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PubMed1996

Growth hormone treatment of subfertile males.

OBJECTIVE: To study spermatogenesis and sperm motility during GH therapy in infertile men. DESIGN: Prospective open study. Each patient was treated with GH for 12 weeks and followed for a total of 36 weeks with sampling of blood and semen. SETTING: Outpatients studied at a clinical research unit of a university hospital. PATIENTS: Nine oligozoospermic (<5 x 10(6) sperm/mL) males and nine asthenozoospermic (percentage motile sperm <30 and >15 x 10(6) sperm/mL) males. The patient groups had a significantly lower GH response to an arginine GH stimulation test as compared with a control group. RESULTS: Serum insulin-like growth factor I (IGF-I) and serum IGF-binding protein 3 (IGFBP-3) levels increased significantly during GH treatment, as did seminal IGF-I. Serum E2, T, PRL, FSH, LH, and GH-binding protein were unchanged during the study. Sperm motility was increased significantly during GH treatment in both patient groups. There was no difference in sperm count during the treatment. There were three pregnancies in the nine couples from the asthenozoospermic group and no pregnancies in the oligozoospermic group. CONCLUSION: The biologic and clinical results in this study encourage the initiation of double-blind, placebo-controlled trials.

Adult↗
PubMed1996

Value of clinical diagnosis in predicting the quality of cryopreserved sperm from cancer patients.

PURPOSE: Cryopreservation of semen from patients with malignant diseases (primarily testicular cancer, Hodgkin's disease and lymphoma) before specific chemotherapy, radiation or surgical therapy has become a realistic option to preserve fertility. Nevertheless, the post-thaw sperm quality in these patients is poor. Whether the clinical diagnosis of malignancy affects the cryopreservation of human sperm is unknown. Such an association could allow post-thaw sperm quality to be predicted. MATERIALS AND METHODS: We compared the effect of cryopreservation on sperm motility, velocity, linearity, amplitude of lateral head movement, motility index and motile sperm count in specimens from 30 normal men, 34 with testicular cancer, 39 with Hodgkin's disease, and 19 with leukemia and lymphoma who were referred for sperm banking during a 5-year period. RESULTS: The pre-freeze and post-thaw motility, motility index and motile sperm count were significantly lower in patients than in normal men (p < 0.01). No significant differences were noted among the patient groups. Similarly, no significant differences were noted after cryopreservation. Our results indicate that the poor pre-freeze sperm quality in some patients with malignancies explains the poor post-thaw results. However, the deterioration in sperm function after cryopreservation of semen among patients with different malignancies and normal donors appears to be similar. CONCLUSIONS: Clinical diagnosis of malignancy is not an adequate predictor of the effect of cryopreservation on human semen.

Cryopreservation↗
PubMed2006

Does exposure to di(2-ethylhexyl) phthalate in pre-pubertal boars affect semen quality post-puberty?

Di(2-ethylhexyl) phthalate (DEHP), a plastic softener used in polyvinyl chloride (PVC) products, has been ascribed to have toxic effects on animal reproduction. The present study aimed at determining potential late effects of pre-pubertal oral exposure to DEHP on semen quality in young pigs. Ten pairs of cross-bred male siblings were used. One brother in each pair became, at random, the test animal while the other acted as control. Test males were exposed to 300 mg/kg body weight (bw) of DEHP administered orally three times a week from 3 to 7 weeks of age. The control group was given placebo (water). Semen analyses started when the boars reached 6 months of age, with semen collected twice weekly, until animals were 9 months of age. Semen was evaluated for ejaculate volume, sperm concentration, total sperm count, sperm motility, sperm morphology (including presence of cells other than spermatozoa) and sperm plasma membrane integrity. Total sperm motility tended to be lower while local motility was higher in the DEHP-exposed group compared with controls (p = 0.07) when assessed by computer-assisted sperm analysis. The DEHP-exposed group had a significantly (p < 0.05) lower percentage of spermatozoa with tailless, defective heads (at 7-8 months of age) and double-folded tails (at 6-7, 7-8 and 6-9 months of age), compared with controls (albeit always under 5%). In summary, there were no obvious adverse effects of early oral exposure to 300 mg/kg bw of DEHP on sperm output and sperm quality in post-pubertal young boars.

Administration, Oral↗
PubMed2005

Breeding soundness evaluations of Senepol bulls in the US Virgin Islands.

The breeding soundness evaluation (BSE) was used to evaluate Senepol (Bos taurus) bulls (n = 495) on St. Croix over a 7-year period. Young, unproven bulls (10-26 months of age) and breeding bulls (16 months to 8.5 years) were tested prior to sale or use in breeding. Inbreeding coefficients were determined for a subset of bulls (n = 290). The percentage of bulls passing the BSE increased (P < 0.0001) with age. Bulls that passed had a higher percentage (P < 0.0001) of normal and motile sperm as well as a larger (P < 0.0001) scrotal circumference than bulls that failed. No bulls failed the BSE for physical soundness traits or other health reasons. The incidence of testicular hypoplasia was 2.5 and 3.3% and the incidence of cryptorchidism was 1.4 and 0.9% in 12- and 16-month-old bulls, respectively, with no occurrence in bulls >20 months. The proportion of all bulls that failed the BSE and received an Unsatisfactory rating for scrotal circumference or sperm motility decreased (P < 0.0001) from >90 to <25% with age. The proportion of all bulls that failed the BSE and received an Unsatisfactory rating for sperm morphology decreased (P < 0.0001) from 99 to 83.3% with age. The inbreeding coefficient was higher (P < 0.03) in bulls that failed the BSE than in those that passed (2.24 +/- 0.19% versus 1.40 +/- 0.32%, respectively). There was a tendency for bulls with testicular hypoplasia or cryptorchidism to have a higher (P = 0.09) inbreeding coefficient than bulls with normal testes (2.90 +/- 0.46% versus 2.13 +/- 0.11%, respectively). In conclusion, Senepol bulls raised under tropical conditions had a low probability of passing the BSE at young ages, but the passing rate increased with age. Older Senepol bulls were more likely to fail the BSE due to abnormal sperm morphology than due to inadequate testicular size or sperm motility. To prevent unnecessary culling, a BSE should not be performed on Senepol bulls <16 months old.

Animals↗
PubMed2003

Evaluation of the one-step eosin-nigrosin staining technique for human sperm vitality assessment.

BACKGROUND: The one-step eosin-nigrosin staining technique for assessment of sperm vitality was developed in the 1950s for various mammalian species. Although commonly used on human sperm in semen, a validation for this use has not previously been published. METHODS: The technique was evaluated on 1235 consecutive semen samples. RESULTS: The one-step eosin-nigrosin staining technique gave valid results when evaluated with sperm motility data obtained according to World Health Organization standard (1992, 1999). The mean for the sums of stained (i.e. supposedly dead) and motile sperm using the one-step eosin-nigrosin technique was 91% (SD +/- 10%). The distribution of sums for percentage stained and percentage motile sperm was similar, regardless of whether the samples had many or few dead sperm. CONCLUSIONS: Standardization and quality control of basic semen analysis demands robust, reliable and simple techniques that are easy to learn, and easy to continue to perform in the same way. The one-step eosin-nigrosin technique does not need negative phase contrast optics but can be run with ordinary bright-field microscopy. Since it also includes fewer methodological steps to control, it seems preferable in terms of standardization and quality control management. It should therefore be recommended in the basic semen analysis when sperm vitality is to be assessed.

Aniline Compounds↗
PubMed1993

Effect of sucrose, trehalose, hypotaurine, taurine, and blood serum on survival of frozen bull sperm.

Factorially arranged experiments were conducted to study the effects of adding sucrose and trehalose, known to have cryoprotective properties, blood serum, and the antioxidants taurine and hypotaurine on sperm motility after freezing and thawing at different rates. At sugar concentrations of 0.1 M, osmolality of the whole milk (WM) or egg yolk-Tris freezing medium (without glycerol) was about 370 mOsmol. Survival following freezing and thawing was reduced unless osmolality was corrected by adding pure water to reduce osmolality to about 280 mOsmol. Then the post-thaw percentages of motile sperm for control WM, and WM with 0.05 or 0.10 M sucrose, or 0.05 or 0.10 M trehalose, respectively, were 62, 55, 61, 57, and 62%. Thawing semen at 5 degrees C, versus 25 degrees C resulted in 64 versus 70% motile sperm (P < 0.05). Post-thaw survival of sperm stored at 25 degrees C for 24 h in trehalose-treated WM was superior to that in WM (P < 0.05). Hypotaurine and taurine had little effect on sperm survival. Up to 10% (v/v) of heated blood serum was generally beneficial, but gave more variable responses with different bulls. Sperm survival after cooling at -25 degrees C/min was slightly superior to that after cooling at -15 degrees C/min to -100 degrees C. The effects of the compounds studied on motility of frozen-thawed sperm were small, but if they protect sperm cell membranes, as reported for other types of membranes, they may assist sperm in surviving in the reproductive tract of the cow prior to fertilization.

Animals↗
PubMed2001

Storage of Chinchilla lanigera semen at 4 degrees C for 24 or 72 h with two different cryoprotectants.

The objectives of this study were to: (a) test the functional activity of Chinchilla lanigera spermatozoa suspended in either glycerol or ethylene glycol, cooled to 4 degrees C, and stored for 24 or 72 h and (b) investigate, after these cooling periods, the effects of incubating sperm at 37 degrees C (for 4 h) upon sperm functional activity. The ejaculate was mixed with the cryoprotectant medium (at 1 M final concentration) and cooled to 4 degrees C. After warming, sperm motility, sperm viability, hypoosmotic swelling test results, and acrosomal integrity were significantly higher for samples containing ethylene glycol than for those in glycerol, stored for 24 or 72 h, and then assayed after 0 or 4 h incubation at 37 degrees C. A significant reduction of sperm motility and viability was detected only when the glycerol cryoprotectant agent was employed, compared to the fresh samples. These results clearly indicate that under our experimental conditions, ethylene glycol is a better protectant for sperm storage than glycerol.

Animals↗
PubMed1997

The hypo-osmotic swelling test in recurrent miscarriage.

OBJECTIVE: To determine whether there is any association between sperm membrane integrity as determined by the hypo-osmotic swelling test score and unexplained recurrent miscarriage. DESIGN: Prospective observational study. SETTING: Tertiary referral center for recurrent miscarriage. PATIENT(S): Semen samples from 20 male partners of women who had had three or more first trimester miscarriages of unexplained etiology and semen samples from 20 prospective semen donors of unknown fertility potential. MAIN OUTCOME MEASURE(S): Sperm density, sperm motility, sperm morphology, and hypoosmotic swelling test score. RESULT(S): There was no difference in the median sperm density, the mean sperm motility, or the mean sperm morphology between the two groups. However, the recurrent miscarriage group had a significantly lower hypo-osmotic swelling test score than the control group. CONCLUSION(S): The hypo-osmotic swelling test score is significantly lower in samples from men whose partners have had unexplained recurrent spontaneous abortions. With the exception of cytogenetic abnormalities in peripheral blood karyotype, this is the first study to identify a male factor component in recurrent miscarriage.

Abortion, Habitual↗
PubMedDate not supplied

Effects of temperature on sperm motion characteristics and reactive oxygen species.

OBJECTIVE: To evaluate the effect of temperature on sperm motility, sperm motion characteristics, and production of reactive oxygen species (ROS) in semen. METHODS: We collected semen samples from 12 infertile patients and 12 healthy donors and divided each sample into four aliquots just after liquefaction. One aliquot was analyzed immediately after liquefaction and the remaining three were analyzed after one hour of incubation at 4 degrees C, 25 degrees C, and 37 degrees C. Sperm motility and motion characteristics were measured by computer-assisted sperm analysis (CASA). Levels of ROS were determined in washed sperm suspensions using a chemiluminescence assay and results were expressed as x10(6) counted photons per minute (cpm)/20 million sperm/mL. RESULTS: Motility, curvilinear velocity, straight-line velocity, average path velocity, and amplitude of lateral head displacement decreased significantly from baseline values in both the patients and donors after incubation at 4 degrees C. Percentage motility was highest and ROS levels were lowest in the samples that were incubated at 37 degrees C. CONCLUSION: Based on our results, we suggest that semen samples be stored at 37 degrees C after collection and during transportation and processing.

Adult↗
PubMed1990

[Studies on pyospermia in male infertility].

Among 670 infertile men, 72 were diagnosed as pyospermia according to our criteria. i.e., WBC greater than or equal to 10/hpf semen. The sperm motile efficiency index (SMEI) which indicates the rate of progressively motile sperms, was significantly low in pyospermic group compared with that of non-pyospermic men (WBC less than 5/hpf semen). From the result of split ejaculation, a major cause of pyospermia was supposed to be chronic prostatitis. The SMEI was decreased immediately after addition of the neutrophils and granulocyte elastase to semen. The mean value of granulocyte elastase in pyospermic group was 2859.6 micrograms/L, whereas that of non-pyospermic men was 131.6 micrograms/L. In summary, granulocyte elastase in seminal plasma may be a cause of inhibition of sperm motility in pyospermic state.

Chronic Disease↗
PubMed1986

Season and social environment influence the membrane integrity of ejaculated boar spermatozoa as assessed by ouabain sensitivity.

Eight adult Landrace boars were housed for 12 months in one of two social environments. Socially nonrestricted boars were penned near estrual females and socially restricted boars were penned behind solid walls to eliminate visual and physical contact with other pigs. All animals were subjected to natural changes in day length. The sensitivity of ejaculated spermatozoa to ouabain (in inhibitor of Na+-K+ ATPase) was determined on 4 consecutive weeks in November, March-April, and July-August. Semen was diluted in Tyrode's solution (pH 7.4) with and without 10(-3) M ouabain. Duplicate samples of control and ouabain-treated spermatozoa were incubated at 37 degrees C for 4 h, and percent motile sperm, motility type, and motility index (combination of percent and type) were determined at hourly intervals. Ouabain-induced decreases in most motility parameters varied with season (season X treatment, P less than 0.05). At hour 4, induced decreases in percent motile sperm were more pronounced in November and July-August than in March-April for socially nonrestricted boars. Decreases in motility type were greater (P less than 0.05) in November and July-August than in March-April for socially nonrestricted boars and were greater (P less than 0.01) in November than in July-August for restricted boars. In March-April motility type decreased (P less than 0.01) to a greater extent for socially restricted vs. nonrestricted boars. Similar season and social environment differences were observed for motility index values. Given the interrelationships between ouabain sensitivity, the functional integrity of sperm cells, and fertilizing capacity, season and social environment differences in ouabain-induced motility depression probably reflect qualitative changes in boar spermatozoa.

Animals↗
PubMed1982

Assessment of fertility after testicular torsion: an experimental study.

The spermatic cord was ligated in Charles River adult rats producing viable reversible and non viable damaged testes. Weight of the rats, weight of the testes, weight of the epididymis, sperm motility, sperm concentration and fertility were the different parameters assessed. The results showed that there is a high percentage of subsequent atrophy in the ligated testes, and that there is a definite correlation between the length of the occlusion and the amount of testicular injury sustained. Leaving a ligated testis in situ for three months provoked a severe reduction in fertility and some lesser reduction in the sperm motility in the contralateral testis. This damage was not observed in the rats where the ligated testes were removed.

Animals↗
PubMed1998

Survival of bull sperm frozen at different rates in media varying in osmolarity.

The effects of freezing procedures, osmolarity, trehalose, and sucrose on survival of bull sperm in whole milk (WM) and egg yolk-Tris (EYT), semen extenders used worldwide, were studied. Sperm were added to extenders at 25 degreesC, cooled slowly to 5 degreesC, glycerolated, packaged in 0.5-ml straws, and frozen. Different freezing rates were accomplished in two steps. Straws were transferred from +5 degreesC to nitrogen vapor at temperatures ranging from -10 to -100 degreesC in the first step and to liquid nitrogen in the second step. Straws were thawed in water at 35 degreesC. A substantial decrease in sperm motility occurred between -10 and -20 degreesC, as abrupt nucleation occurred following supercooling to -13 degreesC. To study the interactions between osmolarity x cooling rate, WM and EYT extenders were prepared to yield media measuring 220 to 420 mOsm/L. The optimal first-step range of cooling in the two-step procedure was -30 to -70 degreesC, and the highest proportions of motile sperm after freezing and thawing were 61 to 62 in 260 to 300 mOsm/L WM and 63 to 64% in 300 to 340 mOsm/L EYT, equivalent to the results with the control procedure used commercially. As the cooling rate increased (first step to -100 degreesC) sperm motility was much higher in hypertonic than in hypotonic extenders (P < 0.05), indicating the importance of partial dehydration before rapid cooling. Replacing part of EYT and WM with equivalent solutions (same mOsm/L) of sucrose or trehalose had no appreciable effect. These results provide a basis for further investigating simple freezing systems that might be more effective in preserving bull sperm than those currently available.

Animals↗
PubMedDate not supplied

Discriminant analysis indicates a single sperm protein (SP22) is predictive of fertility following exposure to epididymal toxicants.

In a previous study, we found that ethane dimethanesulphonate (EDS) compromised the fertilizing ability of proximal cauda epididymal sperm from the rat within 4 days of exposure, an effect that persisted in castrated, testosterone (T)-implanted animals, establishing direct action on the epididymis. This EDS-induced reduction in fertilizing ability was highly correlated with a quantitative decrease in specific sperm protein. Here we sought to determine whether the fertility of proximal cauda epididymal sperm recovered from animals exposed to a variety of male reproductive toxicants could be predicted by assessing quantitative changes in specific sperm protein(s), or whether more common endpoints (e.g., sperm motility, sperm morphology, serum and epididymal tissue T, cauda epididymal sperm reserves) also are required to predict fertility. Intact adult male rats were dosed with EDS (25 or 50 mg/kg), chloroethylmethanesulphonate (CEMS; 12.5 or 18.75 mg/kg), or epichlorohydrin (EPI; 3 or 6 mg/kg) daily for 4 days. Castrated, T-implanted rats were dosed with hydroxyflutamide (HFLUT; 12.5 or 25 mg/kg) daily for 5 days. On day 5, proximal cauda epididymal sperm were inseminated in utero into receptive, cervically stimulated adult females, and on day 9, fertility (implants/corpora lutea) was assessed. Fertility-was decreased by the higher dose of each toxicant (P < 0.05) and also by the lower dose of EPI and HFLUT. Likewise, an acidic 22 kDa sperm protein (SP22) was decreased quantitatively (P < 0.05) in silver-stained two-dimensional gels by the higher dose of each toxicant as well as by the lower dose of EPI and HFLUT. Although sperm motility and serum T were altered by specific exposures, these endpoints were not useful in predicting fertility. In contrast, SP22 was highly correlated (P < 0.0001; r2 = 0.83) with fertility. Indeed, the amount of SP22 correctly predicted 90% and 94% of the fertile (> 50% fertility) and subfertile (< 50 fertility) animals, respectively, when discriminant analysis was performed. Thus, the amount of SP22 in a cauda epididymal sperm sample may be a useful predictor of fertility in toxicant-treated animals.

Animals↗
PubMed2003

Relationship between reproductive success and male plasma vitellogenin concentrations in cunner, Tautogolabrus adspersus.

The gene for vitellogenin, an egg yolk protein precursor, is usually silent in male fish but can be induced by estrogen exposure. For this reason, vitellogenin production in male fish has become a widely used indicator of exposure to exogenous estrogens or estrogen mimics in the aquatic environment. The utility of this indicator to predict impacts on fish reproductive success is unclear because information on the relationship between male plasma vitellogenin and reproductive end points in male and female fish is limited. In the research reported in this article, we investigated whether the presence of male plasma vitellogenin is a reliable indicator of decreased reproductive success in mature fish. Adult and sexually mature male and female cunner (Tautogolabrus adspersus) were exposed to 17ss-estradiol, ethynylestradiol, or estrone, three steroidal estrogens that elicit the vitellogenic response. Data were gathered and pooled on egg production, egg viability, egg fertility, sperm motility, and male plasma vitellogenin concentrations. All males, including two with plasma vitellogenin levels exceeding 300 mg/mL, produced motile sperm. Neither percent fertile eggs nor percent viable eggs produced by reproductively active fish demonstrated a significant correlation with male plasma vitellogenin concentrations. Male gonadosomatic index and average daily egg production by females showed significant, but weak, negative correlation with male plasma vitellogenin concentrations. Results suggest that male plasma vitellogenin expression is not a reliable indicator of male reproductive dysfunction in adult cunner exposed to estrogens for 2-8 weeks during their reproductive season, at least in relation to capacity to produce motile sperm or fertilize eggs. Male plasma vitellogenin expression may serve as an indicator of reduced female reproductive function caused by estrogen exposure.

Animals↗
PubMed1997

Effects of locoweed (Oxytropis sericea) on growth, reproduction, and serum hormone profiles in young rams.

Sixteen ram lambs (5 mo old, average BW = 49 +/- 1.4 kg) received a control diet (50% concentrate, 13.1% CP) or a diet containing 7, 14, or 21% locoweed (LW, DM basis). Rams were housed in individual pens (2.5 x 3.5 m) in an enclosed facility (14 h light, 10 h dark) with free access to feed and water. After 35 d of LW feeding, rams fed the 21% LW diet ate 1.3 to 1.6 kg/d, whereas rams fed the other three diets ate 1.6 to 2.0 kg/d. On d 35, BW (mean +/- SE) were 58.3, 57.2, 57.2, and 55.4 (+/- 1.5 kg/d) for ram lambs receiving 0, 7, 14, and 21% LW, respectively (P > .20). An inverse relationship for BW (P < .10) and gain (P = .04) was observed 1 mo after LW feeding ended relative to amount of LW in the diet. Serum alkaline phosphatase increased with increasing LW (P < .05), and thyroxine concentration was decreased (P < .05) by LW ingestion. On d 35, serum LH averaged 6.8, 9.9, 11.6, and 9.9 (+/- 1.8) ng/mL (P = .56), whereas testosterone averaged 3.5, 2.6, 3.0, and 1.6 (+/- .5) ng/mL (linear effect of LW, P = .05) over a 5-h period after GnRH injection (i.m., 50 microg) in lambs fed 0, 7, 14, and 21% locoweed, respectively. Semen volume, sperm motility, sperm cell concentration, and percentage of abnormal cells did not differ (P > .50) on d 35. One month after the end of LW feeding, a linear (P = .06) decrease in sperm motility and scrotal circumference was observed relative to percentage of prior dietary LW. These data suggest that LW exerts adverse effects on the testes of young rams that may not be evident until several weeks after LW consumption ends.

Alkaline Phosphatase↗
PubMed1981

Mitomycin C- and streptozotocin-induced motility and numerical sperm variants in mice.

Groups of HA (ICR) albino male mice were injected once i.p. with o, 2.5 or 5.0 mg/kg mitomycin C (MC), or 2.65 or 26.5 mg/kg streptozotocin (SZ). 4 weeks after treatment each male was mated with 2 untreated CF1 albino females. 5 weeks after treatment the males were killed for determination of sperm motility and count from the cauda and vas. Both agents decreased sperm motility and count in a dose-dependent manner in the treated males. The effect is believed to be caused primarily by interference with gene expression involved in spermatogenesis. The females mated to treated males showed decreased percentage pregnancy and had few progeny. Mean percentage motility and sperm count was significantly lower in the F1 male progeny of MC- or SZ-treated males compared to control males. 50-60% of F1 males form treated male parents had percentage sperm motility below control range. Decreased motility and count in the F1 progeny of treated males may have been caused by gene mutations and/or chromosomal aberrations. The method which is presented may be useful for detecting mutations and/or developmental effects.

Animals↗
PubMed2006

The outermost layer of egg-jelly is crucial to successful fertilization in the newt, Cynops pyrrhogaster.

The significance of egg-jelly layers in internal fertilization was evaluated in the newt, Cynops pyrrhogaster. In this species, six egg-jelly layers, J1, J2, J3, J4, J5 and the outermost J6 layers, are accumulated on the surface of the fertilizable eggs in pars convoluta of the oviduct. When a large number of sperm (about 6 x 10(5)) were placed on eggs having different numbers of jelly layers, all the eggs were fully fertilized, although many of the eggs developed abnormally. Upon insemination using about 600 sperm, only eggs with the full set of jelly layers were fertilized at a high rate with normal development. Since around 300 (the range of 48-1,192) sperm were observed on and in the egg-jelly in naturally spawned eggs, we conclude that the J6 layer must be present on the outermost surface of the egg-jelly for successful internal fertilization of the newt. Previous studies have suggested that the J6 layer is a prerequisite for the initiation of sperm motility and the acrosome reaction. In the present study, the fertilization rate decreased in eggs with a full set of jelly layers when inseminated using acrosome-reacted and motile sperm. However, the fertilization rate was high when motile sperm with intact acrosome was used. These results suggest that induction of the sperm acrosome reaction in the J6 layer is an important step in the internal fertilization of the newt.

Acrosome Reaction↗