Identification of ectopic ovotestis in a dog with XX ovotesticular, SRY-negative, disorder of sexual development.

A 1-year-old, previously spayed phenotypic female Poodle/Soft-coated Wheaten Terrier (Whoodle) cross was presented for a suspected ovarian remnant. Serum luteinizing hormone (LH) concentration was below the detection limit (<1 ng/ml Witness® LH), and serum progesterone concentration was elevated in the chemiluminescence immunoassay (CLIA; 20 ng/ml), consistent with dioestrus and presence of ovarian tissue. Transabdominal ultrasound revealed a retroperitoneal soft tissue structure suspected to be a gonad. On exploratory laparotomy, a gonad was removed from the cranial retroperitoneum, cranial to the right kidney, after ligation of its primary blood supply. Histological examination proved the gonad to be an ovotestis. Subsequent cytogenetics revealed a 78 XX karyotype, thus confirming the diagnosis of ectopic ovotestis in a XX ovotesticular, SRY-negative, disorder of sexual development in a dog.

The effect of a single administration of cephapirin or cloprostenol on the reproductive performance of dairy cows with subclinical endometritis.

This study examined the effect of a single administration of cephapirin iu or cloprostenol im on the reproductive performance of dairy cows with subclinical endometritis. Cows (n = 228) at 20-33 days in milk (DIM) from two commercial dairy farms, determined to be normal for clinical endometritis (based on absence of abnormal uterine discharge on vaginoscopic examination) were enrolled. At enrollment, a thorough reproductive examination was performed, including rectal palpation, ultrasonography (US) and endometrial cytology (EC). The case definition for subclinical endometritis was the presence of >18% neutrophils on EC examination or fluid in uterus (FIU) on US examination. All cows were randomly assigned to receive one of three treatments: 500 mg benzathine cephapirin iu, 500 microg cloprostenol im, or control (no treatment). Reproductive performance was monitored for a minimum of 8 months after treatment. Cows with subclinical endometritis treated with cephapirin or cloprostenol had a significantly increased relative pregnancy rate compared to control [hazard ratios 1.89 (P = 0.01) and 1.70 (P = 0.05), respectively]. In conclusion, a single treatment with cephapirin or cloprostenol at 20-33 DIM significantly improved the reproductive performance of cows with subclinical endometritis.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses.

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease.

Canine alpha-L-iduronidase (iduronidase) deficiency is a model of the human lysosomal storage disorder mucopolysaccharidosis type I (MPS I). We used this canine model to evaluate the therapeutic potential of hematopoietic stem cell (HSC) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically modified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical improvement. The presence of humoral and cellular immune responses against iduronidase apparently abrogated the therapeutic potential of HSC gene therapy in these experiments. To evaluate HSC gene therapy for canine MPS I in the absence of a confounding immune response, we have now performed in utero adoptive transfer of iduronidase-transduced MPS I marrow cells into preimmune fetal pups. In three separate experiments, 17 midgestation fetal pups were injected with 0.5-1.5 x 10(7) normal or MPS I allogeneic long-term marrow culture (LTMC) cells transduced with neo(r)- or iduronidase-containing retroviral vectors. Nine normal and three MPS I pups survived the neonatal period and demonstrated engraftment of provirally marked progenitors at levels of up to 12% for up to 12 months. However, the proportion of provirally marked circulating leukocytes was approximately 1%. Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS I dog. Humoral immune responses to iduronidase were not detected in neonates, even after "boosting" with autologous iduronidase-transduced LTMC cells. All MPS I dogs died at 8-11 months of age from complications of MPS I disease with no evidence of amelioration of MPS I disease. Our results suggest that iduronidase-transduced primitive hematopoietic progenitors can engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to iduronidase in MPS I dogs. However, the therapeutic potential of HSC gene transfer in this model of iduronidase deficiency appears to be limited by poor maintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes.

Further development of a transcervical technique for artificial insemination in sheep using previously frozen semen.

A transcervical technique (the Guelph System for transcervical AI) was used to inseminate 2060 ewes on 65 farms (average 31 ewes, range 5 to 107) in Ontario, Canada, from October 1990 to September 1992, using previously frozen semen. Estrus was synchronized using progestagen pessaries and PMSG with median inseminations done at 54 h from pessary removal. Maiden ewes were not included. Only ewes in which the cervix could be penetrated were inseminated with 150 million spermatozoa per insemination. A total of 1809 were penetrated and inseminated (penetration rate 87.8%). Success of penetration increased from 76.3% in the first 500 ewes to 97.9% in the last 500 (P=0.01). Cervical penetration was more successful in ewes in the accelerated lambing program (92.3%, average 3.1 mo since the previous lambing) than those in the annual lambing program (82.4%, average 7.0 mo since the previous lambing; P=0.06). The lambing rate for ewes bred during the combined traditional breeding seasons (Fall of 1990, 1991, 1992) was 50.7% compared to 24.4% for ewes bred at other periods (P=0.00001). The average time required for handling and insemination decreased from 8.62 min in the first 500 ewes to 3.62 min in the last 500 ewes. The Guelph System for Transcervical AI was found to be successful for cervical penetration in most ewes. Penetration success was affected by period since the last lambing and by inseminator experience. The lambing rate was higher for ewes bred during the traditional Fall breeding seasons than during other times of the year.

Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows.

The objectives of the study were to validate the use of endometrial cytology (EC) and ultrasonography (US) to diagnose subclinical endometritis in clinically normal postpartum dairy cows, and to measure the impact of subclinical endometritis on reproductive performance. Holstein cows from two dairy farms were examined at Visit 1 (V1) at 20-33 days in milk (DIM), and clinically normal cows (n = 228), based on the absence of abnormal discharge on external inspection and vaginoscopy, were selected. The reproductive tract of selected cows was evaluated by transrectal palpation, US and EC. All cows in the study were re-examined at Visit 2 (V2) at 34-47 DIM (2 weeks after V1) and were subsequently followed for a minimum of 8 months (until pregnant or culled). Survival analysis was used to derive a case definition of subclinical endometritis, based on factors associated with decreased relative pregnancy rate. Positive EC at V1 (>18% polymorphonuclear leukocytes; PMN) or fluid in uterus at V1 (FIU1) were associated with a significant reduction in the relative pregnancy rate and identified cows with subclinical endometritis. Similarly, a positive EC (>10% PMN) at V2 or fluid in the uterus at V2 (FIU2), identified cows with subclinical endometritis. Cows with subclinical endometritis at V1 and at V2 had a relative pregnancy rate of 41 and 51% (hazard ratio for pregnancy of 0.59 and 0.49), respectively, compared to cows without subclinical endometritis. Given EC or US findings, no diagnostic criteria based on transrectal palpation of the uterus had predictive value for risk of pregnancy. In conclusion, subclinical endometritis, diagnosed by EC or US, was associated with reduced relative pregnancy rate.

Hormonal control of estrous cyclicity and attempted superovulation in wood bison (Bison bison athabascae).

The wood bison (Bison bison athabascae) is a threatened Canadian species that has faced extinction twice in the last 100 yr. Development of assisted reproductive technologies could help ensure the long-term propagation and genetic management of this species. The objectives of this study were to refine estrus synchronization techniques and evaluate superovulatory responses after FSH or eCG administration. In Experiment 1, females were fitted with Syncro-mate B (SMB) implants for 9 d and received an injection of either estradiol valerate (E2V; n = 9) or cloprostenol (PGF; n = 9) at implant insertion (Day-9). In Experiment 2, estrus was synchronized with SMB implants and a PGF injection of Day-9, and superovulation was attempted on Day-2 with either 2500 IU eCG (n = 5) or 400 mg Folltropin-V (n = 5). In each experiment, biosin were examined daily for estrual behavior. Ultrasonography was used during the luteal phase to detect ovulation and assess ovarian status; feces were analyzed by ELISA for immunoreactive progestogens (P) to study ovarian endocrine responses. In Experiment 1, a closer synchrony of estrus was observed between Days 2 to 4 among the PGF-treated (77.8%) than the E2V-treated (66.7%) females. Corpora lutea (CL) were detected in 55% of E2V- and PGF-treated females. In Experiment 2, neither treatment successfully induced superovulation, with only a single female per treatment producing > or = 1 CL. In both experiments, progestogen profiles were similar for each treatment (P < 0.05).

Chlamydia psittaci infection and associated infertility in sheep.

Nineteen ewes were injected subcutaneously with the agent of enzootic ovine abortion, Chlamydia psittaci serovar 1, at 50 days gestation. Placental and fetal tissues were examined at 15 days postinfection and thereafter at ten day intervals. Placental infection was detected at 15 days postinfection. Only postinoculation sera collected from postinfected ewes contained antibodies reactive to C. psittaci. Five (26%) chlamydial infected ewes experienced inapparent fetal loss before day 105 of gestation. This finding is significant since C. psittaci infection in sheep is commonly associated with abortion and not infertility.

Correlation of periovulatory serum and fecal progestins in the domestic dog.

This study was initiated to determine the relationship between canine ovarian steroids detected in serum and feces during the periovulatory interval in domestic dogs, and to examine the feasibility of a non-invasive method to estimate the time of ovulation in canid species. When bitches (n = 14) were observed to enter proestrus (based on vulvar enlargement or serosanguineous vaginal discharge), paired daily serum and fecal samples were collected for a 15- to 20-day period and stored at -20 degrees C. After extraction, progestin concentrations in both substrates were measured using an established enzyme immunoassay procedure. All samples were aligned to Day 0, the first day in which fecal progestins reached a sustained rise above 100 ng/g feces. Mean fecal progestin concentrations increased in parallel with mean serum progesterone values (r = 0.78), rising from 44.6+/-2.6 ng/g feces to 409.6+/-90.9 ng/g feces, and 5.4+/-0.9 nmol/L to 81.2+/-18.5 nmol/L, on Day -5 and Day 5, respectively. Individual fecal progestin concentrations varied markedly, but plotted against serum progesterone concentrations demonstrated correlation coefficients ranging from 0.41 to 0.97 (P<0.05). These results demonstrate that sequential changes in domestic dog serum ovarian steroid concentrations are paralleled in the feces, and that it is feasible to non-invasively monitor individual progestin changes in the periovulatory interval using fecal hormone analysis.

Varices with thrombosis in the cervix and uterus of a mare.

Cervical and uterine varices with thrombosis were observed at the necropsy of a virgin 16-year-old Peruvian Paso that had previous episodes of hemorrhage from the uterus. Practitioners and pathologists should be alert to the possibility of ruptured varices in mares with hemorrhage into the uterus or from the vulva.

A comparison of the adrenal cortical response to ACTH stimulation in Angora and non-Angora goats.

The adrenal cortex is believed to be implicated in the high incidence of abortion in the Angora goat. Stimulation testing with adrenocorticotrophic hormone (ACTH) was used to assess the adrenal cortical function in 5 Angora does from herds with a history of abortion and 5 non-Angora does. An acute test involving a single intramuscular (i.m.) injection of 0.25 mg of synthetic ACTH was given during anoestrus, at mid-oestrus, on day 90 and on day 120 of gestation. Blood samples were collected from the jugular vein at 30 min intervals for 1 h before and 5 h after injection. Cortisol concentrations rose within 30 min and returned to baseline values within 3.5 h. Cortisol production was lower (p < 0.01) in the pregnant state compared to the non-pregnant state in both groups. Production of cortisol was consistently lower (p < 0.05) in the Angora does compared to the non-Angora does during anoestrus and pregnancy and marginally so at mid-oestrus. A chronic stimulation test involving once daily injections of 0.5 mg of a depot form of ACTH i.m. for 7 days commencing on day 90 of pregnancy was also conducted. Cortisol concentrations rose to reach a peak on the third day of treatment in both groups. The values then declined in the Angora does despite continued ACTH treatment, while those for the non-Angora does exhibited a second peak. During and following this treatment, two non-Angora does delivered live kids (day 95, day 120). Out of 7 Angora pregnancies, one Angora doe aborted two dead fetuses at day 116. No significant difference in the cortisol response in the acute test was detected between the animals that aborted and their respective cohorts, but the two non-Angora does that aborted had significantly lower cortisol concentrations during depot ACTH administration. Progesterone and oestradiol levels did not differ between Angora and non-Angora animals during pregnancy or on the test days. The results suggest that the steroidogenic response of the adrenal cortex to ACTH stimulation is significantly less in Angora does with a history of abortion than it is in non-Angora does and support the view that the Angora goat would make a more limited adrenal cortical response to a stressful occurrence during pregnancy.

The interbreeding of sheep and goats.

To determine the outcome of interbreeding sheep and goats, ewes and does were bred to rams and bucks, and their embryos recovered. Pregnancy was monitored in 2 does bred to a ram. Fertilization rates in ram X does, buck X does, ram X ewes, and buck X ewes were 72%, 96%, 90%, and 0%, respectively. Ram X doe fetuses died at 5 to 10 weeks.

Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro.

Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ± 1 capsules separated media in the "donor" chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the "recipient" chamber. Concentrations of CPA, determined by gas chromatography, allowed calculation of the capsule's apparent permeability (P(app)) to those CPAs. Permeation of capsule by 1.5 M EG was significantly more rapid than by 0.87 M GLY, or 0.74 M EG; permeation by both CPAs was significantly slower than by SAL. Accumulation of CPA in the recipient chamber depended more on initial donor chamber concentration, rather than type, of CPA. Accumulation rates for CPAs and SAL were linear only when capsule was present, demonstrating that their permeation through capsule was more complex than simple diffusion. Successful cryopreservation of equine expanded blastocysts has been previously linked to lengths of step-wise exposures to CPAs. Based on the present results, we inferred that alternative CPAs, more capable of permeating the capsule, or alternative methods of ensuring CPA entry into the cells, may also be required.

Abortion and subsequent excretion of chlamydiae from the reproductive tract of sheep during estrus.

Chlamydia psittaci serovar 1 infection in pregnant sheep typically causes abortion or the birth of weak lambs. Eight sheep that experienced chlamydia-induced abortion during their first pregnancy were successfully rebred yearly for the past 2 years. Chlamydia-specific lipopolysaccharide was detectable for approximately 3 weeks in vaginal swabs taken from the experimentally infected sheep following abortion. There was no evidence of chlamydiae in vaginal, placental, or neonatal samples obtained immediately after each subsequent successful pregnancy. Sera collected from the experimentally infected sheep had persistent, high antibody levels to C. psittaci, suggesting continued exposure of the immune system to the organism. Examination of vaginal specimens obtained during various stages of the estrus cycle revealed detectable levels of chlamydiae only when the animal was near ovulation. Chlamydiae were not detected in swabs from sheep that did not experience abortion. Enhanced chlamydial excretion during the periovulation period of sheep may provide sufficient stimulation of the immune system to account for the persistent antibody response. Furthermore, the association between estrus and chlamydial shedding has important implications for transmission of infection to other ewes during breeding.

Changes in the number of follicles and of oocytes in ovaries of prepubertal, peripubertal and mature bitches.

Ovaries were collected from prepubertal (< 6 months of age, n = 4 ovaries), peripubertal (6 to 10 months of age, n = 12 ovaries) and mature (> 10 months, n = 12 ovaries) bitches after routine ovariohysterectomies and fixed in formalin. Ovaries were bisected, embedded in paraffin wax and 20 serial sections were made at intervals of 10 microns. Sections were stained with haematoxylin and eosin to examine follicles and oocytes in a cross-section of cortex of known size. Counts were made on all sections, resulting in examination of the entire cortical area present in the sections. Oocytes were counted and classified as nucleate or anucleate. Follicles were counted and classified as large (> 100 microns in diameter) or small (< 100 microns in diameter), containing one oocyte (monovular), more than one oocyte (polyovular) or no oocytes (anovular). It was concluded that at first oestrus there was an increase (P < 0.05) per section in number of total oocytes and small monovular follicles and a significant increase (P < 0.05) in the number of nucleated oocytes in monovular follicles, suggesting that oogenesis or folliculogenesis is still occurring at this age. At pre- and peripubertal ages polyovular follicles were found which persist into maturity. There was no difference in numbers of anovular follicles and total number of polyovular follicles among different age groups.

Magnesium oxide induced metabolic alkalosis in cattle.

A study was designed to compare the metabolic alkalosis produced in cattle from the use of an antacid (magnesium oxide) and a saline cathartic (magnesium sulphate). Six, mature, normal cattle were treated orally with a magnesium oxide (MgO) product and one week later given a comparable cathartic dose of magnesium sulphate (MgSO(4)). The mean percent dry matter content of the cattle feces changed significantly (P<0.001) following administration of both MgO (15.6-8.1) and MgSO(4) (17.0-8.7) but there was no significant difference between treatments. The mean rumen pH values changed significantly (P<0.001) following administration of both MgO (7.-8.7) and MgSO(4) (7.3-8.3) but there was no significant difference between treatments. However, use of the MgO product caused a more severe (P<0.001) metabolic alkalosis as determined by base excess values. The base excess values remained elevated for 24 hours in the MgO treated group compared to only 12 hours after MgSO(4) administration. Following MgO administration, mean hydrogen ion concentration (pH), bicarbonate ion concentration ([HCO(3)-]) and base excess were 7.44, 33.3 mmol/L and +8.0 respectively compared to 7.38, 27 mmol/L and +3.0 after MgSO(4). Since the oral use of MgO in normal cattle causes a greater and more prolonged metabolic alkalosis compared to MgSO(4), MgO is contraindicated as a cathartic in normal cattle or in cattle with abomasal abnormalities characterized by pyloric obstruction and metabolic alkalosis.

Concurrent lymphangioma, immune-mediated thrombocytopenia, and von Willebrand's disease in a dog.

Lymphangioma, immune-mediated thrombocytopenia (IMT), and von Willebrand's disease (vWD) were diagnosed by histology, hematology, and a coagulation profile in a 14-month-old, female dachshund. Clinical and laboratory findings included ecchymotic inguinolabial swelling, thrombocytopenia, positive platelet factor-3 assay, prolonged buccal mucosal bleeding time, and subnormal von Willebrand factor antigen concentration and factor VIII activity. The IMT resolved with immunosuppressive glucocorticoid therapy. Histologic examination identified lymphangioma which was too extensive for surgical excision. The history and the clinical and laboratory findings were consistent with congenital vWD, although acquired vWD secondary to lymphangioma could not be ruled out.

Effects of cooling, freezing and glycerol on penetration of oocytes by spermatozoa in dogs.

A homologous zona penetration assay was used to evaluate dog spermatozoa after selected treatments and associated gamete interaction with sperm characteristics. Canine semen, diluted in egg-yolk extender, was treated to compare samples quickly cooled to 0 degree C (after 0.5 and 3 h), slowly cooled to 0 degree C after 3 h or held for 3 h at room temperature. In a second study, slowly cooled spermatozoa with or without the addition of 4% glycerol were studied. After each treatment, spermatozoa were incubated with canine oocytes for 18 h, stained with Hoechst and examined for bound sperm heads. After 3 h, cells that had been cooled quickly demonstrated lower numbers of spermatozoa per ovum than fresh or slowly cooled treatments (2.4 +/- 0.6 versus 5.2 +/- 1.1 and 3.6 +/- 0.9 sperm per oocyte, respectively) and coincided with a reduction in acrosomal integrity and motility (58.6% fast cool 3 h versus 83.1% fresh). Addition of glycerol effected no change in acrosomal status or motility, but resulted in a decline in spermatozoa bound per oocyte to 1.5 +/- 0.3. While freezing caused a further decline in motility, acrosomal status and oocyte penetration, there was no difference between freezing with and without seeding (0.3 +/- 0.08 versus 0.2 +/- 0.04 spermatozoa per ovum). Evaluation of sperm penetration of homologous oocytes provides new insights into the effects of cooling, seeding and glycerol on canine spermatozoa.

Failure to maintain interspecific pregnancy after transfer of Dall's sheep embryos to domestic ewes.

Over a 3-year period, 32 Dall's sheep (Ovis dalli dalli) embryos were transferred into 24 domestic sheep (O. aries) recipients and 4 were transferred into 2 Dall's sheep recipients. In the first year, none of the 10 O. aries recipients was diagnosed pregnant. In the following 2 years, 9 (37%) of the domestic sheep recipients were pregnant on Day 18, 8 (33%) on Day 40, 6 (25%) on Day 90 and 4 (16%) on Day 120; 1 aborted at Day 125 and another at Day 145. Pregnancies were established only in ewes that had previously been recipients of Dall's sheep embryos. The 2 remaining pregnant sheep were treated with progesterone from Day 125 until the fetuses were determined to be dead at Day 145. Both of the Dall's sheep recipients (Year 2) established pregnancies; 1 live Dall's sheep lamb was born 174 days after mating. No differences in serum progesterone, oestrone, prostaglandin F-2 alpha metabolites or cortisol concentrations could be detected during pregnancy between recipients carrying Dall's sheep embryos, recipients receiving progesterone treatment or domestic ewes carrying domestic sheep pregnancies. Six fetuses were necropsied (1 at Day 125 and 5 at Day 145-146): all fetuses were premature and had various degrees of hydranencephaly. No significant differences were found when cotyledon numbers were compared among domestic ewes carrying Dall's sheep lambs. Dall's sheep ewes lambing naturally and domestic ewes lambing naturally. These results demonstrate that the transfer of Dall's sheep embryos to domestic ewes results in the establishment but subsequent loss of pregnancy and that these losses occur throughout gestation.