Anatomic and neuromuscular characterisation of the equine cricothyroid muscle.

REASONS FOR PERFORMING STUDY: As part of investigation into laryngeal stability and reanimation using functional electrical stimulation, the cricothyroid muscle might be utilised to increase laryngeal cross-sectional area in horses with recurrent laryngeal neuropathy. For optimal electrode placement and muscle recruitment, the neuroanatomy and excitability of the equine cricothyroid muscle needs to be defined. OBJECTIVES: To describe the anatomy, innervation and function of the equine cricothyroid muscle and its contribution to laryngeal diameter. METHODS: Seventeen equine larynges were collected at necropsy and 12 were grossly dissected. Seven larynges (five grade 1, two grade 4) were prepared for immunohistochemistry following gross dissection and 5 larynges were prepared for special staining: acetylcholinesterase staining of motor endplates (n = 3) and Sihler's staining (n = 2). Three larynges were stimulated following in situ cadaver dissection and 2 larynges were removed and stimulated ex vivo. RESULTS: Three neuromuscular compartments, each innervated by a primary nerve branch of the external branch of the cranial laryngeal nerve, were identified in all larynges. Stimulation of each neuromuscular compartment resulted in ventral displacement of the thyroid cartilage with respect to the cricoid cartilage, thereby increasing dorsoventral height of the rima glottis. CONCLUSIONS: The equine cricothyroid muscle has 3 distinct neuromuscular compartments with discrete innervation, fibre type distribution and muscle fibre sizes. All neuromuscular compartments tense the vocal cords by increasing dorsoventral height of the rima glottis through ventral displacement of the thyroid cartilage with respect to the cricoid cartilage. POTENTIAL RELEVANCE: Simultaneous functional electrical stimulation of the cricothyroid and dorsal cricoarytenoid muscles may enhance laryngeal cross-sectional area in horses with recurrent laryngeal neuropathy.

Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo.

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α(+), CD4(+), and CD8α(-)CD4(-) DC and the B220(+) plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8(+) T cells at 24hpi. The CD8α(+) DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4(+) DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α(-)CD4(-) DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8(+) DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8(+) T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α(+) DC subset.

Incidence of swallowing during exercise in horses with dorsal displacement of the soft palate.

REASONS FOR PERFORMING STUDY: The relationship between dorsal displacement of the soft palate (DDSP) and swallowing is unclear. OBJECTIVE: To quantify the relationship between DDSP and swallowing in horses at exercise. HYPOTHESES: The frequency of swallowing increases immediately prior to DDSP in horses at exercise. METHODS: Videoendoscopic and upper airway pressure data were collated from horses with a definitive diagnosis of DDSP at exercise. Horses with no upper airway abnormalities were matched by age, breed and sex and used as controls. Sixty-nine horses were identified with a definitive diagnosis of DDSP during the study interval. Airway pressure data were available for 42 horses. RESULTS: The majority of horses displaced at high exercising speeds while accelerating; a smaller number displaced during deceleration after peak speed had been reached. Horses swallowed significantly more frequently in the 1 min immediately preceding DDSP than in the control horses at equivalent speeds. DDSP at exercise results in a significant increase in tracheal expiratory pressure, a significant decrease in pharyngeal expiratory pressure and a significantly less negative pharyngeal inspiratory pressure compared to matched controls and compared to the pressures during the 1 min interval prior to DDSP. There was no significant difference between any measure of airway pressure before or after a swallow when examined at each time interval in the DDSP population. CONCLUSIONS: The frequency of swallowing decreases with increasing speed in normal horses. In contrast, the frequency of swallowing increases immediately prior to onset of DDSP. This is not a result of pharyngeal and tracheal pressure changes. POTENTIAL RELEVANCE: The increased frequency of swallowing observed prior to DDSP may be related to the aetiology of the disease.

Considerations for pacing of the cricoarytenoid dorsalis muscle by neuroprosthesis in horses.

REASONS FOR PERFORMING STUDY: The success rate of prosthetic laryngoplasty is limited and may be associated with significant sequelae. Nerve muscle pedicle transplantation has been attempted but requires a year before function is restored. OBJECTIVE: To determine the optimal parameters for functional electrical stimulation of the recurrent laryngeal nerve in horses. METHODS: An experimental in vivo study was performed on 7 mature horses (2-21 years). A nerve cuff was placed on the distal end of the common trunk of the recurrent laryngeal nerve (RLN). In 6 horses the ipsilateral adductor branch of RLN was also transected. The electrodes were connected to programmable internal stimulator. Stimulation was performed using cathodic phase and then biphasic pulses at 24 Hz with a 0.427 ms pulse duration. Stimulation-response experiments were performed at monthly intervals, from one week following implantation. The study continued until unit failure or the end of project (12 months). Two of the horses were stimulated continuously for 60 min to assess onset of fatigue. RESULTS: Excellent arytenoid cartilage abduction (mean arytenoid angle of 52.7 degrees, range 48.5-56.2 degrees) was obtained in 6 horses (laryngeal grades I or II (n = 3) and III (n = 2). Poor abduction was obtained in grade IV horses (n = 2). Arytenoid abduction was maintained for up to a year in one horse. Technical implant failure resulted in loss of abduction in 6 horses at one week to 11 months post operatively. Mean tissue impedance was 1.06 kOhm (range 0.64-1.67 kOhm) at one week, twice this value at 2 months (mean 2.32, range 1.11-3.75 kOhm) and was stable thereafter. Maximal abduction was achieved at a stimulation range of 0.65-7.2 mA. No electrical leakage was observed. Constant stimulation of the recurrent laryngeal nerve for 60 min led to full abduction without evidence of muscle fatigue. CONCLUSIONS: Functional electrical stimulation of the recurrent laryngeal nerve leading to full arytenoid abduction can be achieved. The minimal stimulation amplitude for maximal abduction angle is slightly higher than those for man and dogs. CLINICAL RELEVANCE: This treatment modality could eventually be applicable to horses with recurrent laryngeal neuropathy.

Successful treatment of persistent dorsal displacement of the soft palate and evaluation of laryngohyoid position in 15 racehorses.

REASONS FOR PERFORMING STUDY: Caudal descent of the larynx has been proposed to be associated with intermittent dorsal displacement of the soft palate (DDSP) as it leads to a loss of the seal between the epiglottis and soft palate but further investigation of this theory is required. OBJECTIVES: To evaluate laryngohyoid position of horses with persistent DDSP in comparison to horses with intermittent DDSP and evaluate the outcome of treatment. HYPOTHESES: Horses with persistent DDSP have a different laryngohyoid position compared to those with intermittent DDSP. Horses with persistent DDSP can be returned successfully to racing with a laryngeal tie-forward procedure. METHODS: Medical records of 15 racehorses presented for persistent DDSP between 2002 and 2007 were reviewed. Age, sex and breed matched horses diagnosed with intermittent DDSP were used as a comparison group. Treatment of all horses was performed by laryngeal tie-forward, 8/15 horses with persistent DDSP had a subsequent laser staphylectomy. Preoperative laryngohyoid position was compared between the 2 groups using a radiographic reference system. Surgical effect on position was assessed by comparing pre- and post operative radiographic measurements. Outcome was assessed by return to racing and comparison of pre- and post operative race earnings ($). RESULTS: Thirteen of 15 horses with persistent DDSP returned to racing. Seven of 15 horses were treated with laryngeal tie-forward alone while 8/15 horses were also treated with a laser staphylectomy. Horses with persistent DDSP had a more caudal larynx (ossification of the thyroid cartilage) (13 mm, P = 0.014), a more caudal (10 mm, P = 0.044) and dorsal (7 mm, P = 0.01) basihyoid bone, and a more dorsal thyrohyoid-thyroid articulation (10 mm, P = 0.002) than horses with intermittent DDSP. CONCLUSIONS AND POTENTIAL RELEVANCE: Racehorses with persistent DDSP can be treated successfully by laryngeal tie-forward or by laryngeal tie-forward followed by laser staphylectomy. Horses with persistent DDSP have a more caudal larynx and more caudal and dorsal basihyoid bone than horses with intermittent DDSP.

Contractile behavior of the forelimb digital flexors during steady-state locomotion in horses (Equus caballus): an initial test of muscle architectural hypotheses about in vivo function.

The forelimb digital flexors of the horse display remarkable diversity in muscle architecture despite each muscle-tendon unit having a similar mechanical advantage across the fetlock joint. We focus on two distinct muscles of the digital flexor system: short compartment deep digital flexor (DDF(sc)) and the superficial digital flexor (SDF). The objectives were to investigate force-length behavior and work performance of these two muscles in vivo during locomotion, and to determine how muscle architecture contributes to in vivo function in this system. We directly recorded muscle force (via tendon strain gauges) and muscle fascicle length (via sonomicrometry crystals) as horses walked (1.7 m s(-1)), trotted (4.1 m s(-1)) and cantered (7.0 m s(-1)) on a motorized treadmill. Over the range of gaits and speeds, DDF(sc) fascicles shortened while producing relatively low force, generating modest positive net work. In contrast, SDF fascicles initially shortened, then lengthened while producing high force, resulting in substantial negative net work. These findings suggest the long fibered, unipennate DDF(sc) supplements mechanical work during running, whereas the short fibered, multipennate SDF is specialized for economical high force and enhanced elastic energy storage. Apparent in vivo functions match well with the distinct architectural features of each muscle.

Superficial digital flexor tendon lesions in racehorses as a sequela to muscle fatigue: a preliminary study.

REASON FOR PERFORMING STUDY: Racing and training related lesions of the forelimb superficial digital flexor tendon are a common career ending injury to racehorses but aetiology and/or predisposing causes of the injury are not completely understood. OBJECTIVES: Although the injury takes place within the tendon, the lesion must be considered within the context of the function of the complete suspensory system of the distal limb, including the associated muscles. METHODS: Both muscle and tendon function were investigated in vivo using implanted strain gauges in 3 Thoroughbred horses walking, trotting and cantering on a motorised treadmill. These data were combined with assessments of muscle architecture and fibre composition to arrive at an overview of the contribution of each muscle-tendon unit during locomotion. RESULTS: The superficial digital flexor muscle has fatigue-resistant and high force production properties that allow its tendon to store and return elastic energy, predominantly at the trot. As running speed increases, deep digital flexor tendon force increases and it stabilises hyperextension of the fetlock, thus reinforcing the superficial digital flexor in limb load support. The deep digital flexor muscle has fast contracting properties that render it susceptible to fatigue. CONCLUSION: Based on these measurements and supporting evidence from the literature, it is proposed that overloading of the superficial digital flexor tendon results from fatigue of the synergistic, faster contracting deep digital flexor muscle. POTENTIAL RELEVANCE: Future research investigating distal limb system function as a whole should help refine clinical diagnostic procedures and exercise training approaches that will lead to more effective prevention and treatment of digital flexor tendon injuries in equine athletes.

Can an external device prevent dorsal displacement of the soft palate during strenuous exercise?

REASONS FOR PERFORMING STUDY: Dorsal displacement of the soft palate (DDSP) is a common condition in racehorses for which various surgical treatments are often performed. In light of recent findings that suggested the position of the larynx may influence the occurrence of DDSP, we investigated whether a noninvasive mean of affecting the position of the larynx could be effective in the management of DDSP. HYPOTHESIS: An external device (laryngohyoid support; LHS) positioning the larynx in a more rostral and dorsal location and preventing caudal displacement of the basihyoid bone would be effective in preventing DDSP during strenuous exercise. METHODS: Ten horses were exercised on a high-speed treadmill under 4 different treatment conditions: control (n = 10); control with external device (n = 10); after bilateral resection of thyrohyoid (TH) muscles (n = 7); and after bilateral resection of TH muscles with external device (n = 7). Two trials were performed randomly for each of the 4 conditions. In Trial 1, videoendoscopic images of the upper airway, pharyngeal and tracheal static pressures, and arterial blood gases were collected. In Trial 2, airflow measurement combined with mask and tracheal static pressure was obtained, and upper airway impedance calculated. The trials allowed calculation of airway impedance and respiratory frequency, and assessment of ventilation using arterial PO2 and PCO2. RESULTS: Under control conditions, none of the 10 horses developed DDSP. There was no statistically significant effect from the LHS on airway impedance or respiratory frequency, nor on arterial PO2 and PCO2. Seven of the 10 horses developed DDSP during exercise after resection of the TH muscles. None of these 7 horses continued to experience DDSP during exercise with the external device. In the latter group and condition, the LHS significantly improved inspiratory and expiratory flow and impedance. CONCLUSIONS: The LHS helped prevent experimentally induced DDSP at exercise, probably by statically positioning the larynx in a more rostral and dorsal position. POTENTIAL RELEVANCE: Field studies are required to investigate whether the LHS can successfully prevent DDSP in horses with naturally occurring disease.

Investigations into the role of the thyrohyoid muscles in the pathogenesis of dorsal displacement of the soft palate in horses.

REASONS FOR PERFORMING STUDY: Contributes to the understanding of the pathogenesis of dorsal displacement of the soft palate during exercise so that management of this condition could be enhanced. HYPOTHESIS: That the thyrohyoid muscles play an important role in the stability of the laryngo-palatal relationship and that dysfunction of these muscles leads to dorsal displacement of the soft palate (DDSP) during exercise. METHODS: Ten horses were exercised on a high-speed treadmill under 4 different treatment conditions: control conditions (n = 10), after resection of thyrohyoid muscles (TH, n = 10), after sham-treatment (n = 5), or after restoration of function of the thyrohyoid muscles with surgical sutures (prosthesis-treatment, n = 6). During trials, the following determinations were made: videoendoscopy of the upper airway, gait frequency and pharyngeal and tracheal static pressures. RESULTS: None of the 10 horses developed DDSP during 2 separate treadmill-exercise trials under the control conditions. Seven of the 10 horses developed DDSP after resection of the TH muscles, 4 of 5 of these horses still experienced DDSP after sham-treatment, but 5 of 6 horses no longer experienced DDSP at exercise after the prosthesis-treatment. There were significant anomalies in airway pressures, respiratory frequency, and occurrence of DDSP in both the TH resection and sham-treatment conditions compared to control conditions. In contrast, no statistical differences were noted in any of the parameters measured between the prosthesis-treatment and control conditions. CONCLUSIONS: That the function of the TH muscles is important to the stability of the laryngo-palatal relationship and plays a role in the pathophysiology of exercise-induced DDSP. POTENTIAL RELEVANCE: Management of horses with DDSP could be enhanced by restoring the function of the TH muscles.

Effect of diet and GnRH administration on post-partum ovarian cyclicity in autumn-lambing ewes.

Using autumn-lambing ewes, this study investigated (i) the effects of diet on gonadotrophin secretion and responsiveness of the hypothalamic-pituitary-ovarian axis to exogenous GnRH during the early post-partum period; and (ii) whether ovulation prior to completion of uterine involution results in an increased incidence of aberrant ovarian cycles. Thirty-two ewes rearing 1.9+/-0.12 lambs were equally allocated to two dietary treatments at lambing (22 October +/-0.2 day). Diets comprised ad libitum hay and 1.5 kg per ewe per day of one of two concentrates (11.5 MJ ME, 195 g CP per kg) containing 300 g kg(-1) cracked maize grain (M) or 300 g kg(-1) sugar beet pellets (S). Half of the ewes on each diet (G) received 25 i.v. injections of 250 ng GnRH in 2 ml 0.9% saline at 2 h intervals from days 12-14 post-partum while remaining ewes (N) were monitored for the resumption of spontaneous ovarian cyclicity. Blood samples were obtained from all ewes throughout the study (lambing to 18 December) for measurement of circulating hormone concentrations and the uteri and ovaries of all ewes were examined via laparoscopy on day 21 post-partum. There were no effects of dietary treatment on ewe daily live weight loss, lamb daily live weight gain or the immediate post-partum increase in circulating FSH concentrations. Diet did not affect insulin concentrations or LH pulse frequency on day 12 post-partum but LH pulse amplitude was lower in ewes fed concentrate M compared to concentrate S (1.4+/-0.10 versus 1.7+/-0.12 ng ml(-1), respectively, P<0.05) and this was associated with an increased interval to the resumption of spontaneous ovarian cycles (35+/-3.1 versus 26+/-2.1 day, respectively, P<0.05). Administration of exogenous GnRH increased (P<0.05) the proportion of ewes on both diets that ovulated within 20 days of parturition and advanced the onset of ovarian cyclicity in ewes fed concentrate M by 9.5 days (significance of interaction, P<0.05). Four ewes, all of which ovulated before day 22 post-partum, had extended luteal activity while in remaining ewes, duration of the first luteal phase was inversely related to the time of first ovulation (r(2)=0.16, P<0.05). Results demonstrate that (i) the onset of ovarian cyclicity is influenced by diet and can be advanced by administration of exogenous GnRH; and (ii) ovulation during the early post-partum period is associated with an increased incidence of extended luteal activity.

Season affects characteristics of the pre-ovulatory LH surge and embryo viability in superovulated ewes.

The aim of this study was to determine whether there are seasonal shifts in ovulatory response, and in the viability of ova recovered from superovulated ewes. Fifty mature ewes underwent a standard oestrous synchronisation (CIDR), superovulation (oFSH) and artificial insemination procedure during October (peak breeding season) and April (transition to anoestrus). In each month peripheral LH and progesterone concentrations were measured around the time of ovulation and embryos were recovered, graded and cryopreserved on day 6 after insemination. During the subsequent breeding season, grade 1 and 2 morulae and unexpanded blastocysts were thawed and transferred singly to synchronous recipients (October, n = 40; April, n = 40) or cultured in vitro for 18-20 h (October, n = 107; April, n = 98). Following culture, viable embryos were stained to count cell nuclei or assayed to measure their capacity for glucose metabolism ([3H]glucose) and protein synthesis ([35S]methionine). Peak LH concentrations were higher in October than in April (38.2 +/- 3.26 ng ml(-1) versus 25.7 +/- 1.99 ng ml(-1), respectively; P < 0.01) and the pre-ovulatory LH surge was advanced by approximately 3 h (P < 0.05). Progesterone concentrations at CIDR withdrawal were lower in October than in April (3.1 +/- 0.16 ng ml(-1) versus 4.3 +/- 0.19 ng ml(-1), respectively; P < 0.001) but were not different at embryo recovery. Season did not affect the numbers of corpora lutea per ewe or the numbers of ova recovered but the proportion of recovered ova that was unfertilised/degenerate was lower in October than in April (0.43 versus 0.58, respectively; P < 0.001). For embryos containing more than 16 cells, there was no effect of season on the median stage of development or morphological grade. The proportions of October and April embryos that established pregnancy following transfer to recipient ewes were 0.78 and 0.70 (not significantly different), and that were viable after in vitro culture were 0.66 and 0.37 (P < 0.05), respectively. Season did not affect the number of nuclei per viable embryo or the capacity for protein synthesis but the glucose uptake of October embryos was approximately double that of April embryos (3163+/-293.4 dpm versus 1550+/-358.9 dpm, respectively; P < 0.05). Results indicate that during the late compared to peak breeding season, there is an increased incidence of fertilisation failure as a possible consequence of seasonal shifts in LH secretion and (or) associated effects on follicular function. Frozen-thawed embryos produced at contrasting stages of the breeding season are equally viable in vivo but those produced during the late, as opposed to the peak breeding season have lower viability following in vitro culture.

Seasonal differences in lamb birthweight do not arise from inherent differences in the oocyte and/or early embryo.

The aim of this study was to determine whether previously observed seasonal differences in conceptus development in ewes are attributable to inherent differences in the oocyte and/or early embryo. Day 6 embryos were recovered from 50 ewes subjected to a standard oestrus synchronization, superovulation and laparoscopic artificial insemination protocol during October (peak breeding season) and April (transition to anoestrus). During the following October, 40 grade 1 and 2 embryos from each month, which had been cryopreserved at the late morula or unexpanded blastocyst stage, were thawed and transferred in singleton to synchronous recipients. Resulting pregnancies were monitored to term. For ewes receiving October- and April-produced embryos, overall mean +/- SEM liveweight at the time of embryo transfer was 72 +/- 0.7 kg, body condition score was 3.1 +/- 0.04 units, and the number of corpora lutea on the ovaries was 2.7 +/- 0.11 per ewe. Thirty-one and 27 ewes, respectively, became pregnant and their gestation lengths were 147 +/- 0.2 and 147 +/- 0.3 days. There was no effect of month of embryo production on peripheral ovine pregnancy-associated glycoprotein concentrations during pregnancy or on fetal and placental characteristics at term, but, for each month, male lambs were heavier than females and were associated with larger placentae. Lamb birthweight was positively correlated with placental weight (r2 = 0.474, P<0.001) and the total weight of cotyledonary tissue (r2 = 0.429, P<0.001), but not to the number of cotyledons. Results demonstrate close relationships between fetal and placental weights at term, and that seasonal effects on conceptus development in ewes do not arise from inherent differences in the oocyte and/or early embryo.

Decay acceleration of the complement alternative pathway C3 convertase.

An ELISA-based method is described for analyzing the mechanism by which the decay of the alternative pathway C3 convertase is accelerated by C3 regulatory proteins. Using this assay, we show that human decay-accelerating factor (DAF) and factor H are active on mature convertase complexes (C3bBb) but not on their nascent precursor (C3bB). This finding has implications on the mechanisms of action of these two regulators. The complement convertases cleave the serum protein C3, and the resulting C3b activation fragments covalently attach to nearby targets where they direct antigen selection, immune clearance, and cell lysis. Several proteins, including the membrane protein DAF, and the serum protein factor H, limit convertase activity by promoting their irreversible dissociation. An understanding of the biochemical mechanisms providing for their activities would be helpful for the therapeutic control of the complement response.

Ovulation, fertilization and lambing rates, and peripheral progesterone concentrations, in ewes inseminated at a natural oestrus during November or February.

The objective of this study was to determine the relative importance of seasonal changes in ovulation rate, fertilization rate and embryo survival as the cause of reduced lambing rates in ewes mated in February compared with those mated in November. The study was conducted at 57 degrees N using mature Mule ewes and Suffolk rams. Sixty ewes were allocated equally to five groups: unbred (UB) or mated at a natural oestrus during November (N) or February (F) by natural (N) or cervical artificial (A) insemination. Groups were maintained separately at pasture supplemented with hay. A raddled vasectomized or non-vasectomized ram was present with UB, NN and NA groups from 26 October 1995 to 1 January 1996 and with UB, FN and FA groups from 25 January 1996 to 31 March 1996. Ewes marked by the ram were recorded twice a day, and those in groups NN, NA, FN and FA were inseminated at their second behavioural oestrus. For all ewes, blood samples were obtained once a day from introduction of the vasectomized rams until 30 days after mating (groups NN, NA, FN and FA) or 20 days after the first oestrus (group UB), and ovulation rate was measured by laparoscopy 7 days after the first oestrus. For ewes in groups NN, NA, FN and FA, ovulation rate was measured again after the second oestrus and ova were recovered from six ewes per group for assessment of fertilization before autotransfer. Pregnancy and lambing rates were recorded at term. Mean (+/- SE) dates of the first recorded oestrus for ewes in groups NN, NA and UB, and FN, FA and UB were 4 +/- 1.1 November and 4 +/- 0.9 February, respectively, and intervals between the first and second oestrus were 16 +/- 0.2 and 17 +/- 0.3 days (P < 0.01), respectively. Ovulation rates were 2.6 +/- 0.08 and 2.0 +/- 0.05 (P < 0.001), and peripheral progesterone concentrations during the luteal phase were 8.5 +/- 0.25 and 7.6 +/- 0.31 ng ml-1 (P < 0.05), for November and February, respectively. The difference in peripheral progesterone concentration was not solely attributable to the difference in ovulation rate. There was no significant effect of month or method of insemination, or of embryo recovery and autotransfer procedures on pregnancy rates and the proportion of ewes that became pregnant were NN 0.92, NA 0.83, FN 0.67 and FA 0.75. For ewes undergoing embryo recovery and autotransfer, ova recovered per corpus luteum were 1.00, 0.93, 1.00 and 0.92, fertilized ova per ovum recovered were 0.69, 0.92, 1.00 and 0.83, and lambs born per corpus luteum were 0.62, 0.79, 0.78 and 0.58 for NN, NA, FN and FA groups, respectively. There were no significant seasonal effects on fertilization rate or embryo survival. It is concluded that a seasonal decline in ovulation rate is the primary cause of reduced lambing rates in ewes mated in February compared with those mated in November. Pregnancy rates were high after mating in both periods and were not enhanced by the use of cervical insemination.

Mutations of the type A domain of complement factor B that promote high-affinity C3b-binding.

Factor B is a zymogen that carries the catalytic site of the complement alternative pathway convertases. During C3 convertase assembly, factor B associates with C3b and is cleaved at a single site by factor D. The Ba fragment is released, leaving the active complex, C3bBb. During the course of this process, the protease domain becomes activated. The type A domain of factor B, also part of Bb, is similar in structure to the type A domain of the complement receptor and integrin, CR3. Previously, mutations in the factor B type A domain were described that impair C3b-binding. This report describes "gain of function" mutations obtained by substituting factor B type A domain amino acids with homologous ones derived from the type A domain of CR3. Replacement of the betaA-alpha1 Mg2+ binding loop residue D254 with smaller amino acids, especially glycine, increased hemolytic activity and C3bBb stability. The removal of the oligosaccharide at position 260, near the Mg2+ binding cleft, when combined with the D254G substitution, resulted in increased affinity for C3b and iC3b, a C3b derivative. These findings offer strong evidence for the direct involvement of the type A domain in C3b binding, and are suggestive that steric effects of the D254 sidechain and the N260-linked oligosaccharide may contribute to the regulation of ligand binding.

Pulmonary capillary pressure in horses undergoing alteration of pleural pressure by imposition of various upper airway resistive loads.

We hypothesized that changes in pleural pressure induced by resistive breathing would affect transmural pulmonary artery, pulmonary capillary, and pulmonary wedge pressures. Seven horses were assigned to exercise with each of 4 upper respiratory resistive loads in random order at intervals of at least 2 days: 1) control--no added resistive loads; 2) inspiratory resistive load (Iobst)--left laryngeal hemiplegia; 3) expiratory resistive load (Eobst)--one-way valve in the right nostril; and 4) combined inspiratory and expiratory resistive loads (CIEobst)--left nostril occlusion. On each occasion, the horses performed an incremental exercise protocol consisting of exercise episodes of 3 min duration at 75, 90, and 100% of maximal heart rate (HRmax). Pulmonary artery and oesophageal pressures were recorded continuously. Subsequent analysis was carried out on the pulmonary arterial pressure signal with the oesophageal pressure signal subtracted, hence the pulmonary vascular pressures in this paper approximate transmural pressures. Pulmonary vascular pressures, heart rate, and arterial blood gas tensions were measured at each level of exercise. Pulmonary capillary and pulmonary wedge pressures were determined from the pulmonary artery waveform after dynamic occlusion of a branch of the pulmonary artery. During exercise, peak expiratory oesophageal pressure was more positive in horses with Eobst and CIEobst (adjusted means = 43, and 39 mmHg, respectively) compared with control (adjusted mean = 23 mmHg) (P = 0.0001). Peak inspiratory oesophageal pressure was more negative in horses at exercise with Iobst and CIEobst (adjusted means = -42 and -39 mmHg, respectively) compared with control (adjusted mean = -26 mmHg) (P = 0.0012). Eobst was associated with an increase in mean oesophageal pressure while Iobst was associated with a decrease in mean oesophageal pressure. There were significant increases in mean pulmonary artery pressure in horses with CIEobst (adjusted means = 82 mmHg) and in pulmonary wedge pressure in horses with CIEobst and Iobst (adjusted means = 51, and 55 mmHg, respectively) when compared to control (73 and 42 mmHg, respectively) (P = 0.0001). Pulmonary capillary pressure was significantly increased in horses with CIEobst or Iobst (adjusted means = 61 mmHg, 63 mmHg, respectively) when compared to control (adjusted mean = 50 mmHg)(P = 0.0001). At maximal exercise intensity with inspiratory obstruction, the mean oesophageal (pleural) pressure was -17 mmHg while the mean pulmonary capillary pressure was 77 mmHg. The latter exceeds the reported 75 mmHg threshold for capillary failure in horses. We conclude that inspiratory resistive breathing can lead to a significant increase in transmural pulmonary capillary pressure which may contribute to loss of capillary integrity and rupture.

Effects of frusemide on pulmonary capillary pressure in horses exercising on a treadmill.

We hypothesised that frusemide would decrease pulmonary capillary pressure in horses during strenuous exercise. Seven horses were tested after receiving saline or frusemide (2 mg/kg bwt) in random order with an interval of at least one week. Measurements were made with the horses standing, exercising at 75, 90 and 100% HRmax (maximal heart rate), and then walking 2 min after cessation of 100% HRmax. The exercise tests lasted for approximately 3 min with an interval of walking between them. Pulmonary artery and oesophageal pressures were recorded continuously and subsequent analysis of the pulmonary artery pressure signal was carried out after subtraction of the oesophageal pressure signal. Pulmonary arterial pressure, pulmonary capillary pressure, pulmonary artery wedge pressure, breathing rate, heart rate and arterial blood gas tensions were recorded at each level of exercise. Pulmonary arterial wedge and pulmonary capillary pressures were determined from the pulmonary arterial waveform after dynamic occlusion of a branch of the pulmonary artery. The resulting decay in pressure was submitted to exponential curve fitting and the amplitude on this curve at the moment of occlusion was recorded as pulmonary capillary pressure. When adjusted for horse and exercise intensity, horses receiving frusemide had lower pulmonary capillary and wedge pressures (adjusted least-squares means = 36 mmHg and 28 mmHg, respectively) when compared with control values (adjusted least-squares means = 41 mmHg (P = 0.042) and 35 mmHg (P = 0.002), respectively). Pulmonary arterial pressure, breathing rate, heart rate and arterial blood gas tensions did not differ between treatments at any exercise intensity. We conclude that frusemide reduces pulmonary capillary and wedge pressures. This is compatible with reduced transcapillary filtration and, therefore, reduced accumulation of lung water at exercise. It may also account for the putative protective effect of frusemide against exercise-induced pulmonary haemorrhage.

Oral nitroglycerin paste did not lower pulmonary capillary pressure during treadmill exercise.

We hypothesised that 22.5 mg of oral nitroglycerin would cause pulmonary vasodilation and therefore decrease pulmonary capillary pressure in horses during strenuous exercise. Six horses were assigned to exercise twice, once with no medication (control) and once with nitroglycerin (22.5 mg orally) in random order. Horses were exercised for 3 min each at 75, 90 and 100% of maximal heart rate (HRmax) with a 2 min period of walking between each period of exertion. Pulmonary artery and oesophageal pressures were recorded continuously. Subsequent analysis was carried out on the pulmonary arterial pressure signal with the oesophageal pressure subtracted, hence pulmonary vascular pressures reported in this paper approximate transmural pressures. Pulmonary arterial pressure, pulmonary arterial wedge pressure, pulmonary capillary pressure, heart rate and arterial blood gas tensions were determined for each level of exercise. Pulmonary arterial wedge and pulmonary capillary pressures were determined from the pulmonary artery waveform after dynamic occlusion of a branch of the pulmonary artery. The resulting decay in pulmonary artery pressure was submitted to an exponential curve fitting and the amplitude at the moment of occlusion on this curve was recorded as pulmonary capillary pressure. The effects of nitroglycerin on the various parameters were evaluated using a 3-way ANOVA blocked on horse treatment, and exercise intensity, followed by Tukey's multiple comparison procedure. Resting pulmonary artery pressure decreased from mean +/- s.e. 34.0 +/- 5.5 mmHg to 24.0 +/- 3.9 mmHg 5 min after administration of nitroglycerin (P < 0.05) but there were no significant effects on pulmonary capillary or wedge pressures. Nitroglycerin at this dose resulted in no significant differences in pulmonary artery, pulmonary capillary, and pulmonary wedge pressure, heart rate, arterial oxygen tension or arterial carbon dioxide tension at 75, 90 and 100% of HRmax. This dose of nitroglycerin does not appear significantly to protect the pulmonary vascular bed from exercise-induced hypertension. These data do not support the use of this dose of oral nitroglycerin in the prevention of EIPH.

A conserved element in the serine protease domain of complement factor B.

Factor B and C2 are serine proteases that carry the catalytic sites of the complement C3 and C5 convertases. Their protease domains are activated by conformational changes that occur during convertase assembly and are deactivated upon convertase dissociation. Factor B and C2 share an 8-amino acid conserved sequence near their serine protease termini that is not seen in other serine proteases. To determine its importance, 24 factor B mutants were generated, each with a single amino acid substitution in this region. Whereas most mutants were functionally neutral, all five different substitutions of aspartic acid 715 and one phenylalanine 716 substitution severely reduced hemolytic activity. Several aspartic acid 715 mutants permitted the steps of convertase assembly including C3b-dependent factor D-mediated cleavage and activation of the high affinity C3b-binding site, but the resulting complexes did not cleave C3. Given that factor B and C2 share the same biological substrates and that part of the trypsin-like substrate specificity region is not apparent in either protein, we propose that the conserved region plays a critical role in the conformational regulation of the catalytic site and could offer a highly specific target for the therapeutic inhibition of complement.