Effect of black garlic (Allium sativum) on gonadosomatic index, follicle-stimulating hormone level and spermatozoa quality: A study in monosodium glutamate-exposed rat model.

Infertility rates have risen significantly, one of which is due to monosodium glutamate (MSG) consumption. Recent studies have shown that flavonoids in black garlic (Allium sativum) act as antioxidants. The aim of this study was to assess the effect of black garlic extract (BGE) on gonadosomatic index, follicle-stimulating hormone (FSH) levels, and spermatozoa quality in rats exposed to MSG. Twenty-five healthy rats, aged ten to twelve weeks, were divided equally into five experimental groups: (1) negative control (NC), no intervention; (2) positive control (PC), fed with MSG 8 mg/kg; and (3) fed with MSG + BGE 200 mg/kg; (4) fed with MSG + BGE 400 mg/kg; and (5) fed with MSG + BGE 600 mg/kg. Oral MSG was administered once a day for two weeks before BGE administration was started for two weeks. The measured endpoints were gonadosomatic index, FSH levels, and spermatozoa concentration and quality (spermatozoa motility and abnormality). Analysis of variance (ANOVA) followed by Duncan's post hoc analysis was used to assess the measurement differences. The result suggested that the administration of BGE did not significantly affect the gonadosomatic index (p=0.513). Significant decreases in FSH levels (p=0.005) and spermatozoa concentration were observed in the PC group compared to other groups (p<0.001). Additionally, spermatozoa motility was significantly lower in the PC group compared to NC, BGE200, BGE400, and BGE600 (p<0.001), with higher motility noted in BGE200, BGE400, and BGE600 compared to PC (p<0.001). Furthermore, PC had significantly higher spermatozoa abnormalities compared to NC, BGE200, BGE400, and BGE600 (p<0.001). In conclusion, administration of BGE had a significant effect on the improvement of FSH levels and the quality of spermatozoa in rats exposed to MSG.

Quercetin in semen extender improves frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats.

This study investigated the antioxidant effect of quercetin-treated semen on frozen-thawed spermatozoa quality and in-vivo fertility in crossbred Kamori goats. In total, 32 ejaculates from four fertile bucks were diluted in Tris-based egg yolk extender with varying levels of quercetin (0, 1, 5, 10, and 15 µM). Qualified semen samples were pooled and frozen in French straws. The results revealed that the addition of quercetin in the semen extender increased (p < 0.05) frozen-thawed sperm total motility (TM), progressive motility (PM), rapid velocity (RV), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL), and amplitude of lateral head (ALH) displacement in contrast to the control group. Quercetin supplementation had no effect on beat cross frequency (BCF), straightness (STR), and linearity (LIN) (p > 0.05). Quercetin showed significantly higher (p < 0.05) plasma membrane and acrosome integrity and viability (p < 0.05) of spermatozoa in contrast to the control group. Quercetin in the semen extender significantly increased (p < 0.05) superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and total antioxidant capacity (TAC) levels while reduced (p < 0.05) the contents of total oxidant status (TOS) and malondialdehyde (MDA), which were in contrast to the control group. Ultrasound results revealed that 24 out of 30 (80%) goats were found pregnant when semen was treated with 5 µM quercetin while the control group showed 18 out of 30 (60%) animals were pregnant. Thus, the study concluded that 5 µM quercetin-treated semen was found to be efficient, showed increased antioxidant status, and reduced oxidant production, leading to improved spermatozoa quality and in-vivo fertility in goats.

Methylprednisolone improves the quality of liquid preserved boar spermatozoa in vitro and reduces polymorphonuclear neutrophil chemotaxis and phagocytosis.

After artificial insemination, immune cells such as polymorphonuclear neutrophils will be recruited into the genital tract and induce endometrial inflammation, adversely affecting the spermatozoa. This study aimed to analyze the effect of methylprednisolone (MPS) on boar spermatozoa quality of in vitro liquid preservation and chemotaxis and phagocytosis of polymorphonuclear neutrophils toward boar spermatozoa. Various concentrations of MPS were added to the extender and analyzed for their effects on spermatozoa motility, kinetic parameters, abnormality rate, total antioxidant capacity (T-AOC) levels, H2O2 content, mitochondrial membrane potential and acrosome integrity. Testing of MPS on chemotaxis and phagocytosis of polymorphonuclear neutrophils toward spermatozoa induced by lipopolysaccharide (LPS). The results showed that an extender containing 2 × 10-7 mol/mL MPS was the most effective for preserving boar spermatozoa during in vitro liquid preservation at 17°C. It effectively improved spermatozoa motility, kinetic parameters, T-AOC levels, mitochondrial membrane potential and acrosome integrity, reducing the abnormality rate and H2O2 content. Meanwhile, the chemotaxis and phagocytosis of polymorphonuclear neutrophils toward spermatozoa under LPS induction were inhibited in a concentration-dependent manner. In conclusion, MPS has positive implications for improving in vitro liquid preserved boar spermatozoa quality, inhibiting chemotaxis and phagocytosis of polymorphonuclear neutrophils toward spermatozoa.

A systematic review and meta-analysis of spermatozoa cryopreservation, in vitro and in vivo fertility practices in water buffalo.

We explored different aspects of buffalo spermatozoa during cryopreservation. The meta-data comprised of 285 studies, published from January 2008 to March 2020. A free web tool CADIMA as well as PRISMA 2009 Flow Diagram were used for carrying out this study. The inter-reviewer agreement among studies allocated was satisfactory for criteria A (selection bias), B (performance bias), C (detection bias) and D (attrition bias), respectively. India led the percent (%) research ladder with 34.4, followed by Pakistan (29.5), Egypt (12.3), Iran (7.7), Italy (5.6), Indonesia (3.2), China (2.1), Brazil (1.4), Thailand (1.1), Philippines and Bulgaria (0.7 each), Saudi Arabia, Turkey, Vietnam, and USA (0.4 each). Among four categories of studies, Group-1 evaluated only supplements/additives/media in the freezing semen extender (n = 191/285; 67.02%); Group-2 conducted in vivo fertilization (n = 62/285; 21.75%) and Group-3 conducted in vitro fertilization/ cleavage rate/penetration rate/ blastocyst yields (n = 28/285; 9.82%) with their specific cryodiluents/media, respectively. Group-4 conducted different experimental supplements/additives/media and carried out both in vitro and in vivo fertilization simultaneously (n = 4/285; 1.40%). Conventional spermatozoa cryopreservation was reported by 51.9% studies followed by programmable fast freezing by 20.7% studies. A few leading extender types included BioXcell (3.9%); Soyamilk-skim (3.5%); and Andromed (2.1%). The study also describes French straws for semen filling, cooling temperatures, extension time, equilibration time, cryopreservation stages, thawing temperatures, seasons, thawing time, and stains used during semen evaluation assays. The study concludes that the research on spermatozoa cryopreservation of buffalo is largely conducted at quality level and a need of applying these findings for evaluation of fertility potential (in vivo and in vitro) is indispensable for effective genetic improvement.

The Effects of Antifreeze Protein III Supplementation on the Cryosurvival of Goat Spermatozoa During Cryopreservation.

Antifreeze protein (AFP) has been shown to have beneficial effects on frozen mammalian spermatozoa. However, rare reports have been published regarding the use of AFPs in storage of goat spermatozoa. The aim of this study was to investigate the effects of AFPIII on the quality of goat semen during cryopreservation. Ejaculates were collected from six Yunshang black goats through an artificial vagina. The collected semen was pooled, divided into five aliquots, and diluted with the commercial bull semen extender containing: no AFPIII (AFP-0, control), 1 µg/mL AFPIII (AFP-1), 10 µg/mL AFPIII (AFP-10), 50 µg/mL AFPIII (AFP-50), and 100 µg/mL AFPIII (AFP-100), respectively. Spermatozoa motility, membrane integrity, acrosome integrity, mitochondrial function, distribution of phosphatidylserine, and formation of reactive oxygen species (ROS) were measured after the freezing and thawing process. The results showed that the spermatozoa motility, membrane integrity, acrosome integrity, and mitochondrial function were significantly higher in frozen spermatozoa using the extender containing 1 µg/mL AFPIII as compared with the other groups (p < 0.05). Furthermore, the extender supplemented with 1 µg/mL of AFPIII resulted in higher viable and lower nonviable spermatozoa compared with the other treated groups (p < 0.05), after staining using Annexin V-fluoresceine isothiocyanate (Annexin V-FITC) and Propidium Iodide. No significant differences were found between these groups in relation to viable cells with lower ROS production. In conclusion, the addition of AFPIII to the freezing extender improved the post-thaw quality of goat semen. The optimal concentration used in this study was 1 µg/mL. However, excessively high concentrations of AFPIII were unable to exhibit their cryoprotective effects on goat spermatozoa. However, the presence of AFPIII cannot mitigate oxidative stress caused by the freezing and thawing process. In addition, in vitro fertilization or artificial insemination can further evaluate the effects of AFPIII on frozen-thawed goat spermatozoa.

Peritoneal fluid from women with endometriosis impairs human spermatozoa functionality.

The success of mammalian fertilization depends largely on spermatozoa physiological events. However, the manner in which endometriosis influence morpho-functional spermatozoa biomarkers is poorly defined. Here, we studied in vitro the effect of peritoneal fluid (PF) from women with endometriosis (PFE) and non-endometriosis (PFNE) on spermatozoa parameters. This research was prospective and double-blind. A total of 45 PF samples were collected from women with (n = 25) and without endometriosis (n = 20). Semen samples were obtained from normozoospermic donors (n = 15) and cultured with 20 % (v/v) of PF or commercial culture medium (controls) during 0, 24, and 48 h. The outcome measures were spermatozoa/viability, motility, tyrosine phosphorylation (TP) and spontaneous acrosomal reaction. In addition, plasma membrane sugars were characterized by lectins [Aleuria aurantia agglutinin (AAA), Concanavalin A (ConA), Peanut agglutinin (PNA), and Wheat germ agglutinin (WGA)]. After a 24 -h culture, results reported a significant decrease in motility in cells cultured with PFE compared to the control, together with differences in the AAA and WGA-binding sites. Moreover, spermatozoa in contact with PFNE presented a significantly lower level of acrosome spontaneous reaction. At 48 h, no differences were observed in the biomarkers studied between the PFNE and the control, excluding ConA-binding sites. On the other hand, cells cultured with PFE exhibited significantly less motility, TP, and differences in the relocation of spermatozoa surface sugars. Viability was not affected in any culture condition. Overall, the effects of PFE could negatively affect spermatozoa quality, contributing to explain and diagnose the infertility associated to male partners of women with endometriosis.

Taurine Increases Spermatozoa Quality and Function in Asthenospermia Rats Impaired by Ornidazole.

Asthenospermia has been considered as one of the crucial causes of male infertility, which was closely related to epididymal dysfunction. Lots of documents have revealed that taurine palys an important role in male reproduction, including antioxidation, membrane stabilization, stimulation of sexual hormone secretion and elevation of sperm quality. The objective of this study was to expose the effect of taurine on spermatozoa quality and function in ornidazole-induced asthenospermia rats. We found that taurine treatment could obviously recover the decline of cauda epididymal sperm count, viability and motility, and the elevation of sperm abnormality in asthenospermia animals. Spermatozoa acrosin, LDH-X, SDH and CCO activities of model rats also were notably increased by taurine administration. The present data indicated that taurine could raise spermatozoa quality and function by elevating mitochondrial energy metabolism. Notably, taurine supplementation markedly raised serum GnRH, LH and T levels in asthenospermia rays, suggesting taurine rescued asthenosperm by means of stimulating hypothalamic-pituitary-testicular axis secretion. We also found that concentrations of asthenospermia epididymal carnitine, SA, α-Glu and ACP, and mRNA expression levels of MMP7 and IDO2 were significantly rised by taurine administration, indicating taurine may protect epididymal epithelium structure, improve secretion activity, and maintain intraluminal microenvironment homeostasis. Finally, the present results showed taurine effectively increased cauda epididymal SOD, GSH and γ-GT levels in model rats, reduced ROS and MDA production, suggesting epididymal antioxidant ability of asthenospermia rats could be elevated by taurine treatment. To sum up, our results indicated that taurine can promote spermatozoa quality and function in ornidazole-induced asthenospermia rats by facilitating epididymal epithelium secretion and luminal microenvironment homeostasis.

Relation between nitric oxide (NO) level in semen and certain properties of boar spermatozoa stored at 17°C.

The aim of this study was to measure the NO level in boar semen held in a liquid state and to determine its putative relation to spermatozoa motility, plasma membrane integrity, mitochondrial membrane potential and ATP content. Generally, the percentage of spermatozoa which generated nitric oxide gradually increased, while NO level in the surrounding medium declined during the liquid preservation. NO generation in semen preserved in BTS was higher as compared to those in Androhep®Plus. We demonstrated the positive correlation between the NO level in fresh spermatozoa and their quality. We also showed negative correlation between nitric oxide level in spermatozoa preserved in BTS and sperm cells motility as well as plasma membrane integrity. Results obtained in this study confirm that NO may affect sperm physiology in a dualistic manner.

Candidates for reproductive biomarkers: Protein phosphorylation and acetylation positively related to selected parameters of boar spermatozoa quality.

Protein post-translational modifications (PTMs) have been reported to be involved in various functions of sperm, yet the exact correlation between PTMs and sperm motility remains unclear. With the goal of contributing to this subject, motility variables were measured by computer-assisted sperm analysis system (CASA), and the amount of PTMs were evaluated using Western blot and immunofluorescence in fresh sperm and liquid stored sperm. Results of the present study indicate that the amount of the phosphorylated substrates of PKA (P-PKAs), protein tyrosine phosphorylation (PTP), global protein acetylation (Pan-Kac) and α-tubulin acetylation (Tub-Kac) was greater in sperm of fresh semen samples with relatively greater motility than in sperm of fresh semen samples with relatively lesser motility. Similarly, the amounts of phosphorylation and acetylation gradually decreased with the reduction in the motility of sperm in liquid stored semen samples. Interestingly, the P-PKAs (r = 0. 634, P < 0. 01) and Pan-Kac (r = 0. 380, P < 0. 05) were positively correlated with sperm motility in fresh semen, whereas only P-PKAs (r = 0.607, P < 0. 01) were positively correlated with sperm motility during liquid storage. Furthermore, it is noteworthy that the amounts of phosphorylation and acetylation were positively correlated with the acrosome integrity and mitochondrial membrane potential of fresh sperm and liquid stored sperm. This study is the first to explore the correlation between PTMs and sperm motility, and it may provide a new reproductive biomarker for evaluating semen quality and predicting sperm capacity for enhancing reproductive performance, which is meaningful for the pig breeding industry.

Optimization of the incubation time and temperature for spermatozoa extraction in freshwater crayfish Pontastacus leptodactylus (Eschscholtz, 1823).

Determination and control of spermatozoa quality in crustacean aquaculture is an important issue for successful and controlled reproduction. Investigation of spermatozoa number in spermatophores is a basic and common parameter for determining the reproductive quality in farmed decapods. In the present study, spermatozoa extraction from spermatophores located in the ductus deferens was conducted in Pontastacus leptodactylus using different incubation times and temperatures. The results indicate that the duration of incubation and temperature affected (P < 0.05) spermatozoa extraction. Greater temperatures (40 and 75 °C) resulted in a reduction (P < 0.05) in number of extracted spermatozoa. In contrast, more spermatozoa were extracted when the 4 and 23 °C temperatures were imposed. After 4 h of incubation, the number of extracted spermatozoa were greatest in number at 23 °C. In conclusion, the greater numbers of crayfish spermatozoa can be obtained when the ductus deferens containing spermatophores is incubated at 23 °C for 4 h as compared with other temperatures and incubation durations. The results of present study are useful for assessing spermatozoa quality in aquaculture as well as the extraction of spermatozoa for research purposes.

Effect of glutamine supplementation and replacement of tris-egg yolk based extender with defatted cow milk on spermatozoa quality after equilibration and thawing.

AIM: The present study was designed to evaluate the effect of supplementation of glutamine and replacement of Tris-egg yolk (TE) based buffer with defatted cow milk on the spermatozoa quality after equilibration and thawing. MATERIALS AND METHODS: Semen was collected from five Bhadawari bulls biweekly, and a total of 30 ejaculates were taken. The semen ejaculates were pooled and divided into three equal parts. The pooled semen was diluted by TE based extender (control), TE + glutamine (8 mM) (T1) and 50% TE + 50% deffated cow milk + glutamine (8 mM) (T2). At two stages viz. after equilibration and after 12 h of cryopreservation (thawed samples), progressive motility, percent live, and percent acrosomal damage of the spermatozoa was assessed. RESULTS: Supplementation of glutamine improved (p<0.05) the spermatozoa quality with respect to the progressive motility, percent live and acrosomal damage both post-equilibration and post-thaw. T2 improved (p<0.05) the spermatozoa quality as compared to control, however; it was less (p<0.05) effective as compared T1 both post-equilibration and post-thaw. CONCLUSION: From the results of present study it can be concluded that glutamine supplementation was effective in maintaining post-equilibration and post-thaw spermatozoa quality whereas defatted cow milk was not as effective as TE based buffer in the extender in improving the spermatozoa quality.