A missense mutation in the transcription factor Foxo3a causes teratomas and oocyte abnormalities in mice.

An N-ethyl-N-nitrosourea random mutation screen was used to identify recessive modifiers of gene silencing in the mouse using an epigenetically sensitive reporter transgene. One of the mutant lines, MommeR1, was identified as a suppressor of variegation and it showed female-specific age-associated infertility in homozygotes. Linkage analysis identified a region on chromosome 10, containing the Foxo3a gene, previously shown to play a critical role in female gametogenesis. Foxo3a is a transcription factor with roles in cell cycle control, apoptosis, neural and hematopoietic cell differentiation, and DNA repair. Sequencing of the Foxo3a gene in MommeR1 mice revealed a point mutation that causes an amino acid substitution in the highly conserved Forkhead DNA-binding domain. In vitro transcription assays showed that the point mutation causes loss of FOXO3a transactivation activity. Compound heterozygotes made with Foxo3a-null mice (carrying the targeted deletion of exon 2) displayed complementation with respect to both the activation of the reporter transgene and defects in folliculogenesis similar to those seen in MommeR1 homozygotes, supporting the conclusion that this is the causative mutation. Approximately one in six female MommeR1 homozygotes develop teratomas, a phenotype not reported in Foxo3a-null mice. Ovulated oocytes from MommeR1 homozygotes display a number of abnormalities. The MommeR1 mice provide a novel platform to investigate teratocarcinogenesis and link Foxo3a with parthenogenesis and ovarian cancer. The finding of Foxo3a as a modifier of epigenetic reprogramming is discussed.

The effect of parity of the dam on sexual maturation, serum concentrations of metabolic hormones and the response to luteinizing hormone releasing hormone in bull calves.

The objectives of this study were to determine the effect of parity of the dam on age at which a scrotal circumference (SC) ≥ 28 cm was attained and the LH response to Luteinizing Hormone Releasing Hormone (LHRH) in bull calves. We also wanted to confirm, in a large group of bull calves, that the LH response to LHRH could be used to select early maturing bulls. Body weight and SC of the bull calves were measured every other week. At 15, 20 and 25 weeks of age, calves received 4.12 nm/kg body weight of LHRH ischio-rectally and blood samples were taken every 15 min for 4 h. Calves from primiparous and multiparous dams were separated into two sub-groups based on age at which an SC ≥ 28 cm was attained (early or late). An SC ≥ 28 cm was attained earlier in calves born to multiparous as compared with primiparous dams (p < 0.05). At 20 and 25 weeks of age, peak serum LH concentrations (LH-peak) and area under the LH response curve (LH-AUC) in response to LHRH were higher (p < 0.01) in calves born to multiparous as compared with primiparous dams. In calves born to multiparous dams the LH-peak at 15 and 25 weeks of age and the LH-AUC at 15 weeks of age were lower (p < 0.05) in calves that attained an SC ≥ 28 cm early as compared with late. The LHRH-challenge test sensitivity and specificity ranged from 46% to 86%. We concluded that parity of the dam affected age at which SC ≥ 28 cm was attained and the LH response to LHRH in bull calves. Serum LH responses to LHRH at 15 and 25 weeks of age, in calves born to multiparous dams, show some promise for development into a test to select early maturing bull calves.

Public health use of surveillance for West Nile virus in horses: Saskatchewan, 2003-2005.

West Nile virus (WNV) infection in horses was first reported in Canada in 2001 and in the province of Saskatchewan in 2002. This paper outlines the surveillance results of WNV in Saskatchewan horses from 2003 to 2005 and describes the usefulness of its inclusion in an integrated surveillance program in Saskatchewan. The number of human and horse cases was highest in 2003, the epidemic year and then substantially lower in 2004 and 2005. Horses provided additive information about WNV activity in rural areas with low human population, however, this required willingness and active participation by veterinarians and horse owners. Vaccination impedes the future use of horses in WNV surveillance for public health or veterinary purposes; however, for zoonoses where no vaccination is available, domestic animals would be useful components for surveillance. Integration of surveillance data from human and animal health provide the benefit of a more complex epidemiological picture that can be used to improve public health.

Synchronization of follicular wave emergence in the seasonally anestrous ewe: the effects of estradiol with or without medroxyprogesterone acetate.

Fertility is often lower in anestrous compared to cyclic ewes, after conventional estrus synchronization. We hypothesized that synchronization of ovarian follicular waves and ovulation could improve fertility at controlled breeding in anestrous ewes. Estradiol-17beta synchronizes follicular waves in cattle. The objectives of the present experiments were to study the effect of an estradiol injection, with or without a 12-d medroxyprogesterone acetate (MAP) sponge treatment, on synchronization of follicular waves and ovulation in anestrous ewes. Twenty ewes received sesame oil (n=8) or estradiol-17beta (350 microg; n=12). Eleven ewes received MAP sponges for 12d and were treated with oil (n=5) or estradiol-17beta (n=6) 6d before sponge removal. Saline (n=6) or eCG (n=6) was subsequently given to separate groups of ewes at sponge removal in the MAP/estradiol-17beta protocol. Estradiol treatment alone produced a peak in serum FSH concentrations (4.73+/-0.53 vs. 2.36+/-0.39 ng/mL for treatment vs. control; mean+/-S.E.M.) after a short-lived (6 h) suppression. Six of twelve ewes given estradiol missed a follicular wave around the time of estradiol injection. Medroxyprogesterone acetate-treated ewes given estradiol had more prolonged suppression of serum FSH concentrations (6-18 h) and a delay in the induced FSH peak (32.3+/-3.3 vs. 17.5+/-0.5 h). Wave emergence was delayed (5.7+/-0.3 vs. 1.4+/-0.7d from the time of estradiol injection), synchronized, and occurred at a predictable time (5-7 vs. 0-4d) compared to ewes given MAP alone. All ewes given eCG ovulated 3-4d after injection; this predictable time of ovulation may be efficacious for AI and embryo transfer.

A case-control study of factors associated with development of clinical disease due to West Nile virus, Saskatchewan 2003.

REASON FOR PERFORMING STUDY: West Nile virus (WNV) was first diagnosed in Saskatchewan equids in 2002. AWNV epidemic was considered highly likely for 2003, which would provide a unique opportunity to study all aspects of WNV subclinical infection and clinical disease development in a relatively naive population. HYPOTHESIS: There are individual equid attributes and management risk factors associated with development of clinical disease. Specifically, this study could address the question of vaccine efficacy for the prevention of development of clinical disease. METHODS: A case-control study was conducted in the summer of 2003 during a province-wide outbreak of WNV. Between 5 and 10 equids were sampled from each of 23 case premises with clinical disease and 23 control premises with no apparent or confirmed clinical disease. Data were analysed to identify risk factors for the development of clinical disease. RESULTS: The proportion of equids serologically positive for natural exposure to West Nile virus was 64% (193/300). Nonvaccinated equids were 23 times (95%CI limits 3.0, 168.5, P = 0.002) more likely to develop clinical disease than those vaccinated. The estimate of vaccine efficacy in this field study was 96% (95%CI limits 67%, 99%). CONCLUSIONS: The study demonstrated that vaccination was strongly associated with the prevention of clinical disease. POTENTIAL RELEVANCE: Vaccination is an effective, practical method of prevention of clinical disease.

Does injection of prostaglandin F(2alpha) (PGF2alpha) cause ovulation in anestrous Western White Face ewes?

In a previous study in our laboratory, treatment of non-prolific Western White Face (WWF) ewes with PGF(2 alpha) and intravaginal sponges containing medroxyprogesterone acetate (MAP) on approximately Day 8 of a cycle (Day 0 = first ovulation of the interovulatory interval) resulted in ovulations during the subsequent 6 days when MAP sponges were in place. Two experiments were performed on WWF ewes during anestrus to allow us to independently examine if such ovulations were due to the direct effects of PGF(2 alpha) on the ovary or to the effects of a rapid decrease in serum concentrations of progesterone at PGF(2 alpha)-induced luteolysis. Experiment 1: ewes fitted with MAP sponges for 6 days (n = 12) were injected with PGF(2 alpha) (n = 6; 15 mg im), or saline (n = 6) on the day of sponge insertion. Experiment 2: ewes received progesterone-releasing subcutaneous implants (n = 6) or empty implants (n = 5) for 5 days. Six hours prior to implant removal, all ewes received a MAP sponge, which remained in place for 6 days. Ewes from both experiments underwent ovarian ultrasonography and blood sampling once daily for 6 days before and twice daily for 6 days after sponge insertion. Additional blood samples were collected every 4 h during sponge treatment. Experiment 1: 4-6 (67%) PGF(2 alpha)-treated ewes ovulated approximately 1.5 days after PGF(2 alpha) injection; these ovulations were not preceded by estrus or a preovulatory surge release of LH, and resulted in transient corpora hemorrhagica (CH). The growth phase was longer (P < 0.05) and the growth rate slower (P < 0.05) in ovulating versus non-ovulating follicles in PGF(2 alpha)-treated ewes. Experiment 2: in ewes given progesterone implants, serum progesterone concentrations reached a peak (1.7 2 ng/mL; P < 0.001) on the day of implant removal and decreased to basal concentrations (<0.17 ng/mL; P < 0.001) within 24 h of implant removal. No ovulations occurred in either the treated or the control ewes. We concluded that ovulations occurring after PGF(2 alpha) injection, in the presence of a MAP sponge, could be due to a direct effect of PGF(2 alpha) at the ovarian level, rather than a sudden decline in circulating progesterone concentrations.

Effects of a specific endothelin-1A antagonist on exercise-induced pulmonary haemorrhage (EIPH) in thoroughbred horses.

REASONS FOR PERFORMING STUDY: During high intensity exercise, the very high pulmonary artery pressure (Ppa) experienced by Thoroughbred horses is considered a major factor in the aetiology of exercise-induced pulmonary haemorrhage (EIPH). Recently, endothelin-1 (ET-1), a potent vasoconstrictive hormone, has been found to increase Ppa in horses at rest via binding to its ET-1A receptor subtype. In addition, plasma concentrations of ET-1 are increased in horses during and after high intensity exercise. HYPOTHESIS: If ET-1 increases Ppa during exercise in the horse, administration of a specific ET-1A antagonist would decrease Ppa and therefore EIPH. METHODS: Saline (CON) or an ET-1A receptor antagonist, TBC3214 (3 mg/kg bwt i.v.; ANTAG) was administered to horses 1 h prior to maximal incremental exercise on a high-speed treadmill. Gas exchange measurements were made breath-by-breath and blood samples collected during each 1 min stage to determine blood gases, acid-base status and cardiac output. EIPH was determined via bronchoalveolar lavage (BAL) approximately 30 min after exercise. RESULTS: The time to fatigue, gas exchange and cardiovascular responses were not different between groups (P>0.05). Resting and peak Ppa did not differ significantly between treatments. Most importantly, ANTAG did not decrease EIPH. CONCLUSIONS: These results do not support a deterministic role for ET-1 in the increased Ppa and therefore EIPH, during maximal exercise in the equine athlete. POTENTIAL RELEVANCE: Treatment with an ET-1A receptor antagonist does not appear to be a viable therapeutic intervention in the prevention of EIPH.

Exercise-induced pulmonary haemorrhage during submaximal exercise.

REASONS FOR PERFORMING STUDY: Maximally exercising horses achieve mean pulmonary artery pressures (Ppa(mean)) that exceed the minimum threshold (75 mmHg) estimated for pulmonary capillary rupture and exercise-induced pulmonary haemorrhage (EIPH). EIPH is not expected to occur during moderate submaximal exercise (i.e. 40-60% VO2max) since Ppa(mean) remains well below this threshold. HYPOTHESIS: Prolonged submaximal exercise (trotting) would precipitate locomotory respiratory uncoupling and cause EIPH. This would be present as a result of the most negative intrapleural pressures (as estimated by the minimum oesophageal pressure; Poes(min)) occurring simultaneously with the most positive Ppa (Ppa(peak)) to produce estimated maximal pulmonary artery transmural pressures (PATMPmax) that surpass the EIPH threshold. METHODS: Five Thoroughbred horses trotted to fatigue (approximately 25 min) at 5 m/sec on a 10% incline. Ventilation (V(E)), Poes, and Ppa were measured at 5 min intervals, and bronchoalveolar lavage (BAL) red blood cells (RBCs) were quantified 45 min post exercise. RESULTS: BAL revealed an increased EIPH (rest: 2.0 +/- 1 x 10(5), exercise: 17 +/- 10 x 10(5) RBCs/ml BALF; P<0.05), despite the highest Ppamean reaching only mean +/- s.e. 55 +/- 3 mmHg, while V(E), tidal volume and Poes(min) approached 70-80% of the values achieved at maximal running speeds (10% incline: 12-13 m/sec) by these same horses. The resulting PATMPmax was well above the level considered causative of EIPH. CONCLUSIONS: The finding of significant EIPH during submaximal exercise broadens the spectrum of performance horses susceptible to EIPH and supports studies that suggest that extravascular factors are of primary importance in the aetiology of EIPH. POTENTIAL RELEVANCE: Consideration of strategies such as the equine nasal strip for reducing negative extravascular pressures is warranted even for exercise at moderate intensities.

Concerted evolution of mammalian cardiac myosin heavy chain genes.

We have recently determined the complete nucleotide sequences of the cardiac alpha- and beta-myosin heavy chain (MyHC) genes from both human and Syrian hamster. These genomic sequence data were used to study the molecular evolution of the cardiac MyHC genes. Between the alpha- and beta-MyHC genes, multiple gene conversion events were detected by (1) maximum parsimony tree analyses, (2) synonymous substitution analyses, and (3) detection of pairwise identity of intron sequences. Approximately half of the 40 cardiac MyHC exons have undergone concerted evolution through the process of gene conversion with the other half undergoing divergent evolution. Gene conversion occurred more often in exons encoding the alpha-helical myosin rod domain than in the globular head domain, and an apparent directional bias was also observed, with transfer of genetic material occurring more often from beta to alpha.

Structural organization of the human cardiac alpha-myosin heavy chain gene (MYH6).

The human myocardium expresses two cardiac myosin heavy chain (MyHC) isoforms, alpha and beta, that exist in tandem array on chromosome 14q12. We have previously sequenced the entire human cardiac beta-MyHC gene and now report the complete nucleotide sequence of the human cardiac alpha-MyHC, encompassing 26,159 bp as well as the entire 4484-bp 5'-flanking intergenic region. The gene (MYH6) consists of 39 exons, 37 of which contain coding information. The 5'-untranslated region is split into 3 exons, with the third exon containing the AUG translation initiation codon. With the exception of the 13th intron of the human cardiac beta-MyHC, which is not present within the alpha-isogene, all exon/intron boundaries are conserved. Conspicuous sequence motifs contained within the alpha-MyHC gene include four Alu repeats, a single (GT)n element, and a homopurine-homopyrimidine tract containing 23 GAA repeating units followed by 10 GAG repeating units. Comparison of the encoded amino acid sequence with a previously reported human alpha-MyHC cDNA sequence reveals several potential polymorphisms.