Effect of Alcohol on Human Sperm In Vitro

Effect of Alcohol on Human Sperm In Vitro EFFECTS OF ALCOHOL ON HUMAN SPERMATOZOA IN VITRO: SPERM CHROMATIN DISPERSSION TEST AND ROS. Omkar Pokharkar, Dr. Himanshu Patel, Madhuri Patel, Vidisha Bhatt. Abstract: This study was carried out to find the detrimental effect on human spermatozoa when exposed to alcohol/ethanol in vitro. For this study semen samples obtained from 12 healthy individuals were washed using the sperm preparation technique and each washed samples were divided into equal aliquots. Then ethanol at various concentrations of 60 mm and 120 mm were added to the aliquots labeled as test samples and samples without ethanol were considered as control samples. All samples with or without ethanol were incubated for 1 hour (60 minutes) at 370C. These samples when subjected to semen analysis after exposure to ethanol, it was found that samples added with 60 mm ethanol showed a decrease in motility and vitality, sperms in samples with 120 mm ethanol were almost static with minimum motility. Sperms in both test samples revealed degraded vitality. DNA fragmentation test or SCD when carried out it was revealed that ethanol caused DNA fragmentation in sperms and as the concentr ation of ethanol increased, DNA fragmentation also increased, motility of sperms decreased and morphology was affected to some extent such as the head and tail defects which was less in the control samples. The sample aliquots with 120 mm ethanol showed morphological abnormalities. The ROS test performed using Agarose-N-gel tubes on the unwashed/raw samples exposed to ethanol in different concentrations revealed the highest oxidative stress displaying dark purple color and unexposed samples showed light pink color indicating low oxidative stress. Reactive oxygenspeciescan damage DNA bases and can cause lesions that block the progression ofreplication. This study proved that, alcohol when consumed can destroy sperm mobility, vitality and causes high fragmentation levels. Keywords: Ethanol, sperms, DNA fragmentation, motility, vitality, ROS. INTRODUCTION: Alcohol is a drug which is classified as depressant, when consumed in low/controlled volume it can induce the stimulant effect, but if an individual consumes excess alcohol then he experiences the depressant effect. Alcohol is produced by fermentation of yeast, sugars, and starches. Alcohol gets absorbed rapidly from the stomach and small intestine into the bloodstream. Once alcohol enters the stomach, up to 20% of it gets absorbed and directly enters the bloodstream. Within minutes, alcohol reaches to the brain. Even various nutrients are not capable to pull this off. The remaining alcohol enters into the intestines and is absorbed along with the nutrients. Only a small amount of alcohol is excreted from the body through urine, sweat etc which is not enough to get rid of alcohol in the blood stream.When the alcohol is consumed, the human body is hardwired to metabolize alcohol immediately that means the body stops metabolizing all other things just to metabolize alcohol first becaus e protein, carbohydrates, and fat, can be stored whereas alcohol cannot be stored in the body so it has to be metabolized first and excreted as soon as possible. Alcohol can affect every organ in the consumer’s body and can damage the developing fetus in woman who is pregnant. Also alcohol has the potential to severely damage sperms. If alcohol consumption is fairly regular then it will affect sperm motility and vitality in men. Heavy alcohol consumption lowers the sperm count leaving the person infertile and the levels of free radicals generated due to alcohol can break DNA strands in the sperm cells. The aim of this study was focused on the effects of alcohol on sperms and mainly on the level of DNA fragmentation and oxidative stress caused in sperm samples due to alcohol in vitro. MATERIALS AND METHODS: For the study of alcohol effects on sperms in vitro, Semen samples from 12 healthy men (non-alcoholic) aged 21 – 27 were collected in wide mouthed sterile collection containers and Sperm samples were obtained during 3-4 days of sexual abstinence and liquefied for 20-30 minutes at room temperature. The quality of the sperm is judged on the basis of its morphology and movement patterns or motility and vitality. For in vitro study, sperm samples from healthy individuals were washed by swim up from pellet method, which is a very common method for retrieval of viable sperms and has a high success rate. All the washed samples were divided in 3 equal aliquots of 0.5ml. Ethanol in different concentrations of 60mm and 120 mm respectively were added to 2 sample aliquots labeled as test samples and 1 aliquot was un-exposed to alcohol labeled as control sample. These samples were incubated for 60 minutes at 370C and subjected to semen analysis according to the guidelines set by world heal th organization. Sperm count and motility evaluation was done under bright field microscope, morphology was studied by making smears of sperms on the slides and was stained using Giemsa stain. pH was observed by using a pH strip and vitality of the sperms were noted by staining sperms with eosin and nigrosin stain. Morphology was judged by referring the types of sperm deformities proposed by WHO (World Health Organization., 2010). Motility was calculated by using a formula: 100 X (number of motile spermatozoa)/ (total number of spermatozoa counted). Also for calculating vitality percentage: 100 X (number of viable spermatozoa)/ (total number of spermatozoa counted). More than 500 spermatozoa per ejaculate were evaluated for estimation of sperm motility and vitality. The outcome of this analysis was noted. The rest of the control and test samples were used for DNA fragmentation test. SPERM CHROMATIN DISPERSION TEST: The test aliquots were added with various concentrations of alcohol (60mm and 120mm respectively) to see what concentration of alcohol will affect the DNA of the sperms. The control sample (0.5 ml) with no ethanol and 2 test samples (0.5 ml each) mixed with ethanol incubated at 370 C for 60 minutes were subjected to DNA fragmentation test. Requirements per sample were 3 coated slides, 1 for control and other 2 for test. The procedure involved addition of sperm samples of both test and control to agarose tubes which were previously boiled for 2 minutes. After adding and mixing the 40 Â µl droplets of sperms from both control and test samples in the agarose tubes, a total of 150 Â µl droplets of sperm samples from each tube was extracted using micropipette and was laid on pre-coated slides. Cover slips were placed on the droplets for even distribution of sperm sample immersed in agarose matix on the slide. The 3 slides of 1 control and 2 test samples were kept inside a fridge for 6 m inutes to solidify the agarose gel in which sperms were added. After this step, cover slips were removed in such a way that the gel integrity was not disturbed. Acid denaturant (1 ml) was laid on the slide for 7 minutes and was drained. Then 1ml of lysis solution was overlaid on the slide for 20 minutes and was drained. Then both test and control slides were washed with 20 ml of distilled water and were overlaid with 1 ml each of dehydrating solutions 1, 2, 3 for 2 minutes each. After disposing or draining the dehydrating solutions, the slides were air dried for few minutes. Stain was prepared by mixing stain solution and stain diluting solution. The slides prepared for each of the 12 samples were stained for 3 minutes and were washed in a beaker filled with tap water to remove excess stain. Slides were again air dried for few minutes before observing under bright field microscope for halos. For calculating percentage of DNA fragmentation: 100 X (Number of spermatozoa with fragmente d DNA)/ (Total number of spermatozoa counted). More than 450 spermatozoa per ejaculate were evaluated for estimation of sperm DNA fragmentation (Omkar pokharkar et al., 2015). For this study, A Sperm DNA Fragmentation Detection Kit called ‘CANfrag’ [REF No.CA-001], provided by ‘CANdORE Bioscience’, Ahmedabad, Gujarat was used to assess the levels of sperm DNA fragmentation in the samples. This entire procedure was followed for all 12 samples and it took around 18 days (10th February –27th February, 2015) for completion of the study and to arrive at results. ROS (reactive oxygen species) test: A Reactive Oxygen Species detection kit called ‘CANros’ [REF NO.CA-002] also provided by ‘CANdORE Bioscience’ was employed for mapping oxidative stress in exposed and un-exposed samples. Along with washed samples of test and control, 2 aliquot (test) of raw samples of 0.5 ml each was also added with ethanol in various concentration of 60mm and 120 mm respectively and control raw sample of 0.5ml was un-exposed to alcohol. After incubation of both control and test samples for 60 minutes in an incubator at 370C, ROS test was conducted by heating the Agarose N-Gel tubes in boiling water at 90-100Â ºC for 2 minutes to melt the gel. The tubes were cooled down for 2 minutes at room temperature before addition of the sample. 0.2 ml of semen sample was added and mixed thoroughly with the melted agarose gel and air bubbles were avoided. The Agarose-N-gel tubes were then placed in an incubator at 37Â ºC for 60 minutes. After incubation the color changes were observed immediately and were compared with the color code mentioned in the ROS kit to determine the level of oxidative stress present in the sample (Omkar pokharkar et al., 2015). The results of this test were noted. RESULTS: The pH of both exposed samples (60mm and 120 mm) was in the abnormal range. Alcohol affected the pH of the samples in which it was added and increased with the increase in the concentration of alcohol. Morphology of sperms was affected by alcohol to some extent only in samples with 120 mm ethanol/ 0.5 ml of sperm sample. The sperm count in all the samples remained in normal range at these concentrations. The average sperm count for all samples throughout the study was in the range of 60 to 70 million sperms/per ml. The average percentages of motility and vitality obtained for all 12 samples were calculated and framed in the table below. Results are mentioned in the chart and table form below: Table I. Semen parameters for both alcohol exposed and un-exposed samples Chart I. Effect of alcohol on sperm motility The motility in sperm samples was decreased due to alcohol exposure. Motility in samples un-exposed to alcohol showed excellent motility and samples exposed to the alcohol showed a decrease in the motility as the concentration of alcohol increased. Chart II. Effect of alcohol on sperm vitality The chart mentioned above indicates that, the sperm un-exposed to the alcohol showed an excellent range of vitality percentage while the exposed samples at various concentration of alcohol showed a constant drop in the vitality percentage. The table mentioned below represents the average DNA fragmentation percentages obtained for all 12 samples. Table II. DNA fragmentation in exposed and un-exposed samples Chart III. Sperm DNA fragmentation due to alcohol Figure I. Original picture of DNA fragmentation slide un-exposed to alcohol (control slide) It is evident from the picture above that, the samples un-exposed to ethanol showed maximum sperms with intact DNA displaying large halos. On the other hand, the samples exposed to ethanol in concentrations of 60mm and 120mm showed maximum sperms with fragmented and degraded DNA and revealed minimum sperms with intact DNA displaying small and no halos. Below is the picture showing fragmented and degraded sperms. Figure II. Original picture of DNA fragmentation slide exposed to alcohol [60mm and 120mm (test slide)] ROS (reactive oxygen species) TEST. ROS test were performed using freshly collected semen samples. Test aliquot of core samples exposed to alcohol at concentration of 60mm/0.5 ml and the 2nd test aliquot exposed to alcohol at concentration of 120mm/0.5 ml of semen sample produced dark purple colour. On the other hand control aliquot un-exposed to alcohol showed light pink colour. Low levels of ROS (un-exposed samples). light pink Table III. Reactive oxygen species result for alcohol Dark purple color was obtained for both test samples which indicated that highest level of oxidative stress was induced in the sample due to alcohol. Whereas samples un-exposed (control) to alcohol showed light pink color. Alcohol, at even low concentrations can achieve highest levels of ROS. CONCLUSION: The study revealed that alcohol in high concentrations can damage the sperms in every possible way and make it non-viable. The results mentioned above point out to the fact that as the alcohol/ethanol concentration increases, the sperm quality decreases. Motility in control samples was greater as compared to both the test samples (see Chart I). Vitality in sperm samples went on degrading as the ethanol concentration increased (see Chart II). Morphological abnormalities were mostly scored on the test slides with 120 mm ethanol and samples with 60 mm ethanol and control samples with no ethanol showed a normal range of morphological defects and pH of samples also increased with the increase in concentration of alcohol (see Table I). This indicated that the alcohol in high concentrations can damage the physical appearance of sperms. The number of sperms with fragmented DNA was most prominent in the test samples with 120 mm ethanol and 60 mm ethanol. On the other hand, control samples sho wed fewer fragmentations (see Table II and Chart III). The sperms with fragmented DNA disperse small halos and most of the time no halos were observed (see Figure II) and sperms with intact DNA disperse big halos (see Figure I). By this method, it becomes easy to map the fragmented and non-fragmented DNA of the sperms. ROS test was conducted to evaluate the findings and it gave the further evidence of fragmentation caused due to alcohol in vitro by achieving dark purple color in the Agarose-N-gel tubes by reduction of nitro blue tetrazolium and un-exposed samples managed to display light pink color indicating low levels of free radicals (see Table III). Reactive oxygenspecieshas the potential to damage DNA bases and can cause lesions that blocks progression ofreplication. Damaged sperm chromatin contains base adducts and the prominent adducts found in human sperm DNA are 8OHdG (8-hydroxy-2-deoxyguanosine or 8-oxo-7,8-dihydro-2–deoxyguanosine) present in nuclear and mitochondr ial DNA is one of the predominant forms of free radical-induced oxidative lesions, and is widely used as a biomarker for oxidative stress and carcinogenesis also two ethenonucleosides such as 1,N6-ethenoadenosine and 1,N6-ethanoguanosine are found in sperm DNA. Single strand breaks are the direct consequences of oxidative attacks on sperm DNA. DISCUSSION: As the coin has 2 sides, alcohol too have a good and a bad side. It depends how the individual use the alcohol and for what purpose. Alcohol is in use for scientific purposes as well as for production of beverages such as beer and wine for centuries. Applications of Alcohol in the field of science date back to 18th century when it was used for dressing up the wounds and to sterilize the equipment for surgery (civil war in U.S. 1850’s). Today along with these applications, alcohol is also used as antifreeze, preservatives, fuels, and also as solvents. But high consumption of alcohol is not beneficial in any way; Addiction to alcohol could severely damage the reproductive system of that individual making him infertile. A few studies around the world indicated that consumption of alcohol every day can effectively reduce the sperm count and quality of the sperm degrades as the consumption of alcohol increases. The people with disorders such as ‘auto brewery syndrome’ (gut fermentation syndrome) have very less reproductive capabilities. In this type of syndrome body produces alcohol through endogenous fermentations within the digestive system. A fungal infection of ‘Saccharomyces cerevisiae’ in gastrointestinal tract is responsible for this condition. This constant production of alcohol leads to several other body problems such as kidney failure, liver dysfunction and leydig cells in the testes are damaged which are responsible for production of testosterone and the levels of testosterone in blood drops and also Sertoli cells present in the testes are affected which interferes with sperm maturation. Immature sperms present in the ejaculateare not capable to move and fertilize the egg. The aim of this study was focused on in vitro effects of ethanol which involved direct exposure of ethanol to sperms which caused more damaging effect. However, in vivo study of effects of alcohol on sperms would produce more promising and accurate resu lts as compared to in vitro study because alcohol undergoes elimination by various metabolic mechanisms that occur in the body. The enzymes involved are aldehyde dehydrogenase, alcohol dehydrogenase, cytochrome P450 and catalase. The interaction occurring between alcohol byproducts obtained after metabolism and other cell components, leads to the formation of dangerous compounds such as reactive oxygen species (ROS). Alcohol metabolism occurs primarily in the liver. Alcohol undergoes detoxification procedure and gets eliminated from the blood by the process termed as oxidation. Oxidation inhibits accumulation of alcohol and prevents destruction of cells and other organs in the body to some extent. So the in vivo results would be much different than in vitro study as the alcohol undergoes metabolic paths. In in vitro study direct exposure of ethanol caused high fragmentations and more damage was inflicted to the sperm parameters (motility, vitality, morphology, and pH). It can be con cluded that, consumption of alcohol is associated with degradation of sperm quality which can be reversed to some extent upon alcohol consumption discontinuation which would reduce the blood alcohol concentration (BAC) and intake of antioxidants on a regular basis would maintain low concentration of free radicals and will protect the DNA of sperms from damage. AKNOWLEDGEMENT: Authors would like to thank the entire team of stem cure pvt ltd, center for reproductive medicine and stem cell development – Ahmedabad, India, for their contribution whenever needed. Authors would also like to thank Komal Patel for providing laboratory equipments to carry out this study. REFERENCES: [1] World Health Organization. (2010). WHO laboratory manual for the Examination and processing of human semen (5 ed.). (D. T. Cooper, Ed.) geneva, Switzerland. [2] Omkar pokharkar et al. (2015). Effect of Wi-Fi radiations on sperms in vitro:Sperm DNA fragmentation test and ROS. International journal of healthcare sciences, III(1), 26-30.

River Road Community

We re all going to have to learn how to walk again. After a nuclear holocaust devastates the country of the United States, the people of the River Road Community have to work through adversity and strive for the survival of every family. Households have to do without the things they would usually have taken for granted. They have to bond together to fight the trials of learning to walk. Frank s quote demonstrates his understanding of the task ahead of them as they start to learn to live again. They would have to begin by crawling before they can once more stand-up on both feet. Frank s quote is manifested throughout the story by the observed changes in the lifestyle of the community, the ways in which the diminishment of supplies are dealt with, and how the loss of community members forces others to take on new roles. After the nuclear holocaust, the lifestyle of the River Road community changed drastically. Before The Day, many residents of the community were wastrels. These rejects of society lived each day to eat, drink, and sleep. They lacked the drive to succeed in everyday life. After the bomb drop, everything changed. These blots on modern society crawled out of their shells in order to contribute something to a struggling society. They worked hard on construction and other things that would help the community. This strenuous work gave these once slobs a new outlook on life as well as the regular exercise they lacked. But, this sudden increase in work was not the only variable playing a role on them. After the bomb, anarchy broke out among community and surrounding towns. Highwaymen robbed the innocent, and people were taken advantage of. They had to start over. Randy gained leadership for the community. The next part was to gain control. He and other community members formulated a plan in which to regain control. Once it was obtained, laws were made that would govern the growing community. The government had started all over again. Another lifestyle serve was in the economy. The US currency no longer had value. With no gold to back up the paper money, it was worthless (except as toilet paper). Instead, the River Road community relied on the barter system. Trades were made for the items that were needed. If one person needed a spoon, they could trade some honey for it. Because of the nuclear holocaust, the lifestyle of the River Road community changed. Another way in which the community had to learn how to walk again, arose from the lack of supplies. Before The Day, many residents live on a diet of beer and nuts. After the bomb incident, many of the foods found in the pre-bombed world were no longer available. During chapter 666, the food supply runs terribly low. Helen and Lib create a new salad using leaves and other greenery found around the house. This new salad would never have been attempted had it not been for the lack of food due to the holocaust. The nutritional value of this green salad, compared to the beer and nuts, shows a positive improvement in nutritional content of their diet. Another loss came as a result of the loss of running water after the nuclear holocaust. Not long after the water was cut off the tub supplies and other rations of water, which had been stored up, were running out. The community had to limit its use of water and cut back on things that on a pre-bomb day would have seemed natural (like flushing the toilet or taking a bath). The community adapted the Artesian water to use as their source for water. This water was not as nice as the purified water they had previously had, but it was water none-the-less. The harnessing of this water required the help of many. LALALAALA. Also, when medical supplies ran low, things had to be improvised. With just the aid of a billiard table, steak knives, darning needles, hair curlers, and nylon line, Dan operated on Ben Franklin, removing his appendix. This is just one example where things had to be invented due to a lack of supplies. They also decided to use hypnotism as a form of anesthesia. Dan could speak a few sentences, and they would fall into malleable trance. He would then be able to operate without the patient feeling the procedure. Another example of the River Road community having to learn to walk again would be with food preservation. Without the use of refrigerators and other systems for keeping food fresh, there was a great need for salt. The salt could be used to preserve most meats. An animal could be slaughtered, and part eaten for a meal. The remaining portions of the animal could be salted and kept for a while. Towards the end of July, Randy noticed the salt supplies running dangerously low. He and many other members of the community had to set sail in search of a source for salt. They did return triumphantly with many bags of salt. These are all ways in which the community has adapted to a loss of supplies and had to work through the adversity and learn to walk again. The last way in which the River Road community had to learn to walk again was how the loss of community members forces the others to take on new jobs. When the towns were bombed, many skilled people were lost. Jobs needed to be filled and the residents of the River Road community rose to the challenge. Helen served as the barber for the group, cutting everyone s hair. Randy was the leader. He took control of the group and returned order to an anarchical society. Dan took over as the full time doctor, and Lib took over most of the cooking. Many Frank s quote, we re all going to have to learn how to walk again†¦, is manifested throughout the story by the observed changes in the lifestyle of the community, the ways in which the diminishment of supplies are dealt with, and how the loss of community members forces others to take on new roles. The great disaster affected everyone, and everyone pulled together to strive through the hard times. The community, crippled by the nuclear blast, regained strength and managed to walk again. The road there was filled with hardship and pain, but the path made them stronger. The River Road Community survived the nuclear holocaust, even though they had to learn how to walk all over again.

Top Tips of What Are Good Argumentative Essay Topics for Teen Girls

Top Tips of What Are Good Argumentative Essay Topics for Teen Girls What Are Good Argumentative Essay Topics for Teen Girls Ideas Teens should have to take parenting classes. Debate topics don't always need to be serious they are able to decidedly be humorous too! Choosing topics can be difficult. Controversial topics aren't a warm-up exercise! The What Are Good Argumentative Essay Topics for Teen Girls Chronicles The intent of assigning an essay to middle school students is to make awareness and permit them to develop writing skills. Going to a debate, students have to take into account their degree of education to select an appropriate topic that suits both your requirements and your level. Another reason is to observe how well students argue on unique views and demonstrate understanding of the studied subject. Students with a high degree of proficiency relish the chance to express personal points of view and to speak about something from the ordinary. Top What Are Good Argumentative Essay Topics for Teen Girls Choices Inspiration to make your own advertising or media argumentative essay topics isn't tricky to discover. You may continue to keep your argumentative essays for your upcoming job portfolio in case they're highly graded. In fact, many topics for research papers are much too big for a couple pages. A trusted writing services business can assist you with all that. Argumentative writings is a particular kind of a paper. To locate argumentative essay topics easy on various platforms, you will need to comprehend about the argumentative essay. An argumentative essay is a certain sort of academic writing. It is a type of academic papers that students write in the middle school. You don't need to find super technical with legal argumentative essays, but make certain to do your homework on what the present laws about your preferred topic actually say. Actually, you can make up any topic you want all on your own, as long because there is unlimited amount of those. Argumentative essay is about arguing and debating on a subject, which is debatable. The success of your essay is in the perfect selection of the topic. Researching the topic will permit you to find out more about what fascinates you, and should you pick something you really like, writing the essay will be more enjoyable. If you're, then you'll be glad to know there are lots of great debate topics touching on technological innovations. Sometimes you'll get a chance to set your creativity free and just take pleasure in the process as you're participating in a debate on funny or only intriguing debate topics. Therefore, the topic ought to be debatable! Quite frequently, the ideal topic is one which you truly care about, but you also will need to get ready to research it. Therefore, it's important to pick a topic that wouldn't be difficult to study. You will likely find a different and terrific topic you will love to write about. In choosing your topic, it's frequently a good idea to start out with a subject which you already have some familiarity with. The most frequently encountered difficulty is to settle on a debate topic for students. Deciding on an emotional topic is also an excellent idea. You should participate in far-reaching studying to cover all facets of the subject and all point of views by different men and women.

Example Sentences of the Verb Hit

Here are examples of sentences using the verb to hit in all tenses including active and passive forms, as well as conditional and modal forms. Base Form hit / Past Simple hit  / Past Participle hit / Gerund hitting Present Simple:  He often hits balls with his son in the park.Present Simple Passive:  Home runs are often hit out of this ballpark.Present Continuous:  He is hitting the ball hard today.Present Continuous Passive:  The ball is being hit hard today by all the players.Present Perfect:  She has hit fifteen home runs this season.Present Perfect Passive:  Fifteen home runs have been hit by Susan this season.Present Perfect Continuous:  Jack has been hitting that punching bag for the last twenty minutes.Past Simple:  Mary hit a home run out of the park yesterday.Past Simple Passive:  A home run was hit out of the park during yesterdays game.Past Continuous:  Hank was hitting the punching bag when his trainer walked into the gym.Past Continuous Passive:  The punching bag was being hit when the trainer walked into the gym.Past Perfect:  She had already hit four home runs when she hit the grand slam!Past Perfect Passive:  A grand slam had already been hit when the g ame was canceled due to the rain.Past Perfect Continuous:  Alan had been hitting the punching bag for twenty minutes when the trainer walked into the gym.Future (will):  She will hit the roof when she finds out!Future (will) passive:  The ball will be hit out of the park.Future (going to):  Jack is going to hit the ball out of the park today.Future (going to) passive:  The ball is going to be hit out of the park today.Future Continuous:  This time tomorrow we will be hitting the punching bag in the gym.Future Perfect:  William will have hit three home runs before the game is finished.Future Possibility:  She might hit him if he doesnt shut up!Real Conditional:  If she hits a home run, the team will celebrate.Unreal Conditional:  If she hit a home run, the team would celebrate.Past Unreal Conditional:  If she had hit a home run, the team would have celebrated.Present Modal:  You should hit a punching bag to get rid of your anger.Past Modal:  She must have hi t a home run. Quiz: Conjugate With Hit Use the verb to hit to conjugate the following sentences. Quiz answers are below. In some cases, more than one answer may be correct. Mary _____ a home run out of the park yesterday.She _____ fifteen home runs this season.He _____ the ball hard today.The punching bag _____ when the trainer walked into the gym.A grand slam _____ already _____ when the game was canceled due to the rain.She _____ the roof when she finds out!William _____ three home runs before the game is finished.If she _____ a home run, the team would have celebrated.A homerun _____ out of the park during yesterdays game.He often _____ a punching bag to relax. Quiz Answers hithas hitis hittingwas being hithad been hitwill hitwill have hithad hitwas hithits