Ultrastructural characterization of in vivo-produced ovine morulae and blastocysts.

The ultrastructure of in vivo-produced ovine embryos, at the morula, early blastocyst and late blastocyst stages, was evaluated using transmission electron microscopy. Embryonic cells were characterized by the presence of intact intercellular junctions, numerous mitochondria, smooth endoplasmic reticulum cisternae and light vesicles. Polyribosomes, rough endoplasmic reticulum cisternae, secondary lysosomes, Golgi complexes and lipid droplets were also observed in the cytoplasm. The nucleus was well defined and organized, with an intact envelope rich in nuclear pore complexes, and one or more reticular nucleoli. Microvilli were present in external blastomeres of morulae and became more abundant in trophectoderm cells of early and late blastocysts. Light vesicles seemed to be associated with small cisternae of Golgi and endoplasmic reticulum origin. These cisternae fused and created light vesicles with engulfed heterogeneous cytosolic structures, small cisternae and vesicles. Their labile membrane enabled them to rapidly coalesce into medium-sized vesicles that began to engulf mitochondria and lipid droplets, forming giant vacuoles mostly filled with fat. Incomplete matured secretory vesicles were observed to exocytose into the perivitelline space of morulae, whereas fully matured secretory vesicles appeared only in trophectoderm cells, being exocytosed into the blastocoelic cavity. These observations suggested that these endoplasmic-/Golgi-derived vesicles behave as active autophagic organelles presenting probably a maturation process from compact morulae to blastocyst.

Ultrastructural characterization of fresh and cryopreserved in vivo produced ovine embryos.

Controlled slow freezing and vitrification have been successfully used for ovine embryo cryopreservation. Selection of embryos for transfer is based on stereomicroscopical embryo scoring after thawing, but the subjectivity inherent to this selection step has been demonstrated by ultrastructural studies of controlled slow frozen, in vivo produced ovine morulae and blastocysts. These studies have shown that certain abnormalities remain undetected by stereomicroscopy only. In the present study, using ovine in vivo produced morulae and blastocysts, we have studied the ultrastructural alterations induced by open pulled straw vitrification (OPS) and controlled slow freezing, compared stereomicroscopical embryo scoring with light microscopy evaluation of embryo's semithin sections, and related the ultrastructural cellular damage with the embryo classification by stereomicroscopical embryo scoring of embryos' and semithin section evaluation by light microscopy. The ultrastructural lesions found for OPS-vitrified and controlled slow frozen embryos were similar, independently of embryo stage. A significant higher number of grade 3 embryos was found at stereomicroscopical scoring after controlled slow freezing (P=0.02), and a significant higher number of grade 3 blastocysts was found at semithin sectioning after OPS vitrification (P=0.037). The extension of ultrastructural damage, especially of mitochondria and cytoskeleton, was related to the semithin classification but not to stereomicroscopical scoring at thawing. This suggests that semithin scoring is a useful tool for predicting ultrastructural lesions and new improvements in cryopreservation and thawing methods of ovine embryos are still warranted, including in the case of blastocysts cryopreserved by OPS vitrification.

Effects of ivermectin on reproductive functions in ewes.

In response to producers' concerns about possible detrimental effects of ivermectin on ewes during the breeding season, an evaluation of its effects on endocrinological, physiological, and behavioral measures of reproductive performance was made. Twenty cycling ewes were randomly assigned on d 10 (d 0 = estrus) to receive a single recommended oral dose of 200 micrograms/kg of BW of ivermectin or a control volume of water. Twelve hours after treatment, ewes received a luteolytic injection of 5 mg of PGF2 alpha and were introduced to fertile rams. On d 10 after mating, laparoscopies were performed to assess ovulation rate, and on d 18 conceptuses were surgically recovered. Blood samples were collected during the 92-h interval beginning immediately before ivermectin and control treatments and were assayed for LH. It was determined that 1) interval from PGF2 alpha-induced luteal regression to a) onset of estrus (36.1 +/- 1.4 vs 36.3 +/- 1.4 h) and b) onset of the preovulatory surge of LH (38.2 +/- 2.6 vs 44.2 +/- 2.6 h), 2) magnitude of the surge of LH (275.6 +/- 38.9 vs 199.8 +/- 38.9 ng/mL), 3) duration of the surge of LH (9.4 +/- .4 vs 9.0 +/- .4 h), 4) area under the curve of the surge of LH (54,321 +/- 7,419 vs 38,138 +/- 7,419 arbitrary units), 5) ovulation rate (2.1 +/- .4 vs 2.0 +/- .3 ovulations/ewe), 6) pregnancy rate (8/10 vs 5/10 ewes pregnant), and 7) conceptuses per ewe (1.75 +/- .37 vs 1.60 +/- .33) did not differ (all P > .1) between ivermectin- and control-treated ewes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine and endometrial secretory protein changes associated with uterine receptivity in sheep.

Crossbred ovariectomized ewes were treated with steroid therapies determined previously to be adequate (progesterone-primed) or inadequate (unprimed) for embryonic development in order to determine actual serum concentrations of replaced steroid hormones achieved by such treatments and to identify secreted endometrial proteins that might mediate uterine receptivity. Ewes received estradiol-17 beta on day 0, and on days 1-4, either vehicle (unprimed; N = 16) or progesterone (primed; N = 16) daily. All ewes then received "estrus estradiol" (at 8 hr-intervals), followed by "maintenance progesterone" (at 12 hr-intervals), to mimic endocrine profiles of intact ewes at and following estrus. Jugular blood samples were obtained at 4-hr intervals from 6 ewes/treatment on day 0-15 to determine serum progesterone, estradiol, and PGFM concentrations. Endometrium from two ewes/treatment on days 11-15 was cultured in vitro with [3H]leucine and radiolabeled proteins in media were analyzed electrophoretically. Results demonstrated that 1) treatments generated transient serum estradiol levels slightly greater than those reported in intact animals at estrus, 2) serum progesterone concentrations due to treatments were similar to those reported in the luteal phase of intact ewes, 3) progesterone-priming was specifically associated with a small, sustained (24-36 hr) elevation in serum PGFM, and that 4) priming was not associated with the presence or absence of major, secreted endometrial proteins that might act either as factors required for development or as embryotoxins. These results suggest that positive effects of progesterone-priming on embryo survival are not due to pharmacological doses of exogenously administered hormones, nor are due to changes in secretion of hormonally-regulated, major endometrial proteins.

Active immunization of ewes against prostaglandin F2 alpha to control ovarian function.

The hypothesis that pregnancy success could be improved in early postpartum ewes by prolonging the lifespan of the corpus luteum via active immunization against prostaglandin F2 alpha (PGF2 alpha) was tested. Further experiments in ewes immunized against PGF2 alpha investigated the effects of exogenous PGF2 alpha on the preovulatory follicle and the effects of PGF2 alpha and oestradiol benzoate on corpus luteum function. Four weeks pre-partum, 39 ewes bred to lamb during seasonal anoestrus received either 5 mg PGF2 alpha-ovalbumin conjugate (n = 20; immunized) or ovalbumin (n = 19; control) in Freund's complete adjuvant. Treatments were repeated on day 5 post-partum with reagents emulsified in Freund's incomplete adjuvant. On day 17 post-partum, ewes received 500 iu pregnant mares' serum gonadotrophin (PMSG) and 48 h later 50 micrograms gonadotrophin-releasing hormone (GnRH). Laparoscopy was performed 36 h after GnRH to assess ovarian activity and ewes with recent ovulations were inseminated into the uterus. No immunized ewes had ovulated, but ten had follicles that luteinized and secreted progesterone during the 8 weeks studied. Eighteen of 19 control ewes ovulated and 15 of 18 had increased progesterone concentration for at least 21 days. By day 70 post partum, progesterone had returned to basal values in all control ewes. In a second study, 24 immunized ewes bearing persistent corpora lutea, and for which the interval from the previous parturition was greater than 90 days, received 15 mg PGF2 alpha and 500 iu PMSG followed 48 h later by 50 micrograms GnRH. PGF2 alpha induced corpus luteum regression in all ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Lack of effects of indomethacin on estradiol feedback control of luteinizing hormone in ovariectomized ewes.

We tested the hypothesis that indomethacin, a potent inhibitor of prostaglandin synthesis, would modify estradiol's effects on tonic and surge concentrations of LH in chronically ovariectomized ewes during the anestrous season. Ewes (n = 21) were assigned randomly to one of four treatments: Vehicle+Blank (n = 5); Indomethacin+Blank (n = 6); Vehicle+Estradiol (n = 5); or Indomethacin+Estradiol (n = 5). On d=0 (hr = 0), ewes began to receive i.m. injections of either indomethacin (4 mg/kg body weight) or corn oil every 8 hr for 9 d. Blood samples were collected every 12 min for 6 hr beginning at -6 hr, +18 hr, and on day 8 (relative to initial injections of indomethacin or vehicle) to assess tonic patterns of secretion of LH. At +24 hr, ewes received blank- or estradiol-containing Silastic implants and were bled hourly for 48 hr. On day 9, ewes received 50 micrograms of GnRH i.v. and were bled hourly for 8 hr. Serum samples were assayed for LH. Indomethacin had no effect on the following parameters of LH secretion: 1) mean concentrations (ng/ml; 8.4 +/- .7 vs 8.9 +/- .8; P > .1), 2) pulse frequency/6 hr (4.5 +/- .4 vs 4.1 +/- .4; P > .1) or 3) pulse amplitude (ng/ml; 15.3 +/- 1.1 vs 14.9 +/- 1.2; P > 1). Estradiol elicited a surge of LH which began 18.9 +/- 1.7 hr after implant insertion, reached a mean peak concentration of 95.3 +/- 20.1 ng/ml, and did not differ with respect to indomethacin treatment (P > .1).(ABSTRACT TRUNCATED AT 250 WORDS)

Using work simulation to treat adults with back injuries.

Occupational therapy at the Liberty Mutual Medical Service Center, Boston, Massachusetts, offers a diverse variety of modalities for the treatment of patients with low back pain. Treatment may include the use of a balance monitor, a multiwork station, a pneumatic lifting-lowering device, a computerized upper extremity work simulator, and a truck-driving simulator. The primary objective of occupational therapy in this setting is to provide a supportive environment where patients can practice and improve the execution of the work-related activities they need to perform their jobs while they are learning to live with or control their symptoms.