2 ± 7.1%, and 94.2 ± 2.5% for the FER1L5, ABCA16, and WT, respectively. Cleavage rate differed among groups (P < 0.0001), with 42.0 ± 6.4%, 54.0 ± 6.5%, and 78.6 ± 4.5% for the FER1L5, ABCA16, and WT, respectively. Blastocyst formation was reduced in FER1L5 variant (11.6 ± 1.6%) and ABCA16 variant (15.4 ± 1.9%) versus WT (26.7 ± 3.3%). The proportion of cleaved embryos developing to blastocysts was lower in FER1L5 variant (26.6 ± 2.4%) than WT (35.7 ± 2.5%), while the ABCA16 variant (30.1 ± 2.2%) was intermediate. Blastocysts had similar total cell numbers, although mutations affected lineage proportions. During extended culture, mutations affected embryo survival, with reduced survival in the ABCA16 variant compared with the FER1L5 variant and WT. Embryo attachment was not different among groups, but trophectoderm outgrowth area was affected by the genotype. Overall, the candidate causal mutations identified on FER1L5 (BTA11) and ABCA16 (BTA25) impair sperm fertilization competence and early embryo development and may contribute to reduced sire fertility, highlighting the importance of identifying and managing deleterious variants in dairy cattle breeding programs.

Heat stress (HS) is a major environmental challenge that compromises fertility in breeding bulls by disrupting physiological homeostasis and reproductive function. This study evaluated the potential of dietary copper nanoparticles (CuNPs) to improve reproductive resilience and thermotolerance in Sahiwal breeding bulls exposed to summer HS. Eighteen clinically healthy bulls were randomly assigned to three dietary treatments (n = 6/group): control (no CuNPs), S1 (10 mg CuNPs/kg dry matter (DM), and S2 (20 mg CuNPs/kg DM), and maintained under peak HS conditions for 16 weeks. The supplementation of CuNPs significantly improved thermoregulation by reducing rectal temperature, pulse rate, respiration rate, and circulating cortisol while preserving rumen motility. Supplemented bulls exhibited greater semen quality and sperm functional competence, including improved plasma membrane integrity, acrosome integrity, and DNA stability. These modifications were accompanied by enhanced testicular blood perfusion and preservation of ultrasonographic echotexture. Oxidative stress was also attenuated by CuNPs, as indicated by reduced malondialdehyde concentrations and enhanced antioxidant defenses, including superoxide dismutase, catalase, glutathione peroxidase, and total antioxidant capacity. Improved redox status was associated with higher reproductive hormone levels and enhanced immune function. During in vitro fertilization, semen from supplemented bulls achieved higher cleavage and blastocyst rates and produced a greater proportion of transferable grade embryos than the control group. The beneficial effects were more pronounced in the S2 group. These findings demonstrate that dietary copper supplementation in nanoparticle form enhance thermotolerance, redox homeostasis, testicular function, and in vitro sperm fertilizing capacity in heat-stressed Sahiwal breeding bulls.