Effect of Dipeptides on In vitro Maturation, Fertilization and Subsequent Embryonic Development of Porcine Oocytes.

The effects of amino acids and dipeptides on in vitro production of porcine embryos and accumulation of ammonia in culture medium during developmental stages were examined in this study. The maturation, fertilization and development of embryonic cultures were performed in modified Tissue culture medium (mTCM)-199 supplemented with 10% (v/v) porcine follicular fluid, modified Tyrode's albumin lactate pyruvate (mTALP) medium, and modified North Carolina State University (mNCSU)-23 medium, respectively. In addition, amino acids and dipeptides of different concentrations and combinations were used to treat the embryos. The addition of L-alanyl-L-glutamine (AlnGln)+L-glycyl-L-glutamine (GlyGln) significantly (p<0.05) improved oocyte maturation, fertilization and the incorporation and oxidation of (14)C(U)-glucose when compared to the control group and other treatment groups. Additionally, 2-4 cell, 8-16 cell, morula and blastocyst development increased significantly (p<0.05) following treatment with AlnGln+GlyGln when compared to the control group and other treatment groups, while this treatment reduced the accumulation of ammonia. Taken together, these findings suggest that treatment with AlnGln+GlyGln may play an important role in increasing the rate of porcine oocyte maturation, fertilization and embryonic development by reducing the level of accumulated ammonia measured in the culture media.

Selenium and vitamin E improve the in vitro maturation, fertilization and culture to blastocyst of porcine oocytes.

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<0.05) compared with the control and other treatments. In addition, embryo development, specifically in terms of the numbers of morulae and blastocysts, significantly increased (P<0.05) with SeMet and SeMet+Vit-E. In contrast, the accumulation of ammonia was reduced with SeMet and SeMet+Vit-E compared with other treatments. These findings indicate that SeMet and SeMet+Vit-E may play important roles in reducing the accumulation of ammonia and subsequently in increasing the rate of maturation of porcine oocytes and fertilization, as well as development of the blastocyst and utilization of glucose in in vitro maturation, fertilization and culture to blastocysts of porcine oocytes.

Dietary Karaya Saponin and Rhodobacter capsulatus Exert Hypocholesterolemic Effects by Suppression of Hepatic Cholesterol Synthesis and Promotion of Bile Acid Synthesis in Laying Hens.

This study was conducted to elucidate the mechanism underlying the hypolipidemic action of karaya saponin or Rhodobacter (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 (P < .05). 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 and R. capsulatus (P < .05). The supplementation of karaya saponin, R. capsulatus, and the combination of karaya saponin and R. capsulatus suppressed the incorporation of (14)C from 1-(14)C-palmitic acid into the fractions of total lipids, phospholipids, triacylglycerol, and cholesterol in the liver in vitro (P < .05). These findings suggest that the hypocholesterolemic effects of karaya saponin and R. capsulatus are caused by the suppression of the cholesterol synthesis and the promotion of cholesterol catabolism in the liver.

Metabolism of exogenous fatty acids, fatty acid-mediated cholesterol efflux, PKA and PKC pathways in boar sperm acrosome reaction.

PURPOSE: 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. METHODS: 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. RESULTS: Incorporation rate of 14C-oleic acid into the sperm lipids was significantly higher than that of 3H-linoleic acid (P < 0.05). The oxidation of 14C-oleic acid was higher in combined radiolabeling rather than in one. The highest amounts of 3H-linoleic acid and 14C-oleic acid were recovered mainly in the triglycerides and phospholipids fraction, and 14C-oleic acid distribution was higher than the 3H-linoleic acid in both labeled (P < 0.05) sperm lipids. In the 3H-linoleic and 14C-oleic acid combined radiolabeling, the incorporation rate of the radioactive fatty acids in all the lipid fractions increased 15 times more than the alone radiolabeling. Boar sperm utilize oleic acid to generate energy for hyperactivation (P < 0.05). Supplementation of arachidonic acid significantly increased (P < 0.05) cholesterol efflux in sperm. When spermatozoa were incubated with PKA or PKC inhibitors, there was a significant reduction of arachidonic acid-induced acrosome reaction (AR) (P < 0.05), and inhibition by PKA inhibitor is stronger than that by PKC inhibitor. CONCLUSIONS: Incorporation of unsaturated fatty acids, especially oleic acid, into triglycerides and phospholipids provides prerequisite energy for AR. Cholesterol efflux by arachidonic acid triggers AR. Arachidonic acid activated PKA and PKC pathway participate in induction of the AR.

Effect of selenium and vitamin E on acrosome reaction in porcine spermatozoa.

PURPOSE: 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. METHODS: 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. RESULTS: In experiment 1, the sperm motility, viability, AR and incorporation of 14C(U)-glucose increased significantly (P < 0.05) in SS, SeMet and Vit-E (5, 5 µg/l and 1.0 mM, respectively) compared with the control. In experiment 2, treatment of the sperm with SeMet and SeMet + Vit-E in the presence of 300 µM ammonia also resulted in a significant increase (P < 0.05) in the rate of motility, viability, AR and incorporation of 14C(U)-glucose. In contrast, the accumulation of ammonia was reduced by SeMet and SeMet + Vit-E compared with the other treatments. CONCLUSIONS: These findings indicate that SeMet and SeMet + Vit-E may play an important role in reducing the accumulation of ammonia and subsequently in increasing the rate of AR and the utilization of glucose in porcine spermatozoa.

Karaya root saponin exerts a hypocholesterolemic response in rats fed a high-cholesterol diet.

Different sources of saponins are known to have hypocholesterolemic activity with varying degrees of efficacy. We hypothesize that karaya root saponin would efficiently reduce cholesterol. The aim of this study is to examine the comparative hypocholesterolemic effect of karaya root saponin in rats fed a high-cholesterol diet. Sixty male Wister-Imamichi rats were divided into 5 groups of 12 rats each constituting of the following: control group, soybean saponin-supplemented group, karaya root saponin-supplemented group, quillaja saponin-supplemented group, and tea saponin-supplemented group. Compared with the control diet, both the karaya root- and quillaja saponin-supplemented diets significantly reduced (P < .05) serum cholesterol and atherogenic index. Karaya root saponin significantly increased the serum high-density lipoprotein cholesterol, high-density lipoprotein cholesterol/cholesterol ratio, and fecal cholesterol concentrations (P < .05). The triacylglycerol concentration was significantly reduced only in the quillaja saponin-supplemented rats (P < .05). All the tea, soybean, karaya root, and quillaja saponins significantly reduced low-density lipoprotein cholesterol, and the greatest reduction was observed with karaya root saponin. Highest fecal bile acid concentration was found with quillaja saponin, whereas highest liver bile acid concentration was observed with karaya root saponin-supplemented rats (P < .05). These results collectively suggest that karaya root saponin can efficiently reduce serum cholesterol concentration in rats.

Fatty acid composition of lipids in day 7-13 blastocysts, serum and uterine fluid of rabbits.

Purpose: The fatty acid composition of rabbit blastocysts, blood serum and uterine fluids were analyzed to study embryonic lipid metabolism. Methods: Embryos were collected from Japanese white rabbits and fatty acids were analyzed by gas chromatograph. Results: 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. Conclusion: Palmitic acid is the most abundant fatty acid in the blastocysts, serum and uterine fluids of rabbit during days 7-13 of pregnancy.

Effect of relaxin and IGF-I on the pre-implantation development of Mongolian gerbil (Meriones unguiculatus) embryos in vitro.

PURPOSE: 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. METHODS: 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. RESULTS: Relaxin (200 ng/ml) increased total, TE and ICM cell numbers of blastocyst (P < 0.05) when it was compared with the control. IGF-I (150 ng/ml) also has influence on total and ICM cell numbers of blastocyst when compared with control. Apoptosis incidence was relatively low, and a significant difference was not observed between each group. The effect of relaxin on incorporation of 3H-methionine was higher than the control group (P < 0.05). Relaxin increased the developmental rate from the eight-cell stage to blastocyst (P < 0.05). CONCLUSIONS: In conclusion, relaxin and IGF-I stimulated protein synthesis and increased cell numbers of blastocysts, promoting development of the gerbil embryo in vitro culture.

Effect of relaxin on the decidual cell reaction in the Mongolian gerbil (Meriones unguiculatus).

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. METHODS: Ten-to-twelve-week-old female Mongolian gerbils were ovariectomized, treated with estradiol, progesterone, and relaxin, and the uterine horn was stimulated. On day 10, uterine horns were measured for weight, protein concentration, and the incorporation of 14C-methionine; tissue sections were examined. Interleukin-11 (IL-11) primers were used for RT-PCR to confirm decidualization. RESULTS: Decidualization can be induced artificially in gerbils. In general, the histological observations of gerbil decidual cells were very similar to those of rats. The uterine horn weight, protein content, and protein synthesis from 14C-methionine significantly increased in the relaxin-treated gerbils (P< 0.05). Mast cells in the relaxin-treated uterus had proliferated more than those of the non-relaxin-treated group, which was confirmed by IL-11 expression. CONCLUSIONS: We conclude that decidualization can be induced artificially, and relaxin increased weight of uterine horn, protein concentration, protein synthesis and IL-11 expression in gerbils.

Effects of relaxin and IGF-I on capacitation, acrosome reaction, cholesterol efflux and utilization of labeled and unlabeled glucose in porcine spermatozoa.

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. Methods: Swim-up separated spermatozoa that had been washed twice were incubated at 37°C for 1 or 4 h in modified Tyrode's albumin lactate pyruvate (mTALP) medium supplemented without (control) or with relaxin (20 ng/mL) or IGF-I (20 ng/mL) or both (10 + 10 ng/mL). Results: Progressive motility and the induction rate of capacitation and acrosome reaction were increased (P < 0.05) by relaxin and IGF-I alone or in combination, especially after 4 h of incubation. Relaxin alone or combined with IGF-I enhanced (P < 0.05) the cholesterol efflux after 4 h, whereas IGF-I alone did not show any significant effect on the cholesterol efflux compared with the control at any time point. The utilization rates of labeled and unlabeled glucose increased (P < 0.05) in spermatozoa incubated with relaxin or IGF-I alone or in combination compared with the control. Conclusion: Thus, supplementation of relaxin alone or combined with IGF-I into the medium possibly plays a beneficial role in porcine spermatozoal prefertilization events in vitro. (Reprod Med Biol 2008; 7: 29-36).

Effect of amino acids and dipeptides on the acrosome reaction and accumulation of ammonia in porcine 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. Methods: 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. Results: 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. Conclusion: 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. (Reprod Med Biol 2008; 7: 123-131).

Effect of fatty acids bound to bovine serum albumin-V on acrosome reaction and utilization of glucose in boar spermatozoa.

Aim: The present study has been designed with the objective of determining if fatty acids bound to bovine serum albumin-V (BSA-V) can improve motility, viability, and increase acrosome reaction (AR) and utilization of glucose in boar spermatozoa. Methods: Boar spermatozoa were washed, swum-up and incubated at 37°C for 6 h in TALP medium supplemented with fatty acids bound to bovine serum albumin fraction V (BSA-V), fatty acid free BSA (BSA-FAF), polyvinyl alcohol + main fatty acids bound to BSA-V (PVA + FA) and PVA. Sperm motility, viability, AR, and the incorporation and oxidation of 14C-glucose were evaluated during 6 h of incubation. Results: The results show that the BSA-V was superior to BSA-FAF and PVA in improving motility and AR. Viability was significantly increased (P < 0.05) by only BSA-V compared with PVA. When the main fatty acids compound of BSA-V were added to PVA, the sperm motility, viability and AR became almost the same as with BSA-V. The rate of incorporation and oxidation of 14C-glucose were significantly increased (P < 0.05) by BSA-V compared with BSA-FAF and PVA. Fatty acids bound to BSA-V are important for improvement of sperm functions. Conclusions: The present study postulates that fatty acids bound to BSA-V are important to acrosome reaction and the utilization of glucose in boar spermatozoa.

Spermatogenesis in immature mammals.

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).

Effect of amino acids and dipeptides on accumulation of ammonia in the medium during in vitro maturation and fertilization of porcine oocytes.

Aim: The present study was designed to investigate the effect of amino acids and their dipeptides on the accumulation of ammonia in the medium during in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes. Methods: The IVM and IVF media were modified North Carolina State University-37 solution and modified Tyrode's albumin lactate pyruvate, respectively. Porcine oocytes were matured in IVM medium containing 75-2400 µmol ammonia. Amino acids (1.0 mmol) or their dipeptides (2.0 mmol) related to the urea cycle were added individually to the IVM and IVF media containing 300 µmol ammonia. Oocyte maturation and fertilization were assessed using acetic-orcein staining, and the accumulation of ammonia in the media was measured using the indophenol method. Results: Percentages of metaphase II (MII) were adversely affected (P < 0.05) by ≥300 µmol concentrations of ammonia in the IVM medium. In the presence of 300 µmol ammonia in the IVM and IVF media, glutamic acid, l-alanyl-L-glutamine (AlaGln), l-glycyl-L-glutamine (GlyGln) and AlaGln + GlyGln showed the highest rate (P < 0.05) of MII, monospermic fertilization, and the lowest rate (P < 0.05) of ammonia accumulation in the media. Conclusion: AlaGln and GlyGln in IVM and IVF media were more stable and effective than the individual amino acids in reducing the accumulation of ammonia, and increased the rate of porcine oocyte MII and monospermic fertilization in vitro. (Reprod Med Biol 2007; 6: 165-170).

Effect of fatty acids on boar sperm motility, viability and acrosome reaction.

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 < 0.05) the motility and viability of boar spermatozoa. The AR was significantly improved (P < 0.05) by oleic and arachidonic acid in almost all incubation periods. When combinations of oleic, linoleic and arachidonic acid were studied for motility, viability and AR, it was found that oleic plus linoleic acid significantly increased (P < 0.05) motility, whereas arachidonic plus oleic acid significantly increased (P < 0.05) AR. Conclusion: Unsaturated fatty acids, especially arachidonic acid, can improve boar sperm motility and AR. A combination of arachidonic and oleic acid is important for inducing boar sperm AR. (Reprod Med Biol 2007; 6: 235-239).

Effect of relaxin on acrosome reaction and utilization of glucose in 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<0.05) during 1-4 h of incubation. The percentage of acrosome-reacted live spermatozoa was higher (P<0.05) when the spermatozoa were treated with 20 or 40 ng/ml relaxin. The rate of incorporation, and oxidation of glucose were also greater (P<0.05) in the spermatozoa incubated with relaxin compared to the control spermatozoa. The rate of incorporation and oxidation of (14)C-glucose were increased in correlation with acrosome reaction up to 4 h of incubation and then decreased in line with the increasing incubation period. In conclusion, the present study demonstrates that relaxin accelerates not only motility but also the acrosome reaction and utilization of glucose in boar spermatozoa.

Effect of relaxin on in vitro fertilization of porcine oocytes.

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<0.05) sperm motility and increased the percentage of acrosome-reacted live spermatozoa during 1-4 h of incubation, although viability was not significantly improved. Significantly (P<0.05) the highest percentage of monospermic (31.7%) and lowest percentage of polyspermic (16.5%) fertilization was achieved from the sperm-oocyte co-culture group treated with 20 ng/ml relaxin as compared to other groups. The percentage of male pronucleus formation was significantly (P<0.05) greater in the 20 ng/ml relaxin-treated sperm-oocyte co-culture group than in the other groups. These results indicate that supplementation with relaxin is capable of improving sperm function and fertilization of porcine oocytes in vitro.

Effect of fructose on motility, acrosome reaction and in vitro fertilization capability of boar spermatozoa.

Aim: 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. Methods: 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. Results: 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. Conclusion: 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. (Reprod Med Biol 2006; 5: 255-261).

Effect of relaxin on motility, acrosome reaction and viability of cryopreserved boar spermatozoa.

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. Methods: 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 0-4 h of incubation. Results: The results show that the supplementation of relaxin (especially at 20 ng/mL) in the thawing solution improved sperm motility significantly (P < 0.05) at 1-3 h of incubation. The percentage of acrosome reacted live spermatozoa was improved significantly (P < 0.05) when the spermatozoa were treated with 20 ng/mL relaxin. Viability was not 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. Conclusion: We conclude that relaxin can improve the sperm motility and AR, and enhance the glucose metabolism of cryopreserved boar spermatozoa. (Reprod Med Biol 2006; 5: 215-220).

Evaluation of testicular toxicity and sperm morphology in rats treated with methyl methanesulphonate (MMS).

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.