A cooperative action of endothelin-1 with prostaglandin F(2alpha) on luteal function in the cow.

Prostaglandin F(2alpha) (PGF(2alpha)) is the primary luteolysin in the cow, and luteal endothelin-1 (ET-1) interacts with PGF(2alpha) during the process of luteolysis. In contrast, a developing corpus luteum (CL) is refractory to exogenous administration of PGF(2alpha). Thus, the present study was aimed to investigate the functional relationship between ET-1 and PGF(2alpha) in the mid-CL (PGF(2alpha)-sensitive) and early-CL (PGF(2alpha)-refractory). In the mid-CL model, cows (n = 6/treatment) were assigned to receive one of five types of treatments on day 10 of the estrous cycle: (1) an injection of saline; control, (2) a 500 microg of PGF(2alpha) analogue (sufficient dose to induce luteolytis); full-PG, (3) an intraluteal injection of 0.25 mg ET-1; ET-1, (4) a 125 micro g of PGF(2alpha) (insufficient dose to induce luteolytis); 1/4PG or (5) an intraluteal injection of 0.25 mg ET-1 after administration of a insufficient dose of PGF(2alpha) analogue; 1/4PG/ET. In the early-CL model, cows were assigned to receive one of two types of treatments on day 5 of the estrous cycle: (1) a sufficient dose of PGF(2alpha) analogue; PG (n = 5) or (2) an intraluteal injection ET-1 after a sufficient dose of PGF(2alpha); PG/ET (n = 7). In the mid-CL model, 1/4PG/ET resulted in a rapid reduction of progesterone (P) concentrations similar to that in full-PG from the next day. However, the levels of P in 1/4PG/ET (1.5-2.5 ng/ml) kept significantly higher than that in full-PG (< 0.5 ng/ml). ET-1 or 1/4PG did not decrease plasma P concentrations (4-6 ng/ml). The plasma ET-1 levels increased with the full-PG administration. In the early-CL model, both treatments had no effect on plasma P increase and ET-1 levels. The overall results indicate that the intraluteal ET-1 injection after administration of insufficient dose of PGF(2alpha) induces the depression of P secretion in vivo during the mid luteal phase in the cow, supporting the concept that ET-1 is one of a local mediator of functional luteolysis in the cow. The result further indicates that the early-CL is not only PG-refractory but also ET-1-refractory.

Blood flow: a key regulatory component of corpus luteum function in the cow.

Prostaglandin F2alpha (PGF2alpha) is the primary luteolysin in the cow. During the early luteal phase, the corpus luteum (CL) is resistant to the luteolytic effect of PGF2alpha. Once mature, the CL becomes responsive to PGF2alpha and undergoes luteal regression. These actions of PGF2alpha coincide with changes in luteal blood flow (BF): PGF2alpha has no effect on BF in the early CL, but acutely increases BF in the peripheral vasculature of the mature CL within 30 min of PGF2alpha injection. During spontaneous luteolysis, luteal BF increases on Days 17-18 of the estrous cycle, prior to any decrease in plasma progesterone (P). The increase in luteal BF is synchronous with an increase in plasma PGFM levels, suggesting that pulsatile release of PGF2alpha from uterus stimulates the increase in luteal BF. Serial biopsies of these CL showed that mRNA expression for endothelial nitric oxide synthase (eNOS) together with endothelin-1 (ET-1) and angiotensin converting enzyme (ACE) increases on Days 17-18 when the luteal BF is elevated. On Day 19 when plasma P level firstly decreases, eNOS mRNA returns to the basal level whereas ET-1 and ACE mRNA remains elevated. Cyclooxygenase-2 (COX-2) mRNA expression increases on Day 19. In support of these data, an in vivo microdialysis study revealed that luteal ET-1 and angiotensin II (Ang II) secretion increases and precedes PGF2alpha secretion during spontaneous luteolysis. In conclusion, we show for the first time that an acute increase of BF occurs in the peripheral vasculature of the mature CL together with increases in eNOS expression and ET-1 and Ang II secretion in the CL during the early stages of luteolysis in the cow. We propose that the increase in luteal BF may be induced by NO from large arterioles surrounding the CL, and simultaneously uterine or exogenous PGF2alpha directly increases ET-1 and Ang II secretion from endothelial cells of microcapillary vessels within the CL, thereby suppressing P secretion by luteal cells. Taken together, our results indicate that an acute increase in luteal BF occurs as a first step of luteolysis in response to PGF2alpha. Therefore, local BF plays a key role to initiate luteal regression in the cow.

Local interaction of prostaglandin F 2alpha with endothelin-1 and tumor necrosis factor-alpha on the release of progesterone and oxytocin in ovine corpora lutea in vivo: a possible implication for a luteolytic cascade.

Endothelin-1 (ET-1) and tumor necrosis factor-alpha (TNFalpha) participate in the cascade of luteolysis. Thus, in the present study the interactions of ET-1 and TNFalpha with prostaglandin F(2alpha) (PGF(2alpha)) on the release of progesterone and oxytocin (OT) within the corpus luteum (CL) were investigated. A microdialysis system (MDS) was surgically implanted in ovine CL (one MDS line/CL; 5-10 lines/ewe) formed after super-ovulation. A 4-h perfusion with PGF(2alpha) (0.01-1 micromol l (-1)) induced no clear effect on progesterone release, but acutely stimulated OT release in a dose-dependent manner. A perfusion of PGF(2alpha) (1 micromol l (-1)) increased ET-1 release over a period of 12 h. Two perfusions of ET-1 (0.1 micromol l(-1)) or a perfusion of ET-1 followed by TNFalpha (200 ng ml(-1)) decreased progesterone release (56-64% at 36-48 h). When the CL were pre-perfused with PGF(2alpha) (1 micromol l(-1)), two consecutive perfusions of ET-1 decreased progesterone release more rapidly. Similarly, a pre-perfusion with PGF(2alpha) followed by consecutive perfusions of ET-1 and then TNFalpha rapidly decreased progesterone release, with the inhibition most pronounced (35%) at 36-48 h. The simultaneous infusion of ET-1 with PGF(2alpha) induced a rapid decrease in progesterone release (36% at 36-48 h). In a further study, the possible second messenger systems involved in PGF(2alpha) action on the release of progesterone, OT and ET-1 were investigated. A perfusion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 10 micromol l(-1)), A23187 (10 micromol l(-1)), or PGF(2alpha) + A23187 increased progesterone release during infusion, but decreased it after perfusion. All treatments induced a massive release of OT during infusion, and increased ET-1 release after infusion. These results show that ET-1 is capable of suppressing progesterone release in the PGF(2alpha)-primed ovine CL in vivo and thus ET-1 works as a local luteolysin together with PGF(2alpha) during the process of functional luteolysis. During structural luteolysis, TNFalpha may interact with PGF(2alpha) and ET-1 to cause a rapid drop in progesterone release and accelerate the process of luteolysis. This result supports the contention that ET-1 and TNFalpha interact with PGF(2alpha) as local luteolytic mediators in the ewe as previously suggested.

Angiotensin II and atrial natriuretic peptide in the cow oviductal contraction in vitro: direct effect and local secretion of prostaglandins, endothelin-1, and angiotensin II.

Angiotensin II (Ang II) and atrial natriuretic peptide (ANP) may be involved in local regulation of the oviductal contraction during the estrous cycle. Thus, the in vitro effects of Ang II and ANP on the secretion and contraction of bovine oviduct during the follicular, postovulatory, and luteal phases were investigated. An in vitro microdialysis system (MDS) was utilized to determine the intraluminal release of prostaglandins (PGs), Ang II, and endothelin-1 (ET-1) from the bovine oviducts as well as to observe the effect of Ang II and ANP on the local secretion of these substances. The basal release of PGs, ET-1, and Ang II was higher (P < 0.05) during the follicular and postovulatory phases than during the luteal phase. Stimulation by infusion of Ang II (10(-6) M) or ANP (10(-7) M) into the MDS was carried out for 4 h between 4 and 8 h of incubation. In the oviducts from the follicular and postovulatory phases, the infusion of ANP increased the release of Ang II, but not of ET-1. Infusion of Ang II stimulated the release of ET-1. Both Ang II and ANP increased PGE(2) and PGF(2alpha) release. In the contraction study, direct administration of Ang II (10(-7) M) or ANP (10(-8) M) into the medium during the follicular and postovulatory phases increased the amplitude of oviductal contraction. In contrast, these substances did not show any effect in the contraction and secretion of oviducts from cows during the midluteal phase. These results indicate that during the periovulatory period, Ang II and ANP stimulate the contractile amplitude of the oviduct in vitro. In addition to their direct action on oviductal contraction, Ang II may activate oviductal secretion of ET-1 and PGs. Likewise, ANP stimulates oviductal secretion of PGs and Ang II. Hence, the overall results suggest the existence of a functional endothelin-angiotensin-ANP system in the bovine oviduct during the periovulatory period, which may regulate the oviductal contraction to ensure maximum efficiency of gamete/embryo transport through the oviduct.

In vitro regulation of local secretion and contraction of the bovine oviduct: stimulation by luteinizing hormone, endothelin-1 and prostaglandins, and inhibition by oxytocin.

The precise regulatory mechanisms of cyclic oviductal contraction in the cow are unclear. The purpose of this study was to evaluate the effect of luteinizing hormone (LH), steroids, prostaglandins (PGs) and peptides on the oviductal contraction and secretion of PGs and endothelin (ET-1). In addition, the cyclic expression of mRNA for ET-1 and its receptors (ET-R) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). In the in vitro microdialysis study, an infusion of LH alone or in combination with progesterone (P(4)), estradiol-17beta (E(2)) and/or ET-1 stimulated pronounced release of PGE(2), PGF(2alpha) and ET-1 in the oviducts from cows in the follicular and postovulatory phases. The addition of LH, LH+P(4)+E(2) and/or ET-1 to the medium increased the amplitude of oviductal contraction. However, oxytocin (OT) completely blocked the responses of oviductal secretion and contraction. In contrast, these substances did not show any effect in the oviducts from cows in the mid luteal phase. Similar expression patterns of mRNA encoding for ET-R type A and type B were found, which were highest during the postovulatory phase, lower during the luteal phase, with the lowest expression during the follicular phase. We suggest that the preovulatory LH surge, together with increasing E(2) levels from the Graafian follicle and a basal P(4) from regressing corpora lutea (CL), stimulates maximum oviductal production of PG and ET-1, resulting in oviductal contraction for a rapid transport of gametes. OT released from the newly-formed CL may block these mechanisms, and slow contractions for transport of the embryo to the uterus.

Development of in vitro matured and fertilized bovine embryos cocultured with bovine oviductal epithelial cells obtained from oviducts ipsilateral to cystic follicles.

The present experiment was conducted to clarify the effect of bovine oviductal epithelial cells (BOEC) collected from oviducts ipsilateral to cystic follicles (CFs) using an in vitro coculture system on the development of in vitro matured/fertilized (IVM/IVF) bovine embryos. In the first comparison, the effect of the presence of CF on the development of the embryos cocultured with BOEC derived from the cows with CF (n = 18) and corpus hemorrhagicum (CH, n = 10) was examined. In the second comparison, the effect of the type of cyst [progesterone (P4)-dominant; n = 9, estradiol-17beta (E2)-dominant; n = 5] on the development of the embryos cocultured with BOEC derived from the cystic cows was examined. No difference was observed between CF and CH (control) groups in the mean developmental rates of embryos developed to > or =2-cell (86.3% vs. 86.4%), 8-16 cells (53.0% vs. 56.2%), blastocyst (24.2% vs. 24.8%) and hatched blastocyst (12.0% vs. 14.6%). However, the blastocyst production rate was significantly different (P<0.05) between the P4-dominant (19.8%) and E2-dominant (32.6%) groups. The rate of development from cleavage stage embryo to blastocyst was significantly different between P4-dominant (22.9%) and E2-dominant (37.9%) groups. Moreover, the blastocyst rate from 8-16 cells of E2-dominant group (61.6%) was significantly higher than that of P4-dominant one (39.5%). These results indicate that the effects of BOEC collected from oviduct ipsilateral to CFs on embryo development are variable, and the variability is closely associated with the steroid hormone profiles of the follicular fluid.

Effect of ovarian steroids and oxytocin on the production of prostaglandin E2, prostaglandin F2alpha and endothelin-1 from cow oviductal epithelial cell monolayers in vitro.

Cyclic physio-anatomical variation in the oviducts is mediated by the local countercurrent transfer of ovarian products. Thus, in this study cow oviductal epithelial cells (COEC) culture were utilized to investigate the effects of ovarian products such as progesterone (P4), estradiol 17beta (E2) and oxytocin (OT) on local oviductal prostaglandin E2 (PGE2), F2alpha (PGF2alpha) and endothelin-1 (ET-1) production. COEC were collected from non-pregnant Holstein cows (n = 8) during the follicular phase and cultured in M199 under standard culture conditions until monolayer formation. Cells in first passage were incubated for 24 or 48 h with P4 (500 ng/ml), E2 (1 ng/ml), OT (10(-9) M) or combination of E2 + P4. Administration of E2 significantly increased the production of PGE2, PGF2alpha and ET-1. However, simultaneous administration of P4 blocked the effect of E2. OT did not show any effect on oviductal productions of either PGs or ET-1. The results of this study show that E2 stimulates PG and ET-1 production by COEC in vitro. Thus, it can be suggested that locally transferred E2 from the ovarian follicles may be important for oviductal contraction and gamete/zygote transport during the peri-ovulatory period.

Prostaglandin E2, prostaglandin F2 alpha and endothelin-1 production by cow oviductal epithelial cell monolayers: effect of progesterone, estradiol 17 beta, oxytocin and luteinizing hormone.

The optimal oviductal environment, including contractile activity for gamete transport, fertilization and early embryonic development, is mediated by physiological and anatomical changes in the oviduct during the estrous cycle. Oviductal epithelial cell culture was utilized to investigate the effect of ovarian steroids (progesterone [P4] and estradiol 17 beta [E2]), oxytocin (OT) and luteinizing hormone (LH) on the local production of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha) and endothelin-1 (ET-1) in the cow oviduct. Epithelial cells isolated from oviducts collected during the follicular phase were cultured in M199 under standard culture conditions until monolayer formation. Then the cells were trypsinized and plated at a density of 3 x 10(4)/mL/well and cultured again until subconfluency, at which time the cells were incubated for 4 or 24 h with M199 only (control), high P4 (H-P4; 1 microgram/mL), low P4 (L-P4; 10 ng/mL), E2 (1 ng/mL), LH (10 ng/mL), OT (10(-9) M) ET-1 (10(-9) M), PGE2 (10(-8) M) PGF2 alpha (10(-9) M) or their combination (H-P4 + E2, L-P4 + E2, LH + E2, ET-1 + E2, L-P4 + E2 + LH and H-P4 + E2 + LH). The production of both PG and ET-1 was increased by E2 + low P4 and LH + E2 + low P4 (P < 0.05), while LH + E2 enhanced the production of PGF2 alpha and ET-1 (P < 0.05). Moreover, E2 + ET-1 stimulated PG production (P < 0.05). However, OT had no effect on the production of any of these substances. These results suggest that the preovulatory LH surge, together with locally re-circulated high levels of E2 from the Graafian follicle and basal P4 from regressing corpus luteum (CL), induces the maximum stimulatory effect on oviductal PGE2, PGF2 alpha and ET-1 production during the periovulatory period. Consequently, the elevated local ET-1 concentration during periovulatory period may induce the high contractile activity of the oviduct and, at the same time, the stimulation of PG production. Thus, ET-1 may act as a local amplifier for oviductal PG production stimulated by LH and ovarian steroids.

Local release of steroid hormones, prostaglandin E2, and endothelin-1 from bovine mature follicles In vitro: effects of luteinizing hormone, endothelin-1, and cytokines.

Local regulation of ovulation involves the interaction of LH and intrafollicular factors including steroids, prostaglandins, and peptides derived from endothelial cells, leukocytes, fibroblasts, and steroidogenic cells. To estimate the intrafollicular role of endothelin-1 (ET-1) and its possible interaction with LH, tumor necrosis factor alpha (TNFalpha), and interleukin-1beta (IL-1beta), a microdialysis system was implanted into the theca layer of preovulatory bovine follicles that were maintained in organ culture chambers. The effects of LH, ET-1, TNFalpha, and IL-1beta on the local release of steroids, prostaglandin E2 (PGE2), and ET-1 from the cells surrounding the implanted capillary membrane were investigated. Each preovulatory follicle (selected based on the concentrations of steroids and PGE2) was dissected from surrounding stromal tissue and implanted with 4 capillary dialysis membranes (control, LH, cytokines or ET-1, and LH+cytokine or LH+ET-1) into the theca layer. They were then incubated in organ culture chambers and perfused with Ringer's solution for 14 h after pre-perfusion for 2 h. The stimulation with LH (5 microg/ml) between 4 and 6 h increased the release of progesterone (P4), androstenedione (A), estradiol-17beta (E2), PGE2 (p < 0.001), and ET-1 (p < 0.05). The infusion of ET-1 (250 ng/ml) between 8 and 10 h inhibited P4 and A release but stimulated E2 release (p < 0.05). The infusion of TNFalpha (100 ng/ml) between 8 and 10 h after LH exposure suppressed the release of A and E2 (p < 0.05). IL-1beta (10 ng/ml) between 8 and 10 h stimulated E2 release but inhibited A release (p < 0.05). Moreover, ET-1 and cytokines clearly stimulated PGE2 release (p < 0.05). ET-1 and TNFalpha induced further release of PGE2 stimulated by LH (p < 0. 05). Also, TNFalpha and IL-1beta induced further release of ET-1 stimulated by LH (p < 0.05). These results show that ET-1 is released from the theca layer of mature bovine follicles in vitro and that it affects follicular steroids and PGE2 secretion. The overall results suggest that interactions among ET-1, PGE2, and cytokines may have key roles in a local intermediatory/amplifying system of the LH-triggered ovulatory cascade in the bovine preovulatory follicle.

Oocyte quality of small antral follicles coexisting with cystic follicles in the ovaries of the cow.

The present study was conducted with ovaries to evaluate the effect of the presence of a cystic follicle and its steroid hormone concentration on the oocyte recovery rates, oocyte morphology and in vitro maturation of the oocytes from coexisting small follicles. Ovaries, each bearing a follicular cyst (containing > 10 ml of follicular fluids, > 3 cm in diameter, and without a functional corpus luteum), were collected from each of the 26 Holstein cows from a local slaughterhouse. Small follicles (1-7 mm) from these ovaries were aspirated and their cumulus-oocyte complexes (COCs) classified into one of 5 groups (A to E), depending on oocyte and cumulus cell morphologies. Those oocytes with compact cumulus cells were cultured and their maturation rates determined. Concentrations of estradiol-17 beta (E2) and progesterone (P4) were measured in cystic follicular fluid using double antibody enzyme immunoassays (EIA). The morphology of the COCs and maturation rates of the oocytes were then evaluated using two comparisons. In first comparison, the left and right ovaries obtained from an individual cow were considered as a pair, with each pair being divided into two groups depending on the presence or absence of an E2 dominant or P4 dominant cystic follicle in one or another of the ovaries: E2 dominant cows; and P4 dominant cows. Oocytes collected from the ovaries of cyclic cows at follicular, luteal and post-ovulatory stages served as controls. The oocyte recovery rates, oocyte morphologies and oocyte maturation rates were independent of the presence or absence of a coexisting cystic follicle, or its steroid hormone classification or concentration. In the second comparison, each ovary was consider individually and divided into one of the two classes, depending on the presence or absence of a cystic follicle. Those ovaries with cystic follicles were then divided into three subclasses on the basis of E2 and P4 concentrations in the cystic follicular fluids: P4 dominant (P4/E2 ratio > 1); E2 dominant (P4/E2 ratio < 1); and both P4 and E2 dominant follicles present. The numbers of oocytes obtained from an ovary, their morphology and their maturation rates were not significantly different in the presence or absence of a coexisting cystic follicle. Moreover, the number of the oocytes aspirated from an ovary, their morphology and their maturation rates in small follicles coexisting with P4 dominant, E2 dominant and both P4 and E2 dominant cystic follicles were also not different. These results suggested that neither the presence of a cystic follicle in a cow's ovary nor the cyst's steroid hormone concentrations affected the oocyte recovery rate, oocyte morphology and maturation rates of the coexisting small follicles.

Developmental capacity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro.

This study was conducted to assess the ability of the follicular fluid derived from large and small follicles to support the in vitro oocyte maturation and subsequent fertilization and developmental capacity. Oocytes were cultured in bovine follicular fluid aspirated from small (SFF; 2-5 mm in diameter), large (LFF; 10 to 20 mm in diameter) follicles and TCM199 as a control under 5% CO2 in air. All maturation media were supplemented with 1 IU ml-1 pregnant mare serum gonadotropin. After 24 h culture, oocytes were fertilized in vitro with frozen-thawed and heparin-treated (10 micrograms ml-1, 15 min) bull spermatozoa and cultured in TCM199 with bovine oviductal epithelial cells (BOEC) for 7 days. Maturation of bovine oocytes cultured in LFF was inhibited and the low of male pronucleus formation was observed when compared with that of SFF (maturation rate: 69 vs. 78%; P < 0.05; male pronucleus formation rate: 58 vs. 80%; P < 0.05). Developmental capacity of bovine oocytes cultured in SFF was significantly (P < 0.05) higher than that of LFF (15 vs. 5%), but significantly (P < 0.05) lower than that of the control. There were no differences in the number of nuclei per blastocyst obtained after each treatment. These results indicate that the inhibitory action of follicular fluid on in vitro maturation, male pronucleus formation and developmental capacity of bovine oocytes is dependent on the developmental stage of the follicles from which fluid was obtained.

Local distributions of oviductal estradiol, progesterone, prostaglandins, oxytocin and endothelin-1 in the cyclic cow.

The cyclic patterns of hormones which regulate the activity of the oviduct in the cow have not been adequately reported. We studied progesterone (P4), estradiol 17 beta (E2), prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), oxytocin (OT) and endothelin-1 (ET-1) concentrations in the cow oviduct. Reproductive tracts from cyclic Holstein cows in the follicular phase (n = 5), post ovulation phase (n = 5) and luteal phase (n = 5) were collected at a slaughterhouse. Oviducts were separated from the uterus, the lumen vas washed with physiological saline, and the enveloping connective tissues were removed. The fimbria was then separated at first and then the rest was divided into 2 parts of equal length (proximal and distal). After extraction, levels of different hormones in the tissues were measured using double antibody enzyme immunoassays (EIAs). There were no differences in any hormone concentration between the 3 parts of the oviduct at any stage of the estrous cycle. The highest concentration of oviductal P4 was observed during the luteal phase and in the oviduct ipsilateral to the functioning CL. Oviductal OT was unchanged throughout the cycle. The highest E2 concentration was observed during the follicular phase in the oviduct ipsilateral to the dominant follicle. The oviduct ipsilateral to the dominant follicle during the follicular phase and ipsilateral to the ovulation site post ovulation showed higher levels of PGE2, PGF2 alpha and ET-1 than those on the contralateral side or during the luteal phase. The highest PGE2 was observed in the oviduct ipsilateral to the ovulation site during the post ovulation phase. The results suggest that the ovarian products (P4, OT and E2) and the local oviductal products (PGE2, PGF2 alpha, and ET-1) may synergistically control oviductal contraction for optimal embryo transport during the periovulatory period, and provide further evidence for the local delivery of ovarian steroids to the adjacent reproductive tract.

Effects of follicular fluid on fertilization and embryonic development of bovine oocytes in vitro.

This study was conducted to evaluate the effect of bovine follicular fluid (BFF) on fertilizability and developmental capacity of bovine oocytes matured in vitro. Oocytes were collected from slaughterhouse ovaries, and matured in TCM199 supplemented with 5% superovulated cow serum (SCS), 2 mM pyruvate and 1 IU/mL PMSG. BFF was aspirated from small follicles (1 to 5 mm in diameter). In Experiment 1, BFF was added to the Brackett and Oliphant (BO) fertilization medium at concentrations of 0, 1, 5, 10 and 20%. After insemination with frozen-thawed and heparin-treated (10 micrograms/mL, 15 min) bull spermatozoa for 18 h, some of the oocytes were fixed and stained to evaluate the fertilization rate. The rest of the oocytes were co-cultured in serum-free embryo culture medium (ECM; TCM199 supplemented with 5% SCS, 2 mM pyruvate and 5 micrograms/mL insulin) with bovine oviductal epithelial cells (BOEC) at 38.5 degrees C under 5% CO2 in air, and the developmental capacity of embryos was examined at 2, 7 and 9 d. In Experiment 2, BFF was added to the serum-free ECM with BOEC at 0, 5, 10 and 20% concentrations, and embryos were cultured for 9 d. Fertilization rates and blastocyst rates in low (1 and 5%) BFF in fertilization medium were not significantly different from the control (without BFF). However, high concentrations of BFF (10 and 20%) in the fertilization medium suppressed both fertilization rates and development. Large vesicles with fast monolayer formation were observed at all concentrations of BFF added to ECM with BOEC. There were no significant differences in cleavage or development to blastocyst in different concentrations of BFF added to ECM. However, the rate of development to hatched blastocysts in 20% BFF was significantly lower than that of the control (P < 0.05). The results of the present study indicate that BFF addition to fertilization medium and ECM with BOEC does not improve fertilizability or developmental capacity and that high concentrations of BFF reduce the rate of both fertilization and development.

Evaluation of fluids from cystic follicles for in vitro maturation and fertilization of bovine oocytes.

Follicular cysts are defined as cystic structures derived from unovulated follicles. The formation of the cysts appears to be related to failure of the oocyte to resume meiosis. The aim of this study was to evaluate in the bovine: 1) the ability of the fluid from cystic follicles to promote in vitro oocyte maturation and fertilization, 2) the predictive value of the morphology of oocytes derived from cystic follicles on the ability of the follicular fluid to promote in vitro maturation/fertilization as well as the oocytes to undergo maturation and fertilization. In Experiment 1, the ability of fluid from cystic (and normal) follicles from live and slaughtered cows (to promote) in vitro maturation and fertilization of bovine cumulus-oocyte-complexes (COC's) was assessed by cumulus expansion, sperm penetration, male pronucleus formation and polyspermy rates. Concentrations of progesterone (P4) and estradiol-17 beta (E2) were measured in the fluid from cystic follicles collected from live and slaughtered cows. In Experiment 2, we investigated the relationship of the morphology of COC's from cystic follicles, and the effect of the follicular fluids on oocyte maturation as well as P4 and E2 concentrations. In Experiment 1, although sperm penetration and male pronucleus formation were inhibited significantly by fluid from some cystic follicles collected from live and slaughtered cows, there were no significant differences in sperm penetration, male pronucleus formation and polyspermy rates between fluid from cystic follicles collected from live cows, from slaughtered cows and from control groups, regardless of the P4/E2 ratio. In Experiment 2, the morphology of cumulus-oocyte complexes from cystic follicles varied and the pronucleus formation of oocytes after in vitro fertilization was abnormal. On the other hand, the male pronucleus formation rates were not significantly different between the cystic follicular fluids and control, regardless, of the P4/E2 ratio. The results of this study suggest that many of the bovine follicular fluids from cystic follicles possess the ability to induce cumulus expansion, nuclear maturation and male pronucleus formation following in vitro maturation and fertilization of bovine oocytes. The morphology of the cumulus-oocytes complexes from cystic follicles seems not to relate to the ability of the cystic follicular fluids to induce oocyte maturation, and oocytes from cystic follicles possess the ability to form male pronucleus even though most were abnormal after in vitro fertilization.

The effect of viscosity of semen diluents on motility of bull spermatozoa.

The effect of egg yolk extender on semen viscosity and bull sperm motility of fresh and cooled or deep frozen semen was determined by a computer-assisted system. Viscosity of the extender was determined by flow time. Based on the sperm velocity (velocity of the average path), individual spermatozoon were classified into groups of progressively motile (>==30 microm/sec) and immotile (<10 microm/sec) spermatozoa. The average velocity of progressively motile spermatozoa (VPM), the velocity of linear progressively motile spermatozoa (VLP) and the percentage of linear swimming spermatozoa (LIN) were evaluated. The addition of 10, 20 or 30% egg yolk to Tris buffer (pH 6.5) resulted in a linear decrease of VPM and a decrease in the percentage of progressively motile spermatozoa, but it increased the relative rate of LIN in fresh diluted semen. Increasing the levels of egg yolk in the diluent resulted in higher viscosity. The VLP was significantly higher than the VPM. In refrigerated or frozen semen samples, extender with 30 and 20% egg yolk had a similar effect on the VPM but not on the percentage of progressively motile sperm cells. Freezing of egg yolk (30%) extender to -20 degrees C resulted in a significant increased flow time and higher viscosity. Dilution of semen samples with high viscosity extender decreased the VPM in fresh and chilled semen. Freezing semen of high viscosity extender with glycerol had no apparent effect on the percentage of progressively motile spermatozoa compared with that of non-glycerinated egg yolk extender. The results suggest that different concentrations of egg yolk in the extender can influence the parameters of semen viscosity and sperm motility evaluated by a computer-assisted system.

Oviductal progesterone concentration and its spatial distribution in cyclic and early pregnant cows.

Changes and local distribution of oviductal progesterone (P(4)) concentration during the estrous cycle and early pregnancy in cows were investigated. Intact reproductive tracts were collected from 16 Holstein cows at an abattoir. Samples were classified in to 4 stages (follicular, postovulatory, luteal and early pregnant,< 20 d) based on visual observation of corpus luteum (CL), uterine characteristics and luteal P(4) concentrations. Oviducts were separated from the uterus at the utero-tubal junction and divided into 4 parts: fimbriae, proximal, medial and distal parts. Luteal tissue samples were also collected. Progesterone levels in oviductal and luteal tissues were determined by radioimmunoassay (RIA). Comparatively higher (P < 0.001) P(4) levels were found in stages with a functioning CL ( luteal phase and early pregnancy) than in those with a regressing CL (follicular phase and post ovulation). The oviduct ipsilateral to the CL bearing ovary during the luteal phase and early pregnancy showed higher ( P < 0.001) P(4) concentrations than the contralateral side. Such a difference was not observed during the follicular phase or post ovulation. The ipsilateral oviduct to the functioning CL at early pregnancy showed higher (P <0.05) P(4) levels than at the luteal phase, while no significant difference in luteal P(4) levels between these 2 stages was observed. Neither were any differences in P(4) concentration within the oviduct observed during any phase of the estrous cycle or during early pregnancy. A positive relationship between luteal and oviductal P(4) concentrations was noted. In conclusion, changes in P(4) levels in the oviduct depend on the location and functional stage of the CL. Localized levels of P(4) in the oviduct may be due to local delivery of P(4) from the CL.