ResearchThe lipid content and fatty acid composition of fresh immature and in vitro matured bovine oocytes cultured in media with or without serum, and also those of frozen-thawed immature oocytes were analysed. All oocytes were ranked (A or B) on the basis of their cytoplasmic quality. Fatty acid composition (mol %; w/w) in the total lipid fraction was analysed by gas chromatography. Triglyceride, total cholesterol, phospholipid (phosphocholine-containing phospholipid) and non-esterified fatty acid contents of immature and in vitro matured oocytes were determined using lipid analysis kits. Phosphocholine-containing phospholipid and non-esterified fatty acid contents were determined in frozen-thawed immature bovine oocytes. Palmitic acid was the most abundant fatty acid in immature oocytes (A: 35%, B: 36%), and in in vitro matured oocytes cultured in the medium containing serum (A: 36%, B: 35%) or polyvinyl alcohol (A: 33%, B: 36%). Oleic acid was the second most abundant fatty acid in all A ranked oocytes, whereas stearic acid was the second most abundant fatty acid in all B ranked oocytes. There were significant differences (P < 0.05) in linoleic and arachidonic acid fractions between A and B ranked immature oocytes. In vitro matured oocytes had significantly (P < 0.05) lower proportions of linoleic and arachidonic acids, and significantly (P < 0.01) lower contents of triglyceride and total cholesterol compared with those of immature oocytes. The fatty acid composition of in vitro matured oocytes cultured in medium containing fetal calf serum or polyvinyl alcohol was similar, but significant differences (P < 0.01) in triglyceride and the total cholesterol content were observed. There was a significant decrease (P < 0.05) in the arachidonic acid proportion in frozen-thawed immature oocytes compared with that in fresh immature oocytes. In addition, significant (P < 0.05) decreases in both phospholipid (15.8-10.6 pmol) and non-esterified fatty acid (11.0-4.1 pmol) were found in frozen-thawed immature oocytes. The results indicate that lipids are available for use as an energy source for maturation and that serum lipids are incorporated into the oocyte cytoplasm during in vitro maturation. The changes in the lipid content (mainly phospholipid) and fatty acid composition were also observed in frozen-thawed immature oocytes. The study indicates that the alteration of fatty acid composition in bovine oocytes might improve maturation and cryopreservation.

Prostaglandin F2 alpha (PGF2 alpha) is a primary luteolysin in the cow. Although the mechanisms involved in luteolysis are thought to be a complex of its direct action on luteal cells and indirect effect on luteal blood flow, the detailed mechanisms remain to be elucidated. This study focuses on the possible interaction of endothelial cells-derived endothelin-1 (ET-1) with PGF2 alpha in the rapid suppression of progesterone release from the bovine corpus luteum (CL). In in vitro microdialysis system (MDS) of CL, PGF2 alpha acutely stimulated the release of progesterone and oxytocin during infusion and ET-1 release after infusion. Moreover, PGF2 alpha induced slight decrease of progesterone release during the last period of the experiment (8-11 h after PGF2 alpha exposure). Two 1 h-perfusions of ET-1 at 3 h intervals induced only a slight decrease of progesterone release after the second perfusion. This treatment also affected the oxytocin release; the first ET-1 perfusion produced an acute stimulation, whereas the second ET-1 perfusion inhibited the release to below 50%. When the CL pieces were pre-perfused with PGF2 alpha for 2 h, the two consecutive perfusion of ET-1 at 3 h intervals induced drastic decrease in progesterone and oxytocin release only after the second ET-1 perfusion. Thus, a pre-exposure with PGF2 alpha clearly potentiated the inhibiting activity of ET-1 in the progesterone release. These results suggest a physiological impact of PGF2 alpha and ET-1 in the rapid cascade of functional luteolysis in vivo, and a possible interaction between endothelial cells and luteal cells.

Recent observations suggest that the endothelial cell-derived vasoconstrictive peptide endothelin-1 (ET-1) interacts with prostaglandin F2alpha (PGF2alpha) and that luteal ET-1 participates in the rapid cascade of functional luteolysis in vivo. Thus, the present study aimed to determine in detail the real-time changes in ET-1, oxytocin (OT), and progesterone (P4) concentrations within the regressing corpus luteum (CL), along with the changes in ovarian venous plasma (OVP) ipsilateral to the CL as well as in jugular venous plasma (JVP) in the cow. In the first study, peripheral plasma from daily sampling during the estrous cycle (n = 6) showed clear changes in ET-1 concentration with the stage of the cycle (p < 0.05). ET-1 remained at basal concentrations (23.2+/-1.3 pg/ml) on Days 2-12, increased (p < 0.05) on Days 13-19 (33.5+/-2.6 pg/ml), and reached the highest (p < 0.001) concentrations (45.6+/-4.4 pg/ml) on Days 20-22 after estrus. These data indicate that plasma ET-1 concentration increases around luteolysis and estrus. In the second study, a microdialysis system (MDS) was surgically implanted into the CL of 11 cows in the midluteal phase. In 4 of the 11 cows, the catheter was also fitted to the ovarian vein ipsilateral to the CL at surgery. A PGF2alpha analogue (cloprostenol; 500 microg) was then injected (designated as 0 h) i.m. to induce luteolysis. In the cows fitted with an MDS, the PGF2alpha injection clearly induced a rapid decrease in intraluteal P4 release within 4 h (p < 0.05), and the levels decreased to 20% of the baseline after 24 h. Intraluteal release of ET-1 increased (p < 0.05) to 160% within 4 h after PGF2alpha injection, when an enormous OT release (to 950%) occurred, which reached a plateau of 250% after 20 h that persisted until 72 h. ET-1 release into the ovarian vein began to increase at 2 h after PGF2alpha injection, when the acute OT release almost dropped to the baseline. The ET-1 concentration was temporarily (between 0 and 24 h after PGF2alpha) 2-3 times higher in OVP than in JVP (p < 0.05), and increased again to higher levels than in JVP from 32 to 64 h (p < 0.05). ET-1 concentrations in JVP gradually increased from 10 pg/ml to 30 pg/ml during PGF2alpha-induced luteolysis (p < 0.05). In conclusion, PGF2alpha injection rapidly increased ET-1 release within the regressing CL as well as into the ovarian venous blood in the cow. The overall results strongly support the hypothesis that luteal ET-1 is a local luteolytic mediator/promotor in the regressing bovine CL.

Soy isoflavone supplementation for four weeks showed potentially beneficial effects on bone metabolism and on serum lipids in perimenopausal women. These effects could have the potential to reduce the risks of postmenopausal osteoporosis and of cardiovascular diseases in such women.

Abstract. In this study, two following experiments were performed to improve post-thaw motility and viability of frozen-thawed ram spermatozoa. We examined i) the effects of different concentrations of bovine serum albumin (0, 0.3, 1, 5, 10 and 15% BSA) in semen diluents lacking egg yolk and ii) the effects of four semen diluents, fructose (F: control) and trehalose (T) in semen diluents containing egg yolk, 15% BSA in semen diluents without egg yolk (BSA), and modified phosphate buffered saline (m-PBS). Frozen-thawed spermatozoa were examined for progressive sperm motility, viability, morphological abnormality, sperm tail swelling test, and sperm acrosome integrity. In Experiment 1, the rates of sperm motility immediately after thawing (0 h) were significantly (P<0.05) higher in the 10 and 15% BSA groups (55.0 ± 2.9 and 58.3 ± 6.7%, respectively) than in the positive control (F) group (41.7 ± 4.4%). The rate of sperm viability in the negative control (0% BSA) group (80.2 ± 3.3%) was significantly (P<0.05) lower than in the positive control (F) group (89.8 ± 1.5%), but when compared with the F group, no significant differences were found among the 0.3, 1, 5, 10 and 15% BSA groups at 0 h. The rates of sperm morphological abnormality of the 10 and 15% BSA groups (6.5 ± 1.3 and 6.3 ± 1.1%, respectively) were significantly (P<0.05) lower at 0 h than that in the 1% BSA group (16.3 ± 5.2%). In Experiment 2, T addition improved (P<0.05) the post-thaw motility compared with the F and BSA groups. Furthermore, at 3 and 6 h, the post-thaw motility of the T group (36.3 ± 2.4 and 25.0 ± 2.0%, respectively) was significantly (P<0.05) higher than in the BSA (26.3 ± 2.4 and 18.8 ± 1.3%, respectively) and F (28.8 ± 3.8 and 18.8 ± 2.4%, respectively) groups. The post-thaw sperm motility and viability in the m-PBS group were significantly (P<0.05) lower than those of the control (F), T, and BSA groups throughout all observation points. These results indicate that 10 and 15% BSA can be substituted for egg-yolk for ram semen diluent and that the addition of trehalose enhances motility and viability of ram spermatozoa after freezing and thawing. Key words: Bovine serum albumin (BSA), Semen diluent, Sheep, Spermatozoa, Trehalose (J. Reprod. Dev. 52: [675][676][677][678][679][680][681][682][683] 2006) t has been reported that frozen-thawed spermatozoa are greatly damaged during the freezing and thawing processes [1][2][3][4][5]. This damage may decrease sperm motility and the fertilization rate after artificial insemination (AI). Several semen analysis methods have been developed to evaluate the functional parameters of spermatozoa more objectively. The hypo-osmotic swelling test (HOS-test) has been applied to various species, such as dogs [6,7], cattle [8,9], horses [10][11][12], rams [13], minke whales (Balaenoptera bonarensis) [13] and humans [14,15]. It has been recognized that the HOS-Test is a simple and useful method with good reliability and repeatability for examination of the sperm plasma membrane.