Effect of Methanol Extract, Methanol Fraction, and N-Hexane Fraction of Parijoto Fruit (Medinilla speciosa) on Seminal Parameters and Testicular Weight of Male Sprague Dawley Rats

Infertility occurs in 2 million couples or 17% of couples who are married more than 2 years but are not pregnant or have signs of pregnancy. In couples who do not have children, 50% of male infertility factors are caused by abnormalities in semen. Empirically, parijoto fruits (Medinilla speciosa Blume) are used by the people of Kudus, Central Java, Indonesia to increase fertility. The purpose of this research was to find out the effect of methanol extract, methanol fraction, and n-hexane fraction of parijoto fruit (Medinilla speciosa) on seminal parameters and testicular weight of male Sprague Dawley rats. This research used 36 two-month-old Sprague Dawley rats with 200-300 gram of body weight which were divided into 6 groups. Group 1 (normal); groups 2, 3, and 4, used parijoto fruit methanol extract at 100mg/kgBW, 250mg/kgBW, and 500mg/kgBW doses respectively; groups 5 and 6 used methanol fraction and n-hexane fraction of parijoto fruit at 500 mg/kgBW dose respectively for 14 days. Rats were dissected and had examinations on sperm motility, morphology, abd testicular weight. The data were analyzed using Kruskal Wallis and Mann Whitney. The results found that groups 1, 2, 3, 4, 5 and 6 had mean spermatozoa motility (%) that were 56.5±2.43, 72.5±6.89, 77.6±12.99, 83.3±7.53, 84.7±3.98, and 74.2±11.58, mean spermatozoa morphology (%) that were 95.5±3.67, 95±2.76, 92.6±5.13, 96.5±3.27, 94.3±4.37, and 93.2±6.11, and mean testicular weight (gram) that were 1.08±0.10, 0.90±0.14, 0.98±0.10, 1.18±0.21, 1.28±0.43, and 1.02±0.13. There were significant differences between the normal group and all treatments (P <0.05) on spermatozoa motility. There was no significant difference on spermatozoa morphology and testicular weight. Based on the results, it can be concluded that methanol extract, methanol fraction, and n-hexane fraction of parijoto fruit can increase spermatozoa motility of male Sprague Dawley rats.

Evaluation of seminal characteristics of Pelibuey and East Friesian rams at two different times of the year

Objective: To determine the changes in seminal quality of Pelibuey and East Friesian rams during the non-breeding (long days; March-June) and breeding seasons (short days; September-December) at 19° north latitude. Design/methodology/approach: To determine changes in seminal quality over time, seminal parameters of rams, collected with an artificial vagina were evaluated over 32 weeks. An analysis of variance was performed with a completely randomized design in a 2 x 2 factorial arrangement (breed and season). Results: No differences were found within breeds or between breeds in the same season in the evaluated seminal parameters; however, differences were reported between seasons in the live weight parameters, lower in the non-reproductive season, in addition to an increased scrotal circumference and mass motility during the reproductive season. Study limitations/implications: Semen parameters estimation, in field trials, is subjective compared to computerized semen evaluation systems, it is therefore desirable to have extensive experience in semen evaluation at the field level before starting the study. To confirm the results obtained in this study, a new experiment with a larger number of experimental units is suggested. Findings/conclusions: It is concluded that in the environmental and management conditions, where the seminal evaluation took place, no differences were found between breeds, suggesting that the Pelibuey and East Friesian breeds at 19° north latitude do not decrease their seminal parameters during the non-breeding season compared to the breeding season. This suggest that these two sheep breeds are able to reproduce, in such conditions, all year round.

Effect of Apatyakara Ghrita Followed by Koshtha Shuddhi with Haritakyadi Churna in the Management of Ksheena Shukra w.s.r. to Oligozoospermia: A Case Study

Oligozoospermia is a condition where sperm count is below 15 million/ml and a common finding which is related to male infertility. Some of the known responsible factors for male infertility are poor semen quality, genetic factors, testicular dysfunction and endocrine inter relationship etc. According to Ayurvedic classics, Oligozoospermia can be correlated with Ksheena Shukra which is due to increased Vata and Pitta Dosha being more prevalent in Madhyama Vayas. According to WHO, in India, the overall prevalence of primary infertility ranges between 3.9% and 16.8%. Methods: In this study, a male patient aged 40 years was presented with a desire to get a child and other associated symptoms were Daurbalya (weakness), Mukha Shosha (dryness of the mouth), Sadana (loss of rigidity), Shrama (post act exhaustion), Alpachestata (early ejaculation), Maithune Ashakti (problematic or not satisfactory coitus), Rati Anabhimukhata (loss of sexual desire) etc. The patient was managed with Apatyakara Ghrita followed by Koshtha Shuddhi with Haritakyadi Churna. For the assessment of the effect of therapies on sexual parameters, the scoring pattern prepared by Mehra & Singh, 1995 and Seminal Parameters has been adopted. Results and Conclusion: Apatyakara Ghrita is found effective to increase sperm count from 12 million/ ml to 45 million/ml and motility from 20% to 75% and no adverse effect of the adopted management was seen during treatment and even after the 2 months of follow-up.

Varicocele and male infertility: Historical, anatomical and urological perspectives – a mini review

Varicocele is defined as an abnormal dilatation of the pampiniform plexus of the spermatic cord. This is seen in 10 to 20% of general population and is one of the most common correctable causes of male infertility. The history of varicocele dates back to the era of Ancient Greeks to about 600 BC, who had sculptured varicocele in their statues as a tortuous bag of wormsin the scrotum. It was Ambrose Pare who gave a poetic description to the pooling of blood in the spermatic cords and subsequently Curling coined the term Varicocele The intricacy of the venous drainage of the scrotum and its contents and the criss crossing of the veins makes the thermo-regulatory mechanism regulating spermatogenesis, even more complex. The Cremaster and Dartos greatly aid in facilitating the thermo-regulatory mechanism of scrotal wall, which forms the anatomical basis of oligospermia in patients with varicocele. Apart from a visible change in seminal parameters, the benefits of varicocelectomy have extended on to an improvement in serum testosterone and enhancement of erectile and ejaculatory functions. With a greater knowledge of the pathophysiology of varicocele and with better understanding of the roles of gene polymorphisms and reactive oxygen species, the indications for varicocelectomy gain more support and evidence. A sound anatomical knowledge and an in-depth understanding of the events that take place at molecular level might shift the treatment focus in future from varicocelectomy to molecular targeted therapies.

The Effect of Glyphosate on Human Sperm: In Vitro Approximation

Introduction Glyphosate is an herbicide used to eradicate illicit crops; however, its use is controversial due to different health problems associated with it. The present study aims to evaluate the effects of glyphosate on human sperm in vitro. Methods Twenty-two semen samples from healthy normozoospermic men were included; 11 semen samples were incubated with Panzer (INVESA S.A., Antiquia, Colombia) and 11 with Roundup (Monsanto Company, MO, USA). The changes in motility and viability were observed. Functional seminal parameters were evaluated as well. Results The samples exposed to glyphosate showed less motility and viability; a decrease in the potential of the mitochondrial membrane was observed, and an increase in the lipoperoxidation of the membrane was evidenced. Conclusion Based on the present results, we concluded that glyphosate has cytotoxic potential for exposed people and may affect their fertility.

SNP Identification in Sperm Associated Antigen 11B Gene and Its Association with Sperm Quality Traits in Murrah Bulls

Background: The beta-defensin family antigens present on sperm surface suggestively play pivotal role in sperm fertility by aiding in various steps of sperm maturation, motility, capacitation, immune recognition and sperm-oocyte interaction in female tract. It is imperative to explore genetic polymorphisms to build a better understanding of individual variation in male fertility. The experiment was designed to identify DNA polymorphism in Sperm Associated Antigen 11B (SPAG11B) gene and to analyze association between genetic variants with sperm quality traits in buffalo bulls (Murrah) in ICAR-National Dairy Research Institute, Karnal. Methods: Genomic DNA was extracted from hundred and thirty Murrah bulls. A 395 base pair region covering partial Intron 2, Exon 3 and Partial Intron 3 of bovine SPAG11 gene was amplified and genotyped using PCR-RFLP method. The PCR amplified product was purified, sequenced and further ClustalW analysis was done to align edited sequence with reported Bos taurus sequence (AC_000158.1). Gene and genotype frequencies, effective number of alleles, level of heterozygosity and polymorphic information content of various genotypes were estimated by standard procedure. Seminal parameters (Post thaw sperm motility, sperm viability, acrosomal integrity, HOST and abnormality) were estimated and statistical analysis was carried out. Result: A novel SNP (G greater than A substitution) at 2266 base of the SPAG11B gene was identified by sequencing. The fragment of SPAG11 gene containing 395 base pairs was amplified using PCR and digested with restriction enzyme i.e. MunI which showed three distinct genotypes viz., AA (266 bp and 107 bp fragment), AG (307 bp, 266 bp and 107 bp) and GG (373 bp fragment). Least squares means of seminal parameters for the SNP was estimated and compared after correction for non-genetic factors. Between genotypes, significant differences were found only for acrosomal integrity and was highest in AG (74.22±0.72) followed by AA (72.6±0.89) and GG (71.12±0.97), respectively. The identified novel SNP of SPAG11 gene showed significant association with acrosome integrity with genotype AG being superior to other genotypes. However, an association cannot be established with other seminal parameters with this SNP, further studies are required in order to validate the impact of g.2266G greater than A on sperm quality traits in a large population.

​Association of Bio-acoustic Features of Vocal Signals with Age and Semen Quality in Sahiwal Bulls

Background: Bio-acoustic features of animal’s voice can provide meaningful information about their biological and physical characteristics. The present study was conducted to get indicators of age from voice analysis and explore the relationship between voice features and seminal parameters in Sahiwal bulls. Methods: Voice samples were collected from healthy bulls (n=20), maintained at ICAR-NDRI, Karnal. Bulls were classified into two groups i.e. young bulls and adult bulls. The voice signals were analyzed by Adobe Premium software and acoustic features were extracted by using PRAAT software. Result: The mean of acoustic features viz. call duration (sec), mean intensity (dB), total energy (P2S), amplitude (P), pitch (Hz), unvoiced frame (%), jitter (%), bandwidth (Hz) mean N/H ratio (%) have been found significantly different while mean H/N ratio (dB), shimmer (%) and pulses were not found statistically (P greater than 0.05) different between adult and young bulls. The seminal parameters viz mass activity (0-5 scale), individual progressive motility (%), live sperm count (%) and total sperm abnormality (%) were found significantly different between adult and young bulls. No significant association between voice features and semen quality of bulls was observed. Hence, voice signals of male might provide some clues about their age but for semen quality, there is further need to explore the interesting relationship between voice features and fertility of breeding bulls.

P–074 Chromatin Maturity Index (CMI) in unfixed and live spermatozoa and Aniline Blue (AB) stained as an additional evaluation parameter in idiopathic male infertility

Study question To investigate whether idiopathic male infertility may be due to the presence of histones in motile spermatozoa using a modified AB staining protocol. Summary answer No correlation between CMI in live motile spermatozoa, DNA Fragmentation Index (DFI) and other conventional seminal parameters were found in male infertile patients. What is known already The AB stain discriminates between lysine-rich histones and arginine/cysteine-rich protamines. Transition from histones to protamines during spermatogenesis remodels chromatin packaging and abnormalities in the substitution of those proteins maybe interfere with seminal parameters and affect male infertility. The correlation between CMI and seminal parameters is known, but little is knowledge about live and motile spermatozoa associated to CMI because literature report only spermatozoa fixation before staining. Sperm chromatin carries half of the genomic material to offspring. Spermatozoa nuclear status is crucial for balanced transmission to future generations, and histones modifications are directly involved in epigenetic mutations. Study design, size, duration Retrospective observational study of 77 men underwent to standard semen analysis, including the evaluation of CMI and DFI, enrolled from January to December 2020. Mean age of the men was 36.63±8.26 years old, sperm concentration 46.69±37.23 mill/mL, linear progressive motility 39.35±15.31%, normal morphology 6.42±3.40%, DFI 25.91±10.29%. 200 spermatozoa for evaluation of CMI and 300 for DFI were analyzed respectively. Participants/materials, setting, methods Semen samples of 77 patients were collected and analyzed according to 5th edition of WHO guidelines (2010) for examination of human semen. For the evaluation of CMI we performed a new modified protocol for AB stain directly in live spermatozoa. Dilution 1:1 fresh semen and Aniline Blue colorant were mixed and placed on a slide and examined in bright field microscopy x1000 magnification. DFI was evaluated using Sperm Chromatin Dispersion (SCD) test. Main results and the role of chance Of all spermatozoa analyzed, 82.58±29.98% were white, 17.17±17.21% were pale blue, and 28.53±21.09% were dark blue. By our modified protocol, directly in live spermatozoa, we correlated AB staining with motility and , surprisingly, all motile spermatozoa observed were not stained (white), while pale or dark blue spermatozoa resulted always immotile. For this reason, we have considered pale blue spermatozoa as AB positive, in disagreement with some authors. So, maybe, we should reconsider pale blue stained spermatozoa as abnormal. We also observed AB negative spermatozoa with morphological head, neck and tail defects, underlining the independence of these two parameters: nuclear status and morphology. We have observed no statistically significant differences between conventional semen parameters, DFI and CMI, so nuclear analysis seems to be independent parameters. The statistical analysis was performed by Matlab statistical toolbox version 2008 (MathWorks, Natick, MA, USA) for Windows at 32 bit; finally all tests with p-value (p) &lt; 0.05 were considered significant. Attention should be paid to the evaluation of CMI not only in astenozoospermic patients, where a lower CMI is known, but also in normozoospermic infertile patients. Limitations, reasons for caution This is a preliminary observational study on a small number of normozoospermic or mild asthenozoospermic patients. The study should be considered as a pilot study. Future studies with higher number of samples are necessary in order to confirm the results obtained. Wider implications of the findings: This is the first study that reports AB staining on unfixed live spermatozoa with a modified protocol. Our study underlines the necessity of classify pale blue spermatozoa as AB positive. Further investigations are necessary. This is a starting point for future analysis to be carried out under the project EcoFoodFertility. Trial registration number Not applicable