Sperm Cryopreservation

It was 2012 when the first case of corona virus noticed by the Saudi Authorities which spread through the Asian countries, also flown to European countries. In 1960 revealed the presence of the corona virus when it was declared as the reason of common cold in human beings. On January 30, 2020, the WHO declared the COVID-19 outbreak a global health emergency. On March 11, 2020, the WHO declared COVID-19 a global pandemic. There are three brands of vaccines available that are Pfizer, Moderna and Johnson & Johnson (Janssen). A mild difference may present as post vaccination effects. The Pfizer–BioNTech COVID-19 vaccine is based as mRNA and marketed as Comirnaty. Pfizer reported a 91 percent efficacy rate after six months, while Moderna reported a 94 percent efficacy rate. Moderna, we are pioneering a class of medicines based on messenger RNA (mRNA). The Janssen COVID-19 Vaccine may not protect everyone. Mostly 2 doses of the vaccine are required to protect and these are intramuscular i...

Cryopreservation is the process of freezing and storing biological material at ultra-low temperatures
(such as -196⁰C) in liquid nitrogen (LN2) for unlimited periods. Thanks to the application
of this biotechnology, all biological activities including the biochemical reactions
leading to cell death and DNA degradation discontinue. From this point of view, cryopreservation
biotechnology focuses on preservation of cells that have many applications in the
fields of human and veterinary medicine, agriculture and aquaculture. In addition, this form
of biotechnology also has many applications in biomedical research, specifically in the areas
of immunology, virology, neurobiology, toxicology and the pharmaceutical industry.
Cryopreservation Biotechnology in Biomedical and Biological Sciences describes principles and applications
of cryopreservation biotechnology in different research areas and includes seven
chapters that have been written by experts in their research fields. The book chapters are
divided into four sections.
Section I, "Mechanism of Cryopreservation" contains one chapter entitled "Cryoprotectants
and their usage in cryopreservation process", which describes the importance and mechanism
of the cryopreservation process and also its comparison to vitrification. In addition, the
functions and physical and chemical properties of cryoprotectants are discussed.
Section II, "Application of Cryopreservation in Human Medicine Researches" is divided into
four chapters. The first chapter, "Clinical outcomes of assisted reproductive techniques using
cryopreserved gametes and embryos in human medicine", provides valuable information
regarding fertility treatments in human reproductive medicine, focusing on
cryopreservation of spermatozoa, oocytes and embryos. The second chapter, "Cryopreservation
of platelets: advances and current practices" focuses on the long-term preservation of
platelets, which is necessary for the coagulation of blood and stopping bleeding. The third
chapter, "Cryopreservation of preantral follicles", provides valuable information regarding
the structure and development of ovarian follicles. Beside this, advantages and necessities of
using oocytes in preantral follicles for the aim of cryopreservation are explained in humans
and mammals. The fourth chapter, "Vitrification: Fundamental principles and its application
for cryopreservation of human reproductive cells", is related to mechanisms of cryopreservation
in terms of application of this technique in human medicine with emphasis on
reproductive cells. In this way, mechanisms underlying the problem of the intra- and extracellular
ice formation, the role of cryoprotectants, stages of the warming and cooling process
and use of this biotechnology in the field of IVF industry are explained.
Section III, "Application of Cryopreservation in Veterinary Medicine Research", contains one
chapter entitled "Biological signals of sperm membrane resistance to cryoinjury in boars",
discusses biological mechanisms before the cryopreservation/vitrification process causing
cryoinjuries in sperm cells in boars.
Section IV, "Application of Cryopreservation in Agricultural Research", contains one chapter
entitled "Cryopreservation protocols for grapevine shoot tips", which presents valuable information
regarding cryopreservation of shoot tips of grapevines, which is an economically
important agricultural product.
This book covers different applications of cryopreservation biotechnology. I hope that this
book will be helpful for researchers studying cryobiology and related issues. I would like to
thank all the authors for their distinguished contributions, IntechOpen Publishing Company,
and its Author Service Manager Ms. Dolores Kuzelj for her help in publishing this book.

This study was primarily designed to evaluate the effect of different concentrations of ultraviolet (UV)-C–irradiated chicken egg yolk plasma (EYP; v:v; 10%, P1; 15%, P2; 20%, P3) or 20% (v:v) of whole chicken egg yolk (WCEY) in tris-citric acid (TCA) extender on water buffalo sperm quality during cryopreservation (postdilution, PD; postequilibration, PE; post-thawing, PT). Also the effect of best evolved concentration of UV-C–irradiated EYP in extender on in vivo fertility of buffalo spermatozoa was evaluated. At PE and PT, computer-assisted sperm analysis progressive motility (PM, %) was significantly higher in P3 compared with P1 and WCEY. Rapid velocity (RV, %) was higher (P < 0.05) in P3 compared with P1 and WCEY during cryopreservation (PD, PE, and PT). Average path velocity (mm/s) and straight line velocity (mm/s) were higher (P < 0.05) in P2 and P3 than WCEY at PE and PT. The decline percentage (%, longevity) in PM and RV was lower (P < 0.05) in P3 compared with WCEY during 2 hours incubation under in vitro condition at PT. Supravital plasma membrane integrity (%) was higher (P < 0.05) in P2 and P3 compared with control at different stages (PE and PT). Mitochondrial transmembrane potential (%) was higher (P < 0.05) in P2 and P3 compared with P1 and WCEY at different stages (PD and PT). Percentage of viable sperm with intact acrosome, and sperm DNA integrity (%) were higher (P < 0.05) in P2 and P3 compared with WCEY at PT. The in vivo fertility rate (%) was significantly higher with P3 compared with WCEY (76.61 vs. 64.49). In conclusion, WCEY (20%) can be replaced with UV-C–irradiated chicken EYP (20%) in TCA extender for cryopreser-vation of water buffalo spermatozoa.

(38.3%) were sampled, and they had a mean live weight of 19.9 ± 6.10 kg (range 9 - 32 kg) and 20.1 ± 6.08 kg (range 8 - 37 kg), respec- tively. The NIP had a longer snout, wider head and longer erect ears than CBP (P < 0.0001). Morphological variables that were highly correlated with LW included neck circumference (NC), breast height (BH), rump height (RH), body length (BL), interorbital width (IW), paunch girth (PG), hearth girth (HG), tail length (TL) and length of snout (LS) with Spearman correlation coefficients (R2) of 0.97, 0.92, 0.96, 0.97, 0.91, 0.97, 0.97, 0.90, and 0.93, respectively (all P < 0.0001). Teat number for NIP ranged from 5 to 14 while the CBP had a range of 10 to 16 teats. For male NIP, HG and TL best-fit in the model for body weight prediction (LW = −25.71 + 0.43 HG + 2.21 TL; R2 = 0.93; P < 0.0001), while HG and IW had the best-fit for the female NIP (LW = −28.27 + 0.50 HG + 2.22 IW; R2 = 0.96; P < 0.0001). Models for male and female CBP were LW = −8.89 + 0.32 RH + 0.34 BL (R2 = 0.84; P < 0.0001) and LW = −13.01 + 0.44 RH+0.27 BL (R2 = 0.94; P < 0.0001), respectively. Thus, for these populations of pigs, LS and TN differentiated NIP from CBP, because the NIP consistently re- corded longer LS and les- ser TN.

To study the effects of Citrullus colocynthis on testicular function and fertility, 70% methanolic extract of C. colocynthis was fed orally to male albino rats at the dose levels of 75 and 150 mg/kg body wt/day for 60 days. Oral administration of C. colocynthis caused a significant decrease in testicular weight and sperm concentration. The level of serum testosterone, LH and FSH also declined and spermatogenesis was impaired. The histoarchitecture of the testes in the rats receiving C. colocynthis methanolic extract showed degenerative changes and disruption of spermatogenesis and the number of spermatogonia remained significantly decreased. Thus the antifertility effects of C. colocynthis seemed to be mediated by disturbances in structure and testicular function including leydig and Sertoli cells resulting in an alteration in physio-morphological events of spermatogenesis.

Cryoprotectants are basically some chemical compounds which prevent cells or tissues from damage due to freezing. Mostly vitrification and thawing process are mostly used in cryopreservation. Now a day’s cryopreservation is becoming an apex technology for Health sector, as many organ and tissue transplant methods has been developed in recent time. Organs or tissues need to preserve properly before implantation, hence cryoprotectants plays a key role modern cryonics. This article seeks to provide answers for how cryoprotectants works and prevent tissue damage from shrinking ice crystals during freeze drying or vitrification. The various animal tissues, organs, lymphocytes, cartilages, bone marrow, proteins can be stored by using proper cryoprotectants. Optimization of cryoprotectants is a challenging task, as elevated concentration may cause cytotoxicity. This article also highlights the implication, challenges and recent advances of cryoprotectants along with its types. The importance of cryobank system and its importance were also being emphasized in this article. Further, importance of cryoprotectants in nano suspension preparation was also been discussed in this article.

The present study aimed to explore effect of different ionic and sugar based extenders and cryoprotectants on motility, motility duration and fertilization ability of cryopreserved scaly carp (Cyprinus carpio) sperm. For cryopreservation experiments, three different extenders (E-1:75mmol/l NaCl, 70 mmol/l KCl, 2 mmol/l CaCl 2 , 1 mmol/l MgSO 4 , 20 mmol/l Tris, pH: 8; E-2: 350 mM glucose, 30 mM Tris, pH: 8;E-3: 300 mM glucose, 10% egg yolk, pH: 4.5) containing different CPAs such as DMSO, DMA and glycerol at 10% concentration were used. Following dilution of sperm at ratio of 1:3, the samples were transferred into 0.25-ml straws and frozen in liquid nitrogen vapour (for 10 min at-120°C) and finally stored in liquid nitrogen (-196°C) tank. The frozen spermatozoa were thawed in a water bath at 30°C for 20s. Cell motility, motility duration and also fertilizing ability were determined in post-thawed samples. In spite of highest mean post-thaw motility (40.00±5.77%) was determined with extender E-3 supplemented with DMSO, the best mean motility duration was determined with DMSO containing E-2 extender as 106.67±43.33s. On the other hand, DMA supplemented E-2 extender provided highest mean fertility as 99.66±0.33%. According to results of the experiment, scaly carp sperm can be successfully cryopreserved with sugar based extenders supplemented with DMSO and DMA at 10% concentration.

It was 2012 when the first case of corona virus noticed by the Saudi Authorities which spread through the Asian countries, also flown to European countries. In 1960 revealed the presence of the corona virus when it was declared as the reason of common cold in human beings. On January 30, 2020, the WHO declared the COVID-19 outbreak a global health emergency. On March 11, 2020, the WHO declared COVID-19 a global pandemic. There are three brands of vaccines available that are Pfizer, Moderna and Johnson & Johnson (Janssen). A mild difference may present as post vaccination effects. The Pfizer-BioNTech COVID-19 vaccine is based as mRNA and marketed as Comirnaty. Pfizer reported a 91 percent efficacy rate after six months, while Moderna reported a 94 percent efficacy rate. Moderna, we are pioneering a class of medicines based on messenger RNA (mRNA). The Janssen COVID-19 Vaccine may not protect everyone. Mostly 2 doses of the vaccine are required to protect, and these are intramuscular injections. Its quantity would be changed in different age groups. Some common side effects include fatigue, headache, and pain in joints, fever, chills, and muscle pain. However, everyone should vaccinate for protection of corona virus disease.

Sperm survival depending upon integral membranes and function is imperative for fertilization. This study was designed to augment survival of buffalo spermatozoa using alpha-lipoic acid (ALA) and cholesterol-loaded cyclodextrin (CLC) during cryo-preservation. Semen was frozen using 0, 0.5, 1, 1.5, 2 and 2.5 mmol L −1 ALA (experiment 1) and ALA or CLC separately or together (experiment 2). Semen was assessed for post-thaw motility, plasma membrane integrity (PMI), intact acrosome and plasma membrane (IACR-IPM) and DNA integrity at 0, 1.5, 3 and 4.5 hr of incubation. In experiment 1, use of 0.5 mmol L −1 ALA enhanced the sperm cryosurvival and post-thaw longevity than other groups up to 4.5 hr of incubation, and this concentration of ALA was used in second experiment with CLC. The results revealed higher (p < .05) sperm survival function and time of sperm attributes due to use of ALA than CLC and control. However, the sperm quality did not improve (p > .05) when ALA was combined with CLC. In conclusion, survival of buffalo bull spermatozoa during freeze-thawing and post-thaw incubation can be enhanced more with ALA than CLC or control, followed by CLC than control. However, there is no synergistic effect on survival of buffalo bull spermatozoa due to ALA and CLC. K E Y W O R D S alpha-lipoic acid, buffalo sperm survival, cholesterol-loaded cyclodextrin, cryopreservation, synthetic oviductal fluid

a b s t r a c t
The objectives of the present study were to investigate the effects of three equilibration
times (0, 2, and 4 h) and two extenders (TRIS or Bioxcell®) for cryopreservation of bull
semen. Semen from 12 Gyr bulls was cryopreserved using an automated freezing machine.
There were significant interactions between equilibration times and extenders for sperm
motility and membrane integrity. The control treatment (0 h equilibration) had the lowest
values (P < 0.05) for total (MOT) and progressive motilities (PROG), and percentage of
sperm with intact plasma and acrosomal membranes (IPIA), with no significant differences
between extenders. Extender TRIS had greater cryoprotective action than Bioxcell®, with
greater MOT, PROG, IPIA at 2 and 4 h, as well as the lowest proportion of damaged plasma
membrane (DPM, 72.2% vs. 85.8%) for all times. Equilibration for 4 h yielded the most desirable
(P < 0.05) for MOT, PROG, and IPIA, and the least DPM percentage (86.5, 78.0, and 72.6%
for 0, 2, and 4 h, respectively). Overall, the combination of TRIS and 4 h of equilibration
was the most desirable semen cryopreservation method, with greatest MOT, PROG, and
IPIA (TRIS-T4 = 26.8%; BIO-T4 = 18.3%) and the least DPM. In conclusion, based on objective
analyses, equilibration during cryopreservation was essential for maintaining motility and
integrity of sperm membranes; equilibration for 4 h yielded the greatest sperm survival,
independent of the extender used.

The effect of supplementing L-glycine and L-carnitine in cryopreservation mixture on post-thaw semen and fertility parameters was studied. Semen from adult PD6 line roosters was collected, pooled and used. The samples after equilibration at 5°C for 30 minutes were mixed with the diluent with the final concentration of supplements L-glycine of 5 or 15 mM and L-carnitine of 1mM along with a cryoprotectant (4 % dimethylsulfoxide; DMSO), and the semen was cryopreserved in 0.5 ml French straws. Different in vitro semen quality parameters, fertility and hatchability were assessed in post-thaw samples. Post-thaw sperm motility, live sperm, MTT dye reduction test and seminal plasma lipid peroxidation were significantly lower (P < 0.05) in the cryoprotectant only (4 % DMSO) as well as in the L-glycine-and L-carnitine-supplemented treatments. Fertility was significantly (P < 0.05) lower the L-glycine-and L-carnitine-supplemented treatments compared to fresh semen, however, hatchability on fertile egg set was similar in all the treatments. The post-thaw semen parameters, fertility and hatchability in L-glycine-and L-carnitine-supplemented treatments were similar to that of the cryoprotectant only treatment. In conclusion, L-glycine and L-carnitine supplementation did not improve post-thaw semen parameters or fertility and hatchability. Thus, inclusion of these compounds in the chicken semen cryopreservation mixture may not provide advantage during the cryopreservation process.

The aim of this study was to determine the effect of different extenders, glycerol levels, and thawing rates on post-thaw sperm motility and fertilization ability of cryopreserved African catfish (Clarias gariepinus) sperm. Having determined the main spermatological properties (volume, motility, motility duration, spermatozoa concentration and pH), the pooled ejaculates were diluted with 3 different extenders containing different glycerol levels (5, 10 and 15%) respectively. Dilution ratio was 1:10 and the diluted sperm was packaged in 0.25 ml straws and left for 10 min equilibration at 4ºC. Following equilibration, the straws were exposed to liquid nitrogen vapor for 10 min and plunged into liquid nitrogen (-196ºC), and then exposed to different thawing rates (30ºC/20s and 40ºC/20s) to determine sperm motility and post-thaw motility duration. The highest post-thaw sperm motility, motility duration, and fertilization rate was 85%, 81s, and 95% respectively when sperm was frozen with the extender (ACSE 3) containing 15% glycerol (p<0.05). The protocol reported in this study can be successfully used for cryopreservation of African catfish sperm.

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85 ± 4% motility and 160 ± 2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44 ± 9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58 ± 14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5 ± 6% live cells, while the cryopreserved sperm only contained 26.6 ± 14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.

Genetic information is essential for conservation and future aquaculture development of the endangered catfish Rita rita (Hamilton, 1822). Two hundred catfish, R. rita, 50 from four rivers, the Old Brahmaputra, Jamuna, Meghna and Kangsa were collected and analysed to evaluate the genetic diversity and population structure using five microsatellite primers (Cba06-KUL, Cba08-KUL, Cba09-KUL, Phy03-KUL and Phy07-KUL). Four of the five amplified loci were found polymorphic (P95) in all the populations and 46 alleles were recorded with 9 to 14 alleles per locus. Differences were observed in the total number of alleles ranging from 41 to 44, effective number of alleles from 29.96 to 37.46, observed heterozygosity from 0.57 to 0.76, Shannon’s information index from 2.09 to 2.30 and polymorphic information content from 0.84 to 0.88 among the four populations. Results exposed the highest levels of genetic diversity in the Meghna population while the lowest in the Kangsa population of R. rita. All the populations were significantly deviated (P < 0.001) from the Hardy-Weinberg equilibrium for all the loci. Nei’s genetic distance between populations ranged 0.007 to 0.017 with low overall genetic difference FST = 0.011 and high gene flow Nm = 24.333, indicating that R. rita populations were not subdivided. This study revealed a high level of gene diversity with deficiency in genetic heterogeneity in all the populations of R. rita, emphasising natural management, conservation and rehabilitation measures of this species.

Drug metosartan the combination of both metoprolol and telmisartan has proved effective in the treatment of hypertension but the present study deals with the invivo effects of metosartan on testis tissue. It causes release of cytc from mitochondria in both fresh and treated invivo tissue but the marked effect was seen in invivo treated tissue and mitochondrial viability comparision in both drug treated and RNaseA+ drug treated tissue. DNA fragmentation in both RNaseA + drug treated tissue was markedly shown. The combined effects of drug and RNaseA has shown that the apoptosis occurs through extrinsic pathway in testis as DNA is fragmented which is the main cause of apoptosis. Introduction Mitochondria plays a major role in most of the cellular process namely redox maintainance, energy metabolism, ion balance and apoptosis. Apoptosis should be clearly distinguished from the another cellular death process namely necrosis. In case of necrosis the mitochondria lyse where as in case of apoptosis the cells shrink with intact cellular organelles and also mitochondria [1, 2]. Cyt C and AIF (Apoptosis Inducing factor) are the two major proteins release from the mitochondrial membrane but the present study focus on Cyt C. Cyt C is a 12.3 Kda protein involved in the electron transport between complex III and complexIV in mitochondria. The precursor protein of Cyt C the apocytochrome C was synthesised in cytoplasm on free ribosomes and gets inserted in to mitochondrial membrane through non receptor mediated process. The mechanism of activation of apoptosis through Cyt C was clearly known now. The apoptosome Apaf known to contain the CARD domain [3] (Caspase Recruitment Domain) at N-terminal which is shared by caspases and ced-4 and WD-40 repeats at C-terminal regions. The process starts by binding of ATP or dATP to the walker boxes present in ced 4 and cyt c binds to WD-40 domains which exposes the CARD region of the Apaf. The Apaf doesn't have the proteolytic activity so the caspase undergoes autocatalysis. Caspase 9 causes cleavage of caspase-3 which causes cleavage of caspase-6, DNA fragmentation factor [4] and poly (ADP-Ribose) polymerase leading to nuclear lamin cleavage and DNA fragmentation leading to the other changes in apoptosis [5]. So, Cyt C release is a irreversible damage to the cell which is mediated in the form of the cell death and present study deals with the effect of drug metosartan through cyt C and RNase A. Figure: 1

Each amphibian species is evolutionarily distinct, having developed highly specialized and diverse reproductive strategies in both terrestrial and aquatic environments. These unique reproductive patterns and mechanisms, key to species propagation, have only been explored in a limited number of laboratory models. Although the development of applied reproductive technologies for amphibians has proven useful for a few threatened species, the real benefit of this technology has been new insights into the reproductive adaptations, behavior, endocrinology, and physiological mechanisms that have evolved over millions of years. As the basic fundamental database on amphibian reproductive physiology has grown, so has the applied benefit for species conservation. In particular, technologies such as non-invasive fecal and urinary hormone assays, hormone treatments for induced breeding or gamete collection, in vitro fertilization, and the ability to establish genome resource banks have all playe...

This paper is about cryopreservation technology which is the preservation technology currently use in the medical field in order to preserve the cells, tissues, organs and other biological samples for long time in ultra-low temperature. Word "Cryo" comes from Greek language which represent the ice cold or frozen "Preservation" is the process of keeping save or alive for long period of time. Cryopreservation is basically done under liquid Nitrogen which maintains the temperature at-196 ˚C without disturbing to the viability of the cells, tissues or organ. Stem cells which are mainly used in drug discovery process and I the organ transplantation process we have to keep those testing samples and organs alive for long period of time and due to the ice crystal formation, disrupting of cell membrane, osmotic pressure are the main possible reasons can caused to destroy the cell viability during the freezing. These injuries occur during the freezing is commonly use to called as cryoinjury and it can be avoid using cryoprotectants and proper temperature controlling. This paper contains the basic process of cryopreservation along with the technique behind the process.

An experiment was conducted to develop cryopreservation protocol for spermatozoa of stinging catfish, Heteropneustes fossilis and to use the cryopreserved sperm in its breeding trials. The activation of sperm motility at various concentrations of NaCl solution was tested and complete activation and inhibition of sperm were obtained at 0.4% and 0.9% to 1% NaCl solution respectively. In toxicity test, sperms were incubated with DMSO, methanol and ethanol at 5, 10, and 15% concentrations where DMSO and methanol produced better motility at 5 and 10% concentration with Alsever&#39;s solution and egg-yolk citrate at 5 and-10 min incubation period. Three extenders-Alsever&#39;s solution, egg-yolk citrate and Ginsburg Fish Ringer solution and three cryoprotectants-DMSO, methanol and ethanol were used for cryopreservation of sperm, and among the diluents, Alsever&#39;s solution with 10% DMSO showed best performance producing 77.50±3.22% post-thaw motility. On the other hand, egg-yolk citrate...

To our knowledge, this study discover First-Ever: Successful Vitrification of Human Sperm in an optimum quantity. Multiple attempts taken by many researchers to vitrify sperm of different living organism. But resulted either in partial success with controversy or failure. Moreover in our present work we got success in Vitrifying Human Sperm in an optimum quantity of 0.6 ml. Most importantly we performed it in a closed cryovial. This is effective enough to prevent cross contamination through liquid nitrogen. We used only three culture media for whole process. All three media and plastic disposable prepared & developed by Frozen Cell itself. 1. VITRI MEDIA-developed & named by Frozen Cell. 2. Den-1 (75% density gradient)-for sperm separation. 3. Enhance-sperm washing media. VITRI MEDIA (Vitrification Medium) The newly developed VITRI MEDIA does not have any macro-molecule like human serum albumin. Molecular weight of all chemicals are within limit (less than 200 millimoles) + Glycerol 6% (V/V). Out of 200 millimoles sucrose contributes only 20 millimols. Therefore osmotic pressure of VITRI MEDIA is not high enough to harm human sperm. We also tested VITRI MEDIA as sperm preparation medium & sperm culture medium. We noted, sperm can be separated effectively using VITRI MEDIA either by swim up or swim down method. Following washing we used VITRI MEDIA as nutritional media. We got an acceptable survival of sperm in VITRI MEDIA even after 72 hour and more in some case. It indicates that VITRI MEDIA provide optimum environment to sperm. Additionally I mention again that VITRI MEDIA does not contain human serum albumin or any substance derived from human, animal, bird, aquatic or plant origin. In other words it is 100% chemically defined. Failure of previous attempt of sperm vitrification. Many researchers tried to vitrify human / animal sperm by different method. But resulted in either partial success or complete failure. Main reason behind seems

El propósito del presente trabajo fue estudiar las condiciones para la adecuada criopreservación de espermatozoides y larvas de los erizos de mar Tetrapygus niger y Loxechinus albus. Muestras de semen se cargaron en pajuelas plásticas de 0.25 cc y equilibradas por 15 minutos a 20º C en 1.0 M DMSO y congeladas a una tasa de enfriamiento de 6º C/min hasta –20º C y a una tasa de enfriamiento de l9º C/min hasta –80º C, después de lo cual se transfirieron directamente al nitrógeno líquido. La tasa de descongelamiento fue de 1.070º C/min y la tasa de fecundación de estas muestras fue de 77% (rango 52-90,8) comparadas con 100% en los controles. Setenta y dos horas después de la fecundación, la tasa de sobrevida para los experimentales fue de 96% y de 95% para los controles. Para L. albus la tasa de fecundación de los experimentales fue de 88.5% (rango 84.5-95.5%) comparada con 100% de los controles. Aproximadamente 40 larvas pluteus de Tetrapygus niger se cargaron en cada pajuela plástica de 0,25cc y equilibradas por 20 minutos a 0ºC en 1.0M DMSO; congeladas a una tasa de enfriamiento de 16ºC/min hasta -11ºC y a una tasa de enfriamiento de 20ºC/min hasta –85ºC. Después las pajuelas se transfirieron a nitrógeno líquido. La tasa de descongelamiento fue de 420ºC/minutos. Sobrevida evaluada con diacetato de fluoresceína (FDA) fue de 31% al momento de descongelar y de este 59% a 3 días. Algunas larvas pluteus sobrevivieron hasta 29 días.

The possibility of cryopreserving animal gametes and embryos provides a most useful tool for breeding and genetic management. The purpose of the present work was to study the conditions such as the type of cryoprotectant, temperature and time of equilibrium, cooling and thawing rates, that might be used for adequate cryopreservation of sea urchin gametes and embryos. Early prisrn stages of T. niger were treated for 15 minutes at 20ºC in 1.0M solutions of Dimethyl sulfoxide (DMSO), Propylene Glycol (PG) or Ethylene Glycol (EG). Control experiments were carried out in Millipore Filtered Sea Water (MFSW) and artificial sea water (ASW). Survival rate assayed at 1.5 and 10 hours respectively was 95 and 81% in PG, 91 and 68% in DMSO and 97 and 76% in EG. Control experiments had a 100% survival. In subsequent experiments using unfertilized oocytes, 90% of treated and 98% of control oocytes were fertilized. Of these, 0.2% of experimentals and 95% of controls developed to the pluteus stage. When cytotoxicity was studied on later stages of development we found that 3% of two cell stage, 37% of morulae, 56% of gastrulae and 88% of prism reached the larvae pluteus stage. Seventy seven percent of pluteus stages survived for additional 20 hours after treatment. Cytotoxicity studies carried out with T. niger spermatozoa using in 1.0, 1.5 and 2.0M solutions showed that the rate of fertilization was greater in DMSO than in EG. For this reason semen samples were equilibrated for 15 or 45 minutes at 18ºC in 1.0 M DMSO and frozen at a cooling rate between 6 to 9ºC/min to -20ºC and from 19 to 39ºC/min to -80ºC. The thawing rate was of 1070ºC/min. The fertilization rate of the thawed spermatozoa was 76% and 63% for 15 and 45 min of equilibrium. The control fertilization rate was 100%. Seventy two hours after fertilization, the survival rate was 96% and 98% for the experimentals and 95% for the controls. In addition, L. albus frozen�thawed spermatozoa fertilized 91% of L. albus eggs.

La inseminacion artificial bovina ha sido la tecnica de reproduccion asistida de mayor difusion por las ventajas que presenta en el mejoramiento genetico, la prevencion y control de enfermedades de transmision sexual, la posibilidad de emplear toros con facilidad de parto y la optimizacion del manejo reproductivo del rodeo. La Inseminacion Artificial a Tiempo Fijo (IATF) suma beneficios tales como evitar la deteccion de celo, acortar el periodo de anestro post-parto y concentrar los partos en un periodo breve. El exito de la IATF depende de multiples factores entre los que se encuentra la calidad seminal. Ningun examen de semen in vitro presenta una correlacion alta con la fertilidad, por lo cual debe desarrollarse un protocolo de control de calidad que estudie el mayor numero posible de caracteristicas de los espermatozoides. La seleccion de mejores muestras para IATF permitiria evitar la repeticion en las inseminaciones o el repaso con monta natural. El objetivo del presente traba...

It was 2012 when the first case of corona virus noticed by the Saudi Authorities which spread through the Asian countries, also flown to European countries. In 1960 revealed the presence of the corona virus when it was declared as the reason of common cold in human beings. On January 30, 2020, the WHO declared the COVID-19 outbreak a global health emergency. On March 11, 2020, the WHO declared COVID-19 a global pandemic. There are three brands of vaccines available that are Pfizer, Moderna and Johnson & Johnson (Janssen). A mild difference may present as post vaccination effects. The Pfizer-BioNTech COVID-19 vaccine is based as mRNA and marketed as Comirnaty. Pfizer reported a 91 percent efficacy rate after six months, while Moderna reported a 94 percent efficacy rate. Moderna, we are pioneering a class of medicines based on messenger RNA (mRNA). The Janssen COVID-19 Vaccine may not protect everyone. Mostly 2 doses of the vaccine are required to protect, and these are intramuscular ...

The aim of the article is to report a review on different sperm cryopreservation techniques, various stress-related freeze-thaw damages altering sperm structure and function during conventional cryopreservation, and strategies to minimize these stresses. Sperm cryopreservation has allowed indefinite storage and successful transportation of valuable germplasm from proven sites at distant locations, for genetic upgradation through implementation of reproductive techniques, such as artificial insemination. Different techniques for sperm cryopreservation have been proposed such as conventional freezing techniques, directional freezing, and sperm vitrification. Drawbacks related to conventional freezing methods, such as heterogeneous ice nucleation and repeated freeze-thaw cycles at the ice front that disrupts and kill sperm cells, led to the emergence of the directional freezing technique. Sperm vitrification is advantageous as there is no ice crystal-induced physical damages to sperm. However, sperm vitrification has less applicability as encouraging results are only reported in human, dog, and cat. In spite of several drawbacks, conventional freezing techniques are still most widely used for sperm cryopreservation. Spermatozoa experience stresses in the form of cold shock, osmotic stress, and mainly oxidative stress during conventional cryopreservation ultimately reduces the sperm viability and fertility. Several attempts have been made in the past to minimize all these stresses individually or in combination. Membrane fluidity was increased to prevent the cold shock and cryocapacitation-like changes by the addition of cholesterol to the membrane. Antifreeze proteins were added in semen extender to minimize freeze-thaw damages due to heterogeneous ice nucleation and ice recrystallization. Oxidative stress was reduced either by neutralizing reactive oxygen species (ROS) through enzymatic, nonenzymatic, plant-based antioxidants or reductants; or by minimizing the level of sources like the semen radiation exposure, leucocytes, and dead and defective spermatozoa, which lead to ROS production during the semen cryopreservation process. A novel approach of minimizing oxidative stress was to reduce the oxygen tension in sperm microenvironment that is, extender by partial deoxygenation process, as a number of literatures pointed out direct link of O 2 with ROS production. When compared with other strategies, partial deoxygenation of semen extender with N 2 gassing is found as a cost-effective, comparatively easy and a potential approach to large-scale frozen semen production.

The effects of freezing methods (FR1, nonprogrammable/static, 5 cm above liquid nitrogen [LN 2 ] for 10 min, plunging in LN 2 ; FR2, programmable medium, +4°C to −15°C at 3°C min −1 , from −15 to −80°C at 10°C min −1 and final holding for 1 min at −80°C, plunging in LN 2 ; FR3, programmable fast, from initial holding at +4°C for 2 min, from +4°C to −20°C at 10°C min −1 , from −20°C to −100°C at 30°C min −1 , final holding for 1 min at −100°C and plunging in LN 2) were assessed on post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa. Mean sperm progressive motility (%), rapid velocity (%), average path velocity (μm s −1), straight line velocity (μm s −1), curved line velocity (μm s −1), integrities (%) of plasmalemma, mitochondrial transmem-brane, DNA and acrosome were higher (p < .05) in samples cryopreserved with FR3 compared to FR1 and FR2. Similarly, in vivo fertility (%) of buffalo spermatozoa was higher (p < .05) with FR3 than FR1 (%; 68.0 versus 50.0). We concluded that program-mable fast-freezing method (FR3) improves the post-thaw in vitro quality and in vivo fertility of water buffalo spermatozoa.

Abstract
The paper discusses the issue when a potential biological father is against the transfer after the creation of embryo(s) (artificial fertilization), before having the embryo transferred into the body of a surrogate mother, and/or when he requests the medical facility destruct the embryo(s), but the potential biological mother is willing to carry out the above-mentioned transfer.
In this case any action from the medical facility is facing a certain legal gap, as far as the embryo(s) has(have) already been created, and a written consent of both potential parents (parties of the agreement) is necessary for their destruction. Besides, the written consent is also necessary for the transfer of embryo(s) into the body of a surrogate mother. What should a medical facility and/or a court do in such and/or similar legal situation?
The paper addresses the above-mentioned issues, and the discussion is taking place in the light of philosophy, law, medicine and bioethics, and guiding proposals are offered for the subjects involved in the disputable issue(s). The discussion and applicability of reached conclusions are not limited to any particular country, and it can be applied to various countries worldwide, and it may represent a source of formation of a future positive law.