The objective of the present study was to evaluate changes in equine follicular fluid insulin-like growth factor binding protein (IGFBP) proteolytic activity as well as steroid, IGF, and IGFBP concentrations during follicular development in the mare. Mares (n = 14) were classified as either in the follicular phase (n = 8) or luteal phase (n = 6). Follicles (n = 92) were categorized as small (6 to 15 mm; n = 54), medium (16 to 25 mm; n = 23), or large (> 25 mm; n = 15), and follicular fluid was collected. Estradiol and androstenedione levels in follicular fluid were greater (P < 0.05), and IGFBP-3 concentrations tended to be greater (P < 0.10) in large than in small or medium follicles, whereas IGFBP-2, -4, and -5 levels were less (P < 0.05) in large than in small or medium follicles. Estradiol and androstenedione concentrations were negatively correlated (P < 0.01) with IGFBP-2, -4, and -5 but not IGFBP-3 concentrations. To evaluate proteolysis of IGFBP, follicular fluid was incubated with human 125I-labeled IGFBP-2, -3, and -5 and protein separated by 12% SDS-PAGE. Follicular fluid caused little or no proteolysis of 125I-lableled IGFBP-2 or -3, and the small amount of proteolysis of IGFBP-2 and -3 did not differ (P > 0.10) among follicle classes. However, more 125I-labeled IGFBP-5 was cleaved (P < 0.05) by follicular fluid from large follicles collected during the follicular phase than large follicles during the luteal phase, and small or medium follicles from follicular and luteal phase mares indicating that a protease to IGFBP-5 exists in estrogen-dominant equine follicles. This IGFBP-5 protease was inhibited by kallikrein/serine protease and metalloprotease inhibitors. We conclude that the tendency of estrogen-dominant follicles of mares to have greater levels of IGFBP-3 and lesser levels of IGFBP-2 does not appear to be due to differences in proteolysis, whereas changes in IGFBP-5 levels are likely due to changes in activity of a serine protease or metalloprotease. Changes in IGFBP may alter levels of bioavailable IGF that stimulate steroidogenesis and mitogenesis in developing mare follicles.

Little is known regarding the hormonal regulation of granulosa cell steroidogenesis and the ovarian insulin-like growth factor (IGF) system in the mare. The objectives of this study were to determine, first, if estradiol, insulin, and/or FSH affect steroid production by equine granulosa cells (experiment 1) and, second, if the components of the IGF system are produced by equine granulosa cells in culture as well as whether estradiol, insulin, and/or FSH affects IGF and/or IGF-binding protein (IGFBP) production by equine granulosa cells (experiment 2). Granulosa cells from small (6-15 mm), medium (16-25 mm), and large (25-48 mm) follicles were collected from cyclic mares (n = 14), cultured for 2 days in medium containing 10% fetal calf serum, washed, and then treated for an additional 2 days in serum-free medium with or without added hormones. In experiment 1, large-follicle granulosa cells produced less progesterone and more estradiol than did medium- and/or small-follicle granulosa cells (P < 0.05). Progesterone production was inhibited (P < 0.05) by FSH and insulin in small- and medium- but not in large-follicle granulosa cells; estradiol was without effect. Insulin increased (P < 0.05) estradiol production in small- and medium-follicle granulosa cells but had no effect in large-follicle granulosa cells. In experiment 2, IGF-I production was inhibited (P < 0.05) by insulin across all follicle sizes but was not affected by estradiol or FSH. Granulosa cells of medium and large follicles produced more IGF-II than did granulosa cells of small follicles (P < 0.05). Insulin and FSH inhibited (P < 0.05) IGF-II production by granulosa cells of large and medium but not of small follicles; estradiol was without effect. Only IGFBP-2 and -5 were produced by equine granulosa cells. Production of IGFBP-2 was less (P < 0.10) in granulosa cells of large versus those of small and medium follicles, whereas medium-follicle granulosa cells produced more (P < 0.05) IGFBP-5 than did small- or large-follicle granulosa cells. Averaged across follicle sizes, estradiol increased (P < 0.05) IGFBP-2 production, FSH increased (P < 0.10) IGFBP-2 and -5 production, and insulin was without effect. These results indicate that IGF-I, IGF-II, IGFBP-2, and IGFBP-5 are produced by equine granulosa cells and that insulin, FSH, and estradiol play a role in the regulation of steroidogenesis and the IGF system of equine granulosa cells.

No abstract

Embryo CultureReproduction, Fertility and Development 215 GOT (91.73 ± 3.59 µ/L) and GPT (16.09 ± 3.23 µ/L) activities were recorded in high yielder and the lowest mean values of GOP (72.58 ± 4.79 µ/L) and GPT (13.05 ± 1.99 µ/L) activities were observed in low yielder cows. Based on the findings of this study, it was concluded that although the occurrence of moderate fatty liver was 41.67%, this could be associated with the infertility condition of crossbred dairy cows. It is quite possible that cows showing mild liver damage were in recovery stage after severe or moderate liver damage, as the postpartum period in cross bred cows under study varied from 1.5 to 8 months. Oxygen consumption is a ubiquitous parameter which can provide valuable information about metabolic mechanisms and embryo quality. Recently, we succeeded in non-invasively and quantitatively determining oxygen consumption of individual bovine embryos by the scanning electrochemical microscopy (SECM). The aim of this study was to assess by SECM the oxygen consumption of individual bovine embryos at different developmental stages cultured in serum-free and serum-supplemented media. Bovine oocytes were matured in IVMD101 medium [Research Institute for the Functional Peptides (IFP), Shimojo, Yamagata, Japan] and inseminated in BO-based medium. For serum-free culture, inseminated ooocytes were cultured to the blastocyst stage in IVD101 medium in an atmosphere of a low oxygen condition (5% CO 2 /5% O 2 /90% N 2 ) at 38.5 • C. For serumsupplemented culture, inseminated oocytes were cultured in HPM199 medium (IFP) supplemented with 5% calf serum (HPM199 + CS) in the presence of bovine cumulus/granulosa cells in a humidified atmosphere of 5% CO 2 in air. Oxygen consumption by individual bovine embryos was non-invasively quantified by the SECM measuring system. Some embryos were prepared for transmission electron microscopy. The oxygen consumption rates are presented in the table. Oxygen consumption rates (F) of the single embryos were low from 2-cell to 8-cell stages (0.45-0.52 × 10 −14 mol s −1 ). In serum-free culture, an increase in oxygen consumption rate was found at the morula (1.03 × 10 −14 mol s −1 ) stage, and blastocysts showed an even higher oxygen consumption rate (1.86 × 10 −14 mol s −1 ). On the other hand, the oxygen consumption of morulae and blastocysts produced in serum-supplemented medium was lower than that of embryos cultured in serum-free medium. Electron microscopic study demonstrated that many of the mitochondria of morulae and blastocycts cultured in HPM199 + CS medium were an immature form, indicating a correlation between respiration activity and development of mitochondria. These results suggest that the culture conditions affect the respiration activity of bovine embryos. The SECM procedures may have a wide application for judging embryo quality and culture conditions for embryos. Embryo Culture RESPIRATION ACTIVITY OF BOVINE EMBRYOS CULTURED IN SERUM-FREE AND SERUM-CONTAINING MEDIA THE EFFECT OF ALTERED ENERGY SUBSTRATE CON...

The objective of this study was to analyze factors affecting the pregnancy rates after transfer of IVF-derived Japanese Black embryos. Holstein cows and heifers (n = 7250) were selected as recipients, and embryo transfers were performed for 3 yr (between 1998 and 2000). The IVM-IVF procedure was performed according to a method previously described (Hamano S and Kuwayama M 1993 Theriogenology 39, 703-712). IVF-derived embryos that developed into expanded blastocysts (grade 1, manual of IETS) after 7 to 8 days (insemination = Day 0) were used for this study. Some of these embryos were frozen in TCM-199 supplemented with 1.4 M glycerol, 20% calf serum, and 0.25 M sucrose. The embryos were seeded at −6 • C, held at −6 • C for 10 min, and then cooled to −25 • C at a rate of 0.33 • C min −1 . Frozen embryos were thawed in a 30 to 35 • C water bath after 10 s of air thawing. Fresh (n = 3952) or frozen-thawed (n = 3298) embryos were nonsurgically transferred to recipients on Days 6 to 9 of the estrous cycle. Data collected at the time of embryo transfer included recipient parity (cow or heifer), whether recipient estrus was natural or synchronized with PGF 2α , cloprostenol or CIDR, methods of estrous confirmation (showing standing heat, rectal palpation of ovary without standing heat, or showing only mucous vulvular discharge), number of examinations of the CL by palpation per rectum (twice on the day before embryo transfer and the day of embryo transfer, or once on the day of embryo transfer), type of embryos (fresh or frozen), and day of the estrous cycle at the time of embryo transfer. CATMOD procedures of SAS were used to determine the factors affecting the pregnancy rate. Overall pregnancy rates were 37.3% (n = 2704). Whether recipient estrus was natural or synchronized and the type of embryos did not influence the pregnancy rates. Heifers had significantly higher pregnancy rates than cows (44.0% v. 33.0%, respectively, P < 0.05). Pregnancy rates among the subset of heifers and cows showing standing heat were significantly higher than those showing only mucous vulvular discharge (39.5% v. 33.5%, respectively, P < 0.05). Examining the CL twive had a significantly higher pregnancy rate than did a single examination of the CL (41.1% v. 35.6%, respectively, P < 0.05). Pregnancy rate on Day 8 (38.4%, 1358/3533) of the estrous cycle at the time of embryo transfer was significantly higher than on Days 6 (27.7%, 23/83) and 7 (36.2%, 1235/3408) (P < 0.05), and the pregnancy rate on Day 6 of the estrous cycle at the time of embryo transfer tended to be lower than on Day 9 (38.9%, 88/226) (P < 0.08). These results demonstrate that confirming standing heat, performing CL examination twice before embryo transfer, freezing high quality embryos, and performing embryo transfers on Day 8 resulted in an improved pregnancy rate for the transfer of IVF-derived embryos. Commercial bovine IVP is being extended into new markets and regions worldwide. This study presents pregnancy results from 457 OPU (n = 2860 oocytes) sessions performe...