Meiotic studies in infertile domestic pig-babirusa hybrids.

Mating of a babirusa (Babyrousa babyrussa) boar and a domestic sow (Sus scrofa) resulted in the birth of 5 live domestic pig-babirusa hybrid piglets. Chromosome analysis of one of the surviving males confirmed that they were domestic pig-babirusa hybrids by revealing the presence of a complete haploid set of 19 porcine chromosomes as well as a complete haploid set of 19 babirusa chromosomes in the karyotype. None of the surviving piglets, two males and one female, had shown signs of sexual maturity at age 27 months. Histological examination of gonadal biopsies from the 2 males revealed that both were azoospermatic. Immunostaining revealed SCP3-positive axial elements in the nuclei of primary spermatocytes, indicating that they were progressing through leptotene and zygotene of meiotic prophase. However, the presence of multiple short stretches of axial elements in pachytene nuclei indicated that this phase was blocked, probably due to aberrant chromosome pairing. Histological examination of the ovaries revealed follicular structures, but oocytes within them were generally degenerated. We conclude that both male and female pig-babirusa hybrids were infertile, most likely due to germ cell death resulting from abnormalities of chromosome pairing during meiotic prophase.

Jugular venous pooling during lowering of the head affects blood pressure of the anesthetized giraffe.

How blood flow and pressure to the giraffe's brain are regulated when drinking remains debated. We measured simultaneous blood flow, pressure, and cross-sectional area in the carotid artery and jugular vein of five anesthetized and spontaneously breathing giraffes. The giraffes were suspended in the upright position so that we could lower the head. In the upright position, mean arterial pressure (MAP) was 193 +/- 11 mmHg (mean +/- SE), carotid flow was 0.7 +/- 0.2 l/min, and carotid cross-sectional area was 0.85 +/- 0.04 cm(2). Central venous pressure (CVP) was 4 +/- 2 mmHg, jugular flow was 0.7 +/- 0.2 l/min, and jugular cross-sectional area was 0.14 +/- 0.04 cm(2) (n = 4). Carotid arterial and jugular venous pressures at head level were 118 +/- 9 and -7 +/- 4 mmHg, respectively. When the head was lowered, MAP decreased to 131 +/- 13 mmHg, while carotid cross-sectional area and flow remained unchanged. Cardiac output was reduced by 30%, CVP decreased to -1 +/- 2 mmHg (P < 0.01), and jugular flow ceased as the jugular cross-sectional area increased to 3.2 +/- 0.6 cm(2) (P < 0.01), corresponding to accumulation of approximately 1.2 l of blood in the veins. When the head was raised, the jugular veins collapsed and blood was returned to the central circulation, and CVP and cardiac output were restored. The results demonstrate that in the upright-positioned, anesthetized giraffe cerebral blood flow is governed by arterial pressure without support of a siphon mechanism and that when the head is lowered, blood accumulates in the vein, affecting MAP.

Ultrastructure and electrolyte transport of the epithelium of coprodeum, colon and the proctodeal diverticulum of Rhea americana.

The structure and function of the lower intestinal tract of Rhea americana were characterized to evaluate the evolutionary relationship to other struthioniform and avian species. In 5 rheas the gross anatomy and the light and transmission electron microscopy were studied in parallel to in vitro electrophysiological measurements of ion transport. The mucosa in the colon was amplified with villi, often branched, and in the coprodeum with folds. In both tissues the epithelium was a monolayer composed of columnar absorptive cells, goblet cells and mitochondria-rich cells. Colon and coprodeum appeared to produce large amounts of mucus. The proctodeal diverticulum was rich in lymphoid tissue arranged into lobuli bursales, and it was concluded that this structure is a modified bursa of Fabricius. The sparse interlobular epithelium of the diverticulum resembled that of colon and coprodeum. Baseline short circuit currents (I(SC)) averaged 114.5+/-13.8 microA/cm(2) in colon, 193.1+/-30.3 microA/cm(2) in coprodeum and 60.4+/-9.1 microA/cm(2) in the diverticulum. Amiloride sensitive Na+-transport amounted to 31, 88 and 38% of the baseline I(SC) in these three tissues, respectively. In all tissues, there was also a modest, theophylline activated chloride secretion response, and ouabain, the Na+/K+-ATPase inhibitor, abolished most of the I(SC). The transepithelial resistance (TER) of the diverticulum was much higher than the other tissues. Upon dissection, urate from ureteral urine was observed in the lower third of the colon and to a lesser extent in the proctodeal diverticulum, indicating retrograde peristalsis of the urine. Thus, unlike the ostrich, there is no sphincter separating colon and coprodeum. On the other hand, a thick mucus layer was seen overlying the mucosa in both colon and coprodeum, as in the ostrich. This may help to prevent osmotic water loss, despite the presence of hyperosmotic urine (up to 800 mOsm) in the lower intestine. Both morphological and electrophysiological data from the rhea support the hypothesis that the rhea lower intestine contributes to post-renal modification of ureteral urine and to the regulation of osmotic balance, as also seen in domestic fowl and other avian species. The proctodeal diverticulum functions mainly as an immune organ, with only limited transport capability.

Long-term treatment with carprofen of 805 dogs with osteoarthritis.

The pain-relieving effect of carprofen and tolerance to the drug were investigated in 805 dogs that were lame as a result of osteoarthritis. The dogs were of different breeds, ages and bodyweights and of both sexes, and were selected from 51 veterinary clinics. Each dog was treated orally by its owner with 4 mg/kg carprofen for 84 consecutive days. Twenty-four dogs were removed from the study because of side effects, and 55 left the study for reasons unrelated to the treatment. The condition of the dogs and the benefit of the treatment were evaluated by the veterinary surgeons and the owners after 14 days, and at the end of the period of treatment, when 194 of the dogs (26.7 per cent) were no longer lame, and 357 (49.2 per cent) had improved. The period for which the dogs had been lame before entering the study significantly (P<0.01) affected the results and the rate of improvement. Too much exercise during the 84 days of treatment caused some dogs to relapse.

Persistent BVDV infection in mousedeer infects calves. Do we know the reservoirs for BVDV?

Bovine virus diarrhoea virus (BVDV)-1f was isolated from a Lesser Malayan Mousedeer in Copenhagen Zoo during a routine screening. Analysis of animals related to the Copenhagen mousedeer revealed that its mother and all siblings were virus positive, a pattern also seen for persistently infected (PI) cattle. BVDV could be transmitted from the PI mousedeer to a calf after indirect contact. The host spectrum for BVDV seems to be even wider than expected; the implications for BVDV control are discussed.

The giraffe kidney tolerates high arterial blood pressure by high renal interstitial pressure and low glomerular filtration rate.

BACKGROUND: The tallest animal on earth, the giraffe (Giraffa camelopardalis) is endowed with a mean arterial blood pressure (MAP) twice that of other mammals. The kidneys reside at heart level and show no sign of hypertension-related damage. We hypothesized that a species-specific evolutionary adaption in the giraffe kidney allows normal for size renal haemodynamics and glomerular filtration rate (GFR) despite a MAP double that of other mammals. METHODS: Fourteen anaesthetized giraffes were instrumented with vascular and bladder catheters to measure glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). Renal interstitial hydrostatic pressure (RIHP) was assessed by inserting a needle into the medullary parenchyma. Doppler ultrasound measurements provided renal artery resistive index (RI). Hormone concentrations as well as biomechanical, structural and histological characteristics of vascular and renal tissues were determined. RESULTS: GFR averaged 342 ± 99 mL min(-1) and ERPF 1252 ± 305 mL min(-1) . RIHP varied between 45 and 140 mmHg. Renal pelvic pressure was 39 ± 2 mmHg and renal venous pressure 32 ± 4 mmHg. A valve-like structure at the junction of the renal and vena cava generated a pressure drop of 12 ± 2 mmHg. RI was 0.27. The renal capsule was durable with a calculated burst pressure of 600 mmHg. Plasma renin and AngII were 2.6 ± 0.5 mIU L(-1) and 9.1 ± 1.5 pg mL(-1) respectively. CONCLUSION: In giraffes, GFR, ERPF and RI appear much lower than expected based on body mass. A strong renal capsule supports a RIHP, which is >10-fold that of other mammals effectively reducing the net filtration pressure and protecting against the high MAP.

Acrosomal status in fresh and frozen/thawed stallion spermatozoa evaluated by scanning electron microscopy.

Semen from stallions with equal fertility at natural services, but yielding semen with either satisfactory or poor fertilizing capability after freezing and thawing, was processed for scanning electron microscopy before and after freezing and thawing. In all fresh semen samples the following three categories of acrosomal defect were noticed: (1) minor fenestrations of the plasma membrane (PM) and outer acrosomal membrane (OAM), (2) complete vesiculation and loss of PM and OAM and (3) lack of a large circular part of PM and OAM. The frequency of these defects ranged from 15% to 27%. All frozen and thawed samples displayed defects of categories 1-3 at similar frequencies as the fresh ones. However, additional defects categorized as: (4) major fenestrations of PM and OAM and (5) complete vesiculation of PM and OAM without loss of the vesicles were noted upon freezing and thawing. The total frequency of defects categorized as 4 and 5 ranged from 8% to 21%, and they seemed to be more frequent in stallions with poor fertility after freezing and thawing although the difference was not significant. Moreover, a particular defect categorized as (6) loosening of the whole acrosomal cap was found exclusively in stallions yielding semen with poor freezability.

Impact of mixed strongyle infections in foals after one month on pasture.

Twelve Standardbred foals (age 3-6 months), with little previous exposure to parasites, were allocated to 2 groups and put onto pasture with low (Group L) or high (Group H) levels of larval contamination of large strongyles and cyathostomes. After 4 weeks grazing in September, the foals were housed indoors until necropsy 15 weeks later. Foals in Group H became clinically more affected than those of Group L in that they showed loss of vigour, weight gain depression, intermittent soft faeces and inappetence. One foal of Group H had persistent diarrhoea and was subjected to euthanasia 12 weeks after housing. Signs of colic were not observed. Faecal egg counts were significantly higher in Group H than in Group L (P<0.05). At necropsy, the mean number of S. vulgaris and cyathostomes was 20 and 18,000, respectively, in Group L, and 167 and 25,000 in Group H. Routine blood chemistry did not specifically reveal presence of S.vulgaris in pre-patency. A transient neutrophilia and eosinophilia, most prominent in Group H, was seen 2-8 weeks after start of exposure and anaemia was observed later in Group H. Serum albumin and albumin/globulin ratio were reduced, particularly in Group H, and a marked hyperbetaglobulinaemia was observed at 16-20 weeks in Group H. In conclusion, heavy infections with strongyles including S. vulgaris may become established in weaned foals after a brief period on pasture. Infections may be expressed clinically as debilitation, inappetence and intermittent diarrhoea without colic, and the need for control is imperative.

Vertical transmission of bovine viral diarrhoea virus (BVDV) in mousedeer (Tragulus javanicus) and spread to domestic cattle.

This study investigates the transmission of bovine viral diarrhoea virus (BVDV) 1f from a persistently infected (PI) lesser Malayan mousedeer to two bovine calves. Different contact routes to two calves were analysed: 1) aerosol contact between two adjacent pens without physical contact; 2) indirect contact by use of common utensils; 3) direct nose-to-nose contact for 30 seconds. One of the calves was infected either by aerosol or indirect contact. The virus sequence in 247 nucleotides in the 5'-UTR was 100% identical in mousedeer and calf. To elucidate the distribution of BVDV within the affected mousedeer family the captive population in a Zoo was analysed. The maternal line of PI animals was maintained, whereas a PI male was able to reproduce and have a non-PI calf. As a consequence of this, six female PI mousedeer were killed; subsequent autopsies did not reveal any lesions. Sequencing mousedeer BVD virus in the E2 region (420 nucleotides) through 4 generations showed only 7 mutations, which were maintained from mother to offspring.

Nucleologenesis and ribonucleic acid synthesis in preimplantation equine embryos.

The nucleolus is believed to be the active site of rRNA synthesis in all eukaryotic cells. In preimplantation embryos, the embryonic genome is apparently more or less silent up to a species-specific developmental stage at which a major burst of transcription occurs. Here we report on nucleologenesis and some ultrastructural aspects of the onset of RNA synthesis in equine embryos during in vivo development. The zygotes and embryos up to blastocyst stages were surgically recovered from normally cycling mares. Mares were induced to ovulate by treatment with 3000 IU hCG and inseminated 20 and 34 h later. At different time intervals postovulation, mares were anesthetized and ova were collected from oviducts removed through a flank incision. The presumptive fertilized ova were incubated for 20 min with [3H]uridine and processed for light microscopy, transmission electron microscopy (TEM), and TEM autoradiography. Ultrastructurally, electron-dense nucleolus precursor bodies were observed in zygotes and 2- and 4-cell embryos. In 6- and 8-cell embryos, reticulated fibrillo-granular nucleoli displaying both fibrillar and granular components were observed. At this stage of development, the first autoradiographic labeling was observed over the dense fibrillar component of the nucleoli as well as over the nucleoplasm in the 8-cell embryos. In the 16-cell embryos and beyond, fully transcriptionally active compact fibrillo-granular nucleoli displaying granular and fibrillar components as well as fibrillar centers were observed, and autoradiographic labeling was detected over the dense fibrillar component of the nucleolus as well as over the nucleoplasm. In conclusion, nucleolar activation, including transcription of presumptive rRNA and heterogeneous nuclear RNA, is initiated during the fourth cell cycle.

Graafian follicles are cooler than neighbouring ovarian tissues and deep rectal temperatures.

In order to establish appropriate culture temperatures for in-vitro maturation of pig ovarian oocytes, large Graafian follicles (7-10 mm diameter) were sensed by infra-red technology during the latter part of a spontaneous oestrous cycle. Temperatures were measured under systemic anaesthesia almost instantaneously upon revealing the ovaries at mid-ventral laparotomy. Temperature differentials were observed within all 16 ovaries sensed in 14 animals. Ovaries were always cooler than deep rectal temperatures (mean rectal temperature was 38.0 +/- 0.4 degrees C; range 37.5-38.6 degrees C) and mature follicles always cooler than ovarian stroma (35.6 +/- 0.3 degrees C versus 37.3 +/- 0.2 degrees C respectively; P < 0.01). Such follicles were frequently 1.5-1.8 degrees C cooler than the adjacent stroma, the mean being 1.7 +/- 0.4 degrees C. Small Graafian follicles (< 5-6 mm diameter) and recent ovulations did not show this differential. The control experiment of excising an ovary, deep freezing it in liquid nitrogen, and then restoring it to the body cavity before further sensing indicated that intra-ovarian temperature gradients depended on the activity of living tissues and/or a functional blood supply. Furthermore, calculation of anticipated rates of cooling for exposed Graafian follicles strongly suggested that artefacts could not have been solely responsible for the observed temperatures. Endothermic reactions within mature follicles were thus brought into focus. It is concluded that follicular temperatures may influence the meiotic progression and cytoplasmic maturation of oocytes and act to regulate enzymatic activity in the biosynthetic pathways for steroid and/or peptide hormones.

Autoradiographic localization of specific binding of meiosis-activating sterol to cumulus-oocyte complexes from marmoset, cow, and mouse.

The sterol, 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS), isolated from human follicular fluid, can induce resumption of meiosis in denuded and cumulus-enclosed mouse oocytes inhibited by hypoxanthine, IBMX, or dibutyric cyclic adenosine monophosphate. In this study the distribution of FF-MAS binding sites in denuded oocytes and in cumulus-oocyte complexes (COCs) was studied using light microscopic (LM) and transmission electron microscopic (TEM) autoradiography in marmoset, cow, and mouse oocytes. Denuded (n = 39) and cumulus-enclosed (n = 28) marmoset, cow, and mouse oocytes were cultured in the presence of [3H]FF-MAS with and without excess of unlabeled FF-MAS, respectively. In denuded oocytes LM autoradiography demonstrated specific binding to the oolemma and zona pellucida and, to some extent, the cytoplasm. In the nucleus, no specific binding of [3H]FF-MAS was demonstrated. In some COCs the labeling was dispersed throughout the zona pellucida, the oolemma, and the cytoplasm as well as the cumulus cells; whereas in others, only the outer part of the cumulus cells were labeled. TEM autoradiograms of denuded cow oocytes (n = 6) demonstrated that specific [3H]FF-MAS binding was closely related to the oolemma and that a low level of [3H]FF-MAS binding to cumulus cell remnants was present. In conclusion, specific binding of FF-MAS is predominant at the oolemma of denuded oocytes, suggesting the existence of a plasma membrane-associated molecule with affinity for FF-MAS (i.e., a putative FF-MAS receptor).

Action of hypoxanthine and meiosis-activating sterol on oocyte maturation in the mouse is strain specific.

Follicular fluid meiosis-activating sterol (FF-MAS) is regarded as an important compound relevant to meiotic resumption in mammalian oocytes. The objective of this study was to investigate the influence of FF-MAS on germinal vesicle breakdown (GVBD) and first polar body (PBI) extrusion with regard to culture conditions, state of the oocyte and mouse strain. Denuded oocytes (DO) and cumulus-enclosed oocytes (CEO) were retrieved from PMSG-primed Quackenbush or C57BL/6J x DBA/2 (C57) mice and cultured for 20 h in alpha-MEM medium under the following conditions: (i) 250 micromol/l dibutyryl cAMP (dbcAMP) +/- EGF, 1 ng/ml or FF-MAS, 20 micromol/l; (ii) 4 mmol/l hypoxanthine (HX) +/- EGF or FF-MAS; (iii) HX + EGF + FF-MAS; and (iv) HX + FF-MAS 5 h priming and subsequent culture with HX + EGF. Oocyte GVBD and PBI emission were recorded and stained with Hoechst 33342. Very limited meiotic inhibition was observed in Quackenbush mice in comparison with C57 mice. FF-MAS promoted maturation in C57 DO and CEO and Quackenbush DO. In Quackenbush DO and CEO and C57 DO a significant increase in atypical PBI extrusion occurred, but not in C57 CEO as well as in EGF-treated Quackenbush CEO primed or co-cultured with FF-MAS. These results support a meiosis resumption function for FF-MAS and suggest that in its presence, the quality of the MII oocytes retrieved appears to be influenced by the strain of the mice, the state of the oocyte and the presence or absence of growth factors in the culture medium.

Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.

Human oocyte maturation in vitro is stimulated by meiosis-activating sterol.

In-vitro studies in mouse oocytes have shown that the C-29 endogenously occurring sterol FF-MAS (follicular fluid meiosis-activating sterol) is a potent inducer of meiotic maturation leading to increased fertilization rates. We have used synthetic FF-MAS to induce meiotic maturation in immature human oocytes aspirated from polycystic ovarian syndrome patients. The patients were asked to give written consent to donate half of their aspirated oocytes to investigate the influence of culture conditions on maturation kinetics. The oocytes were aspirated from follicles 8-12 mm in diameter under ultrasound guidance after initial treatment with a gonadotrophin-releasing hormone agonist and s.c. injections of recombinant FSH for 3 days. The other half of the oocytes remained outside this present study. They were reserved for the patients' benefit and were fertilized with appropriate embryo stages being transferred. Fertilization and transfer were not attempted for the study oocytes. Synthetic sterol FF-MAS was added to the culture media at a concentration of 20 micromol/l and nuclear maturation was compared to a control group of oocytes cultured in media only supplemented with vehicle (TCM-199 supplemented with 0.2% ethanol v/v); thus no additional hormones, growth factors, serum or follicle fluid were added. In 31 cycles, oocytes were randomly allocated to one of seven treatment groups: fixed immediately upon aspiration (0 h group) or after in-vitro maturation culture in the presence or absence of FF-MAS for 22, 30 or 40 h respectively. A total of 81 oocytes were processed for light microscopy. The optimal timing of maturation was observed following 30 h of in-vitro culture, when 67% of FF-MAS-treated oocytes had completed nuclear maturation to the metaphase-II stage compared to 29% in the control group. The maturation time of 30 h appeared significantly superior to both 22 and 40 h, but only in the presence of FF-MAS. Cumulus expansion was most profound in the FF-MAS group after 30 h whereas all oocytes had shed the cumulus investment after 40 h. Our observations indicate that FF-MAS positively influences the absolute frequency and the kinetics of human oocytes undergoing nuclear maturation.

Structural and endocrine aspects of equine oocyte maturation in vivo.

The objectives were to describe the ultrastructure of equine oocytes aspirated from small and preovulatory follicles, and to relate the ultrastructural features to follicle size and follicular fluid steroid concentrations. Mares were examined every second day by transrectal ultrasonography, and follicles measuring > 30 mm were aspirated (in vivo) using a 20-cm-long 12-gauge needle through the flank. Following slaughter, both large and small follicles were aspirated (in vitro) from six mares. The oocytes were isolated under a stereomicroscope and processed for transmission electron microscopy, and the follicular fluid was assayed for progesterone (P4) amd estradiol-17 beta (E2). A total of 29 oocytes (32% recovery rate) were aspirated in vivo, and 15 oocytes were recovered in vitro. According to the stage of nuclear maturation, the oocytes could be divided into the following six categories: 1) the central oocyte nucleus (CON) stage, 2) the peripheral spherical oocyte nucleus (PON-I) stage, 3) the peripheral flattened oocyte nucleus (PON-II) stage, 4) the oocyte nucleus breakdown (ONBD) stage, 5) the metaphase I (M-I) stage, and 6) the metaphase II (M-II) stage. The maturation of the preovulatory follicle was reflected by alterations in the follicular fluid concentrations of steroid hormones. E2 was high in all preovulatory follicles, whereas P4 concentration exhibited a 10-fold increase during follicle maturation, particularly associated with the progression from M-I- to M-II-stage oocytes. The nuclear oocyte maturation included flattening of the spherical oocyte nucleus, followed by increasing undulation of the nuclear envelope, formation of the metaphase plate of the first meiotic division, and, finally, the extrusion of the first polar body and the subsequent formation of the metaphase plate of the second meiotic division. The cytoplasmic oocyte maturation changes comprised breakdown of the intermediate junctions between the cumulus cell projections and the oolemma, enlargement of the perivitelline space, the formation and arrangement of a large number of cortical granules immediately beneath the oolemma, the rearrangement of mitochondria from a predominantly peripheral distribution to a more central or semilunar domain, and the rearrangement of membrane-bound vesicles and lipid droplets from an even distribution to an often semilunar domain, giving the ooplasm a polarized appearance. It is concluded that the final equine oocyte maturation includes a series of well-defined nuclear and cytoplasmic changes that are paralleled by an increase in P4 concentration in the follicular fluid, whereas E2 concentration remains constantly high.

Intracytoplasmic sperm injection of in vitro-matured equine oocytes.

Intracytoplasmic sperm injection (ICSI) was performed on equine oocytes matured in vitro. The oocytes were aspirated from abattoir ovaries and matured in vitro for 36 h at 38 degrees C. ICSI was performed using frozen/thawed stallion semen after swimup in medium containing human serum albumin. Sperm-injected oocytes were either 1) cultured in vitro for 10, 20, or 72 h; 2) transferred to oviducts of pseudopregnant mice; or 3) transferred to a synchronized mare after initial in vitro culture. The transferred ova were recovered after 72 h, and all ova were subsequently fixed, stained, and processed for light and transmission electron microscopy. Single pronucleus formation was observed in 2 out of 12 presumptive zygotes 10 h postinjection, at which time abundant cortical granules were observed in the subplasmalemmal region. Twenty hours postinjection, however, 2 pronuclei were observed in 6 of 12 injected oocytes (fertilization rate 50%), and almost all cortical granules were released. The cleavage rate in vitro was 16% after 72 h in culture, and the most advanced embryo stages obtained were 6- to 8-cell embryos. The cleavage rate in vivo was very low since only 1 of 10 recovered had cleaved to the 2-cell stage. Thus, in conclusion, ICSI fertilization of equine oocytes did result in fertilization, pronucleus formation, and cortical granule release. However, the observed fertilization rate and oocyte activation was not paralleled by substantial cleavage of the zygotes.

Activation of meiotic maturation in rat oocytes after treatment with follicular fluid meiosis-activating sterol in vitro and ex vivo.

Meiosis-activating sterols (MAS) have been found to induce meiotic maturation in mouse oocytes in vitro. In the present study we have extended these observations by investigating the effects of follicular fluid MAS (FF-MAS) on rat oocyte maturation in vitro and ex vivo. Rat oocytes freed from their follicles were cultured with FF-MAS (0 microM, 1 microM, 3 microM, 10 microM, 30 microM) for 22 h in a medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 250 microM). A dose-dependent significant increase in germinal vesicle breakdown (GVB) was observed after adding FF-MAS to the culture medium in both cumulus-enclosed (CEO) and denuded (DO) oocytes. A time course study (0, 3, 8, 14, and 22 h) showed a significant increase in GVB after 14 h when DO and CEO were cultured in the presence of 10 microM FF-MAS + 250 microM IBMX. Furthermore immature rats were primed with eCG (20 IU) and 48 h later perfused ex vivo for 12 h in a recirculating system with either FF-MAS (0 microM, 10 microM, 30 microM, 60 microM), cholesterol (60 microM), or LH (0.2 microg/ml) in the presence of 200 microM IBMX, respectively. In addition, ovarian perfusion was carried out with FF-MAS (30 microM, 60 microM) or 0.2 microg/ml LH in the absence of IBMX. After 12 h, oocytes were freed from the ovaries and checked for GVB. By using the ex vivo perfused rat ovary, we found that FF-MAS, starting at 30 microM, was dose-dependently able to overcome IBMX-induced meiotic arrest leading to a comparable increase in GVB as was observed for LH. Furthermore, it was found that FF-MAS in the absence of IBMX was also able to induce meiotic maturation. Our data are consistent with the notion that the maturation-inducing effects of FF-MAS are mediated by different mechanisms compared to spontaneous maturation.

Experimental transfer of adult Oesophagostomum dentatum from donor to helminth naive recipient pigs: a methodological study.

This study was carried out to compare potential methods of transplanting adult Oesophagostomum dentatum from experimentally infected donor pigs to helminth naive recipient pigs. The following methods were each tested in five pigs: A. Transfer of worms by stomach tube to the gastric ventricle of pigs per os pretreated with 0.5 mg/kg cisapride to increase gastrointestinal peristalsis; B. Transfer by stomach tube to the gastric ventricle of pigs per os pre-treated with cisapride (0.5 mg/kg) and omeprazol 20 mg which blocks hydrochloric acid secretion; C. Surgical transfer of worms to caecum of pigs. Worms for transplantation to pigs were obtained after slaughter of experimentally infected donor pigs and following isolation from the contents of the large intestine, using an agar gel migration technique. A mean of 1054 nematodes were transferred into each recipient pig within 2 hours. Procedures A and B resulted in establishment rates corresponding to only 0.5% and 7.6% of the transferred worms. In contrast, surgical transfer allowed 74.2% of the transplanted worms to be established. In all groups the transplanted worms migrated to the normal predilection site, i.e. the middle part of the large intestine. More female than male worms established in all groups. It was concluded from this study that surgical transfer was the most reliable of the methods tested for experimental establishment of adult O. dentatum in helminth naive pigs.