Development and standardization of a protocol for sperm cryopreservation of two important commercial oyster species

Dissertação de mestrado, Aquacultura e Pescas, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015Aquaculture activities have a huge contribution for the world food production and their development is extremely necessary to answer to the lack of resources, especially to the demand for seafood. Bivalve production, especially Crassostrea angulata (Portuguese oyster) has been practiced from long ago, and although its production suffered several constraints, in recent years it has been increasing the interests in recovering production and in preserving nature populations. In this sense, new research needs to guarantee an efficient and economically viable production, contributing to a relatively new environmental concern: wild population restoration. Nowadays, pure wild populations of Crassostrea angulata are rare to find due to multiple factors that affected this oyster industry. Cryopreservation technology could promote alternative techniques to contribute for the resource management efficiency of the Portuguese oyster and associated economic activity. In this sense, standardization of procedures is important for Crassostrea genus. At the present there are no cryopreservation reports on Crassostrea angulata sperm, and therefore, one of the objectives of this work is to design a cryopreservation protocol for this species, testing the more adequate cryoprotectant solution, its ideal concentration, different freezing rates and types of containers. In parallel, this stablished protocol was applied in Crassostrea gigas and compared to other previously published for this species. Analysis of motility, viability, agglutination and fertilizations were used as guides for the establishment of the protocol in C. angulata. Moreover, ATP content, DNA fragmentation and lipid peroxidation were done in order to standardize the same protocol for both species. Movement analysis were assessed by CASA system, viability through common staining techniques and flow cytometer, agglutination was quantified according to the scale developed by Dong et al., (2007), ATP content determined by bioluminescence, Comet assay was performed to quantify the DNA fragmentation and lipid peroxidation determined spectrophotometrically by measuring the absorbance of the malondialdehyde (MDA). Significant differences were observed (p<0.05) for lipid peroxidation and fertilization trials whereas ATP content and fragmentation of DNA of the cryopreserved samples did not differ significantly from the control. In C. gigas, the same analysis were performed and did not reveal post-thaw quality differences in the samples cryopreserved with 10% DMSO. The established protocol revealed to be effective and with a low degree of cellular damage on C. angulata sperm and, at the same time, viable to apply in other species, such as Crassostrea gigas

Raw Semen Characteristics of Three Different Indonesian Local Roosters

Indonesia has agreat variety of roosters, either indigenous type as well as exotic and cross breed. The purpose of this experiment was to study the characteristics of semen from three types of Indonesian local roosters such as Merawang, Kampung and crosses Sentul Kampung with Kedu (SK Kedu). A total of 15 roosters consist of Merawang roosters, Kampung, and SK Kedu roosters were 5 each. The semen was collected 3 times a week by dorso-abdominal and cloaca massage method. The parameters evaluation was macroscopic characteristics consist of volume, color, consistency, and pH. Microscopic evaluation of semen such as a mass movement, sperm motility, live sperm, sperm abnormality and sperm concentration. Results of this experiment showed that semen volume of Merawang (0.40±0.26 mL) was higher (p<0.05) compare to Kampung (0.24±0.12 mL) or SK Kedu (0.16±0.10 mL) but no difference on semen color, consistency and semen pH. There were no difference in the mass movement, sperm motility and live sperm as well as on sperm abnormality among three types of roosters. Sperm concentration of Merawang (4490 million mL-1) was significantly higher than Kampung (3245 million mL-1) and the SK Kedu roosters (3751 million mL-1). Its was conclude that Merawang roosters had good semen quality better than Kampung and SK Kedu rooster

Fatherhood and sperm DNA damage in testicular cancer patients

Testicular cancer (TC) is one of the most treatable of all malignancies and the management of the quality of life of these patients is increasingly important, especially with regard to their sexuality and fertility. Survivors must overcome anxiety and fears about reduced fertility and possible pregnancy-related risks as well as health effects in offspring. There is thus a growing awareness of the need for reproductive counseling of cancer survivors. Studies found a high level of sperm DNA damage in TC patients in comparison with healthy, fertile controls, but no significant difference between these patients and infertile patients. Sperm DNA alterations due to cancer treatment persist from 2 to 5 years after the end of the treatment and may be influenced by both the type of therapy and the stage of the disease. Population studies reported a slightly reduced overall fertility of TC survivors and a more frequent use of ART than the general population, with a success rate of around 50%. Paternity after a diagnosis of cancer is an important issue and reproductive potential is becoming a major quality of life factor. Sperm chromatin instability associated with genome instability is the most important reproductive side effect related to the malignancy or its treatment. Studies investigating the magnitude of this damage could have a considerable translational importance in the management of cancer patients, as they could identify the time needed for the germ cell line to repair nuclear damage and thus produce gametes with a reduced risk for the offspring

A Research Documentation On Men's Sexual Health Disclosed

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Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p &lt; 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p &lt; 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF. © 2016 American Society of Andrology and European Academy of Andrology

Comparison of five different methods to assess the concentration of boar semen

Both for research and practical purposes, accurate and repeatable methods are required to assess the concentration of boar semen samples. Since the method which is used may influence the results considerably, the aim of the present study was to compare 5 frequently used techniques to determine boar semen concentration. Fifty ejaculates were collected from 37 different boars at an artificial insemination centre. Subsequently, each ejaculate was analyzed for sperm concentration by means of 2 different types of colorimeters (Colorimeter 1: Model 252, Sherwood Scientific Ltd, Cambridge, UK; Colorimeter 2: Ciba-Corning, Schippers, Bladel, The Netherlands), the Burker counting chamber (golden standard), and the Hamilton Thorne Analyzer (Ceros 42.1) using 2 types of Leja chambers (the 'former' and the 'recently developed'). Each ejaculate was assessed 5 times with each of the 5 methods, and the repeatability, expressed by coefficient of variation (CV), was determined for each method. The different methods were compared using Pearson's correlations and limits of agreement. The colorimeters yielded the lowest CV's (both 3.7%), while the former Leja chamber resulted in the highest CV (12.4%). Moreover, significant (P0.71) were found between the results obtained by the different methods. The limits of agreement plots showed that none of the methods consistently over- or underestimated the sperm concentrations when compared to the Burker chamber, although there was a tendency toward higher over- or underestimation in highly concentrated sperm samples. Based on our results, there were no major differences in the assessment of sperm concentration between the evaluated methods. The choice of method used in a laboratory could therefore be based on factors such as cost, number of samples to be assessed and practical use, without thereby negatively affecting the validity of the results thus obtained

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