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Aim: Relaxin and insulin‐like growth factor (IGF)‐I have pronounced effects on the male and female reproductive tracts. The aim of this study was to investigate the effects of relaxin and IGF‐I on the motility, capacitation, acrosome reaction, cholesterol efflux and utilization of glucose in porcine spermatozoa.

Aim: The present study was undertaken to determine which fatty acids improve motility, viability, and increase acrosome reaction (AR) in boar spermatozoa.

Aim: The present study was designed to investigate the effect of amino acids and their dipeptides in the medium related to the urea cycle on the motility, viability, acrosome reaction (AR) and accumulation of ammonia in the medium over different incubation periods in porcine spermatozoa and to assess the utilization of glucose.

Background and Aims: Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa.

Mammalian spermatogenesis has been studied extensively as a prime theme of male reproductive biology, especially for germ cell production, fertilization and development. Investigation of spermatogenesis has provided us with the opportunity to both study the male germ line stem cells and generate the transgenic animals. Spermatogenesis is conducted in the seminiferous tubules, which end in the rete testis. The organization of spermatogenesis means that the spermatogonia are uniformly distributed around the seminiferous tubules. The pubertal establishment and mature maintenance of spermatogenesis requires precursor cells. In bull testes at 4 weeks postnatal, gonocyte migration occurs and differentiated spermatogonia are recognized after 8 weeks. Within the period of 4–8 weeks of age, spermatogonial stem cell conversion and niche formation must occur. Spermatogonial stem cells are the only cells that can undergo self‐renewal in spermatogenesis. Spermatogonial stem cell transplantation can potentially contribute to studies of gene expression during spermatogenesis and provide genetic progress in domestic animals. Bull spermatogonial stem cells have been demonstrated to be capable of colonizing recipient mouse seminiferous tubules. (Reprod Med Biol 2007; 6: 139–149)

Selenium (Se) and vitamin E (Vit-E), as integral parts of antioxidant systems, play important roles for sperm and embryos in vitro. In this study, the effects of Se and Vit-E on the maturation, in vitro fertilization and culture to blastocysts of porcine oocytes and accumulation of ammonia in the culture medium during different development stages were investigated. The maturation was performed in modified tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, the fertilization medium was modified Tyrode's albumin lactate pyruvate (mTALP), and the embryo culture medium was modified North Carolina State University (mNCSU)-23. Se in the form of sodium selenite (SS) and seleon-L-methionine (SeMet) and Vit-E at different concentrations were also used. The incorporation and oxidation of (14)C(U)-glucose were assessed with a liquid scintillation counter. In this study, SeMet and SeMet+Vit-E increased oocyte maturation, fertilization and incorporation and oxidation of (14)C(U)-glucose significantly (P

The present study was conducted to investigate the effect of dietary Rhodobacter capsulatus on lipid fractions and egg-yolk fatty acid composition in laying hens. Thirty-six laying hens (30 weeks old) were randomly assigned into two dietary groups fed diets with (0.04%) or without (control) R. capsulatus for a 60-day feeding trial. Dietary R. capsulatus decreased (p

Summary The effect of karaya saponin supplementation on the serum and egg yolk cholesterol and fatty acid composition in egg yolk were investigated in Japanese quails. A total of 80 Japanese quails aged 5 weeks were equally divided into four groups of 20. Four levels (0, 25, 50 and 75 mg/kg feed) of karaya saponin were included in the basal diet and experiment was lasted for 6 weeks. The cholesterol fractions in the egg yolk and serum were measured by enzymatic assay, and the fatty acid composition in egg yolk was determined by gas chromatography. The results revealed that the supplementation of 75 mg/kg karaya saponin significantly reduced (p

Mongolian gerbil 1-cell embryos can develop into blastocysts in vitro in a co-culture with oviductal cells in mTCM199 medium. When 1-cell embryos are cultured in a chemically defined medium, they do not develop into blastocysts. Thus, the objective of this study was to examine the effects of amino acids on the in vitro development of Mongolian gerbil embryos. Amino acid depletion/accumulation by the Mongolian gerbil preimplantation embryo was analyzed with an amino acid analyzer. The amino acids of threonine, valine, isoleucine, leucine, lysine, serine, glutamine, glycine, and alanine were depleted in the medium at all the developmental stages. Two-cell stage embryos were cultured in mM16 medium supplemented with each amino acid. We found that 37.1% and 11.4% of 2-cell embryos reached the morula and blastocyst stages, respectively, after supplementation of the medium with valine. Three amino acids (threonine, aspartic acid, and glutamine) promoted development to the 4-cell stage (P < 0.05). Alth...

Mongolian gerbil 1-cell embryos do not develop into blastocysts in vitro because of 8-cell block. Recently, we reported that 2-cell embryos of Mongolian gerbils developed into blastocysts in vitro in mM16 supplemented with valine. However, the role of valine in the in vitro development of Mongolian gerbil embryos is unclear. Thus, the objective of this study was to investigate of the role of valine in the in vitro development of Mongolian gerbil embryos. Mongolian gerbil embryos at the 1-cell, 2-cell, 8-cell, morula, and blastocyst stages were cultured in mM16 medium containing 14C-valine or 14C-glucose. A 2.5 mM NaOH solution was used as a trap for the evolved 14CO2. After incubation, all samples were analyzed using a liquid scintillation counter. The incorporation of 14C-valine significantly increased at the 8-cell stage. The oxidation of 14C-valine significantly increased at the 8-cell and blastocyst stages. The incorporation of 14C-glucose in mM16+valine was significantly higher than that in...

This study was conducted to elucidate the mechanism underlying the hypolipidemic action of karaya saponin orRhodobacter (R.) capsulatus. A total of 40 laying hens (20-week-old) were assigned into four dietary treatment groups and fed a basal diet (as a control) or basal diets supplemented with either karaya saponin,R. capsulatus, or both for 60 days. The level of serum low-density-lipoprotein cholesterol and the levels of cholesterol and triglycerides in the serum, liver, and egg yolk were reduced by all the supplementations (). Liver bile acid concentration and fecal concentrations of cholesterol, triacylglycerol, and bile acid were simultaneously increased by the supplementation of karaya saponin,R. capsulatus, and the combination of karaya saponin andR. capsulatus(). The supplementation of karaya saponin,R. capsulatus, and the combination of karaya saponin andR. capsulatussuppressed the incorporation of from -palmitic acid into the fractions of total lipids, phospholipids, triacylglycerol, and cholesterol in the liver in vitro (). These findings suggest that the hypocholesterolemic effects of karaya saponin andR. capsulatusare caused by the suppression of the cholesterol synthesis and the promotion of cholesterol catabolism in the liver.

Dietary karaya saponin and Rhodobacter capsulatus (R. capsulatus) are known to have hypocholesterolemic actions, as reported in our previous studies. This study examined possible synergistic hypocholesterolemic effects of karaya saponin and R. capsulatus in broilers. A total of 150 broilers were allocated into 10 treatments: control, saponin 25 mg, saponin 50 mg, saponin 75 mg, saponin 25 mg+R. capsulatus 0.2 g, saponin 25 mg+R. capsulatus 0.4 g, saponin 50 mg+R. capsulatus 0.2 g, saponin 50 mg+R. capsulatus 0.4 g, saponin 75 mg+R. capsulatus 0.2 g and saponin 75 mg+R. capsulatus 0.4 g. Feed intake and feed efficiency were improved when karaya saponin and R. capsulatus were synergistically supplemented in the diet. Combinations of karaya saponin, especially supplementation of karaya saponin 50 mg+R. capsulatus 0.4 g were shown to have potential hypolipidemic actions in breast and thigh muscle cholesterol and triglycerides, serum cholesterol, low density lipoprotein-cholesterol and triglycerides, as well as improved high density lipoprotein (HDL)-cholesterol (p, Article, ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES. 23(6):733-741 (2010)

1. It has been reported that karaya saponin and Rhodobacter capsulatus individually have hypocholesterolaemic activity in laying hens. This study focuses on the effect of adding karaya saponin with R. capsulatus to hen's diet with regard to serum and egg yolk cholesterol and triglycerides. 2. A total of 56 Boris Brown laying hens were divided into 7 groups at 20 weeks of age. Combinations of 25, 50, 75 mg kg(-1) karaya saponin and R. capsulatus 200 and 400 mg kg(-1) were used as treatment groups. 3. After 8 weeks of supplementation, the effects of all the combinations of karaya saponin and R. capsulatus on serum and egg yolk cholesterol, triglycerides, and high-density lipoprotein (HDL)-cholesterol were greater than either karaya saponin or R. capsulatus alone. The combination of karaya saponin 50 mg kg(-1)+ R. capsulatus 400 mg kg(-1) exhibited the greatest reduction of serum (325%) and yolk (225%) cholesterol and the greatest increase of faecal, liver bile acids and yolk fatty acid (oleic, linoleic and linolenic) concentrations. In addition, egg production and yolk colour were significantly improved by the combined use of karaya saponin and R. capsulatus supplementation. 4. Therefore, the dietary supplementation of karaya saponin and R. capsulatus may lead to the production of a low-cholesterol egg, with production performance maintained at a standard level.

Immune response and yolk cholesterol are crucial factors for commercial chicken producers. The objectives of this study were to investigate the effect of selenium-enriched Japanese radish sprouts (Se-enriched JRS) and R. capsulatus synergistically on immune response and cholesterol in laying hens. A total of 50 laying hens (20-wk old) were assigned to 5 dietary treatment groups, and fed diets supplemented with 2.5 mu g/kg, 5 mu g/kg, 10 mu g/kg Se-enriched JRS and 5 mu g/kg Se-enriched JRS+R. capsulatus (0.02%). Egg production and yolk color were significantly improved by the supplementation of Se-enriched JRS+R. capsulatus in the layer diet (p, Article, ASIAN-AUSTRALASIAN JOURNAL OF ANIMAL SCIENCES. 23(5):630-639 (2010)

Selenium (Se) and vitamin E (Vit-E), as an integral part of antioxidant systems, play an important role in the motility and acrosome reaction (AR) of mammalian spermatozoa. The purpose of this study was to investigate the effect of Se and Vit-E on motility, viability, AR and accumulation of ammonia in the culture medium during different incubation periods in porcine sperm. Sperm samples were washed, swum-up and incubated at 37°C for 1 and 3 h in Sp-TALP medium supplemented with sodium selenite (SS), seleon l-methionine (SeMet) and Vit-E in the presence or absence of ammonia. Sperm motility was determined on the basis of movement quality examined by phase microscopy. Viability and AR of spermatozoa were assessed by Hoechst 33258 and chlortetracycline (CTC) staining technique, and accumulation of ammonia was measured by the indophenol method. The incorporation of 14C(U)-glucose was assessed with a liquid scintillation counter. In experiment 1, the sperm motility, viability, AR and incorporation of 14C(U)-glucose increased significantly (P

For understanding the roles of fatty acids on the induction of acrosome reaction which occurs under association of cholesterol efflux and PKA or PKC pathways in boar spermatozoa, metabolic fate of alone and combined radiolabeled 14C-oleic acid and 3H-linoleic acid incorporated in the sperm was compared, and behavior of cholesterol and effects of PKA and PKC inhibitors upon fatty acid-induced acrosome reaction were examined. Semen was collected from a Duroc boar, and the metabolic activities of fatty acids in the spermatozoa were measured using radioactive compounds and thin layer chromatography. Cholesterol efflux was measured with a cholesterol determination assay kit. Participation of fatty acids on the AR through PKA and PKC pathways was evaluated using a specific inhibitor of these enzymes. Incorporation rate of 14C-oleic acid into the sperm lipids was significantly higher than that of 3H-linoleic acid (P

In this study, we used SrCl2 treatment to activate mouse oocytes. The objectives of this study were to compare the development of parthenogenic and fertilized mouse embryos, and the effect of insulin-like growth factor II (IGF-II) on their development to blastocysts; glucose and methionine metabolism were also examined. Compared to normal fertilized embryos, parthenogenically developed embryos exhibited delayed development, reduced total cell number, and reduced trophectoderm cell (TC) and inner cell mass (ICM) cell numbers of blastocysts. Supplementation of IGF-II to the culture medium enhanced parthenogenic embryo development, cell number, and TC and ICM cell numbers but not to the same level as that of normal fertilized embryos (P < 0.05). Incorporation and oxidation of glucose and protein synthesis from methionine were lower in parthenogenic blastocysts than in fertilized blastocysts (P < 0.05).

Purpose Differentiation of endometrial stromal cells into decidual cells occurs during embryo implantation and pregnancy. Recently, it has been reported that relaxin affects the decidualization of cultured human endometrial cells in vitro; however, there has been no study on the decidualization of the Mongolian gerbil (Meriones unguiculatus). The authors demonstrated artificially induced decidualization, and the effect of relaxin on decidualization in gerbils.

The fatty acid composition of rabbit blastocysts, blood serum and uterine fluids were analyzed to study embryonic lipid metabolism. Embryos were collected from Japanese white rabbits and fatty acids were analyzed by gas chromatograph. Total amount of fatty acids in blastocysts was higher than that in serum and uterine fluid. The amount of fatty acids in blastocysts markedly decreased during days 7–13 of pregnancy, and in serum had hovered, but in uterine fluid on day 13 was nine times higher than that on day 7 of pregnancy. Palmitic acid predominates in blastocysts, serum and uterine fluid during this period. Palmitic acid is the most abundant fatty acid in the blastocysts, serum and uterine fluids of rabbit during days 7–13 of pregnancy.

Both relaxin and insulin-like growth factor (IGF) are members of the insulin super family. This study aimed to investigate the effect of relaxin and IGF-I on the pre-implantation of Mongolian gerbil of blastocyst development in vitro. Blastocysts and eight-cell stage embryos were collected from female gerbils. Eight-cell embryos and blastocysts were cultured in mM16 medium supplemented with or without relaxin or IGF-I for 24 h. Blastocysts were counted for total, inner cell mass (ICM) and trophectoderm (TE) cell numbers, and assessed apoptosis incidence. In addition, to measure incorporation of 3H-methionine, blastocysts were cultured for 3 h with relaxin or IGF-I, washed with trichloroacetic acid and measured by liquid scintiration counter. Relaxin (200 ng/ml) increased total, TE and ICM cell numbers of blastocyst (P

The present study was designed to investigate the effect of amino acids and their dipeptides in the medium related to the urea cycle on the motility, viability, acrosome reaction (AR) and accumulation of ammonia in the medium over different incubation periods in porcine spermatozoa and to assess the utilization of glucose. Porcine spermatozoa were washed, swim-up and incubated at 37°C for 0–4 h in mTALP medium supplemented with 75–600 μmol/L ammonia. Amino acids (1.0 mmol) or their dipeptides (2.0 mmol) were added individually to the mTALP medium containing either no ammonia or 300 μmol/L of ammonia. The viability and AR of porcine spermatozoa were assessed using the triple-staining technique and the accumulation of ammonia in the medium was measured using the indophenol method. The motility viability and AR were adversely affected (P < 0.05) by concentrations of ammonia ≥300 μmol/L compared with the control. Supplementation of L-alanyl-L-glutamine (AlaGln), L-glycyl-L-glutamine (GlyGln) and AlaGln + GlyGln in the presence of 300 μmol/L ammonia significantly increase (P < 0.05) the rate of motility, viability, AR, incorporation, accumulation of ammonia and oxidation of 14C(U)-glucose compared with the ammonia supplement control. AlaGln and GlyGln in mTALP medium were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and subsequently increasing the rate of AR and the utilization of glucose in porcine spermatozoa.

The study was designed to investigate the effect of Rhodobacter capsulatus on serum and meat cholesterol, fatty acid composition in meat, as well as meat quality of finishing pigs. A total of 16 120-day-old Landrace female pigs of about 60 kg initial body weight were randomly assigned into two groups. The pigs were fed a supplemented diet with 0.04% dietary R. capsulatus or a control diet for 60 days. Total cholesterol and low density lipoprotein-cholesterol concentration in serum was significantly lowered (P

Aim Relaxin and insulin-like growth factor (IGF)-I have pronounced effects on the male and female reproductive tracts. The aim of this study was to investigate the effects of relaxin and IGF-I on the motility, capacitation, acrosome reaction, cholesterol efflux and utilization of glucose in porcine spermatozoa.

Aim: The present study was undertaken to determine which fatty acids improve motility, viability, and increase acrosome reaction (AR) in boar spermatozoa. Methods: Boar spermatozoa were washed, swum-up and incubated at 37°C for 4 h in TALP medium supplemented with myristic, palmitic, stearic, lignoceric, oleic, linoleic, arachidonic, docosahexaenoic and palmitoleic acid. Sperm motility, viability and AR were evaluated during 4 h of incubation. Results: Results show that oleic and linoleic acid significantly improved (P

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen-thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim-up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp-TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of 14C-glucose was assessed by a liquid scintillation counter. Motility was improved (P

This comparative study was to investigate the hypocholesterolemic effects of dietary Rhodopseudomonas palustris and Rhodobacter capsulatus on rats fed a high cholesterol diet. Thirty male Wister–Imamichi rats were assigned to three groups and fed on either a high cholesterol diet, or a high cholesterol diet supplemented with 2.0% R. palustris or R. capsulatus for 4 weeks. Compared to the control diet, both of the R. palustris and R. capsulatus supplemented diets significantly reduced the serum cholesterol, triglycerides, low-density lipoprotein, very low density lipoprotein cholesterol and hepatic triglycerides, but increased hepatic cholesterol in rats. In addition, both of the R. palustris and R. capsulatus supplemented diets may reduce the risk of atherosclerosis, as the ratio of high density lipoprotein cholesterol to the total cholesterol was significantly higher than in the control group (P

Mammalian spermatogenesis has been studied extensively as a prime theme of male reproductive biology, especially for germ cell production, fertilization and development. Investigation of spermatogenesis has provided us with the opportunity to both study the male germ line stem cells and generate the transgenic animals. Spermatogenesis is conducted in the seminiferous tubules, which end in the rete testis. The organization of spermatogenesis means that the spermatogonia are uniformly distributed around the seminiferous tubules. The pubertal establishment and mature maintenance of spermatogenesis requires precursor cells. In bull testes at 4 weeks postnatal, gonocyte migration occurs and differentiated spermatogonia are recognized after 8 weeks. Within the period of 4–8 weeks of age, spermatogonial stem cell conversion and niche formation must occur. Spermatogonial stem cells are the only cells that can undergo self-renewal in spermatogenesis. Spermatogonial stem cell transplantation can potentially contribute to studies of gene expression during spermatogenesis and provide genetic progress in domestic animals. Bull spermatogonial stem cells have been demonstrated to be capable of colonizing recipient mouse seminiferous tubules.

The present study was conducted to investigate the effects of dietary Rhodobacter capsulatus on the laying hen. A total of forty 23-wk-old Hy-Line Brown laying hens were randomly assigned into 4 treatment groups (10 laying hens/group) and fed diets supplemented with 0 (control), 0.01, 0.02, and 0.04% R. capsulatus during the 60-d feeding period. Dietary supplementation of R. capsulatus (0.04%) reduced (P < 0.05) cholesterol and triglycerides concentration in serum (15 and 11%), as well as in egg-yolk (13 and 16%) over a 60-d feeding period. Cholesterol and triglycerides concentrations in serum as well as egg-yolk were changed linearly in accordance with increasing levels of dietary R. capsulatus. Supplementation of R. capsulatus in diets increased high-density lipoprotein cholesterol level and decreased (P < 0.05) atherogenic index in serum. Yolk color was improved (P < 0.05) in the group fed the 0.04% R. capsulatus supplemented diet compared with the control group. Hepatic cholesterol and triglycerides were reduced (P < 0.05) by 0.04% R. capsulatus. Moreover, the supplementation of R. capsulatus in layer diets did not appear to cause any adverse effects on egg production, shell weight, shell thickness, Haugh unit, yolk index, and feed conversion efficiency compared with the same parameters for the control laying hens. It is postulated that known and unknown factors are present in R. capsulatus presumably responsible for the hypocholesterolemic effect on laying hens. Therefore, the dietary supplementation of R. capsulatus may lead to the development of low-cholesterol chicken eggs as demanded by health-conscious consumers.

The present study was carried out to investigate the effects of fructose supplementation in glucose containing mTALP medium on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa. Boar spermatozoa were preincubated, swum-up, resuspended and then incubated for 6 h in mTALP medium supplemented with 0, 0.5, or 1.0 mmol fructose in the presence of 5.0 mmol glucose. After completion of the specified incubation period, motility was determined subjectively on the basis of speed of progression and on the type of forward movement of spermatozoa; acrosome status was evaluated by applying a triple staining technique; and in vitro fertilization capability was assessed by acetic-orcein staining. The combination of fructose and glucose (0.5 + 5.0 mmol) supplements in mTALP medium improved sperm motility significantly (P < 0.05), more than glucose alone (5.0 mmol) at 2–6 h of incubation. Acrosome reaction (live spermatozoa) and the sperm penetration rate was increased significantly (P < 0.05) when the spermatozoa were treated with the combination of fructose and glucose compared with glucose alone, but the incidence of polyspermy was not significantly different between the treatments. These results suggest that the combination of glucose and fructose as supplements in mTALP medium improve the progressive motility, acrosome reaction and fertilization capability of boar spermatozoa.

Relaxin has an important role in stimulating motility and the acrosome reaction (AR) of fresh boar spermatozoa. The objective of the present study was to determine whether relaxin can improve the motility, AR and viability of cryopreserved boar spermatozoa. Cryopreserved boar spermatozoa were thawed, washed and incubated at 37°C for 4 h in modified Beltsville thawing solution supplemented with 0, 20 or 40 ng/mL relaxin. Sperm motility, AR, viability, and incorporation and oxidation of 14C-glucose were evaluated during 0–04 h of incubation. The results show that the supplementation of relaxin (especially at 20 ng/mL) in the thawing solution improved sperm motility significantly (P 0.05) improved by supplementation with relaxin. The rates of incorporation and oxidation of 14C-glucose were increased in correlation with AR up to 4 h of incubation. We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa.

Relaxin is a peptide hormone found in seminal plasma that has a physiological influence on sperm motility in some species. There are no reports on the effect of relaxin on acrosome reaction and utilization of glucose in boar spermatozoa. In this study, to investigate the effects of relaxin on sperm motility, acrosome reaction, and incorporation and oxidation of labeled glucose, boar spermatozoa were washed and preincubated for swim-up and then incubated (0-6 h) with 0, 20, or 40 ng/ml relaxin in mTALP medium. The results indicated that the addition of relaxin stimulated sperm motility significantly (P

Porcine relaxin is a peptide hormone belonging to the insulin super family that has a variety of biological functions. The present experiment was designed to investigate the effects of relaxin on sperm function and on in vitro fertilization (IVF) of porcine oocytes. Porcine spermatozoa were washed, swum-up, and incubated for 1-4 h in mTALP medium supplemented with 0, 20 or 50 ng/ml porcine relaxin. Motility was determined by observing the type of forward movement of the spermatozoa, and acrosome status was evaluated by applying the triple staining technique. Immature oocytes were aspirated from antral follicles and matured in IVM medium (modified NCSU-37). Matured oocytes were co-cultured with spermatozoa in IVF medium (mTALP) supplemented with 0, 5, 10, 15 or 20 ng/ml relaxin. After 6 h of sperm-oocyte co-incubation, putative zygotes were cultured for 18 h in oocyte culture medium NCSU-37 and then assessed for the rates of monospermy, polyspermy, and male pronucleus formation after acetic orcein staining. Relaxin improved (P

An experiment was conducted to investigate pH, osmolality, and the concentration of ammonia, total protein, glutamine and glutamic acid in follicular fluid at different developmental stages (

Methyl methanesulphonate (MMS), a potent alkylating agent and testicular toxicant, was orally administered to rats for 5 days at 40 mg/kg. During the recovery period of up to 5 weeks, males were evaluated for testicular toxicity and sperm morphology. The 5-week recovery period were designated as follows: Day 1 (the day after final treatment); Week 1, Week 2, Week 3, Week 4 and Week 5 (1, 2, 3, 4 and 5 weeks after final treatment). Morphologically abnormal sperm increased beginning in Week 3, peaked in Week 4 and declined slightly in Week 5. Histopathological examinations indicated retention of step 19 spermatids at stage IX from Day 1 through Week 3. Quantitative evaluation of spermatogenic cells indicated a decrease in the number of late pachytene spermatocytes and early spermatids on Day 1. TUNEL examination showed a significantly high frequency of apoptosis in the meiosis cells in Week 1. In the present study, genetic damage induced by treatment with MMS affected spermatogenesis and a wide variety of spermatogenic cells in the testis. Apoptosis in the course of meiosis seemed to be involved in the elimination process of genetically insulted germ cells, and this process seems to play an important role in eliminating and/or decreasing the germ cells with retention of spermatids and the potential to express morphologically abnormal spermatozoa.

Glucose is utilized for oxidation and synthesis of various lipids in cultured rat embryos. The present experiment examined the effect of insulin on the incorporation of glucose into lipid fractions in rat embryos in vitro. Embryos at the 2-cell, 8-cell and blastocyst stages were incubated for 5 h in hamster embryo culture medium (HECM)-1 containing 14C-glucose and 170 nmol/L insulin, or in HECM-1 containing only 14C-glucose, and the oxidation of glucose in these embryos was examined. In addition, the total lipids of blastocysts were separated by thin layer chromatography and the radioactivity of the separated lipid fractions was measured. Oxidation of glucose was significantly increased after insulin treatment compared with that without insulin treatment in 8-cell embryos and blastocysts (P

To investigate the effects of vitamin E on the in vitro development of mouse embryos from 1-cell to blastocysts, embryos were cultured in two media, Brinster's BMOCIII and BMOCIII supplemented with 100 μM vitamin E (BMOCIIIvitE). The incorporation and oxidation of glucose were also compared between the embryos cultured in these media. Both the number of embryos that developed to the 1-cell stage and to the blastocyst stage were much larger for BMOCIIIvitE than BMOCIII. No significant differences in the incorporation of 14C-glucose or in the oxidation of 14C-glucose were found between the 2-cell embryos incubated in BMOCIIIvitE and BMOCIII. On the other hand, the incorporation and oxidation rates of 14C-glucose were significantly higher (p

To develop an in vitro culture system for Mongolian gerbil embryos, we examined the effect of co-culture with oviductal epithelial cells on the development of 1- and 2-cell embryos collected from superovulated gerbils. In the first experiment, 2-cell embryos were cultured in modified TCM 199 supplemented with pyruvate, lactate and fetal calf serum (mTCM199) with or without gerbil or mouse oviductal cells. In the second experiment, in 1-cell embryos were cultured with or without gerbil oviductal cells in mTCM199. Although, 2-cell gerbil embryos did not develop beyond the 16-cell stage without oviductal cells in both TCM199 and mTCM199, co-culture with gerbil or mouse oviductal cells supported the development of 2-cell gerbil embryos to blastocysts. Co-culture with gerbil oviductal cells in mTCM199, but not in TCM199, also supported the development of 1-cell embryos to blastocysts. There were no differences between the in vivo and in vitro developed blastocysts in morphological appearance and cell numbers. This co-culture system can be used for in vitro culture of Mongolian gerbil embryos.

We investigated the relationship between the menstrual cycle and hormone levels in cynomolgus monkeys, and developed a sulpiride-induced hyperprolactinemic anovulation model. On this study, we demonstrated the usefulness of the commercial human prolactin immunoradiometric assay kit for the measurement of cynomolgus monkey serum samples. In the normal menstrual cycle of the cynomolgus monkey, serum prolactin concentrations were not significantly different between luteal and follicular phases. However, the serum prolactin concentration tended to elevate at the ovulation stage. And serum progesterone began to increase after an estradiol surge, and then declined before the ensuing preovulatory rise in estradiol. During the luteal phase, the serum concentration of progesterone was elevated. Moreover, we aimed to develop an anovulation model, using sulpiride-induced hyperprolactinemia in the cynomolgus monkey. The serum prolactin level gradually increased during the twice-daily administration of sulpiride, and the drug produced as big a response at 5 mg/kg. In this study, the length of the menstrual cycle was approximately 29 days in normal cynomolgus monkeys. When treatment with sulpiride had been continued for more than one month, serum progesterone and estradiol levels fell to within the range seen in the follicular phase of the normal cycle, and the absence of ovulation was recognized by laparoscopy. Moreover, in this period we found that amenorrhea or anovulatory menstruation in the experimental animals. We could produce an anovulatory model induced by sulpiride repeatedly administered over a long time period. Our findings suggest that the cynomolgus monkey is useful as a endocrinological model that uses prolactin as a parameter and as an anovulatory model; thus, it could be a useful model for the hyperprolactinemic amenorrhea and/or anovulation seen in humans.

A serum-free culture system was used to allow determination of the fatty acid composition of bovine embryos during in vitro development. The proportion of embryos developing to the blastocyst stage in serum-free medium (TCM199 supplemented with bovine serum albumin, insulin, apotransferrin and transforming growth factor-α; BITTα) was as high as in serum-supplemented medium (TCM199+5% calf serum). Bovine blastocysts grown in serum-supplemented medium contained abundant lipid droplets in the cytoplasm. The fatty acid composition of bovine immature oocytes, embryos at 2-cell and blastocyst stages cultured in serum-free or serum-supplemented medium was determined by capillary column gas chromatography. Additionally, the fatty acid composition of calf serum alone was determined. Myristic acid (59.6%) was the most abundant fatty acid in immature oocytes followed by docosahexaenoic acid (12.3%). The fatty acid profiles of two-cell embryos derived in the serum-free medium were similar to those of immature oocytes. In contrast, two-cell embryos derived in the serum-supplemented medium showed high levels of palmitic (27.6%) and stearic (27.2%) acids and a low level of myristic acid (21.9%). For blastocysts grown in the serum-free medium, myristic acid (35.7%) was high and palmitic (17.0%) and stearic (12.5%) acids were moderately high. On the other hand, blastocysts developed in the serum-supplemented medium retained high levels of palmitic (30.5%) and stearic (24.2%) acid compositions and had considerably increased levels of palmitoleic (16.3%) and oleic (12.1%) acids. High levels of oleic (17.3%), palmitic (16.5%) and stearic (12.3%) acids were detected in calf serum, which were similar to the profile of the 2-cell and blastocyst stage embryos cultured with serum. This study shows that bovine embryos grown in serum-supplemented medium showed different morphology and fatty acid composition when compared to those cultured in serum-free medium.

In the present study, the oxidation of palmitic acid to carbon dioxide and the difference of incorporation rate of the same and oleic acid into embryo lipids were examined. To test the exact role of fatty acids for energy production, the oxidative rates of exogenous palmitic were assessed in M16 medium with and without carbohydrate substrates. The oxidation of palmitic acid in the medium with carbohydrates was lower than that without carbohydrates in the blastocyst stage. Incorporation rates of both palmitic and oleic acids into embryo lipids increased significantly from the 2-cell to the blastocyst stage (P

The oxidation and incorportion of [U-14C] glucose were examined in preimplantation rat embryos. An increasing capacity for incorporation and oxidation of glucose after the 1-cell stage was observed and the oxidative turnover of this substrate at the blastocyst stage was ten times more than that at the 1-cell stage. To evaluate how glucose is utilized for the synthesis of embryo lipids, 2-cell embryos and blastocysts were cultured for 5 h in medium containing [U-14C] glucose, and total lipids extracted from the embryo were separated into various neutral lipids and phospholipids by thin layer chromatography and radioactivities of these lipid fractions were measured. Most of the radioactivity was recovered in triacylglycerols in both stages, and radioactivities were also found in other neutral lipids and phospholipids. These results indicate that [U-14C] glucose was certainly utilized for oxidation and the synthesis of various lipids in embyros at the preimplamtation stage.