Sperm DNA 5-mC and RNA m 6A methylation are differently affected during periods of body weight losses and body weight gain of young and mature breeding bulls

Aiming to characterize the effects of nutritional status on epigenetic markers, such as DNA 5-mC methylation and RNA m 6A methylation, of bovine sperm, twelve Angus × Hereford crossbred breeding bulls were submitted to nutritional changes for a period of 180 days: no change in BW (Phase 1 = 12 d); BW loss (Phase 2 = 78 d); and BW gain (Phase 3 = 90 d) in a repeated measures design. Animals were fed Beardless wheat (Triticum aestivum) hay and mineral mix. Statistical analyses were performed using SAS 9.4 (SAS Inst., Cary, NC). Higher levels of RNA m 6A (P = 0.004) and DNA methylation (P = 0.007) of spermatic cells were observed at Phase 2 compared with Phase 1. In Phase 3, sperm RNA m 6A methylation levels continued to be higher (P = 0.004), whereas the DNA of sperm cells was similar (P = 0.426) compared with the Phase 1. Growing bulls had a tendency (P = 0.109) of higher RNA m 6A methylation levels than mature bulls. Phase 2 altered scrotal circumference (P < 0.001), sperm volume (P = 0.007), sperm total motility (P = 0.004), sperm progressive motility (P = 0.004), total sperm count (P = 0.049), normal sperm (P < 0.001), abnormal sperm (P < 0.001), primary sperm defects (P = 0.039), and secondary sperm defects (P < 0.001). In Phase 3, bulls had scrotal circumference, sperm volume, sperm motility, sperm progressive motility, total sperm count, normal and abnormal spermatozoa, and primary and secondary spermatozoa defects similar to Phase 1 (P > 0.05). Serum concentrations of insulin-like growth factor-1 (IGF-1) and leptin decreased during Phase 2 (P = 0.010) while no differences (P > 0.05) were detected between Phase 3 and 1; growing bulls tended (P = 0.102) to present higher leptin levels than mature bulls. Specific for mature bulls, DNA methylation was positively correlated with leptin concentration (0.569, P = 0.021). Whereas for young bulls, DNA methylation was positively correlated with abnormal spermatozoa (0.824, P = 0.006), primary spermatozoa defect (0.711, P = 0.032), secondary spermatozoa defect (0.661, P = 0.052), and negatively correlated with normal spermatozoa (−0.824, P = 0.006), total sperm count (−0.702, P = 0.035), and sperm concentration (−0.846, P = 0.004). There was no significant correlation (P > 0.05) between RNA m 6A and hormones and semen traits. In conclusion, the nutritional status of breeding bulls alters epigenetic markers, such as DNA methylation and RNA m 6A methylation, in sperm, and the impact of change seems to be age-dependent. These markers may serve as biomarkers of sperm quality and fertility of bulls in the future. Detrimental effects on sperm production and seminal quality are observed at periods and places when and where environmental and nutritional limitations are a year-round reality and may carry hidden players that may influence a lifetime of underperformance.

Tramadol injection as dosage form -Induced deterioration in reproductive performance of male rabbits

Injections are usually more expensive than tablets, but they may be required less often since they are more concentrated, whereas a person may need to take a tablet more regularly, thus consuming a large volume. when we administer a drug via oral rout ,its meant to be absorbed by stomach or intestinal mucosa ,but its so happens that, gastric acid, food ,other drugs and many other factor come into the play and retarded the drug absorption ,metabolized in intestinal mucosal cells even before it can reach to liver or target tissue(first-pass metabolism)thus decrease the effective amount reaching to circulation, Now, a drug given through injection reaches circulation at cent percent level and show prompt action which is earnestly required at the moment ,it also decrease the dose and systemic side effects . also the tablets formulation contain the excipient like filler ,diluents for the purpose of long -term stabilization ,bulking up tablet formulations making concentration of drug less in tablets and need more dose to give the therapeutic action. Tramadol is a potent analgesic effective in the treatment of mild to severe pains. However, the use of the drug can pose a threat to other organs and systems. Therefore, this study was designed to investigate the effect of tramadol injection as dosage form on reproductive parameters in male rabbits. Tenth rabbits were randomly divided into two equal groups (each group five rabbits). The first group was used as a control. The second group was used to study the effect of tramadol (50 mg/kg body weight) for six weeks. Results obtained showed that tramadol significantly (P<0.05) decreased libido (by increasing the reaction time), ejaculate volume, sperm concentration ,total sperm output, sperm motility (%), total motile sperm per ejaculate (TMS), packed sperm volume (PSV), total functional sperm fraction (TFSF), normal and live sperm and semen initial fructose. While initial hydrogen ion concentration (pH) and dead and abnormal sperm were increased (P<0.05). Live body weight (LBW) and relative weights of testes (RTW) were significantly (P<0.05) decreased. Concentrations of thiobarbituric acid-reactive substances (TBARS) were significantly (P<0.05) increased in plasma of rabbits treated with tramadol compared with control. The study showed the harmful effects of tramadol on the reproductive performance on male rabbits. Keywords: Tramadol Injection; Rabbits; Semen; Testosterone

The Effect of Oral Vitamin E on Semen Parameters and IVF Outcome: A Double-Blinded Randomized Placebo-Controlled Clinical Trial

Background. Male infertility is a main clinical problem that affects about 7% of all men worldwide. Many patients with male infertility are caused by a reduced antioxidant capacity of semen. Several antioxidant supplements, especially vitamin E, are proposed to help male infertility treatment. This project was goaled to study the effects of oral synthetic vitamin E (400 IU/day) for eight weeks on betterment of semen parameters and pregnancy rate. Methods. After dropping the cases, 124 infertile couples with a male factor who were admitted to the IVF program were included. The male patients with idiopathic abnormal motility and/or morphology were randomized into two groups: 61 receiving vitamin E and 63 as the control group receiving placebo for eight weeks. The pretreatment semen parameters of both groups were compared with those of posttreatment. The pregnancy outcomes were considered between the two groups. Results. There were no significant differences statistically between before and after treatment in the term of sperm volume, count, motility, and morphology. Furthermore, the IVF outcomes of the two groups were not different significantly, either. Interestingly, the percent of normal sperm in the placebo group was significantly decreased after eight weeks. Conclusion. Vitamin E supplementation might neutralize free radical activity to keep sperm from more oxidative damages. Further studies regarding the influence of higher acceptable doses of vitamin E on semen characteristics and fertility rates are needed. This study was registered as a two-arm, blinded, randomized, placebo-controlled clinical trial (IRCTID: IRCT2014020616506N1, 2014-03-18).

PSXIV-25 Search for the association of SNP in GRM8 gene with sperm quality in stallions

The formation and functioning of the animal reproductive system occurs as a result of the coordinated interaction of a wide range of genes. To search for causal mutations, work was carried out to search for polymorphic variants in the marker regions detected using GWAS. The four single-nucleotide substitutions in the exon of the GRM8 gene identified during the studies and the association of these SNPs with sperm quality was carried out. Semen from 22 stallions was collected. Sperm volume, concentration and progressive motility were assessed. Sequencing of the sections of the candidate GRM8 gene was carried out using an Applied Biosystems 3500 genetic analyzer. For the rs1138419111 genotype, no significant differences were found in the studied parameters. According to the identified single nucleotide substitution rs1147388106, the highest ejaculate volume was in stallions with the GG genotype (55.9±26.5 ml) compared to stallions with the GA genotypes (32.5±13.9 ml) and AA (18.0±33,6) (p &lt; 0.05). When analyzing data on SNP rs395286150, stallions with a heterozygous CT genotype had the best sperm quality. Thus, the cell concentration was 317.0±66.5 million/ml in stallions with the CT genotype, 209.6±58.2 and 189.5±74.9 % with the CC and TT genotypes, respectively (P &lt; 0.05). The progressive sperm motility of stallions with the CT genotype was 65.5±20.5% versus 48.7±22.0% in stallions with the TT genotype and 48.4±18.6% with CC. Analysis of data on SNP rs394524550 revealed a significant effect of the genotype on progressive motility. Stallions with the AG genotype had a progressive motility of 64.6±16.3%, and those with GG and AA 32.7±15.7 and 49.6±18.1%, respectively (P &lt; 0.05). Thus, as a result four single nucleotide polymorphisms were identified in the exon of the GRM gene. Three of them were significantly associated with such indicators of sperm quality as ejaculate volume, concentration and progressive motility. Project No. 0445-2021-0011.

Characteristics of Neurokinin-3 Receptor and Its Binding Sites by Mutational Analysis

NKB (Neurokinin B) is already known to play a crucial role in fish reproduction, but little is known about the structure and function of NKB receptors. Based on an in silico model of the tilapia NKB receptor Tachykinin 3 receptor a (tiTac3Ra) found in the current study, we determined the key residues involved in binding to tilapia NKB and its functional homologue NKF (Neurokinin F). Despite studies in humans suggesting the crucial role of F2516.44 and M2897.43 in NKB binding, no direct peptide interaction was observed in tilapia homologs. In-silico, Ala mutations on residues F2516.44 and M2897.43 did not influence binding affinity, but significantly affected the stability of tiTac3Ra. Moreover, in vitro studies indicated them to be critical to tiNKB/tiNKF-induced receptor activity. The binding of NKB antagonists to tiTac3Ra both in-vitro and in vivo inhibits FSH (follicle stimulating hormone) and LH (luteinizing hormone) release and sperm production in mature tilapia males. Non-peptide NKB antagonist SB-222200 had a strong inhibitory effect on the Tac3Ra activation. SB-222200 also decreased LH plasma levels; two hours post intraperitoneal injection, changed sperm volume and the ratios of the different stages along the spermatogenesis in tilapia testes.

CRYOPRESERVATION OF ALPACA SPERMATOZOA OBTAINED VIA POST COPULA IN A TRIS EXTENDER WITH EGG YOLK FROM THREE AVIAN SPECIES.

The objective of this study was to evaluate the cryopreservation of alpaca spermatozoa obtained via post copula in a Tris extender with egg yolk from three avian species. Forty samples of eight alpacas were collected by the post-copula method. After the collection, we proceeded to evaluate sperm volume, color, motility and concentration. The 25 samples with volume 1 ml and total motility 60% were mixed to form pool (5 samples/pool), divided into three aliquots and diluted in Tris-base with 20% egg yolk from three avian species (hen, quail, paw). These diluted samples were refrigerated for 1,5 h at 5 °C. Once this temperature was reached, the 5% glycerol basic dilutor was added, balanced for 20 min, packed in 0,5 mL straws and frozen in liquid nitrogen vapours for 20 min. The thawed samples were evaluated at different incubation times at 37 °C: 0; 1,5 and 3 h. All parameters of fresh and thawed sperm quality were analyzed using the GLM procedure (ANOVA). The samples collected (fresh) showed a motility of 69,1%, viability of 82,8%, membrane functionality of 77,9% and acrosomal integrity of 85,8%. After the cooling process, no differences were observed between the different egg yolk when comparing the sperm characteristics evaluated (p>0,05). At thawing, motility and acrosomal integrity were superior (p<0,05) when hen and quail were used compared to paw egg yolk. At 1,5 and 3 h of incubation, motility and acrosomal integrity were superior (p<0,05) in the samples with hen and quail with respect to paw. In conclusion, the use of hen and quail provided better cryoprotective action than paw egg yolk in cryopreserved alpaca sperm and incubated at 37°C for 3 h

P–092 The more, the merrier: does ejaculatory frequency influence seminal parameters in oligospermic men?

Study question Does ejaculatory frequency during the three months preceding semen collection influence semen parameters in oligospermic men? Summary answer A frequency of 2–3 ejaculations/week during the three months preceding semen collection significantly optimizes sperm motility, without any reduction in sperm concentration. What is known already Male gametes undergo crucial physiological and biochemical changes during epididymal transit, but a longer storage is known to have negative effects on semen quality, especially on motility. Previous studies focused on abstinence prior to semen collection, while few data are available on the effect of ejaculation frequency. On one hand, a longer storage could increase exposure to reactive oxygen species and a pro-inflammatory environment, with a reduction in vitality and motility. On the other, an increased ejaculation frequency could cause a reduction in sperm volume and concentration. The effects of ejaculatory frequency are particularly understudied in men with oligospermia. Study design, size, duration This is a retrospective study performed at a tertiary level public infertility center. We included all semen samples, collected both for diagnostic purposes and ART cycles between September 2019 and September 2020, with a sperm concentration of 15 million/ml or less, and an abstinence of 3- 5 days. Exclusion criteria were surgically collected or collected for fertility preservation semen samples. Participants/materials, setting, methods Standard demographic and clinical data were recorded, as well as semen parameters. Ejaculation frequency was considered “optimal” (at least 2–3/week) or “reduced” (&lt;1/week). The potential predictive role of ejaculation frequency, age, BMI, smoking habits, previous cryptorchidism, varicocele, days of abstinence on semen parameters was evaluated by univariate and then by multivariate analysis for all factors significant in the univariate models. P &lt; 0.05 was considered statistically significant. Main results and the role of chance: Out of 738 men, 491 reported an optimal ejaculation frequency, 247 had &lt;1 ejaculation/week, no one reported everyday ejaculations. Total sperm mobility (35.91±22.84% vs. 32.28±16.91%, p = 0.02) and sperm rapid progressive motility (5.56±6.09% vs. 4.20±6.1%, p = 0.006) were significantly higher in the group with optimal ejaculation frequency. Ejaculation frequency remained predictive of total mobility (p = 0.04) and rapid progressive mobility (p = 0.03) in a multivariate linear regression model with age and sperm concentration. Sperm volume (2.92±1.56 ml vs. 2.91±1.54 ml, p=NS) and concentration (5.74±5.05 mil/ml vs. 6.05±4.78 mil/ml, p=NS) did not significantly differ depending on the declared ejaculation frequency. Limitations, reasons for caution The study is retrospective and ejaculatory frequency was self-reported as an estimate of the mean of the number of ejaculations per week. Wider implications of the findings: Optimizing ejaculatory frequency may improve ART outcomes as well as success of spontaneous conceptions. There is no reason to limit ejaculatory frequency in oligospermic men for a hypothesized benefic in sperm concentration. Trial registration number Not applicable

Hormonal treatment with Ovopel increases sperm production in lake minnow, Eupallasella percnurus (Pallas, 1814)

The aim of the study was to determine the suitability of Ovopel, which contains an mGnRH analog and metoclopramide (a dopamine inhibitor), for the stimulation of spermiation in the cyprinid fish lake minnow, Eupallasella percnurus (Pallas), which is endangered with extinction in Poland. The hormonal treatment effected an approximate twofold increase in sperm volume with a simultaneous decrease in sperm concentration and negligible differences in motility and straight-line velocity.

REPRODUCTIVE PERFORMANCE OF HATCHERY-BRED, WILD-CAUGHT BROODSTOCK, AND THEIR OUTBREED OF THE AFRICAN CATFISH Clarias gariepinus (Burchell, 1822)

This study was conducted to evaluate the reproductive performance of hatchery-bred and wild-caught broodstock of Clarias gariepinus. Thirty pairs of each hatchery-bred and wild-caught broodstock (15 females and 15 males) was used for the study. Induced breeding was carried out in four groups with three replications at a ratio of 1:1 by hypophysation method. Group A: hatchery-bred male and hatchery-bred female (HBM m + HBF f); B: wild-caught male and wild-caught female (WCM m + WCF f), C: hatchery-bred female and wild-caught male (HBF f+ WCM m) and D: wild-caught female and hatchery-bred male C. gariepinus (WCF f + HBM m). Results showed that egg diameter, sperm motility, sperm density, and male GSI were not significantly different (P>0.05) whereas ovary weight, sperm volume, fecundity, female GSI, and percentage fertilization were significantly higher (P<0.05) in hatchery-bred broodstock than wild-caught broodstock. Hatchability was significantly higher (P<0.05) in Group C and D than A and B. In conclusion, a better reproductive performance in C. gariepinus with an economic advantage could be recorded through the combination of wild-caught and hatchery-bred broodstock.

Semen characteristics and scrotal size of pubertalWest African dwarf rams fed diets containing (Bitter kola) Seed Meal

The effect of theGarciniaKola Seed Meal (GKSM) on the spermatozoa attributes and scrotal characteristics of West African dwarf rams was assessed in a 16 week study. Twelve West Africa dwarf rams weighing between 12.50 and 13.80kg were randomly allotted to four dietary treatments in a completely randomized design. Rams were fed experimental diets for 16 weeks. The diets included; 0% GKSM, 2.5 % GKSM, 5.0% GKSM and 7.5% GKSM inclusion levels as treatments 1, 2, 3 and 4 respectively. Each treatment had three replicates while semen was collected once from all replicates in the treatments using electro ejaculator and assessed for semen colour, semen pH, semen temperature, sperm concentration, mass activity, sperm motility and live sperm cells. The results showed that scrotal circumference ranged from 18.16 to 21.00cm and scrotal length from 8.60 to 10.30cm but did not differ significantly (p >0.05) across treatments. Sperm motility (80.00 – 84.16 %) and sperm volume (0.29 – 0.43 mL) were not significantly different (p >0.05). Rams on treatment I had the highest sperm concentration of 2.59 x 10 . Despite the reproductive potential benefits of Garicinia kola, the inclusion of GKSM up to 7.5% in the diets of rams used in this study did not have any direct or adverse effects on the sperm motility, live sperm cells, sperm volume, and scrotal characteristics of West Africand warf rams.