Quantification of immunoglobulins in respiratory tract secretions of the horse

Lavage techniques were used to obtain secretions from the nasal cavity, trachea and bronchi of conscious horses. The techniques, which utilised fibreoptic endoscopy for recovery of tracheal and bronchial secretions, were well tolerated by the horses. The recovery rates of the lavaged fluids were acceptable, but were lowest for bronchial secretions, and there was minimal contamination by blood. The fluids were analysed for IgG and IgM by single radial immunodiffusion, and for IgA and albumin by rocket immunoelectrophoresis. Relative to albumin there was significantly more IgA and IgM, and significantly less IgG, in the nasal cavity than the trachea. IgA and IgM levels were greatest in the nasal cavity and decreased progressively to the bronchi, whereas IgG levels showed the reverse trend. The immunoglobulin: albumin ratios of secretions taken from many levels of the tract were significantly higher than those of serum, suggesting local production of immunoglobulin in the respiratory tract.     

Ultrasonic evaluation of the reproductive tract of the mare: Ovaries

Ultrasonography is useful for monitoring the dynamic follicular and luteal changes of equine ovaries, since it permits rapid, visual, non-invasive access to the reproductive tract. A 5 MHz transducer has greater resolving power and is far more suitable for evaluation of ovaries than a 3–3.5 MHz transducer. Follicles as small as 2–3 mm can be seen and the corpus luteum can usually be identified throughout its functional life. In a study involving daily ovarian examinations, there was a pronounced change in shape of the preovulatory follicle from a roughly spherical to a pear-shaped or oblong form in 66% of the ovulatory periods, This change usually occurred on the day preceding ovulation. The occurrence of ovulation was detectable by the disappearance of a large follicle. In addition, the ovulation site on day 0 was characterized by an intense echogenic area in 88% of 32 ovulations. The developing corpus luteum retained the echogenicity for a mean of 2.4 days. In a blind study, the location of the corpus luteum, as determined by ultrasound, agreed with a previous independent determination of the side of ovulation by palpation in 88% of the 40 bred mares on days 0–14. In all of the 12 mares that were in estrus, the location of the corpus luteum could not be ascertained. In another study, the corpus luteum was identified for a mean of 16 days in 14 estrous cycles. One or more days before the corpus luteum became ultrasonically unidentifiable, it developed increased echogenicity in 36% of the mares, indicating greater tissue density. It is concluded that ultrasonic evaluation of the corpus luteum is superior to digital evaluation by rectal palpation. Some of the potential applications of ultrasonic examination of the ovaries include: 1) obtaining important, sometimes definitive, information by a single examination for judging whether a mare has entered the ovulatory season, 2) aiding in estimating the stage of the estrous cycle, 3) detecting double preovulatory-sized follicles which are in close apposition and difficult to discern by palpation, 4) detecting failure of ovulation or anovulatory estrus by the absence of a corpus luteum, 5) differentiating a persistent corpus luteum from anovulatory or anestrous conditions, 6) diagnosing certain pathological conditions such as peri-ovarian cysts and ovarian tumors, and 7) diagnosing anovulatory hemorrhagic follicles.     

Measurement of immunoglobulins in reproductive tract fluids of bulls

Seminal, seminal vesicular, urethral and preputial fluids from bulls of two different age groups were assessed for quantitative differences in immunoglobulins. Selected markers were measured in individual samples to differentiate locally derived immunoglobulins from those present as a result of trauma or secretions from other accessory glands.Immunoglobulin levels in reproductive tract fluids from older bulls (5–6 years) were higher than those of younger bulls (3–4 years) and preputial fluids contained the highest concentration of immunoglobulins of all fluids examined. Similarities existed, however, among all fluids in the relative concentrations of immunoglobulins. IgG was generally in highest concentration, though the predominant subclass varied. A marked predominance of IgG₂ over IgG₁ occurred in preputial fluid samples of older bulls. IgA was in second highest concentration, and levels were often equal to or greater than those in serum. IgM was in low concentration and occasionally undetectable. IgG/IgA ratios did not exceed 5 in most of the reproductive fluids, whereas serum ratios were usually over 100. Proportional contents of albumin and immunoglobulin in reproductive tract fluids by comparison with those in serum indicated that substantial quantities of IgG as well as IgA were synthesized locally or derived by selective transport. Increased numbers of plasma cells in the lamina propria of the preputial and penile mucosa of older bulls were correlated with higher immunoglobulin concentrations in preputial fluid from older bulls, suggesting that differences in local synthesis were responsible.     

Acrosin system of dog spermatozoa and reproductive tract secretions

The aim of this study was to determine the activity of proacrosin and acrosin in spermatozoa originating from the sperm-rich fractions (SRF) and whole ejaculates (WE) of dog semen. In addition, experiments were conducted to determine the activity of antitrypsin inhibitors in the fluids of different ejaculate fractions and whole seminal plasma. Ejaculates were collected from five dogs of mixed breed and one Beagle dog (aged from 2 to 9 years). In the SRF, it was confirmed that the activity of the free acrosin form was predominant (acrosin/proacrosin; 2.38 +/- 0.22/1.05 +/- 0.08 mIU/10(6) spermatozoa). On the other hand, spermatozoa originating from the WE exhibited significantly higher (p < 0.05) proacrosin activity (proacrosin /acrosin; 2.19 +/- 0.19/1.30 +/- 0.11 mIU/10(6) spermatozoa). Furthermore, acrosin inhibitor activity was lower in the fluids of the pre-sperm fraction (0.09 +/- 0.006 IU/cm3), whereas it was higher in the fluids of the post-sperm fraction (0.11 +/- 0.007 IU/cm3). Using PAGE analysis, the antitrypsin activity of the enzyme was represented by the presence of one electrophoretic band in the fluids of the pre-sperm and post-sperm fractions and whole seminal plasma. Furthermore, two electrophoretic bands were detected in the fluids of the SRF. The findings of this study indicate that specific proteinase inhibitors present in the individual ejaculate fractions of dog semen may act by stabilizing the sperm acrosin system.     

Immunohistological studies of the local immune system in the reproductive tract of the mare

The immunoperoxidase technique was adapted for the identification of free immunoglobulin and immunoglobulin producing cells in equine tissues. Staining specific for free IgG, IgA and IgM was detected at all levels of the reproductive tract, and secretory component staining was present in the uterine epithelium but not in the oviduct, cervix or vagina. Immunoglobulin producing cells were present at all levels of the tract, with IgG and IgA cells at equivalent concentrations, but with fewer IgM cells. There was no cyclical trend in free immunoglobulin staining, or plasma cell numbers. IgG and IgM plasma cell numbers declined from uterus to vagina, as did epithelial staining, and the ratio of IgG:IgA cells declined from oviduct to vagina.     

Ultrasonic evaluation of the reproductive tract of the mare; principles, equipment, and techniques

Ultrasound scanners have the potential of becoming as important for examination of the reproductive tract of mares, as x-ray machines are for examination of the legs. The principles of ultrasonography are based on the abilities of various tissues and fluid-filled structures to either reflect or propagate high frequency sound waves. A sound beam is emitted from an intrarectal, hand-held transducer. The proportion of the beam that is reflected (echoed) is received by the transducer, converted to electrical impulses, and displayed on a screen as moving images. Liquid-filled structures do not reflect sound waves and appear black on the screen. At the other extreme, dense tissues reflect much of the beam and appear white. Other tissues are seen in various shades of the gray-scale, depending upon their echogenicity (ability to reflect sound waves). Certain tissue formations may cause the sound waves to bend (refract), bounce back and forth (reverberate), or to become weakened (attenuated) or entirely blocked. Therefore artifacts may appear on the screen and must be appreciated by the ultrasonographer. The resolving power of the equipment is dependent upon the frequency of the sound waves measured in megahertz (MHz) units. A 5 MHz transducer is more suitable for examining the reproductive tract of mares than the generally available 3 or 3.5 MHz transducer.

Preparation for intrarectal ultrasound examination is similar to preparation for rectal examination, but there are other considerations, such as protection of the equipment and management of external lighting. A linear-array transducer is hand-held and is generally oriented in the sagittal plane with respect to the mare's body. The images of the cervix and uterine body are therefore longitudinally oriented, and those of the uterine horns are cross-sectional. With sector scanners, however, the sound beam generally travels transversely with respect to the mare's body and images of the cervix and uterine body are cross-sectional and images of the horns are longitudinal or oblique. The availability of ultrasound instruments for examination of the reproductive tract should provide additional encouragement for veterinarians to be as knowledgeable as possible about reproductive anatomy and pathology. The ability to study “living,” detailed, sequential, sectional views of the organs demands a more detailed working knowledge of anatomy than that required for rectal palpation.     

Quantitation of immunoglobulins in ovine sera and secretions by laser nephelometry. Comparison with the radial immunodiffusion (RID) technique

In twenty-five ovine body fluids (serum, lung fluid and cerebrospinal fluid), the concentrations of IgG1I, IgG2, IgM and IgA were determined by laser nephelometry and radial immunodiffusion (RID). When nephelometric assays are carried out, antisera free from any turbidity are essential. Methods ensuring that goat and rabbit anti-sera will satisfy this requirement are described in the present paper. When sheep immunoglobulins were measured by laser nephelometry, adequate and reproducible results were obtained, comparable with those obtained by RID. Advantages of the nephelometric method include the speed of assay and its sensitivity, allowing precise determination of the very low concentrations of immunoglobulins in CSF.     

Recent advances in reproductive endocrinology of the mare

The recent development of radioimmunoassay techniques to determine changes in blood hormone levels has increased our understanding of the endocrinology of the oestrous cycle and pregnancy in the mare. This review describes recent findings concerning the endocrine basis and treatment of some common types of infertility.

Further evidence supporting the concept that prostaglandin F2∝ is the uterine luteolysin in the mare is discussed. Persistent corpus luteum is now recognized as an important cause of infertility, which may be treated by uterine distension or prostaglandin; recommendations for avoiding treatment failures are presented.

The clinical significance of recent findings on the effectiveness of HCG in hastening ovulation, and on the increase in anti-HCG antibodies after repeated injections of this hormone are also discussed.

Although antibiotics successfully control the specific pathogen causing contagious equine metritis, the treatment of common opportunistic organisms with antibiotics appears to give indifferent results. The increased understanding of the role of sex steroids in susceptibility to uterine infection seems likely to lead to improved methods of control.

Although stimulation of folliculogenesis and ovulation in seasonally acyclic mares is possible using extracts of equine pituitary glands, this treatment is impractical on a clinical scale. Gonadotrophin-releasing-hormone provides a more physiological method of stimulation, and results of recent experiments using this hormone are reviewed.

In the pregnant mare, recent studies have shown that the “pregnancy signal” to the mare may be the production of oestrogens from the developing blastocyst from Day 14. Measurement of PMSG as an aid to pregnancy diagnosis is evaluated, and the use of progesterone supplementation to prevent abortion is discussed.     

Immunoglobulin isotype of specific antibodies in reproductive tract secretions and sera in Tritrichomonas foetus-infected heifers

Four virgin heifers were experimentally inoculated intravaginally with 7 x 10(6) Tritrichomonas foetus, and 2 heifers served as uninfected controls. The durations of infection were 13, 20, 21, and 28 weeks, respectively. An ELISA that used whole T foetus antigen was used to detect anti-T foetus immunoglobulins (IgA, IgG1, IgG2, and IgM) in vaginal, cervical, and uterine secretions, and sera during the course of infection. The vaginal and cervical antibody responses were characterized by significantly increased T foetus-specific IgA and IgG1 at 7 to 9 weeks of infection, whereas uterine IgA and IgG1 responses peaked at 10 to 12 weeks. The antibody response in serum was predominantly of the IgG1 and IgG2 subclasses. In all reproductive tract regions, IgA persisted at least until the time of T foetus clearance, and usually longer. The next most persistent isotype was IgG1, lasting longest in the vagina, then cervix, and for the shortest time in the uterus. In local secretions, IgG2 was seen only transiently, increasing at weeks 13 to 15 in the vagina, and at weeks 10 to 12 in the cervix. Little IgM, relative to that present before infection, was detected in any secretion or serum, although cervical secretions had the greatest amount. Eight to 12 weeks after clearance, the 4 experimental heifers were inoculated intravaginally with 1 x 10(5) T foetus; transient infections (2 to 3 weeks' duration) were established in only 2 of 4 heifers, as determined by culturing of reproductive tract secretions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mammary secretions in normal spontaneous and induced premature parturition in the mare

Total calcium, total protein, albumin and globulin content were determined in 49 samples of mammary secretions from 37 crossbred and Thoroughbred mares. The mares were divided into three categories: Group 1--post partum samples from spontaneous full term Thoroughbred deliveries (n = 20); Group 2--pre- and post partum samples from spontaneous full term Thoroughbred deliveries (n = 6); Group 3--pre- and post partum samples from induced premature deliveries in crossbred mares (n = 11). Calcium concentrations of the mammary secretions proved useful in predicting full term and also in assessment of the chances of foal survival in prematurely induced parturition. Mean (+/- se) calcium levels in full term mares that foaled spontaneously (Group 1) were 10.6 +/- 2.7 mmol/litre on the day of parturition. However lower values than this did not necessarily mean parturition would not take place. In Group 2 mares, calcium levels were 4.1 +/- 0.8 mmol/litre just before foaling and rose to 9.2 +/- 1.5 mmol/litre within 20 mins of parturition; an increase of 124 per cent. Group 3 mares, induced prematurely, showed poor foal survival if pre-partum calcium levels were less than 3 mmol/litre. Total protein, albumin and globulin concentrations showed a similar trend but the increase just before or during parturition was much less marked.     

Protective factors in mammary gland secretions during the periparturient period in the mare

Changes in mare mammary secretion composition were characterized prior to and after foaling. Eighteen mares were used for collection of mammary secretions from 23 days prepartum through 44 days postpartum. Concentrations of lactose, total protein, IgG and lysozyme (activity assay) were determined. Considerable variabilty was observed among mares. Mean lactose concentrations by mare were lower (P<.05) in the prepartum period compared with the postpartum period. Mean total protein and immunoglobulin G concentrations by mare were higher (P<.05) in the prepartum period compared with the postpartum period. Mean lysozyme activities by mare tended to be lower (P<.08) during the prepartum period compared with the postpartum period. Mammary secretion protein profiles, characterized by SDS-PAGE, were consistent with changes in concentrations of specific proteins measured in the secretions. Dynamic changes occurring in mammary secretions during the transition from prepartum accumulation of colostrum to postpartum production of milk include factors like IgG and lysozyme which have protective roles in the neonatal foal.     

Daytime management of the mare: 1: Pre-foaling mammary secretions testing

Fifty-six mares of lighthorse breeding were utilized in a controlled management scheme for induced daytime foaling. All mares had pre-foaling mammary secretions sampled for evaluation of water hardness (ppm) or calcium carbonate content (ppm). Sampling began 10 days prior to expected foaling date for each mare and was performed once daily for 3 days followed by twice daily until foaling occurred. Samples were diluted 1:6 in distilled water and tested by each of 3 methods: Sofchek™ Test Strips⁴, Predict-A-Foal™ Test Kit⁵ and Titrets™ Calcium Hardness Test Kit⁶. Mares were then either induced (i) to foal (no.= 33) according to a decision point of readiness for birth as indicated by pre-foaling mammary secretion testing (≥250 ppm water hardness by Sofchek™ test, ≥ 250 ppm calcium carbonate content by Titrets test, ≥ 4 color bar changes by Predict-A-Foal test), or allowed to foal spontaneously (s) (no.=23). There were no differences (p>0.05) in the mean pre-foaling mammary secretion test values (μ_i=293ppm, μ_s=329ppm; μ_i=4.1, μ_s=4.2; and μ_i=281ppm, μ_s=298ppm; for Sofchek, Predict-A-Foal and Titrets, respectively) between mare groups at the time of foaling (time=0) for any of the 3 testing methods employed. Mean intervals to foaling after reaching decision points of readiness for birth were different (p<0.05) between Sofchek and Titrets test for both mare groups, but only in the induced-to-foal group between Sofchek and Predict-A-Foal tests. Probabilities of 79%, 53% and 59% were calculated for mares foaling spontaneously within 24 hours of reaching the decision points used in this trial of readiness for birth on the initial occasion for Sofchek, Predict-A-Foal and Titrets™ tests, respectively. Each test was determined to have the ability to predict readiness of approaching parturition and found to be easily applicable to field use. The Titrets test was found to be least variable in its response to measurement of pre-foaling mammary secretion hardness changes both within and between mares.     

Preliminary studies of mammary secretions in the mare to assess foetal readiness for birth

The status of the mare and foetus in relation to readiness for birth was assessed by measurement of the electrolytes sodium, potassium and calcium in mammary secretions pre-partum. Sixteen Thoroughbred mares were allowed to foal spontaneously and the ionic status of their mammary secretions was measured over three to five weeks pre-partum. From these measurements, a scoring system was developed where an ionic score of 35 points or more suggested that the mare was within 24 h of foaling. On the basis of this ionic score, 10 pony mares were induced with either oxytocin or fluprostenol and assessment of foal maturity was made by physical, behavioural and physiological criteria. Eight pony mares, induced when the ionic score was 35 points or more, delivered full term foals; two mares were induced when their scores were 30 and 20 points and delivered a full term and slightly immature foal respectively. These results suggest that foetal maturity may be related to electrolyte concentrations in mammary secretions and that an ionic score of 35 points or more may indicate that induction would be successful in terms of maturity of the newborn foal.     

REVIEW paper: mare reproductive loss syndrome

An epidemic of early fetal loss (EFL), late fetal loss (LFL), fibrinous pericarditis, and unilateral uveitis which occurred during the spring of 2001, are together now known as the mare reproductive loss syndrome (MRLS). A similar epidemic with less intensity was reported during the same period of time from southern Ohio, West Virginia, and Tennessee. The same syndrome with lesser intensity recurred in 2002. The estimated economic loss from the syndrome in 2001 and 2002 together was approximately $500 million. Both EFL and LFL were characterized by the absence of specific clinical signs in aborting mares. Nonhemolytic Streptococcus spp. and Actinobacillus spp. accounted for 65% of the organisms isolated from fetuses submitted for a postmortem during the MRLS period in 2001 and 2002. The pathologic findings in fetoplacental units of LFL included bronchopneumonia and funisitis, and there were no findings in EFL. Epidemiologic studies conducted in 2001 suggested an association between the presences of eastern tent caterpillars (ETC) in pastures with MRLS. Experimental studies in pregnant mares by exposure to ETC, or administration by stomach tube or with feed material, reproduced EFL and LFL. Similar experimental studies in mouse, rats, and goats with ETC were unsuccessful. Currently, 2 hypotheses are proposed for MRLS. One hypothesis proposes that an ETC-related toxin with secondary opportunistic bacterial invasion of the fetus leads to MRLS. The second hypothesis suggests that a breach of gastrointestinal mucosal integrity by hairs of ETC leads to a bacteremia and results in MRLS. In 2004, a similar equine abortion storm was reported from Australia and caterpillar exposure was identified as a risk factor for the abortion. In 2006, the syndrome was observed in Florida and New Jersey.     

The irritancy of chlorhexidine gluconate in the genital tract of the mare

Uterine irrigation was carried out in eight Welsh pony mares using 50 ml of chlorhexidine gluconate solution diluted to give active ingredient concentrations of 0.25 per cent to 2 per cent. Treatment was repeated up to twice in mares showing no adverse effects and irritancy of treatment judged on clinical symptoms and uterine biopsy. Results indicated the inadvisability of using a higher concentration than 0.25 per cent. Three daily applications of a diluted surgical scub solution containing 2 per cent chlorhexidine gluconate to the penis of an arab stallion failed to produce symptoms or irritation. An in vitro minimum inhibitory concentration test performed with the contagious equine metritis organism confirmed its high sensitivity to both chlorhexidine gluconate preparations.