Sperm Functional Characteristics Research Articles

The impact of autologous platelet-rich plasma supplement on Ossimi ram semen quality and fertility: In vivo fertility of freshly diluted and frozen-thawed spermatozoa

The study aimed to investigate the effects of using the ram's platelet-rich plasma (PRP) as a supplement on the quality, functionality, antioxidant activity, and in vivo fertilization of freshly diluted and frozen-thawed ram semen. Nine Ossimi rams were involved in the analysis, which comprised 162 ejaculates. Each ejaculate was segmented into five parts and mixed with a Tris-based extender containing varying concentrations of autologous PRP (ranging from 0% to 10%) for both freshly diluted and frozen-thawed semen. The evaluation encompassed sperm viability, motility, kinetics, functional traits, antioxidant enzyme activity, lipid peroxidation content, and in vivo fertility. Results showed that supplementing with 5% PRP in both freshly diluted and frozen-thawed semen led to increased total and progressive motility of sperm, improved sperm kinetics and functional characteristics, and enhanced viability compared to the control groups. Notably, concentrations of PRP at 5% and 7.5% reduced malondialdehyde levels while increasing total antioxidant capacity, glutathione reductase, and superoxide dismutase levels compared to the groups supplemented with 10% PRP and the control groups. Furthermore, the in vivo fertilization rate of ram semen significantly improved with 5% PRP supplementation compared to the control groups for both freshly diluted and frozen-thawed semen.In conclusion, the study suggests that incorporating 5% autologous PRP into seminal extenders can protect Ossimi ram sperm and enhance in vivo fertility for freshly diluted and frozen-thawed semen.

Differential sperm histone retention in normozoospermic ejaculates of infertile men negatively affects sperm functional competence and embryo quality.

The unique epigenetic architecture that sperm cells acquire during spermiogenesis by retaining <15% of either canonical or variant histone proteins in their genome is essential for normal embryogenesis. Whilst heterogeneous levels of retained histones are found in morphologically normal spermatozoa, their effect on reproductive outcomes is not fully understood. Processed spermatozoa (n=62) were tested for DNA integrity by sperm chromatin dispersion assay, and retained histones were extracted and subjected to dot-blot analysis. The impact of retained histone modifications in normozoospermic patients on sperm functional characteristics, embryo quality, metabolic signature in embryo spent culture medium and pregnancy outcome was studied. Dot-blot analysis showed heterogeneous levels of retained histones in the genome of normozoospermic ejaculates. Post-wash sperm yield was affected by an increase in H3K27Me3 and H4K20Me3 levels in the sperm chromatin (p<0.05). Also, spermatozoa with higher histone H3 retention had increased DNA damage (p<0.05). Spermatozoa from these cohorts, when injected into donor oocytes, correlated to a significant decrease in the fertilisation rate with an increase in sperm histone H3 (p<0.05) and H3K27Me3 (p<0.01). An increase in histone H3 negatively affected embryo quality (p<0.01) and clinical pregnancy outcome post-embryo transfer (p<0.05). On the other hand, spent culture medium metabolites assessed by high-resolution (800MHz) nuclear magnetic resonance showed an increased intensity of the amino acid methionine in the non-pregnant group than in the pregnant group (p<0.05) and a negative correlation with sperm histone H3 in the pregnant group (p<0.05). Histone retention in spermatozoa can be one of the factors behind the development of idiopathic male infertility. Such spermatozoa may influence embryonic behaviour and thereby affect the success rate of assisted reproductive technology procedures. These results, although descriptive in nature, warrant further research to address the underlying mechanisms behind these clinically important observations.

Review: Evaluation of bull fertility. Functional and molecular approaches.

With the term "assisted reproduction technologies" in modern cattle farming, one could imply the collection of techniques that aim at the optimal use of bovine gametes to produce animals of high genetic value in a time- and cost-efficient manner. The accurate characterisation of sperm quality plays a critical role for the efficiency of several assisted reproduction-related procedures, such as sperm processing, in vitro embryo production and artificial insemination. Bull fertility is ultimately a collective projection of the ability of a series of ejaculates to endure sperm processing stress, and achieve fertilisation of the oocyte and production of a viable and well-developing embryo. In this concept, the assessment of sperm functional and molecular characteristics is key to bull fertility diagnostics and prognostics. Among others, functional features linked to sperm plasma membrane, acrosome and DNA integrity are usually assessed as a measure of the ability of sperm to express the phenotypes that will allow them to maintain their homeostasis and orchestrate-in a strict temporal manner-the course of events that will enable the delivery of their genetic content to the oocyte upon fertilisation. Nevertheless, measures of sperm functionality are not always adequate indicators of bull fertility. Nowadays, advancements in the field of molecular biology have facilitated the profiling of several biomolecules in male gametes. The molecular profiling of bovine sperm offers a deeper insight into the mechanisms underlying sperm physiology and, thus, can reveal novel candidate markers for bull fertility prognosis. In this review, the importance of three organelles (the nucleus, the plasma membrane and the acrosome) for the characterisation of sperm fertilising capacity and bull fertility is discussed at functional and molecular levels. In particular, information about sperm head morphometry, chromatin structure, viability as well as the ability of sperm to capacitate and undergo the acrosome reaction are presented in relation to the cryotolerance of male gametes and bull fertility. Finally, major spermatozoal coding and non-coding RNAs, and proteins that are involved in the above-mentioned aspects of sperm functionality are also summarised.

Multiple flow cytometry analysis for assessing human sperm functional characteristics.

Multiparametric analysis by flow cytometry solves one of the major problems in sperm evaluation, the inability to test multiple attributes simultaneously in a single cell, which would increase the precision to predict fertility potential since several sperm parameters are tested. The association of fluorochromes and compounds conjugated to fluorochromes in multiparametric sperm analysis is well-established in microscopy techniques. However, these techniques are subjective and limit the assessment in small cell numbers, thereby harming analytic accuracy. Therefore, the current study aimed to present new possibilities for assessing the integrity and stability of the sperm plasma membrane, acrosome status, mitochondrial potential, and superoxide anion production in the mitochondrial matrix in only 2 cytometric assays using cytometers equipped with 2 and 3 lasers. For this, human semen samples collected by masturbation and selected by the swim-up technique were divided into 3 treatments: T0 (flash-frozen semen), T50 (flash-frozen semen + fresh semen, V: V), and T100 (fresh semen) for the validation of the multiparametric protocols by flow cytometry. For both protocols, sperm percentage with positive stain for all fluorophores differed significantly between treatments. The determination coefficients presented values close to 1, which validated objective, sensitive, rapid, and reproducible methodologies. Therefore, we concluded that the results reflect the status of analyzed structure, enabling a more accurate diagnosis of male infertility that has become an increasingly prevalent worldwide setback due to exposure to a variety of environmental toxicants.

Analysis of amplification and association polymorphisms in the bovine beta-defensin 129 (BBD129) gene revealed its function in bull fertility

β-defensins are adsorbable on the sperm surface in the male reproductive tract (MRT) and enhance sperm functional characteristics. The beta-defensin 129 (DEFB129) antimicrobial peptide is involved in sperm maturation, motility, and fertilization. However, its role in bovine fertility has not been well investigated. This study examines the relationship between the bovine BBD129 gene and Bos indicus x Bos taurus bull fertility. The complete coding sequence of BBD129 mRNA was identified by RNA Ligase Mediated-Rapid Amplification of cDNA End (RLM-RACE) and Sanger sequencing methodologies. It consisted of 582 nucleotides (nts) including 5' untranslated region (UTR) (46nts) and 3'UTR (23nts). It conserves all beta-defensin-like features. The expression level of BBD129 was checked by RT-qPCR and maximal expression was detected in the corpus—epididymis region compared to other parts of MRT. Polymorphism in BBD129 was also confirmed by Sanger sequencing of 254 clones from 5 high fertile (HF) and 6 low fertile (LF) bulls at two positions, 169 T > G and 329A > G, which change the S57A and N110S in the protein sequence respectively. These two mutations give rise to four types of BBD129 haplotypes. The non-mutated TA-BBD129 (169 T/329A) haplotype was substantially more prevalent among high-fertile bulls (P < 0.005), while the double-site mutated GG-BBD129 (169 T > G/329A > G) haplotype was significantly more prevalent among low-fertile bulls (P < 0.005). The in silico analysis confirmed that the polymorphism in BBD129 results in changes in mRNA secondary structure, protein conformations, protein stability, extracellular-surface availability, post-translational modifications (O-glycosylation and phosphorylation), and affects antibacterial and immunomodulatory capabilities. In conclusion, the mRNA expression of BBD129 in the MRT indicates its region-specific dynamics in sperm maturation. BBD129 polymorphisms were identified as the deciding elements accountable for the changed proteins with impaired functionality, contributing to cross-bred bulls' poor fertility.

Progesterone, Myo-Inositol, Dopamine and Prolactin Present in Follicular Fluid Have Differential Effects on Sperm Motility Subpopulations.

Considering the challenges surrounding causative factors in male infertility, rather than relying on standard semen analysis, the assessment of sperm subpopulations and functional characteristics essential for fertilization is paramount. Furthermore, the diagnostic value of sperm interactions with biological components in the female reproductive tract may improve our understanding of subfertility and provide applications in assisted reproductive techniques. We investigated the response of two sperm motility subpopulations (mimicking the functionality of potentially fertile and sub-fertile semen samples) to biological substances present in the female reproductive tract. Donor semen was separated via double density gradient centrifugation, isolated into high (HM) and low motile (LM) sperm subpopulations and incubated in human tubal fluid (HTF), capacitating HTF, HD-C medium, progesterone, myo-inositol, dopamine and prolactin. Treated subpopulations were evaluated for vitality, motility percentages and kinematic parameters, hyperactivation, positive reactive oxygen species (ROS), intact mitochondrial membrane potential (MMP) and acrosome reaction (AR). While all media had a significantly positive effect on the LM subpopulation, dopamine appeared to significantly improve both subpopulations’ functional characteristics. HD-C, progesterone and myo-inositol resulted in increased motility, kinematic and hyperactivation parameters, whereas prolactin and myo-inositol improved the LM subpopulations’ MMP intactness and reduced ROS. Furthermore, progesterone, myo-inositol and dopamine improved the HM subpopulations’ motility parameters and AR. Our results suggest that treatment of sub-fertile semen samples with biological substances present in follicular fluid might assist the development of new strategies for IVF treatment.

Sperm Functional Genome Associated With Bull Fertility.

Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility.

Freezing-thawing induces deprotamination, cryocapacitation-associated changes; DNA fragmentation; and reduced progesterone sensitivity in buck spermatozoa

In the present study, there was evaluation of cryocapacitation-associated changes, apoptotic-like changes, deprotamination, total antioxidant capacity (TAC), and in vitro sperm functional attributes in Barbari bucks after freezing-thawing. The correlation between deprotamination and sperm functional characteristics was established. Using immunoblotting procedures, there was detection of the presence of a single 28-kDa protein band corresponding to protamine-1. The localization in the head region of the spermatozoa was further validated by an immunofluorescence test. Capacitated (B-) and acrosome-reacted (AR-) pattern spermatozoa, spermatozoa with the externalization of phosphatidylserine and a relatively lesser mitochondrial transmembrane potential, and deprotamination and DNA fragmentation was greater (P < 0.05) after freezing-thawing and indicated there were cryocapacitation- and apoptotic-like changes, respectively. Furthermore, the detection of phosphorylation of tyrosine-containing proteins with use of immunoblotting and immunofluorescence procedures confirmed there were cryocapacitation-like changes in the buck spermatozoa after freezing-thawing. Total antioxidant capacity (TAC), in vitro thermal resistance response, Vanguard distance, progesterone sensitivity, and in vitro capacitation response were less (P < 0.05) in the spermatozoa after freezing-thawing compared with spermatozoa after initial dilution and equilibration. Deprotamination (chromomycin A3-positive cells, CMA3+) and DNA fragmentation (TUNEL+ve) were positively correlated with B- and AR-pattern spermatozoa, while other values for other variables were negatively correlated. In conclusion, the results of this study indicated there was protamine-1 in buck spermatozoa and after freezing-thawing there was a loss of protamine-1 combined with cryocapacitation-associated changes and apoptotic-like changes in buck spermatozoa. Spermatozoa deprotamination might be attributed to increased DNA fragmentation, resulting in compromised fertilizing capacity of buck spermatozoa.

Kolaviron suppresses dysfunctional reproductive axis associated with multi-walled carbon nanotubes exposure in male rats.

Reproductive toxicity associated with excessive exposure to multi-walled carbon nanotubes (MWCNTs), which are commonly used in medicine as valuable drug delivery systems, is well documented. Kolaviron, a bioflavonoid isolated from Garcinia kola seeds, elicits numerous health beneficial effects related to its anti-inflammatory, anti-genotoxic activities, anti-apoptotic, and antioxidant properties. However, information on the role of kolaviron in MWCNTs-induced reproductive toxicity is not available in theliterature. Herein, we assessed the protective effects of kolaviron on MWCNTs-induced dysfunctional reproductive axis in rats following exposure to MWCNTs (1mg/kg) and concurrent treatment with kolaviron (50 or 100mg/kg body weight) for 15 successive days. Results showed that MWCNTs-induced dysfunctional reproductive axis as evidenced by deficits in pituitary and testicular hormones, marker enzymes of testicular function, and sperm functional characteristics were abrogated in rats co-administered with kolaviron. Moreover, co-administration of kolaviron-abated MWCNTs-induced inhibition of antioxidant enzyme activities increases in oxidative stress and inflammatory indices. This is evidenced by diminished levels of tumor necrosis factor-alpha, nitric oxide, lipid peroxidation, reactive oxygen, and nitrogen species as well as reduced activity of myeloperoxidase in testes, epididymis, and hypothalamus of the rats. Biochemical data on the chemoprotection of MWCNTs-induced reproductive toxicity were corroborated by histological findings. Taken together, kolaviron suppressed dysfunctional reproductive axis associated with MWCNTs exposure via abrogation of oxidative stress and inflammation in male rats.

Zika virus in rhesus macaque semen and reproductive tract tissues: a pilot study of acute infection†.

Although sexual transmission of Zika virus (ZIKV) is well-documented, the viral reservoir(s) in the male reproductive tract remains uncertain in humans and immune-intact animal models. We evaluated the presence of ZIKV in a rhesus macaque pilot study to determine persistence in semen, assess the impact of infection on sperm functional characteristics, and define the viral reservoir in the male reproductive tract. Five adult male rhesus monkeys were inoculated with 105 PFU of Asian-lineage ZIKV isolate PRVABC59, and two males were inoculated with the same dose of African-lineage ZIKV DAKAR41524. Viremia and viral RNA (vRNA) shedding in semen were monitored, and a cohort of animals were necropsied for tissue collection to assess tissue vRNA burden and histopathology. All animals exhibited viremia for limited periods (1-11days); duration of shedding did not differ significantly between viral isolates. There were sporadic low levels of vRNA in the semen from some, but not all animals. Viral RNA levels in reproductive tract tissues were also modest and present in the epididymis in three of five cases, one case in the vas deferens, but not detected in testis, seminal vesicles or prostate. ZIKV infection did not impact semen motility parameters as assessed by computer-assisted sperm analysis. Despite some evidence of prolonged ZIKV RNA shedding in human semen and high tropism of ZIKV for male reproductive tract tissues in mice deficient in Type 1 interferon signaling, in the rhesus macaques assessed in this pilot study, we did not consistently find ZIKV RNA in the male reproductive tract.

A Simple, Centrifugation-Free, Sperm-Sorting Device Eliminates the Risks of Centrifugation in the Swim-Up Method While Maintaining Functional Competence and DNA Integrity of Selected Spermatozoa

This pilot study was conducted to explore the benefits of using a centrifugation-free device based on the migration–sedimentation (MS) technique over centrifugation-based techniques in selecting competent spermatozoa, as compared with using split human semen samples. Ejaculates from 35 men undergoing semen analysis were split into four parts where one part was retained as the neat (NE) and the other three parts were subjected to sperm selection by using migration–sedimentation (MS), density gradient (DG) separation, and swim-up (SU) techniques. Sperm functional characteristics along with mitochondrial integrity, tyrosine phosphorylation, acrosome reaction, and ultrastructure were measured. The ability of selection techniques in reducing spontaneous and radiation-induced sperm DNA lesions was assessed by the TUNEL assay. In results, MS-selected spermatozoa had higher viability (P < 0.001), longevity in terms of total motility at the end of 6 and 18 h post-extraction (P < 0.001), and mitochondrial integrity (P < 0.001) compared with those selected by DG. Furthermore, spontaneous DNA lesions were significantly reduced in MS and SU fractions compared with NE (P < 0.001). Similarly, radiation-induced sperm DNA lesions were significantly lower in MS and SU fractions (P < 0.001) compared with DG. Ultrastructural analysis using scanning electron microscopy suggested a moderate, non-significant increase in the number of spermatozoa with normal head and mid-piece in MS fraction compared with other methods. In conclusion, the MS-based device offers a centrifugation-free, efficient, and reliable sperm selection method, making it suitable for partially equipped intra-uterine insemination (IUI) laboratories or office IUI programmes. Further research should focus on the safety and clinical usefulness of the device in assisted conception programmes in general and IUI in specific.

Gallic acid enhances reproductive function by modulating oxido-inflammatory and apoptosis mediators in rats exposed to aflatoxin-B1.

Infertility resulting from reproductive deficiency can be stressful. Exposure to aflatoxin B1, a dietary mycotoxin prevalent in improperly stored grains, is reported to elicit reproductive insufficiencies and infertility. We, therefore, examined the likely beneficial effect of gallic acid (GA) a phytochemical, recognized to exhibit in vitro and in vivo pharmacological bioactivities against oxidative stress and related inflammatory damages in rats, since AFB1 toxicities are predicated on oxidative epoxide formation, in a bid to proffer new evidence to advance the field of nutriceutical application from plant-derived chemopreventive agents. Our findings will advance the field of chemoprevention by presenting data absent in the literature on GA. Our results demonstrate further evidence for GA conferred protection against AFB1-mediated histological lesions in testes, epididymis, and hypothalamus of treated rats; suppresses oxidative damages, relieved inflammatory and apoptotic responses, restored sperm functional characteristics, and hormonal levels relevant for reproductive integrity and function.

Ethanolic extract of Moringa oleifera leaves alleviate cyclophosphamide-induced testicular toxicity by improving endocrine function and modulating cell specific gene expression in mouse testis

Ethnopharmacological relevanceMoringa oleifera Lam. is known for its nutritional and ethno medicinal values due to the presence of wide array of phytochemicals with multiple biological activities. We have previously reported that ethanolic extract of Moringa oleifera leaves (MOE) ameliorated cyclophosphamide (CP)-induced testicular toxicity and improved functional integrity of spermatozoa as well as spermatogenic cells. Aim of the studyThe present study was planned to investigate whether the mitigation of CP-induced testicular toxicity by MOE is mediated via modulation of endocrine profile, genes associated with function of different cell types and enhancement of DNA repair response in spermatogonial cells. Materials and methodsAdult Swiss albino mice (8 week) were injected with CP (100 mg/kg, one dose in a week for 3 weeks) and MOE (100 mg/kg, 5 doses in a week for 4 weeks) either alone or in combination intraperitoneally. At 35 day post CP injection (first dose), the functional characteristics such as count, motility, head morphology and DNA integrity were assessed in epididymal spermatozoa. Key reproductive hormones like testosterone, follicle stimulating hormone (FSH) and Inhibin B concentration were analyzed in serum and testis. In addition, mRNA expression of genes pertaining to the function of Leydig, Sertoli and spermatogonial cells as well as antioxidant enzymes were evaluated in the testis. To understand the DNA damage and repair process in germ cells, prepubertal (2 week) mice were administered with single dose of CP (200 mg/kg) and/or MOE (100 mg/kg) and analyzed for expression of DNA damage (γ-H2AX, P53 and Caspase3) and repair genes (Rad51 and Ku80) in isolated spermatogonial cells at various time points after treatment. ResultsCP administration resulted in decrease in count, motility and increase in morphological defects and DNA damage in spermatozoa. Testosterone level was marginally decreased while there was a significant increase in FSH (p < 0.001) and decrease in inhibin B (p < 0.05) observed in CP treated mice. Administration of MOE prior to CP, improved sperm functional characteristics, decreased FSH and increased inhibin B levels. Expression of Abp was down-regulated while Transferrin, Fshr and Gata4 (Sertoli cell specific genes) were up-regulated in testis treated with CP. Administration of CP down-regulated the expression of Oct4 and Ddx4 (Spermatogonia specific genes). MOE administration was shown to ameliorate CP-induced damage by modulating the expression of genes specific to Sertoli and spermatogenic cells. Furthermore, MOE treatment reduced CP-induced DNA damage as evident from lower percentage of γ-H2AX positive spermatogonial cells. ConclusionAdministration of MOE mitigated CP-induced testicular damage by improving blood and, intra-testicular hormonal milieu as well as modulating the expression of genes pertaining to Sertoli and spermatogonial cells.

Gallic acid and omega-3 fatty acids mitigate epididymal and testicular toxicity in manganese-treated rats.

Environmental contamination by manganese is correlated with diverse health outcomes plus reproductive dysfunction. Dietary gallic acid (GA) and omega-3 fatty acids (ω-3-FA) are well reported to elicit beneficial health effects. Though, information on GA and ω-3-FA effects on manganese-induced reproductive toxicity is absent in literature. We examined the effect of GA or ω-3-FA on manganese-induced epididymal and testicular toxicity in rats, exposed to manganese (15mg/kg b.w.) alone, in combination with GA (30mg/kg b.w.) or ω-3-FA (20mg/kg b.w.) by gavage for 14 consecutive days. GA or ω-3-FA significantly (p<.05) prevented manganese-mediated increase in lipid peroxidation, myeloperoxidase activity, reactive oxygen and nitrogen species production but increased antioxidant enzymes activities and glutathione level in epididymis and testes treated rats. GA or ω-3-FA enhanced the activities of testicular function marker enzymes, namely acid phosphatase (ACP), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and glucose-6-phosphate dehydrogenase (G6PD) in the treated rats. GA or ω-3-FA amelioration of manganese-induced decreases in follicle-stimulating hormone, luteinising hormone, and testosterone levels were complemented by increase (p<.05) sperm functional characteristics in treated rats. Conclusively, GA or ω-3-FA may serve as dietary supplements to improve male reproductive dysfunction associated with manganese toxicity.

Comparative effects of galactose-induced aging on mitochondrial permeability transition in rat liver and testis

This study investigated the status and sensitivity of mitochondrial permeability transition (mPT) pore in testis and liver of rats exposed to doses of galactose, an acceptable model used to mimic natural aging. Male albino rats were divided into five groups of eight animals in each group for in vivo studies and administered distilled water, 50,100, 200 and 500 mg galactose/kgbdwt, respectively, for six consecutive weeks. Mitochondria were isolated from liver and testis by differential centrifugation. Mitochondrial permeability transition (mPT) was assessed as mitochondrial swelling and was monitored spectrophotometrically. Mitochondrial lipid peroxidation, ATPase activity, antioxidant enzymes, caspase activation, interleukins, and sperm functional characteristics were also assessed. Administration of galactose (50–500 mg/kg) to male Wistar albino rats had no effect whatsoever on the testicular mPT pore. However, liver mPT pore was significantly opened. Furthermore, the enhancement of mitochondrial ATPase activity and malondialdehyde generation were observed in the liver of galactose-exposed rats. Significant alterations in antioxidant enzymes were observed in post-mitochondrial fraction (PMF) of liver and testis. There were also increases in serum levels of IL-1β and 6. In addition, caspases 9 and 3 were significantly elevated in PMF of the liver with evidence of DNA fragmentation. However, there was no significant difference in levels of caspases in PMF of testis in model groups of galactose when compared with control. These results provide evidence that testis mitochondria do not readily undergo permeability transition pore upon exposure to doses of D-galactose that induce the opening of the pore in the liver.HighlightsTesticular mitochondria are less sensitive to induction of permeability transition than liver mitochondria in rats exposed to D-galactose for 6 weeks, despite the occurrence of alterations in the antioxidant defense system and generation of ROS in sperm cells as in hepatocytes.The occurrence of mitochondrial permeability transition in liver of galactose-exposed rats is consistent with malondialdehyde production, alteration in antioxidant levels, enhanced ATPase activity, caspases-9 and 3 activation, immune dysfunction, and DNA fragmentation.The study of biochemical basis of reduced sensitivity of testis to permeability transition under conditions which the liver is extremely susceptible may become useful in age associated-neurodegenerative diseases where apoptosis is upregulated and has to be properly managed to achieve downregulation.

Successful sperm cryopreservation in Egyptian spiny mice Acomys cahirinus.

The menstruating Egyptian spiny mouse has recently been proposed as a new animal model for reproductive health research. Unfortunately, little is known about reproduction in males. This study compared several characteristics of sperm function before and after cryopreservation. Epididymal spermatozoa were cryopreserved in different concentrations of raffinose and skim milk and tested for motility and membrane integrity (Experiment 1). Further evaluations of motility, plasma membrane and acrosome integrity, mitochondrial membrane potential and DNA integrity were conducted with the addition of l-glutamine to the extender (Experiment 2). The results show that, following cryopreservation, motility and membrane integrity were reduced, but were better maintained in the presence of l-glutamine (P<0.05). Moreover, although all sperm parameters were significantly reduced following cryopreservation (P<0.05), most cryopreserved spermatozoa retained acrosome, membrane and DNA integrity while also maintaining motility and mitochondrial membrane potential. This study provides a new step towards the development of assisted reproductive techniques and archiving the important genetics of the world's only known menstruating rodent.

Systemic arterial hypertension leads to decreased semen quality and alterations in the testicular microcirculation in rats

Arterial hypertension is a cardiovascular disease that leads to important systemic alterations and drastically impairs normal organ function over time. Hypertension affects around 700 million men of reproductive age and hypertensive men present increased risk for reproductive disorders, such as erectile dysfunction. However, the link between arterial hypertension and male reproductive disorders is associative at best. Moreover, many studies have reported associations between decreased male fertility and/or semen quality and alterations to general male health. In this study we aim to investigate the effect of systemic high blood pressure in sperm quality, sperm functional characteristics and testicular physiology in a rat model. Hypertensive rats presented altered testicular morphology – mainly vascular alterations and impaired testicular vasomotion. Hypertensive rats also presented decrease in sperm concentration, DNA integrity and increased percentages of sperm with dysfunctional mitochondria, intracellular superoxide anion activity and abnormal morphology. This study provides mechanistic insights by which arterial hypertension affects the testes, evidencing the testes as another target organ for hypertension as well as its impact on sperm quality.

Organic Zn and Cu interaction impact on sexual behaviour, semen characteristics, hormones and spermatozoal gene expression in bucks (Capra hircus)

The reproductive performance during the transition from prepubertal to pubertal stage was evaluated in Osmanabadi breed bucks supplemented with organic Zinc (Zn) and Copper (Cu). A total number of 40 bucks aged 20 weeks were randomly assigned to 10 groups (each n = 4). The control group was maintained with basal diet, without any additional mineral supplementation. The treatment groups were supplemented with graded doses of organic Zn (Zn 20 mg, Zn 40 mg and Zn 60 mg), Cu (Cu 12.5 mg, Cu 25 mg and Cu 37.5 mg) and a combination of Zn + Cu (Zn 20 mg + Cu 12.5 mg, Zn 40 mg + Cu 25 mg and Zn 60 mg + Cu 37.5 mg), respectively for a period of 26 weeks (up to the age of 46 weeks). Sexual behaviour and scrotal biometry were recorded periodically. Blood and semen samples were collected and processed for LH estimation in blood plasma, and testosterone, T3 and T4 hormones in the seminal plasma. The mounts with ejaculation were observed earlier (P < 0.05) in the treatment bucks (from 38th week of age) than the control group (43rd week onwards). A positive correlation was observed between blood plasma LH and testosterone with total mounts (r = 0.31, P < 0.05; r = 0.51, P < 0.01) and mounts without ejaculation (r = 0.40, P < 0.01; r = 0.52, P < 0.01). A negative correlation between T4 with sperm number per ejaculation (r = −0.31, P < 0.05) and sperm concentration (r = −0.35, P < 0.05) had been noticed. Different doses of minerals showed positive interaction (P < 0.05) with sperm functional and behavioural characteristics. The spermatozoal gene expression of ODF2 and ZCCHC6 were significantly influenced by the mineral supplementation in all doses. The ZCCHC6 gene expression was positively correlated with testosterone (r = 0.50, P < 0.001) and sperm number per ejaculation (r = 0.42, P < 0.001), and ODF2 gene with T3 hormone (r = 0.34, P < 0.05). The present study indicates that the diet supplemented with organic trace minerals cause intense sexual behaviour, enhancement in sperm number per ejaculate, total motility, spermatozoal genes expression and altered LH, testosterone and T4 hormones.

Dietary protocatechuic acid abrogates male reproductive dysfunction in streptozotocin-induced diabetic rats via suppression of oxidative damage, inflammation and caspase-3 activity

Clinical and experimental studies demonstrated that reproductive dysfunction is a non-lethal complication of diabetes. Protocatechuic acid (PCA) reportedly elicited several pharmacological effects in diabetic animals. However, there is paucity of information on the role of PCA in reproductive dysfunction associated with diabetes. The present study investigated the influence of PCA on the functional changes along the hypothalamic-pituitary-testicular axis in male diabetic rats. Streptozotocin (STZ)-induced diabetic rats were orally treated with PCA at 25 and 50 mg/kg body weight for 45 consecutive days. Results showed that PCA treatment significantly dwindled blood glucose level as well as prevented diabetes mediated decrease in body weight gain and organo-somatic indices of the testes and epididymis in the treated rats. Moreover, PCA increased the reproductive hormone levels, marker enzymes of testicular function and sperm functional characteristics in the treated rats. Further, PCA augmented the antioxidant status, inhibited lipid peroxidation and suppressed pro-inflammatory biomarkers including myeloperoxidase (MPO) activity, nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) levels as well as caspase-3 activity inhypothalamus, testes and epididymis of diabetic rats. Collectively, PCA effectively abrogated reproductive deficits in diabetic rats via mechanisms involving suppression of oxidative stress, inflammation and caspase-3 activity along with enhancement of sperm functional parameters. Thus, PCA may preserve reproductive health in humans suffering from diabetes.

Relationship of organic mineral supplementation and spermatozoa/white blood cells mRNA in goats

The antioxidant properties and the protective role of organic zinc (Zn) and copper (Cu) in white blood cells (WBCs) and spermatozoa were analyzed through quantification of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase 4 (GPx4) and nuclear factor erythroid 2-like 2 (NFE2L2) and correlations were determined with sperm functional characteristics in Osmanabadi bucks. Bucks (aged 5 months; n = 40) were divided into ten groups, and the dietary treatments comprised of a control and nine treatment groups as follows: organic Zn as Zn 20, Zn 40 and Zn 60, organic Cu as Cu 12.5, Cu 25, Cu 37.5 and combined organic Zn and Cu as Zn 20+Cu 12.5, Zn 40+Cu 25, Zn 60+Cu 37.5, respectively per kg dry matter for a period of 8 months. The blood (120 and 240 days) and semen (240 days: 40 × 4 = 160) samples were collected from 40 bucks. In WBCs: the relative abundance of mRNA for SOD1, CAT, GPx4, NFE2L2 was greater (P < 0.05) in (120 and 240 days) in majority of the mineral supplemented animals. In spermatozoa: the relative abundance of SOD1, NFE2L2, GPx4 and CAT mRNA was greater (P < 0.05) in selected treatment groups. The abundance of SOD1 mRNA in WBCs was positively correlated (P < 0.05) with sperm mass motility (r = 0.692, P = 0.027). The abundance of GPx4 mRNA was negatively correlated (P < 0.05) with type A sperm (straightness; STR) > 85% and amplitude of lateral head displacement (ALH) > 2.5 μm/ s) (r = −0.711, P = 0.021) and (P < 0.05) positively correlated with sperm viability (r = 0.669, P = 0.035). Organic Zn and Cu supplementation was associated with an increase in the expression of antioxidant defense enzyme genes in bucks.