BACKGROUND: Various antioxidant substances are added to sperm extenders to protect spermatozoa against oxidative stress and cryodamage.
OBJECTIVE: To investigate the effects of the flavonoid diosmin (DIO) and a flavanone glycoside naringin (NAR) on the freezability of ram semen.
METHODS: In this study, six Merino rams were used during the breeding season. The ejaculates were pooled after collection from the rams. Pooled ejaculates were divided into six groups: control, NAR 1 mM, NAR 2 mM, NAR 4 mM, DIO 2 mM, and DIO 4 mM, and then diluted with a TRIS-based diluent. The pooled semen was equilibrated, placed in 0.25 mL pipettes with 10 × 10 7 sperm cells in each pipette, and frozen in liquid nitrogen vapor. After 24 h, the pipettes were thawed at 37 degree C for 25 s and analyzed in terms of spermatological parameters.
RESULTS: The highest plasma membrane integrity ratio was found in the DIO 4 mM group, whereas a statistically significant difference was found between the NAR 1 mM and NAR 2 mM groups (p < 0.05). While the DIO 4 mM group had the highest acrosome integrity rate, a statistically significant difference was found between the other groups (p < 0.05). Mitochondrial activity was the highest in the NAR 4 mM, DIO 4 mM and DIO 2 mM groups (p < 0.05). In the analysis of the sperm membrane lipid profile, it was observed that the DIO group had the highest lipid-phospholipid ratio. In sperm membrane protein profile analysis, it was found that both additives exerted protective effects at different levels. The highest total protein content was seen in the DIO 4 mM and NAR 4 mM groups. 8-hydroxydeoxyguanosine (8-OhDG) positivity was more common in the control group than in the DIO and NAR groups. Cu-Zn superoxide dismutase (SOD) expression was lower in the control group and more intense in all other groups. Positive results were especially observed in the acrosome of the sperm cells.
CONCLUSIONS: The addition of NAR and DIO to the ram semen extender increased the quality of sperm parameters after the freeze-thaw process. Doi.org/10.54680/fr24510110412.

This study was aimed at evaluating the effects of the commercial formulation Roundup Ultramax® on sperm mortality and viability, in terms of the capacity of spermatozoids (spz) to trigger the acrosome reaction (AR), using the estuarine crab Neohelice granulata as a model. To this, an in vivo assay comprising 100 days duration was carried out, on a control group and two groups exposed to the formulation (0.01 mg/L and 0.2 mg/L of glyphosate) under controlled conditions of photoperiod, feeding, and temperature. At the end of the assay, the right vas deferens (VD, proximal and middle portion) was dissected, and after homogenizing it in calcium-free saline solution, the acrosome reaction was induced in the phase containing the spz. In each treatment, the percentage of spz with total and partial AR was calculated, as well as that of dead spz. Compared to the control, crabs exposed to the herbicide showed a significant decrease in spz with full AR, together with an increase in the percentage of spz with partial AR. Furthermore, spz mortality was significantly higher in both glyphosate concentrations compared to the control, in a concentration-dependent manner. On the other hand, abnormal spermatophores, showing expanded walls and coalescence, were observed in a significant percentage in the left VD of the groups treated with the herbicide. The results obtained are compared with those from other studies on several invertebrate and vertebrate species that found inhibition of the AR and abnormal sperm, together with inhibition of spermatogenesis, endocrine disruption, and reduced sperm motility by effect of pure glyphosate and/or different glyphosate formulations. In summary, the available evidence highlights the possible impact of glyphosate on sperm quality, in a wide variety of species.

Globozoospermia is a form of male infertility characterized by spermatozoa with spherical heads lacking acrosomes. The aim of this study was to evaluate ultrastructural and molecular defects in different types of globozoospermia. Semen samples from 12 infertile patients (9 with complete globozoospermia and 3 with partial globozoospermia) and 10 normozoospermic men (control) were examined by transmission electron microscopy and immunocytochemistry with antibodies against lamin B1. The presence of lamin A and progerin was assessed by reverse transcription-PCR. Whole exome sequencing was performed in three patients. In semen samples with complete and partial globozoospermia, lamin B1 was observed at the periphery of sperm nuclei, whereas lamin A and progerin were absent. Nuclear envelope pores were found in spermatozoa from both patient groups, regardless of morphology and chromatin condensation, in contrast to the control group. Non-condensed chromatin was present in 51%-81% of cases of complete globozoospermia and in 36%-79% of cases of partial globozoospermia. Homozygous DPY19L2 and SPATA16 variants were identified in two patients with partial globozoospermia and one patient with complete globozoospermia. An atypical nuclear membrane with abnormal nuclear pore distribution and lamin B1 localization was observed in spermatozoa from patients with both complete and partial globozoospermia. The genetic defects in the DPY19L2 and SPATA16 genes detected in patients from both globozoospermic groups suggest a generalized disruption of nuclear structure in globozoospermia, highlighting the genetic and phenotypic similarities between complete and partial globozoospermia.

Infertility is a global health problem affecting one in six couples, with 50% of cases attributed to male infertility. Spermatozoa are male gametes, specialized cells that can be divided into two parts: the head and the flagellum. The head contains a vesicle called the acrosome that undergoes exocytosis and the flagellum is a motility apparatus that propels the spermatozoa forward and can be divided into two components, axonemes and accessory structures. For spermatozoa to fertilize oocytes, the acrosome and flagellum must be formed correctly. In this Review, we describe comprehensively how functional spermatozoa develop in mammals during spermiogenesis, including the formation of acrosomes, axonemes and accessory structures by focusing on analyses of mouse models.

The Bachaur is a mediumized draft purpose breed which has been recognized by ICAR-National Bureau of Animal Genetic Resources (NBAGR) Karnal, India, and presently is on the verge of extinction. Since there are no data regarding the seminal parameters of this breed, this work was performed to evaluate seminal parameters of freshly ejaculated semen. A total of three healthy breeding Bachaur bulls aged 2.5-5 years were selected for the study which were maintained under identical managemental conditions. Semen parameters of these bulls were studied across 10 ejaculates. The average scrotal circumference and testicular weight of the three bulls were 27.78 ± 1.2 cm and 400.67 ± 26.6 g, respectively. The average overall volume (mL), pH, concentration (million/mL), liveability (%), abnormality (%), HOST (%) and acrosome integrity (%) were 2.20 ± 0.19, 6.86 ± 0.06, 1245.60 ± 23.49, 85.09 ± 0.91, 4.13 ± 0.06, 81.16 ± 1.18 and 83.54 ± 1.32, respectively. The average overall mass motility of three Bachaur bulls was 3.57 ± 0.06 in 0-5 scale and individual motility averaged 84.78 ± 1.70 per cent. The volume of ejaculates in Bachaur bull seemed to be lower as compared to other exotic and Indian breeds. However, the semen parameters with regard to mass motility, liveability, abnormalities, hypo-osmotic swelling test (HOST) and acrosomal integrity seemed similar to other exotic and Indian breeds.

BACKGROUND: Exposure of humans and animals to heavy metals is increasing day-by-day; thus, lead even today remains of significant public health concern. According to CDC, blood lead reference value (BLRV) ranges from 3.5 µg/dl to 5 μg/dl in adults. Recently, almost 2.6% decline in male fertility per year has been reported but the cause is not well established. Lead (Pb2+) affects the size of testis, semen quality, and secretory functions of prostate. But the molecular mechanism(s) of lead toxicity in sperm cells is not clear. Thus, present study was undertaken to evaluate the adverse effects of lead acetate at environmentally relevant exposure levels (0.5, 5, 10 and 20 ppm) on functional and molecular dynamics of spermatozoa of bucks following in vitro exposure for 15 min and 3 h.
RESULTS: Lead significantly decreased motility, viable count, and motion kinematic patterns of spermatozoa like curvilinear velocity, straight-line velocity, average path velocity, beat cross frequency and maximum amplitude of head lateral displacement even at 5 ppm concentration. Pb2+ modulated intracellular cAMP and Ca2+ levels in sperm cells through L-type calcium channels and induced spontaneous or premature acrosome reaction (AR) by increasing tyrosine phosphorylation of sperm proteins and downregulated mitochondrial transmembrane potential. Lead significantly increased DNA damage and apoptosis as well. Electron microscopy studies revealed Pb2+ -induced deleterious effects on plasma membrane of head and acrosome including collapsed cristae in mitochondria.
CONCLUSIONS: Pb2+ not only mimics Ca2+ but also affects cellular targets involved in generation of cAMP, mitochondrial transmembrane potential, and ionic exchange. Lead seems to interact with Ca2+ channels because of charge similarity and probably enters the sperm cell through these channels and results in hyperpolarization. Our findings also indicate lead-induced TP and intracellular Ca2+ release in spermatozoa which in turn may be responsible for premature acrosome exocytosis which is essential feature of capacitation for fertilization. Thus, lead seems to reduce the fertilizing capacity of spermatozoa even at 0.5 ppm concentrations.

Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

Sperm heads contain not only the nucleus but also the acrosome which is a distinctive cap-like structure located anterior to the nucleus and is derived from the Golgi apparatus. The Golgi Associated RAB2 Interactors (GARINs; also known as FAM71) protein family shows predominant expression in the testis and all possess a RAB2-binding domain which confers binding affinity to RAB2, a small GTPase that is responsible for membrane transport and vesicle trafficking. Our previous study showed that GARIN1A and GARIN1B are important for acrosome biogenesis and that GARIN1B is indispensable for male fertility in mice. Here, we generated KO mice of other Garins, namely Garin2, Garin3, Garin4, Garin5a, and Garin5b (Garin2-5b). Using computer-assisted morphological analysis, we found that the loss of each Garin2-5b resulted in aberrant sperm head morphogenesis. While the fertilities of Garin2-/- and Garin4-/- males are normal, Garin5a-/- and Garin5b-/- males are subfertile, and Garin3-/- males are infertile. Further analysis revealed that Garin3-/- males exhibited abnormal acrosomal morphology, but not as severely as Garin1b-/- males; instead, the amounts of membrane proteins, particularly ADAM family proteins, decreased in Garin3 KO spermatozoa. Moreover, only Garin4 KO mice exhibit vacuoles in the sperm head. These results indicate that GARINs assure correct head morphogenesis and some members of the GARIN family function distinctively in male fertility.

Oligoasthenoteratozoospermia is an important factor affecting male fertility and has been found to be associated with genetic factors. However, there are still a proportion of oligoasthenoteratozoospermia cases that cannot be explained by known pathogenic genetic variants. Here, we perform genetic analyses and identify bi-allelic loss-of-function variants of MFSD6L from an oligoasthenoteratozoospermia-affected family. Mfsd6l knock-out male mice also present male subfertility with reduced sperm concentration, motility, and deformed acrosomes. Further mechanistic analyses reveal that MFSD6L, as an acrosome membrane protein, plays an important role in the formation of acrosome by interacting with the inner acrosomal membrane protein SPACA1. Moreover, poor embryonic development is consistently observed after intracytoplasmic sperm injection treatment using spermatozoa from the MFSD6L-deficient man and male mice. Collectively, our findings reveal that MFSD6L is required for the anchoring of sperm acrosome and head shaping. The deficiency of MFSD6L affects male fertility and causes oligoasthenoteratozoospermia in humans and mice.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.