Cysteamine Supplementation Research Articles

Cysteamine Supplementation of In vitro Maturation Media: A Review

Under in vitro culture conditions, oxidative modifications of cell components via increased reactive oxygen species (ROS) represent a major culture induced stress. Anti-oxidant systems such as glutathione (GSH) can attenuate the deleterious effects of oxidative stress by scavenging ROS. It has been suggested that GSH content in oocytes may serve as a reservoir protecting the zygote and the early embryos from oxidative damage before genomic activation and de novo GSH synthesis occur. Addition of low molecular weight compounds to culture media, such as cysteamine, can increase GSH levels by increasing cysteine uptake. Quite naturally, effects of supplementation of in vitro maturation (IVM) media with low molecular weight thiols have been studied in various species. This article reviews the use of cysteamine supplementation for IVM, its effects on maturation rates and further embryo development.

Mouse Cumulus-Denuded Oocytes Restore Developmental Capacity Completely When Matured with Optimal Supplementation of Cysteamine, Cystine, and Cumulus Cells1

Our objectives were to study how cysteamine, cystine, and cumulus cells (CCs), as well as oocytes interact to increase oocyte intracellular glutathione (GSH) and thereby to establish an efficient in vitro maturation system for cumulus-denuded oocytes (DOs). Using M16 that contained no thiol as maturation medium, we showed that when supplemented alone, neither cystine nor cysteamine promoted GSH synthesis of mouse DOs, but they did when used together. Although goat CCs required either cysteamine or cystine to promote GSH synthesis, mouse CCs required both. In the presence of cystine, goat CCs produced cysteine but mouse CCs did not. Cysteamine reduced cystine to cysteine in cell-free M16. When TCM-199 that contained 83 microM cystine was used as maturation medium, supplementation with cysteamine alone had no effect, but supplementation with 100 microM cysteamine and 200 microM cystine increased blastulation of DOs matured with CC coculture to a level as high as achieved in cumulus-surrounded oocytes (COCs). Similar numbers of young were produced after two-cell embryos from mouse COCs or CC-cocultured DOs matured with optimal thiol supplementation were transferred to pseudopregnant recipients. It is concluded that 1) mouse CCs can use neither cysteamine nor cystine to promote GSH synthesis, but goat CCs can use either one; 2) goat CCs promote mouse oocyte GSH synthesis by reducing cystine to cysteine, but how they use cysteamine requires further investigation; and 3) mouse DOs can use neither cystine nor cysteamine for GSH synthesis, but they restore developmental capacity completely when matured in the presence of optimum supplementation of cysteamine, cystine, and CCs.

Oocyte glutathione and fertilisation outcome of Macaca nemestrina and Macaca fascicularis in in vivo- and in vitro-matured oocytes

Fertilisation and development of IVM non-human primate oocytes is limited compared with that of in vivo-matured (IVO) oocytes. The present study describes the IVM of macaque oocytes with reference to oocyte glutathione (GSH). Timing of maturation, comparison of IVM media and cysteamine (CYS) supplementation as a modulator of GSH were investigated. A significantly greater proportion of oocytes reached MII after 30 h compared with 24 h of IVM. Following insemination, IVM oocytes had a significantly lower incidence of normal fertilisation (i.e. 2PN = two pronuclei and at least one polar body) and a higher rate of abnormal fertilisation (1PN = one pronucleus and at least one polar body) compared with IVO oocytes. Immunofluorescence of 1PN zygotes identified incomplete sperm head decondensation and failure of male pronucleus formation as the principal cause of abnormal fertilisation in IVM oocytes. The IVO oocytes had significantly higher GSH content than IVM oocytes. Cumulus-denuded oocytes had significantly lower GSH following IVM compared with immature oocytes at collection. Cysteamine supplementation of the IVM medium significantly increased the GSH level of cumulus-intact oocytes and reduced the incidence of 1PN formation, but did not improve GSH levels of the denuded oocyte. Suboptimal GSH levels in macaque IVM oocytes may be related to reduced fertilisation outcomes.

Effect of Dietary Supply of Cysteamine on Sites of Digestion, Small Intestinal Amino Acid Disappearance in Growing Goats

Tan, Z.L., Tang, S.X., Liao, Y.P., Zhou, C.S., Jiang, H.L., Sun, Z.H., Tayo, G.O., Xiao, W.J. and Chen, F.H. 2008. Effect of dietary supply of cysteamine on sites of digestion, small intestinal amino acid disappearance in growing goats. J. Appl. Anim. Res., 34: 71–76. Four Liuyang Black growing goats were used to examine the effect of dietary supply of cysteamine (CS) on feed intake, site and extent of digestion and apparent intestinal amino acid (AA) digestibility in a 4x4 Latin square design. The goats were weighed initially at 11.8 kg (SD 1.6), fitted with the permanent ruminal, duodenal and ileal fistulae and fed ad libitum in individual pens. Four treatments consisted of a basal diet only (control) and dietary supply of CS at levels of 20, 40 and 60 mg/kg dry matter, respectively, to the basal diet. The experimental period lasted for 14 d for each treatment. Dietary supply of CS had no effect (P>0.05) on intake of nutrients. Ruminal digestion of ADF and NDF decreased (P=0.01) on higher level of CS supplementation. The small intestinal digestibilities of alanine, methionine, threonine and valine decreased (P<0.05) with the inclusion of CS in the diets. The results imply that CS supplementation did not improve the digestibility and absorption of nutrients in the gastro-intestinal tract of goats.

Effects of dietary cysteamine supplementation on growth performance and whole-body protein turnover in finishing pigs

The objective of this study was to investigate the effects of dietary cysteamine (CS) supplementation on growth performance and whole-body protein turnover in finishing pigs. This experiment contained 2 trials with the first trial of effect of dietary cysteamine supplementation on growth performance in finishing pigs for 47 d. A total of 16 PIC pigs (60.02 ± 1.01 kg; eight gilts and eight barrows) were randomly assigned to one of two dietary groups, with four pens per group (per pen: one gilt, one barrow). In the second trial, a total of eight PIC barrows (60.04 ± 1.02 kg) were randomly assigned to one of the two dietary groups, with four pens/group (one pig/pen) for 37 d and then the eight barrows were housed individually in metabolism crates (four barrows in control group and four barrows in CS group) for 10 d. The pigs of the first and second trials were fed a basal diet containing 0 (control) or 70 mg CS/kg diet. A total of eight barrows of the second trial received a 7-d nitrogen balance trial. 15 N-Glycine (10 mg/kg BW) was gastrically infused after d 5 of the nitrogen balance trial. Feces and urine were collected daily to determine the N output. Results indicated that CS supplementation increased ( P < 0.05) average daily gain (ADG), feed intake (ADFI), nitrogen retention, nitrogen retention efficiency, and efficiency of digestible N utilization. In addition, CS supplementation resulted in a decreased ( P < 0.05) protein breakdown rate. Net protein gain in nitrogen was increased ( P < 0.05) by 63.5%. However, protein synthesis rate, nitrogen flux, and endogenous urinary nitrogen were not affected ( P > 0.05). It is concluded that dietary CS supplementation may improve growth performance in finishing pigs. Protein deposition is increased after dietary CS supplementation. This increase is caused by a decrease in protein breakdown.

Effects of Dietary Supplementation with Cysteamine on Growth Hormone Receptor and Insulin-Like Growth Factor System in Finishing Pigs

The present study was conducted to test the hypothesis that chronic cysteamine (CS) supplementation may affect serum insulin-like growth factor (IGF)-I concentrations and growth hormone (GH) receptor (GHR), IGF-I, IGF-I receptor (IGF-IR), IGF binding protein (IGFBP)-3, and insulin receptor (IR) mRNA levels in different tissues of finishing pigs. A total of 24 finishing pigs (60.05 +/- 1.24 kg; 12 gilts and 12 barrows) were assigned randomly to one of the three dietary groups, with four pens/group (per pen: one gilt, one barrow). The pigs were fed a basal diet containing 0 (control), 70, or 140 mg/kg cysteamine feed additive (containing 28% cysteamine hydrochloride) for 47 days. The results indicated that CS supplementation (70 mg/kg) increased the average daily gain (ADG) and serum IGF-I level, upregulated mRNA levels of GHR and IGF-I (liver, stomach, muscle), IGF-IR (stomach, duodenum, muscle), and IGFBP-3 (liver) but downregulated IGFBP-3 (stomach, duodenum, muscle). CS supplementation (70 mg/kg) did not affect mRNA levels of GHR and IGF-I (duodenum), IGF-IR (liver), and IR (liver, stomach, duodenum, muscle). CS supplementation (140 mg/kg) downregulated GHR (duodenum), IGF-I, and IGF-IR mRNA (liver, stomach, duodenum, muscle) but upregulated IGFBP-3 and IR mRNA (liver, stomach, duodenum, muscle) and did not affect ADG and serum IGF-I concentration. Collectively, the results suggest that dietary CS supplementation modulates the growth rate, serum IGF-I concentrations, and the gene expression of GHR, IGF-I, IGF-IR, IGFBP-3, and IR in a dose-dependent manner. CS supplementation has tissue-specific regulation of GHR, IGF-I, IGF-IR, and IGFBP-3 mRNA levels. Moreover, the results also imply the possible physiologic role of the GH-IGF axis in mediating the dietary CS supplementation-supported growth of finishing pigs.

The interactions between cysteamine, cystine and cumulus cells increase the intracellular glutathione level and developmental capacity of goat cumulus-denuded oocytes

To improve in vitro maturation (IVM) of denuded oocytes (DOs), we observed the interactive effects of cysteamine, cystine and cumulus cells on the glutathione (L-gamma-glutamyl-L-cysteinyl-glycine; GSH) level and developmental capacity of goat IVM oocytes. Cysteamine supplementation increased the GSH level and blastocyst rates of both cumulus-oocyte complexes (COCs) and DOs, while the addition of cystine increased the GSH level and blastulation only in the presence of cumulus cells (COCs or DOs co-cultured on a cumulus cell monolayer). Simultaneous supplementation of cysteamine and cystine increased the GSH content and blastulation of co-cultured DOs to a level similar to that of COCs matured without thiol supplementation. Co-culture without thiol supplementation improved DOs' GSH synthesis but not blastulation. The results suggest that DOs cannot utilize cystine for GSH synthesis unless exogenous cysteamine is supplied by either cumulus cells or supplementation. Thus, while the addition of cystine alone is enough to improve IVM of COCs, improvement of DOs requires supplementation of both cystine and cysteamine. Synergic actions between cysteamine, cystine and cumulus cells restore the GSH level and developmental capacity of goat DOs.

Cysteamine supplementation of in vitro maturation medium, in vitro culture medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryos

The effects of supplementation of in vitro maturation (IVM) or in vitro culture (IVC) or both IVM and IVC media with cysteamine on the yield, hatching rate (HR) and total cell number (TCN) of buffalo blastocysts were examined. Oocytes obtained from slaughterhouse buffalo ovaries were subjected to IVM and IVF. The IVM or IVC media were supplemented with 0, 50, 100 or 200 microm cysteamine. Supplementation of IVM medium with 50 microm cysteamine increased (P < 0.01) the cleavage rate and blastocyst yield without affecting the HR and TCN whereas a higher concentration of 200 microm significantly (P < 0.05) reduced the blastocyst yield but not TCN. Similar increases in blastocyst yield, without any effect on HR and TCN were observed after supplementation of the IVC medium with 100 (P < 0.01) or 50 microm (P < 0.05) cysteamine, whereas 200 microm cysteamine was ineffective. Supplementation of both IVM medium with 50 microm cysteamine and of IVC medium with 100 microm cysteamine increased the yield of blastocysts and hatched blastocyst by over 100% (P < 0.01) compared with the controls without any adverse effects on HR or TCN. The results of the present study suggest that supplementation of both IVM and IVC media improves the yield of blastocysts without compromising their health.

Tissue-Specific Effect of Dietary Cysteamine on Expression of Adiponectin Receptors in Rats

Adiponectin is synthesized by adipocytes and affects glucose and lipid metabolism by binding to its receptors, AdipoR1 and AdipoR2. Cysteamine, a naturally existing intermediate metabolite of sulfur amino acid, has been reported to modulate metabolism and growth in various species of animals; however, whether the action of cysteamine involves adiponectin and its receptors is unknown. The objective of the present study was therefore to investigate the effect of dietary cysteamine on the expression of AdipoR1/R2 in different tissues, in association with the alterations in endocrine and metabolic status. Rats were fed either of the diets supplemented with 0 or 700 mg/kg cysteamine feed additive (containing 30% of cysteamine hydrochloride) for 4 weeks, and the expression of adiponectin and its receptors in adipose tissue, AdipoR1 and AdipoR2 in liver, gastrocnemius, and soleus muscle was determined, in association with the growth performance and serum concentrations of hormones and metabolites. A temporal trend of increase in growth rate and the ratio of feed consumption relative to body weight gain was observed in the second week of cysteamine supplementation. Serum concentrations of insulin and TNF-alpha increased, while serum levels of triglycerides, FFA, and total cholesterol decreased significantly 4 weeks after cysteamine treatment. Leptin and GH remained unaffected. Cysteamine supplementation increased mRNA expression of AdipoR1 in adipose tissue, gastrocnemius, and soleus muscle as well as that of AdipoR2 in soleus muscle and adipose tissue. Nevertheless, hepatic expression of AdipoR1 and AdipoR2 was not influenced. Despite a numeric increase, no significant alteration in adiponectin mRNA expression in adipose tissue was observed. In conclusion, dietary supplementation of cysteamine modulates the endocrine and metabolic status of rats, which may involve the tissue-specific responses of adiponectin receptors at the level of mRNA transcription.

Effect of cysteamine supplementation of in vitro matured bovine oocytes on chilling sensitivity and development of embryos

In vitro techniques for production of bovine embryos including in vitro oocyte maturation (IVM), fertilization (IVF) and culture (IVC) are becoming increasingly employed for a variety of research purposes. However, decreased viability following cryopreservation by conventional methods has limited commercial applications of these technologies. A practical alternative to facilitate transport would be to arrest development by chilling without freezing. The present research was undertaken to evaluate chilling sensitivity of IVM–IVF embryos at different stages of development, and to determine possible beneficial effects of cysteamine treatment during IVM, previously shown to enhance embryo development in culture, on survival following chilling at different stages. Embryos produced by standard IVM–IVF–IVC methods were chilled to 0 °C for 30 min at 2-cell (30–34 h post-insemination, hpi), 8-cell (48–52 hpi) or blastocyst (166–170 hpi) stages. Viability after chilling was assessed by IVC with development to expanded blastocyst stage determined on days 7 and 8 post-insemination (pi) and hatching blastocyst stage determined on days 9 and 10 pi. Control embryos at the same stages were handled similarly, but without chilling, and development during culture similarly assessed. The effect of cysteamine supplementation (100 μM) of the IVM medium was determined for both chilled and non-chilled (control) embryos. Cysteamine supplementation during IVM had no significant effect on oocyte maturation or fertilization, but increased the proportions of oocytes developing to blastocyst stage by day 7 (13.7 ± 0.9% versus 7.2 ± 0.9%; P < 0.05), total blastocysts (20.5 ± 0.9% versus 15.3 ± 1.3%; P < 0.05), and hatching blastocysts (16.8 ± 1.6% versus 12.0 ± 1.5%; P < 0.05). The greater survival in terms of hatching (78.6 ± 7.0) following chilling of blastocysts produced by IVM–IVF of oocytes matured in media supplemented with cysteamine offers promise for applications requiring short-term storage to facilitate transport of in vitro produced bovine embryos.

Effects of dietary supplementation of cysteamine on growth performance, carcass quality, serum hormones and gastric ulcer in finishing pigs

AbstractA study was conducted to determine the effects of graded levels of dietary inclusion of a cysteamine (Cs) preparation on growth performance, carcass quality, plasma hormone levels, gastric pH and occurrence of gastric ulcer in finishing pigs. A total of 384 Landrace × Large White finishing pigs, (192 gilts and 192 barrows) with an average initial body weight of 66.05 ± 0.623 kg (mean ± SEM) were randomly divided into 24 floor pens, with eight gilts and eight barrows in each pen (9.2 m2) as one experimental unit. The 24 pens of pigs were randomly allocated to one of three diets: (1) a maize/soybean meal basal diet; (2) the basal diet plus 30 mg Cs kg−1 diet; and (3) the basal diet plus 50 mg Cs kg−1 diet. Dietary supplementation of Cs had quadratic effects (P &lt; 0.01) on final body weight and average daily gain, with optimal responses occurring at 30 mg kg−1. Dietary supplementation of Cs quadratically improved (P &lt; 0.01) average daily feed intake and feed/gain ratio, with optimal responses occurring at 30 mg kg−1. Dietary supplementation of Cs had a quadratic effect (P &lt; 0.01) on muscle RNA/DNA ratio. Furthermore, dietary supplementation of Cs reduced (P &lt; 0.05) back‐fat thickness. Dietary supplementation of Cs had quadratic effects (P &lt; 0.05) on plasma glucagon and T3 hormone levels, with optimal responses occurring at 30 mg kg−1, but had no effect (P &gt; 0.05) on plasma growth hormone, insulin and T4 levels. There were no apparent pathological changes seen in the stomach mucosa of pigs fed at 30 mg Cs kg−1 compared with the control diet. It is concluded that a low dose of dietary inclusion of Cs at 30 mg kg−1 can improve growth performance and carcass quality without adverse effects on the stomach in finishing pigs. Copyright © 2005 Society of Chemical Industry

Influence of cysteamine supplementation and culture in portable dry-incubator on the in vitro maturation, fertilization and subsequent development of mouse oocytes

The need to transport oocytes and embryos between two laboratories have prompted us to evaluate the effects of in vitro maturation of immature mouse oocytes in a CO 2-deficient dry heat portable incubator and subsequent in vitro development of these fertilized mouse oocytes in a standard CO 2 incubator. In addition, the effects of cysteamine supplementation on maturation rate and embryonic development during in vitro maturation (IVM) and culture of embryos in the portable incubator were also investigated. Germinal vesicle stage mouse oocytes, recovered at 40-h post-FSH from 6- to 8-week-old C57BL/6 × CBA F1 healthy female mice, were matured in vitro in a modified TCM-199 supplemented with or without 100 μM cysteamine in a standard incubator (5% CO 2; 37 °C) or cultured in a CO 2-deficient dry heat portable incubator for 5 h at 37 °C and thereafter transferred to a standard incubator for further culture. The addition of cysteamine in the IVM medium significantly improved maturation rates of the GV mouse oocytes to metaphase II stage. However, cysteamine supplementation in the culture medium did not significantly improve fertilization and blastocyst formation rates of IVM and ovulated oocytes, and in vivo-derived zygotes. Culture conditions in a CO 2-deficient dry heat portable incubator did not adversely affect the developmental competence of in vivo-derived zygotes and in vitro matured mouse oocytes after IVF or parthenogenetic activation. Cysteamine supplement in the IVM medium could enhance nuclear maturation of these immature oocytes during shipment.

Glutathione synthesis during in vitro maturation of buffalo ( Bubalus bubalis) oocytes: effects of cysteamine on embryo development

It was demonstrated that cysteamine supplementation during in vitro maturation (IVM) improves embryo development by increasing glutathione (GSH) synthesis in several species. An improved developmental competence of oocytes matured in the presence of cysteamine was also recorded in buffalo species. The purpose of this work was to investigate (1) if glutathione is de novo synthesized during in vitro maturation of buffalo oocytes, (2) if cysteamine improves buffalo embryo development via an increase in GSH synthesis, and (3) if the inhibition of glutathione synthesis by buthionine sulfoximide (BSO), in the presence or absence of cysteamine, affects subsequent embryo development and GSH synthesis. Cumulus–oocytes complexes (COCs), recovered from slaughtered animals, were matured in vitro in TCM199+10% fetal calf serum (FCS), 0.5 μg/ml FSH, 5 μg/ml LH and 1 μg/ml 17-β-estradiol in the absence or presence of cysteamine (50 μM), with or without 5 mM BSO. Glutathione content was measured by high-performance liquid chromatography (HPLC) and fluorimetric analysis in immature oocytes and in oocytes matured in the different experimental conditions. In a second experiment, the mature oocytes were in vitro fertilized and cultured for 7 days in order to assess development to blastocysts (BLs). It was demonstrated that buffalo oocytes synthesize glutathione during in vitro maturation and that cysteamine increases glutathione synthesis. Furthermore, the promoting effects of cysteamine on embryo development and GSH synthesis were neutralized by buthionine sulfoximide. These results indicate that glutathione plays a critical role on buffalo embryo development.

Developmental competence of prepubertal goat oocytes selected with brilliant cresyl blue and matured with cysteamine supplementation.

The aim of this study was to assess the effect of oocyte selection using the brilliant cresyl blue (BCB) test plus the addition of cysteamine to the in vitro maturation (IVM) medium to improve the in vitro embryo development of prepubertal goat oocytes. The oocytes were exposed to 26 microM BCB and classified according to their cytoplasm coloration: BCB+ (oocytes with blue cytoplasm) and BCB- (unstained oocytes). The oocytes were matured in a conventional IVM medium supplemented with cysteamine 100 microM. The control group consisted of oocytes not exposed to BCB and matured without cysteamine. The IVM-oocytes were inseminated and cultured in synthetic oviductal fluid (SOF) for 7 days. The normal fertilisation rate (oocytes showing 2 pronuclei and 1 sperm tail) of BCB+ oocytes (40%) was higher than those of BCB- (21%) and control oocytes (22%). The percentage of morulae plus blastocysts was higher (P < 0.05) in the BCB+ group than in the BCB- group (23.8 vs. 5.1%, respectively). In conclusion, the integration of the BCB test and the addition of cysteamine in the protocol of in vitro embryo production from prepubertal goat oocytes has improved the developmental rates of embryo development.

Cysteamine supplementation during in vitro maturation and embryo culture: a useful tool for increasing the efficiency of bovine in vitro embryo production.

Cysteamine when added during in vitro maturation (IVM) or in vitro embryo culture (IVC) stimulates glutathione (GSH) synthesis and improves embryo developmental rates. This suggests that GSH synthesis is decreased in the in vitro produced embryo. The present study was carried out to evaluate if addition of cysteamine to culture medium at the same time, during IVM and IVC of bovine oocytes, may promote an overall improvement on the developmental rate and embryo quality. Oocytes were matured in TCM 199 supplemented with 10% (v/v) fetal calf serum, hormones, and 0 or 100 microM of cysteamine for 24 hr. After IVM, the oocytes were fertilized (day 0). Day 2 embryos (2-8 cell) were washed and transferred to fresh IVC medium supplemented with 0, 25, 50, or 100 microM of cysteamine and cultured for 48 hr. After this, embryos were cultured in IVC medium without cysteamine until day 8 of IVC. In the present study, we confirmed our previous results by demonstrating that the percentage of embryos that developed to the blastocyst stage was significantly higher (P < 0.05) when 100 microM of cysteamine was added during IVM, and this was further improved when 100 and 50 microM of cysteamine where present during IVM and IVC, respectively (P < 0.05). After cryopreservation, no differences were observed on embryo development, but a significant increase on embryo hatching was found between unsupplemented and supplemented oocytes with 100 and 50 microM of cysteamine during IVM and IVC, respectively (P < 0.05). We can conclude that GSH synthesis stimulation during bovine IVM with cysteamine, concomitant with GSH stimulation during IVC, will be a useful and simple tool for increasing the efficiency of in vitro bovine embryo production.

Stimulation of glutathione synthesis of in vitro matured bovine oocytes and its effect on embryo development and freezability.

Glutathione (GSH) has been shown to play an important role in embryo development. In a previous study, we demonstrated that cysteamine supplementation of in vitro maturation (IVM) medium increased the intracellular GSH content in bovine oocytes and improved subsequent embryo development to the blastocyst stage. The present study was carried out to evaluate the effect of inhibition by buthionine sulfoximide (BSO) of GSH synthesis during IVM in the presence of cysteamine, on subsequent embryo development, and the effect of cysteamine during IVM on the survival of blastocysts following freezing. The effect of beta-mercaptoethanol and cysteine added to the maturation medium on GSH levels in bovine oocytes, as well as the effect of these compounds on de novo GSH synthesis by oocytes during in vitro maturation, was also studied. The inhibitory effect of BSO during in vitro maturation on GSH synthesis was also evaluated. Evidence was found confirming that GSH synthesis occurs intracellularly during IVM of oocytes and is stimulated by cysteamine, beta-mercaptoethanol and cysteine. Moreover, the present results suggest that the increase in the rate of embryo development exerted by cysteamine, when present during IVM, was due to its stimulatory effect on GSH synthesis. This increase in GSH levels during IVM improves embryo development and quality, producing more embryos reaching the blastocyst stage on day 6, those most suitable for freezing.