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Reproduction
horses during road transportation in relatively hot environmental conditions. Six adult, Thoroughbred horses that were in full training were used in a balanced crossover design. This model was selected to simulate the type of horse that is transported most often over long distances in South Africa. The horses were randomly assigned to one of two treatment groups. One group was transported, while the second group served as controls. A period of one month was allowed before the crossover. Horses in the transport group were transported by tarred road in a circular route over 600 km. This allowed the standardization of measurements and the use of the same instruments (scale & laboratory) in both groups. Data were collected in each treatment group before, and during transportation that lasted for eight hours (transport phase) and for six hours after the transportation (recovery phase). The following data were collected or calculated: Water and electrolyte (sodium, potassium and chloride) intake and output, changes in body weight, hematocrit, serum concentrations of protein, albumin, creatinine, sodium, potassium, chloride, osmolality as well as feed consumption. Although water was always available, the transport group failed to drink during transit. Based on body weight, the transported horses were 3% dehydrated at the end of transit. This loss in body weight was corrected within one hour after their return due to a higher water intake compared to control horses. The feed intake in the transported horses was unaffected during traveling, but was decreased for six hours following transportation. The output of water via the urine remained the same in the two treatment groups during and following transportation. The fecal output of water decreased in the transported horses and remained lower than the control group for six hours following transit. Electrolyte intake was unaffected by transportation. Sodium and potassium loss via the feces and urine were similar in the two treatment groups, whereas potassium output was decreased in the transport group during the recovery period of the study. Apart from the decrease in body weight the dehydration in the transport group could not be detected clinically. The hematocrit and serum potassium values were unchanged, while serum protein, albumin, creatinine, sodium, chloride and osmolality measurements were elevated at the end of transportation. It was concluded that transportation by road affected the water and electrolyte balance of conditioned horses for a period up to six hours after traveling. T O W A R D S SYDNEY 2000, T H E TRANSPORT CHALLENGE M.H. Atock, Peden Bloodstock Ltd. The purpose of the forwarding agent is to make full arrangements to transport horses from their home stables to the site of competition in a foreign country and to return with the minimum of stress and inconvenience to the horses and their owners.
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This involves supervising pre-export disease testing, isolation and/or quarantine where required, completion of all necessary documentat i o n / h e a l t h certification, a r r a n g i n g and co-ordinating transport and liasing with government veterinarians in the home country and that of the country to be visited. Considerable experience of making such arrangements for horses participating at past major events involving, for example, some 250 horses at the Olympic Games in Seoul in 1988, in Barcelona in 1992, and Atlanta in 1996 and some 700 horses at the World Equestrian Games (WEG) in The Hague in 1994 are reviewed. Preliminary plans for the Sydney Olympic Games in 2000 are discussed with particular emphasis on the climate, length of journey involved, travel stress and training facilities during pre- and post-import quarantine. THE PHYSICAL EFFECTS OF T R A N S P O R T ON C O M P E T I T I O N HORSES N. K. Waran Institute of Ecology& ResourceManagement,University of Edinburgh, Scotland, UK Due to the financial and sentimental value attached to horses, they are perceived to enjoy a much higher standard of transport than most farm animals. Although transport may not subject the experienced competition horse to the same psychological stresses that result from fear of a novel experience, recent studies at the University of Edinburgh have shown that the physical effects of road transport on horses include increased heart rate, changes in body posture, and an increase in energy expenditure. These results suggest that for experienced performance horses, transport in a lorry is associated with average heart rates higher than those recorded in a stationary vehicle, and changes in body posture that are thought to be caused by problems of balance experienced in the back of a moving vehicle. Energy expenditure during transport was measured as similar to that required when the horse is walking, and transporting horses facing away from the direction of travel (backward) is associated with lower energy expenditure than transporting them facing in any other direction. This paper discussed the impact these results may have on horses transported for competition. T H E P H Y S I O L O G I C A L E F F E C T S ON H O R S E S OF A I R T R A N S P O R T A T I O N IN AN ENCLOSED C O N T A I N E R J.R. Thornton University of Queensland,Australia An enclosed insect proof container designed and built for the air transportation of horses was evaluated for its physiological effect on the horses during flights of 12 to 24 hours duration. Three horses traveled in the container, had their heart rates monitored continuously and had blood samples collected regularly for hematological and chemical analyses and determination of
g-endorphin. The air within the container was sampled regularly for bacterial and fungal populations and the air temperature and humidity recorded inside and outside the container. On one flight insect traps were installed in the contalner. On the two flights of 12 hours a regular open container with three horses was used as control. During the flights heart rates were regular and generally in the range of 35 to 55 beats per minute with increases to 80 to 100 during take off and landing or when the horses were restrained for blood collections. There were no significant accumulations of bacteria or fungi in the container and the temperature and humidity remained constant or slowly decreased with the duration of the flights. Temperatures inside the container were 3 to 6~ higher than that in the cargo hold. The heart rates and blood analyses, including the g-endorphin showed no evidence of undue stress or infection in the horses and when compared to the controls on the two 12hour flights there was no significant effect of the enclosed container. It was concluded that the enclosed container was a suitable means of transporting horses for plane flights for periods of at least 24 hours duration.
ULTRASONOGRAPHIC EVALUATION OF E N D O U T E R I N E FLUID C L E A R A N C E IN M A R E S A F F E C T E D BY E N D O M E T R I T I S AND T R E A T E D W I T H O X Y T O C I N O R PGF2a S. Barbacini, V. Marchi, G. Zavaglia Studio Veterinario Cristella, S. Daniele Po, Cremona, Italy Among the species of zootechnical interest the equine is undoubtedly the one with the lowest reproductive efficiency. Persistent and recurrent post-insemination endometritis is one of the most important causes of this phenomenon. The latest research has found reduced myometrial activity as one of the major causes responsible for this reproductive pathology. In fact the inability of clearing the inflammatory cells (ex WBC) and the exudate that normally appear in the uterine lumen after an insemination, causes the persistence of this inflammation. Because of this, the embryo, after its descent into the uterus, doesn't find a compatible environment with its survival. The broodmares are at the moment classified as "susceptible" or "resistant" according to their ability of clearing the uterus. Moreover, recent clinical observations have shown a bigger incidence of this pathology when using frozen semen. Hypothetically this is caused by the protein alterations observed in frozen/thawed spermatozoa and by the almost total seminal plasma removal during the freezing procedure. Thirty broodmares with persistent and recurrent postinsemination endometritis, inseminated by means of frozen semen, have been considered. All the broodmares were inseminated between 12 hours before and 6 hours after ovulation. Eight hours after insemination, they were
JOURNAL OF EQUINE VETERINARY SCIENCE
treated with uterine lavage using saline solution until the fluid recovered was completely clear. Immediately after the treatment we checked by uhrasonography if fluid was still present in the uterine lumen. The broodmares with presence of intrauterine fluid, were treated with a placebo (2 ml 0.9% NaCl I.V.). Thirty minutes after the placebo treatment, the ones presenting an amount of intrauterine fluid similar (+_20%) to that found before the injection were divided into two different groups: group OX=20UI I.V. of oxytocin; group PG=0.075 mg I.M. of d-clorprostenoL The amount of uterine fluid in the group treated with OX was ecographically monitored after 1530-60 minutes - - while that in the group treated with PG was checked after 15-30-60 minutes and 5 hours. The broodmares in which the fluid removed from the uterus was less than 90%, were immediately subjected to the opposite treatment to evaluate if there was any different reaction to oxytocin or prostaglandin.
OCCURRENCE AND ENDOCRINE CHARACTERISTICS OF ANOVULATORIC F O L L I C L E S IN THE MARE B. Kaiser1, M. Koene1, J. Swagemakersl , H. Bader2, H.O. Hoppen3 lTier~irztliche Klinik fiir Pferde, M0hlen; 2lnstitut flit Reproduktionsmedizin, Tier~irztliche Hochschlule, Hannover;3ChemischesInstitut,Tier'al'ztlicheHochschule, Hannover, Germany In a clinical study, covering about 1000 broodmares in different AI centers, the occurrence of anovulatoric follicles during the breeding season 1997 was evaluated. All mares were closely monitored gynecologically every other day for follicular dynamics and estrus behavior. Mares with follicles that exceed 50 nun in diameter and do not ovulate during the normal estms period (> 5 days) are divided into three treatment groups. In treatment group A, the follicles are punctured transabdominally through the fossa paralumbaris at the ypsilateral side and the follicular fluid is collected by aspiration. In treatment group B, the follicles are punctured using a laparoscopic technique and a biopsy sample of the follicular wall is taken. In treatment group C, the mares are not treated but routinely checked until the follicle is luteinized or becomes atretic. For a control group D, preovulatory follicles (>35 nun) from mares with normal follicular dynamics and estrus behavior are punctured, using the same technique as in group A. All mares are inseminated in the first established estrus after treatment and a possible pregnancy is confirmed at day 18 after insemination by ultrasonography. From the groups A, B and D, serum samples are taken at the day of puncture and in the first established estrus after the puncture. From group C, serum samples are taken at the day of the confirmed diagnosis and in the first estrus occurring after atresia. The follicular fluid and serum are analyzed for the following parameters; Estradiol, progesterone, E/P-ratio, testosterone, LH, FSH, LH/ FSH-ratio and prolactin; routine diagnosis also includes chemistry panel and electrolytes in follicular fluid and serum and vitamins A, E and
Volume 17, Number 9, 1997
folic acid in the serum. The biopsy of the follicular wall is histologically prepared for detection of cell layers and luteinization. The etiology of erratic follicles, comparison of the components of anovulatoric and physiological follicles and follicle puncture as a therapy to shorten the time ofdiestrus in these mares will be discussed, as most of the mares with erratic follicles do not reset to any hormone treatment, i.e. hCG, GnRII, prostaglandins or long-term gestagen therapy.
T W I N SQUEEZING AT D I F F E R E N T GESTATIONAL AGES: ULTRASONOGRAPHIC TECHNIQUES AND M O N I T O R I N G C. Rognoni,M. Livini A survey on the management of 52 twin pregnancies. Video demonstrations about the use of ultrasonography to separate twins and the selective squeezing of one of the two, the typical ultrasound imaging of twins and rupturing of the wall will be shown in real time. Until day 16 the scanner probe is used to shake and separate adjacent blastocyst, otherwise difficult to distinguish and scan separately. Once separated the blastocysts are placed wide apart within the uterine horus to allow a safe and easy squeezing. After day 18 the blastocyst became more and more difficult to separate and selectively squeeze; during this period; however, it is possible, using the scanner probe, to rupture the blastocyst wall of only one of the two twins without separating them. Success rate is about 30% at gestational age more than 35 days and 70% at a gestational age of less than 16 days. This technique can also be useful to differentiate between endometrial cysts and blastocysts.
FLUID ACCUMULATION IN THE U T E R I N E LUMEN OF MARES F O L L O W I N G INSEMINATION W I T H FROZEN SEMEN
between 100X106 and 175x106 progressively motile, morphologically normal spermatozoa. The volume of inseminate ranged from 0.5 ml to 4.0 ml depending on the stallion. All mares were negative for culture and inflammatory cells on an endometrial swab taken prior to insemination. The depth of any uterine fluid was recorded as the maximal depth, usually in the uterine body during the visits 24 hours before insemination/ ovulation. Mares were treated with uterine layage, oxytocin and intrauterine antibiotics 12 hours after insemination. A small amount of fluid (usually less than 2 cm) was detected before inseminating in 21 (26%) of estrous periods. More mares had fluid detected 12 hours after insemination (57 [80%] of estrous periods, P<0.01). By 24 hours, although this number had decreased to 31 (38%), it was still greater than before insemination (P<0.01). When examined 48 hours after insemination, fluid was only detected in 2 (2.4%) cases. There appeared to be no relationship between fluid production and volume of inseminate within the small range of insemination volumes used in this study. The per cycle pregnancy rate was greater in mares with no visible uterine fluid 24 hours after insemination (71 vs 55%; P<0.05). In conclusion, this study has shown that uterine fluid is a frequent finding 24 hours after insemination with frozen semen, but this fluid rarely persists longer than 24 to 48 hours after insemination. Mares with uterine fluid persisting for at least 24 hours after insemination have a reduced pregnancy rate.
E F F E C T OF PROSTASOMES ON THE F O R W A R D M O T I L I T Y O F HORSE SPERMATOZOA BEFORE AND AFTER FREEZING M. Rubei, M. Moroni1, A. Minelli1, A.M. de Cosmo2, S. Degl'Irmocenti Istitutodi Ostetriciae GinecologiaVeteiinaria-Universit~ degli; Studi di Perugia; 1Departimentodi Biologia Cellulare e Molecolare, Universi~ degli Studi di Perugia; 2Facol~ di Medicina Veterinaria, Universit~degli Studi di Camerino (MC)
C. Bamini, S. Ventura, J.E Pycock There has been a considerable increase in the last few years in the use of frozen equine semen. While it is well known that the breeding of mares is followed by an acute inflammatory response including production of uterine fluid, little is known about this inflammatory response in mares inseminated with frozen semen. In view of the importance of uterine clearance in the persistence of acute post-breeding endometritis, and anecdotal reports of the appearance of uterine fluid following insemination with frozen semen, it was decided to investigate the accumulation of fluid in the uterine lumen of a large number of mares after insemination with frozen semen. During the 1996 breeding season, 59 mares were inseminated with frozen semen from five stallions at 82 estrous periods. All the stallions had, after thawing of a frozen semen sample, > 40% progressively motile spermatozoa. The spermatozoa concentration before freezing varied from 50xl06/ml to 500x 106/ ml and the inseminating volume varied from 0.5 ml to 4 ml to give a final insemination dose of
Seminal fluid contains the secretion of the testis, epididymis and sex accessory glands. Its components can influence the motility and the metabolism of spermatozoa with opposite effects: either a positive or a negative influence on the fertilizing capacity of the sperm has been reported. The presence of membrane particles in the seminal plasma of rabbit, ram, bull and man has been already documented. The vesicles isolated from horse seminal plasma present on their surface enzyme activities which, according to literature, are considered to be markers of human prostasomes. In addition, the anatomy of the horse prostate closely resembles the human gland; therefore, these equine particles might be, unless further investigation will prove otherwise, of prostatic derivation. The particles in rabbit seminal fluid originate from epididymis and cause sperm decapacitation and inhibit the fertility whereas human prostasomes are involved in stimulation of forward motility of the spermatozoa. However, the functional importance of the particles in seminal
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fluid are contradictory since these vescicles differ in their detailed biochemical composition. Electron microscopy of particles present in horse seminal plasma shows vesicular structures (100m diameter) surrounded by a bilaminar membrane containing unevenly distributed floccular material. Our analysis of the motility of horse spermatozoa with and without added prostasomes do not reveal any dramatic change. Preliminary investigation carried out with frozen semen samples seem to lead to different results. In fact, when frozen spermatozoa are thawed in the presence of prostasomes, there is a major change in the sperm motility pattern indicating an induction of the forward motility. Further studies will be needed to assess this preliminary positive effect on the fertilizing capacity of the horse frozen semen. PREGNANCIES AFTER EMBRYO T R A N S F E R IN A N E S T R U S R E C I P I E N T MARES: N A T U R A L AND A R T I F I C I A L M O D I F I C A T I O N S OF OVARIAN AND H O R M O N A L ACTIVITIES D. Lagneaux, E. Palmer Haras Nationaux and INRA Mares in anestrus were used as a recipient for non-surgical embryo transfers. They were treated with 25 mg estradiol-benzoate daily, during three days up to the day of ovulation of the donor, then with 44 mg Altrenogest daily from the day after the ovulation of the donor mare: up to Day 100 in EXP 1 or up to the first day endogenous progesterone levels were analyzed and ovarian activity, follicular growth and luteinized unruptured follicles (LUF) were detected by ultrasound echography. In EXP 1, two groups of pregnant anestrus recipient mares were (I-A): stimulated with lighting procedure (16 hours daily) (N=4) or (I-B): control, under natural lighting conditions (N=7). In EXP 2, mares were allotted in two groups: (2-A): treated daily with eLH, 25 mg CEG (i.m.) (Crude Equine Gonadotrophin) up to day 70 or when progesterone level was higher than 4 ng/ml (N=5), or (2B): control without treatment (N=7). In EXP 3, mares were also allotted in two groups, (3-A): treated two times (Day 40 and Day 47) with 4.5 mg (6 times more than usual) (i.m.) e FSH (purified Crude Equine Gonadotrophin) (N=10), or (3-B) control, without treatment (N=9). Results of these three experiments are showed in this table: groups
I-A
I-B
2-A
2-B
3-A I 3-B
pregnaneie~ I 4 I 7 I s I 7 I 1~ 9 Day 1st ov ]73_+24154_+6158_+16168_+22155_+12170+16 1st Day progt 175-+21158+6163-+21/71_+201 60_+14172_+13 Day 55* 2/4 2/7 4/5 2/7 7/10 I 2/9 Day 70* 2/4 / 7/7 / 4/5 4/7 I 9/10 4/9 Day 85* 2/4 7/7 4/5 5/7 9/10 4/9
Treatmentl-A= 16 h light Treatment2-A=eLH daily Treatment3-A=2X6 eFSH TreatmentsB= controls #= progesterone>4ngper ml *ovulationor LUF Mean of the first days of ovulation and endogenous progesterone level in control groups (1, 2 & 3 - B) suggested that ovarian activity is different from one mare to the other during early
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pregnancy. Stimulated groups (1, 2 & 3 -A) were provided to stimulate ovarian activity and induce ovulation or luteinization. Neither lighting nor long period eLH treatment seem efficient. This suggests that hormonal environment and the response to stimulation of pregnant anestrus mares are different than of normal anestrus mares in which light or eLH are efficient to induce ovulations. The high dose of eFSH (3-A) seems efficient to induce early ovulations or LUF in anestrus recipient mares compared to control group (3-B). Treatment of eFSH stimulates follicular growth and when follicles larger than 25 m m diameter are obtained, eCG induces their luteinization or ovulation (before days 55 or 70 in 7 or 9 cases out of 10). Efficiency of such a treatment is only 90% at day 70 which doesn't allow interruption of exogenous Altrenogest treatment except if previous results showing that exogenous supplementation is not useful up to day 70 are confinned. PROBLEMS ASSOCIATED WITH O V A R I A N ACTIVITY IN B A R R E N AND P O S T P A R T U M M A R E S E A R L Y IN THE BREEDING SEASON R Nagy 1, Gy- Huszenicza1, J. Jnhfisz2 L. Solti 1, M. Kulcsfir1 1Department at'Obstetricsand Reproduction,2Research Insititute, University of Veterinary Science, Budapest, Hungary In two studies ovarian activity was monitored with regular teasing, ultrasonography and blood sampling for progesterone in the winter and spring period. Of the 50 barren mares studied 41 (82%) stopped cycling during the autumn and winter period. The anovulatory season started before December 1 in 61% of the mares (25 animals). In 16 mares (39%) cyclic ovarian activity ceased after December 1. Of these 16 mares 5 (12.2%) stoppedcycling after January 1 (Jan. 4, Jan. 10, Jan. 11, Feb. 7). The lengths of the anovulatory season varied between 20 to 172 days. There was a strong correlation between the time of cessation and the time of resumption of cyclic ovarian activity (P <0.01). Mares that stopped cycling before December 1 resumed cyclic ovarian activity one month later than mares in which the anovulatory season began after December 1 (98.6+-30.5 days and 67.9+-35.0 days after January 1 for mares that stopped cycling before and after December 1, respectively). The first ovulation was not preceded by overt estrous (silent heat) in 64.7% of the cases (22 of 34 mares). Overt estrous was observed without ovulation in 8 mares (22.9%). Thirty-eight mares were followed in the postpartum period. Foalings occurred in the first three months of the year. The season of foaling strongly influenced ovarian activity and sexual behavior. Only 71.1% of the mares (27 animals) ovulated within 20 days post partum. Foal heat was observed in 17 mares (51.5%). The mean intervals from parturition to the 1st and 2nd ovulation and to 1st overt estrous were 25.4+-25.74 days; 52.6+-26.42 days and 35.6_+34.38 days from f o a l i n g . The
interovulatory interval was also longer then expected (32.2+ 19.44 days). In some cases the lutelysis of the first corpus lutenm was followed by delayed follicular growth resulting in a prolonged follicula phase (17.8 +/- 18.06 days). It is concluded that seasonal reproductive activity is one of the most important research and practical problems in equine reproduction. Management of both barren and postpartum mares early in the breeding season gives alot of problems to veterinarians each year. The use of mare-side progesterone assays (like the semiquantitative tube test used by us) can provide a powerful mean to determine the background of anestrous early in the breeding season both in barren and postpartum mares. H A P T O G L O B I N IN T H O R O U G H B R E D MARES ANTE AND POST P A R T U M C. Gothe,A. Vandenhouten,C. Lippeheide, H. Sommer InstituteofAnatomy,Physiologyand Hygieneof Domestic Animals, Dept. Hygiene, Bonn, Germany Parturation is always a critical stage for both mares and foals. Therefore blood parameters are discussed to indicate possible difficulties in time. Concentration levels of Acute-Phase-Proteins change rapidly in case of inflammations, infections or tissue damages. For this reason they are used as inflammation markers to determine the status of health in animals. The aim of the study was to observe the physiological course of haptoglobin around the time of foaling. Furthermore, we investigated whether haptoglobin (Hp) is a suitable parameter in preventive health control in mares during this time. Blood samples were taken from 8 clinically healthy thoroughbred mares four and two weeks ante partum and at day 1,3, 5, 7 and 14 post parturation between 8-9 a.m. For the determination of Hp in the plasma the automated nephelometer analyser 100 (Behring Diagnostics, Germany) was used. Our results show that haptoglobin always increased immediately after foaling. Hp-values at day 1 and 3 p.p. were significantly higher than a.p. and decreased slowly during the following 14 days to their fundamental basis. Values ranged between 9 and 97.4 mg/dl in mares with "normal" parturation and healthy foals (n=6). Hp-values increased up to 130mg/dl in mares with still birth (n= 1) and retained placenta (n= 1). No correlation between haptoglobin and other biochemical parameters (aspartatamino-transferase, lactate-dehydrogenase, creatinkinase, total protein, albumin, globulin, urea, bilirubin, gamma-GT, cholesterol, phosphor, magnesium, calcium) could be found. So, it seems to be useful to investigate haptoglobin as a parameter for ante partum health control in further studies. E F F E C T S OF A N T I S P E R M ANTIBODIES ON THE F U N C T I O N A L I N T E G R I T Y OF S T A L L I O N SPERMATOZOA C. Lee, H.S. Joe, H.W. Moment I, B.G. Crabo,
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OF EQUINE VETERINARY
SCIENCE
Hunter University of Minnesota, 1University of Wisconsin A.G.
Antisperm antibody is one of the factors associated with infertility or subfertility in domestic animals. Antisperm antibody interferes with the reproduction in various ways including sperm motility, binding, capacitation, penetration, and zygote development, etc. Antibody to nine major polypeptide groups (85, 51-59, 34-37, 26, 2324,16-19, 15, 10, and 5-6 kDa) of stallion sperm membrane protein were produced in rabbits. ELISA, indirect immunofluorescent antibody test, sperm motility, Glass wool/Sephadex filtration, zone-free hamster oocytes test were used to characterize and determine the effects of the antisperm antibodies on stallion spermatozoa. Antiserum to 85 kDa polypeptide was localized in the acrosome, to 51-59 kDa in the sharp line on the tail, to 34-37 kDa in the tail midpiece, to 26 kDa in the head, midpiece, and neck, to 23-24 kDa in the head and midpiece, to 16-19 kDa in the head and midpiece, to 15 kDa in the head and midpiece, to 10 kDa in the equatorial and tail, and to 5-6 kDa in the head and tail. Motility data were collected during three periods, before, during and after in vitro capacitation process. In glass wool/Sephadex filtration, the antisera to 85, 34-37, 23-24 kDa increased the filtered stallion spermatozoa, and the antisera to 16-19 and 5-6 kDa decrease the filtered spermatozoa comparing with the control serum produced against PB S. In zone-free hamster oocytes test, the antisera to 23- 24, 16-19, 15, 10, 5-6 kDa showed a decreased rate in the penetration test when spermatozoa were exposed to antibodies before in vitro capacitation. When stallion spermatozoa were exposed to antibodies after in vitro capacitation, the antiserum to 23-24 kDa only decreased the result of penetration. More detail data was shown at the presentation. F E R T I L I T Y AT T H E F I R S T POST P A R T U M ESTRUS C O M P A R E D W I T H F E R T I L I T Y AT T H E F O L L O W I N G E S T R O U S CYCLES IN F O A L I N G M A R E S AND W I T H F E R T I L I T Y IN NON F O A L I N G M A R E S F. Camillo, P.Marmorini,S. Romagnoli,L. Vannozzi, M. Bagliacca Istituto di Patologia Speciale e Clinica Chirurgica Veterinaria, Universith di Pisa, Italia The aim of this study was to compare conception rate (CR) (pregnant mares/inseminated mares) and foaling rate (FR) (foaling mares/ pregnant mares) at the first post partum estrus with fertility at the following estrous cycles in foaling mares and with fertility of non foaling mares (maiden+barren). Estrous Trotter mares were inseminated, through > 6 consecutive breeding seasons, with fresh semen of 3 fertile stallions on alternate days between the ultrasound detection of a follicle 2 35 m m and ovulation. Pregnancy diagnosis was performed with ultrasound at day 14 post ovulation and, when possible, confirmed once a week until about day 35. Parturition was checked by consulting the official Italian Stud Book of Trotters. Results are reported in the following table.
V o l u m e 17, N u m b e r 9, 1997
Cycle
Inseminated
Pregnant mares mares Post Not Post Not partum foa}ing partum foaling % %
3st
251
123
2rid
113
31
3rd
41
13
>3rd
15
12
Foaling mares Post Not partum foalinc % %
78/251 96/123" 97/132" 58/79' (71)a (78) (73) (73) 75/113 19/31 40/56* 12/16' (66) (61) (71) (75) 27/41 8/13 17/19" 4/8 (66) (61) (89) (50) 9/15 3/12 6/8* 3/3 (60) (25)b (75) (100)
* The occurrence of foaling during 1996 has not yet been recorded for some mares a > b (P< 0.01).
This study indicates that CR and FR of Trotter mares inseminated with fresh semen at the first post partum estrus is not different from CR and FR achievable in any other estrous cycles of the mare. THE FUNCTIONAL COMPETENCE E V A L U A T I O N OF P O L Y M O R P H O N U C L E A R N E U T R O P H I L S IN M A R E S R E S I S T A N T AND S U S C E P T I B L E T O CHRONIC ENDOMETRITIS C.M.Q. Barros 1., M.D. Lopes 1, N.P. Rocha 2, H. Langoni 3 1Department of Animal Reproduction and Veterinary Radiology, *MS student sponsored by FAPESP; 2Department of Pharmacology, IB UNESP; 3 D e p a r t m e n t of Public Health, Botucanu, Sao Paulo Chronic uterine infection represents a major cause of infertility in mares. Most younger mares are capable of spontaneously clearing and induced uterine infection within a few days and are classified as resistant to chronic endometritis. In contrast, mares referred to as susceptible lack the ability to spontaneously clear bacterial contamination of the uterus, resulting in a persistent inflammation that interferes with fertility. It has been suggested that susceptibility to chronic endometritis is associated with an impaired local uterine immune response and other causes. The functional competence evaluation of uterine and blood derived polimorphonuclear neutrophils (PMN) from resistant and susceptible mares to uterine infection were measured for migration responsiveness by use of chamber (filter) assay. Uterine infection was induced with 1x 109 of Streptococcus zooepidemicus in mares during estrus. Six (6) hours after inoculation uterine flush was performed and blood samples were collected in order to obtain PMNs and protein concentration was determined. For a sequential analysis of concentration of MMNs and number of bacteria, uterine flushes were obtained 6,30 and 102 hours after inoculation. The chemotactic responsiveness of the uterine PMNs of susceptible mares (12.75+0.96) was lower than resistant mares (61.64+3.45) for p< 0.01. Pheripheral blood PMNs obtained from mares experimentally inoculated (13.23+11.62) and control mares (11.08_+6.53) had similar chemo-
tactic responsiveness for p > 0.50. protein concentration was similar among groups. In the sequential analysis of the concentration of uterine PMNs and number of bacteria from the uterine flushings, differences among groups at fixed moments were observed. Six hours after inoculation there was an increase in the concentration of PMNs and number of bacteria in both groups. Thirty (30) hours after inoculation, there was a decline in the concentration of PMNs in both groups, but a different response with regard to the number of bacteria. At 102 hours after inoculation, the number of bacteria decreased in resistant mares and increased in susceptible mares. The PMNs concentration decreased in resistant mares and remained elevated in susceptible mares. The result of this study suggested that uterine derived PMN obtained from mares susceptible to endometritis have a compromised ability to migrate. L A R Y N G E A L D Y S F U N C T I O N IN HORSES: E F F E C T S ON RESPIRATORY FUNCTION DURING EXERCISE R. Rose, R. Christley, D. Evans, D. Hodgson Equine Performance Laboratory, University of Sydney, Rural Veterinary Centre, Camden, Australia The relationship between different grades of laryngeal function, as assessed by endoscopy at rest, and the measurements of indices of gas exchange and exercise capacity was assessed during a standardized treadmill exercise test in 149 horses. Horses underwent a rapid incremental exercise test with the treadmill set at a slope of+10 per cent. The test commenced at 4 m/s (3 min) with subsequent 60 s steps at each of 6, 8, 10, 11 and 12 m/s or until horses were unable to keep pace with the treadmill. The duration of the run was recorded as an objective measure of exercise capacity. Horses with abnormalities other than idiopathic laryngeal hemiplegia (ILH) were excluded from the study and laryngeal function was graded according to the description given by Lane (1995). There was no significant difference in age, weight, maximum oxygen uptake, maximum carbon dioxide production, maximum respiratory exchange ratio, maximum oxygen pulse, and run time between the different grades of ILH. Blood lactate concentration at 10 m/s was greater in horses with grade 5 laryngeal function than in horses with lesser grades of laryngeal function. Minimum PaO2 and SaO2 w e r e lower, and maximum PaCO2, higher in horses with grade 4 and 5 laryngeal function than other grades. Horses with grade 4 function had a lower minimum CaO 2 than horses with other grades. Minimum PaO2 decreased from grades 1 and 2 to grades 4 and 5. The minimum alveolar ventilation was lower in horses with grades 4 and 5 laryngeal function compared to other grades. There is some difficulty in comparisons across groups of horses with differing abilities and levels of fitness. However, the results of this study indicate that endoscopic assessment of laryngeal function at rest using a simple grading system provides an indication of dynamic changes in ventilation and the effects on gas
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exchange during exercise. From the data we suggest that horses that have some movement of the left arytenoid cartilage but are unable to achieve full abduction have similar ventilatory effects and blood gas responses during maximal exercise to those with complete paralysis. Some horses with grade 3 laryngeal function had blood gas results similar to those of horses with grades 4 and 5 laryngeal function, indicating that discrepancies may occur between the resting assessment of laryngeal function and laryngeal function during strenuous exercise. Arterial blood gas analyses provides functional physiological information that can be useful to supplement or complement resting and exercising upper respiratory tract endoscopy.
PHYSIOLOGICAL PARAMETERS IN O V E R T R A I N E D S T A N D A R D B R E D PACERS M.J. Hamlin, W.G. Hopkins Departmentof Physiology,University of Otago, Dunedin NZ A 34-week longitudinal study was conducted to investigate the effects of overtraining on physiological parameters in 10 standardbred pacers. The first 24 weeks involved jogging, conventional training, and taper training. For the next eight weeks the horses received up to a 61% greater weekly workload, followed by two weeks of reduced training. Horses performed a 2400 m time trial every second week, in which they covered the first 1200 m in 105 seconds and the final 1200 m as fast as possible. Every other week horses performed a sub maximal exercise test, in which they exercised for 3 min at each of three velocities; recordings of heart rate were used to interpolate velocities at l g0 and 200 min-1 (VI80 and V200). Venous blood was sampled at rest, and at 1 and 4 rain after exercise tests. Significant increases in final 1200 m time (p = 0.03- 0.5) and decreases in peak velocity (p = 0.02 - 0.04) by the end of the period of heavy training suggested overtraining had occurred. Body weight showed a trend downward as the study progressed, but during overtraining weight showed a trend downward as the study progressed, but during overtraining weight dropped significantly below the trend line (p=0.03 - 0.05). Other changes in physiological parameters during overtraining included significant increases in blood lactate concentration after the time trial and submaximal test, venous P02, and venous HC03 - (p = 0.001 - 0.05). Significant decreases were found in mean velocity, V 180, V200, plasma cortisol concentration after the time trial, post-exercise hematocrit, and total C02 (p= 0.001 - 0.05). Some of these measures may be useful for detecting overtraining in standardbred pacers. Measures that showed no change with overtraining or were otherwise unreliable included maximal and recovery heart rate, basal plasma cortisol, plasma and red cell volume, creatine kinase, aspartate aminotransferase, and venous
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Na+, K+, pH and PC02. The changes in physiological parameters during this study occurred only after unusually large increases in training. It is not clear whether these markers would be useful in detecting overtraining in more realistic training situations. EXTRAVASCULAR LUNG WATER IN T H E HORSE AT REST A N D EXERCISE P.A. Wilkins, R.D. Gleed, A. Dobson DepartmentsofClinicalSciencesand Physiology,College of Veterinary Medicine, CorneUUniversity, Ithaca, NY, USA Unlike most other athletic species, the maximally exercising horse experiences both pulmonary hypertension and arterial hypoxemia. These findings suggest that the pulmonary vascular bed is at the limit of its ability to accommodate increased cardiac output and oxygen exchange in the maximally exercising horse. We have demonstrated that capillary pressure also increases with exercise in the horse, most closely following pulmonary wedge pressure. The measured values of pulmonary capillary pressure are not as great as those estimated by the midpoint between the pulmonary artery and wedge pressures. Nevertheless, capillary pressure is high enough to cause the formation of interstitial extravascular lung water. Should this accumulate in the lung, it might disrupt blood oxygenation by inhibiting diffusion. We measured extravascular lung water, as the extravascular thermal volume, by a dual indicator dilution technique using electrical conductivity as the intravascular marker and heat as the total water marker in 6 Standardbred horses on a treadmill. Thermal volume determinations were made at rest, 1.8 m/s, 75% heart rate maximum and 100% heart rate maximum. Extravascular thermal volume increased from ~5.9 ml/kg at rest to ~7.7 ml/kg at 75% HRmax, parallel to the increase in pulmonary artery pressure, but was measured at ~5.4 ml/kg at 100% HRmax. We conclude that extravascular thermal volume, a measure of extravascular lung water, increases under conditions of maximal exercise and may contribute to the arterial hypoxemia seen in exercising horses. Supported by the Grayson Jockey Club Research Foundation and Harry M. Zweig Memorial Foundation. T H E USE OF STICK D I A G R A M S IN THE K I N E M A T I C S T U D Y OF LAME HORSES E Audigir, E Pourcelot, C. Deguerce, M.J. Denoix, E.A. Biomrcanique du Cheval EcoleVEtrrinaired"A1fort, Maisons-Alfort Cedex, France Visual examination of the lame horse in motion is difficult because locomotion is a complex phenomenon requiring simultaneous assessment of many events. The Equine Kinematic Analysis System (E.K.A.S.) developed at the veterinary school of Alfort allows to record in outdoor conditions the 3D motion of both sides of the horse. Kinematics
of sound and lame horses were obtained while trotting guided by hand in the conditions of the clinical lameness examination. Each horse was fitted with skin markers. The motion of the markers were collected using specific softwares and presented as stick diagrams. The stick diagrams of a sound trotting horse are shown. Differences are found in the trajectories of the proximal markers between non painful and painful lamenesses. Stick diagrams allow also to quantify the decrease of protection in the lame hind limb, changes in the hoof trajectories and changes with the lameness degree. Providing a general view of limb movement patterns stick diagrams could help to understand the way horses adapt their movements during lameness. SOME E F F E C T S OF E Q U I N E S O M A T O T R O P I N IN HORSES R. Rose, L. Creis, A. Dart, D. Hodgson Rural Veterinary Centre, University of Sydney, PMB, Australia Equine somatotropin or growth hormone has only been studied relatively recently in the horse. A number of experiments have shown that the pattern of secretion of growth hormone is pulsatile in nature. Growth hormone has a range of important physiological effects and studies in many species, including humans, have indicated exogenous administration of growth hormone can have beneficial effects on immune function and body condition. Equine somatotropin (eST) has been shown to be effective in improving the body condition of mares by anticatabolic actions. Somatotropin and associated growth factors such as the insulin like growth factor family of proteins are major determinants of tissue growth and repair. There is evidence in the literature to suggest that somatotropin can increase collagen deposition and maturation rate. Administration of somatotropin releasing factor has been shown to increase cutaneous wound strength and collagen maturation in granulation tissue. Two major studies were undertaken in our laboratory to evaluate possible adverse effects of eST and to assess any beneficial effects in a model of wound healing. Because it is possible that higher doses than those recommended may be inadvertently administered in therapeutic situations, clinical, hematological and biochemical indices were evaluated in Thoroughbred mares administered 2.5 and 5 times the recommended normal dose rate. The highest dose rate of 100 ug/kg was selected because for a 500 kg horse, this dose rate would represent a full vial of the eST, which is the likely maximum that would be inadvertently administered. The only significant effects associated with the eST administration were on plasma glucose and heart rate. However, with both these measurements all values fell within the normal ranges and there was no obvious hyperglycemia or tachycardia following administration of eST. The wound healing study utilized a model of full thickness skin wounds 2.5cm x 2.5 cm (area 6.25cm2), created on the dorsomedial aspect of either the left metacarpus and metarsus or right
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This involves supervising pre-export disease testing, isolation and/or quarantine where required, completion of all necessary documentat i o n / h e a l t h certification, a r r a n g i n g and co-ordinating transport and liasing with government veterinarians in the home country and that of the country to be visited. Considerable experience of making such arrangements for horses participating at past major events involving, for example, some 250 horses at the Olympic Games in Seoul in 1988, in Barcelona in 1992, and Atlanta in 1996 and some 700 horses at the World Equestrian Games (WEG) in The Hague in 1994 are reviewed. Preliminary plans for the Sydney Olympic Games in 2000 are discussed with particular emphasis on the climate, length of journey involved, travel stress and training facilities during pre- and post-import quarantine. THE PHYSICAL EFFECTS OF T R A N S P O R T ON C O M P E T I T I O N HORSES N. K. Waran Institute of Ecology& ResourceManagement,University of Edinburgh, Scotland, UK Due to the financial and sentimental value attached to horses, they are perceived to enjoy a much higher standard of transport than most farm animals. Although transport may not subject the experienced competition horse to the same psychological stresses that result from fear of a novel experience, recent studies at the University of Edinburgh have shown that the physical effects of road transport on horses include increased heart rate, changes in body posture, and an increase in energy expenditure. These results suggest that for experienced performance horses, transport in a lorry is associated with average heart rates higher than those recorded in a stationary vehicle, and changes in body posture that are thought to be caused by problems of balance experienced in the back of a moving vehicle. Energy expenditure during transport was measured as similar to that required when the horse is walking, and transporting horses facing away from the direction of travel (backward) is associated with lower energy expenditure than transporting them facing in any other direction. This paper discussed the impact these results may have on horses transported for competition. T H E P H Y S I O L O G I C A L E F F E C T S ON H O R S E S OF A I R T R A N S P O R T A T I O N IN AN ENCLOSED C O N T A I N E R J.R. Thornton University of Queensland,Australia An enclosed insect proof container designed and built for the air transportation of horses was evaluated for its physiological effect on the horses during flights of 12 to 24 hours duration. Three horses traveled in the container, had their heart rates monitored continuously and had blood samples collected regularly for hematological and chemical analyses and determination of
g-endorphin. The air within the container was sampled regularly for bacterial and fungal populations and the air temperature and humidity recorded inside and outside the container. On one flight insect traps were installed in the contalner. On the two flights of 12 hours a regular open container with three horses was used as control. During the flights heart rates were regular and generally in the range of 35 to 55 beats per minute with increases to 80 to 100 during take off and landing or when the horses were restrained for blood collections. There were no significant accumulations of bacteria or fungi in the container and the temperature and humidity remained constant or slowly decreased with the duration of the flights. Temperatures inside the container were 3 to 6~ higher than that in the cargo hold. The heart rates and blood analyses, including the g-endorphin showed no evidence of undue stress or infection in the horses and when compared to the controls on the two 12hour flights there was no significant effect of the enclosed container. It was concluded that the enclosed container was a suitable means of transporting horses for plane flights for periods of at least 24 hours duration.
ULTRASONOGRAPHIC EVALUATION OF E N D O U T E R I N E FLUID C L E A R A N C E IN M A R E S A F F E C T E D BY E N D O M E T R I T I S AND T R E A T E D W I T H O X Y T O C I N O R PGF2a S. Barbacini, V. Marchi, G. Zavaglia Studio Veterinario Cristella, S. Daniele Po, Cremona, Italy Among the species of zootechnical interest the equine is undoubtedly the one with the lowest reproductive efficiency. Persistent and recurrent post-insemination endometritis is one of the most important causes of this phenomenon. The latest research has found reduced myometrial activity as one of the major causes responsible for this reproductive pathology. In fact the inability of clearing the inflammatory cells (ex WBC) and the exudate that normally appear in the uterine lumen after an insemination, causes the persistence of this inflammation. Because of this, the embryo, after its descent into the uterus, doesn't find a compatible environment with its survival. The broodmares are at the moment classified as "susceptible" or "resistant" according to their ability of clearing the uterus. Moreover, recent clinical observations have shown a bigger incidence of this pathology when using frozen semen. Hypothetically this is caused by the protein alterations observed in frozen/thawed spermatozoa and by the almost total seminal plasma removal during the freezing procedure. Thirty broodmares with persistent and recurrent postinsemination endometritis, inseminated by means of frozen semen, have been considered. All the broodmares were inseminated between 12 hours before and 6 hours after ovulation. Eight hours after insemination, they were
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treated with uterine lavage using saline solution until the fluid recovered was completely clear. Immediately after the treatment we checked by uhrasonography if fluid was still present in the uterine lumen. The broodmares with presence of intrauterine fluid, were treated with a placebo (2 ml 0.9% NaCl I.V.). Thirty minutes after the placebo treatment, the ones presenting an amount of intrauterine fluid similar (+_20%) to that found before the injection were divided into two different groups: group OX=20UI I.V. of oxytocin; group PG=0.075 mg I.M. of d-clorprostenoL The amount of uterine fluid in the group treated with OX was ecographically monitored after 1530-60 minutes - - while that in the group treated with PG was checked after 15-30-60 minutes and 5 hours. The broodmares in which the fluid removed from the uterus was less than 90%, were immediately subjected to the opposite treatment to evaluate if there was any different reaction to oxytocin or prostaglandin.
OCCURRENCE AND ENDOCRINE CHARACTERISTICS OF ANOVULATORIC F O L L I C L E S IN THE MARE B. Kaiser1, M. Koene1, J. Swagemakersl , H. Bader2, H.O. Hoppen3 lTier~irztliche Klinik fiir Pferde, M0hlen; 2lnstitut flit Reproduktionsmedizin, Tier~irztliche Hochschlule, Hannover;3ChemischesInstitut,Tier'al'ztlicheHochschule, Hannover, Germany In a clinical study, covering about 1000 broodmares in different AI centers, the occurrence of anovulatoric follicles during the breeding season 1997 was evaluated. All mares were closely monitored gynecologically every other day for follicular dynamics and estrus behavior. Mares with follicles that exceed 50 nun in diameter and do not ovulate during the normal estms period (> 5 days) are divided into three treatment groups. In treatment group A, the follicles are punctured transabdominally through the fossa paralumbaris at the ypsilateral side and the follicular fluid is collected by aspiration. In treatment group B, the follicles are punctured using a laparoscopic technique and a biopsy sample of the follicular wall is taken. In treatment group C, the mares are not treated but routinely checked until the follicle is luteinized or becomes atretic. For a control group D, preovulatory follicles (>35 nun) from mares with normal follicular dynamics and estrus behavior are punctured, using the same technique as in group A. All mares are inseminated in the first established estrus after treatment and a possible pregnancy is confirmed at day 18 after insemination by ultrasonography. From the groups A, B and D, serum samples are taken at the day of puncture and in the first established estrus after the puncture. From group C, serum samples are taken at the day of the confirmed diagnosis and in the first estrus occurring after atresia. The follicular fluid and serum are analyzed for the following parameters; Estradiol, progesterone, E/P-ratio, testosterone, LH, FSH, LH/ FSH-ratio and prolactin; routine diagnosis also includes chemistry panel and electrolytes in follicular fluid and serum and vitamins A, E and
Volume 17, Number 9, 1997
folic acid in the serum. The biopsy of the follicular wall is histologically prepared for detection of cell layers and luteinization. The etiology of erratic follicles, comparison of the components of anovulatoric and physiological follicles and follicle puncture as a therapy to shorten the time ofdiestrus in these mares will be discussed, as most of the mares with erratic follicles do not reset to any hormone treatment, i.e. hCG, GnRII, prostaglandins or long-term gestagen therapy.
T W I N SQUEEZING AT D I F F E R E N T GESTATIONAL AGES: ULTRASONOGRAPHIC TECHNIQUES AND M O N I T O R I N G C. Rognoni,M. Livini A survey on the management of 52 twin pregnancies. Video demonstrations about the use of ultrasonography to separate twins and the selective squeezing of one of the two, the typical ultrasound imaging of twins and rupturing of the wall will be shown in real time. Until day 16 the scanner probe is used to shake and separate adjacent blastocyst, otherwise difficult to distinguish and scan separately. Once separated the blastocysts are placed wide apart within the uterine horus to allow a safe and easy squeezing. After day 18 the blastocyst became more and more difficult to separate and selectively squeeze; during this period; however, it is possible, using the scanner probe, to rupture the blastocyst wall of only one of the two twins without separating them. Success rate is about 30% at gestational age more than 35 days and 70% at a gestational age of less than 16 days. This technique can also be useful to differentiate between endometrial cysts and blastocysts.
FLUID ACCUMULATION IN THE U T E R I N E LUMEN OF MARES F O L L O W I N G INSEMINATION W I T H FROZEN SEMEN
between 100X106 and 175x106 progressively motile, morphologically normal spermatozoa. The volume of inseminate ranged from 0.5 ml to 4.0 ml depending on the stallion. All mares were negative for culture and inflammatory cells on an endometrial swab taken prior to insemination. The depth of any uterine fluid was recorded as the maximal depth, usually in the uterine body during the visits 24 hours before insemination/ ovulation. Mares were treated with uterine layage, oxytocin and intrauterine antibiotics 12 hours after insemination. A small amount of fluid (usually less than 2 cm) was detected before inseminating in 21 (26%) of estrous periods. More mares had fluid detected 12 hours after insemination (57 [80%] of estrous periods, P<0.01). By 24 hours, although this number had decreased to 31 (38%), it was still greater than before insemination (P<0.01). When examined 48 hours after insemination, fluid was only detected in 2 (2.4%) cases. There appeared to be no relationship between fluid production and volume of inseminate within the small range of insemination volumes used in this study. The per cycle pregnancy rate was greater in mares with no visible uterine fluid 24 hours after insemination (71 vs 55%; P<0.05). In conclusion, this study has shown that uterine fluid is a frequent finding 24 hours after insemination with frozen semen, but this fluid rarely persists longer than 24 to 48 hours after insemination. Mares with uterine fluid persisting for at least 24 hours after insemination have a reduced pregnancy rate.
E F F E C T OF PROSTASOMES ON THE F O R W A R D M O T I L I T Y O F HORSE SPERMATOZOA BEFORE AND AFTER FREEZING M. Rubei, M. Moroni1, A. Minelli1, A.M. de Cosmo2, S. Degl'Irmocenti Istitutodi Ostetriciae GinecologiaVeteiinaria-Universit~ degli; Studi di Perugia; 1Departimentodi Biologia Cellulare e Molecolare, Universi~ degli Studi di Perugia; 2Facol~ di Medicina Veterinaria, Universit~degli Studi di Camerino (MC)
C. Bamini, S. Ventura, J.E Pycock There has been a considerable increase in the last few years in the use of frozen equine semen. While it is well known that the breeding of mares is followed by an acute inflammatory response including production of uterine fluid, little is known about this inflammatory response in mares inseminated with frozen semen. In view of the importance of uterine clearance in the persistence of acute post-breeding endometritis, and anecdotal reports of the appearance of uterine fluid following insemination with frozen semen, it was decided to investigate the accumulation of fluid in the uterine lumen of a large number of mares after insemination with frozen semen. During the 1996 breeding season, 59 mares were inseminated with frozen semen from five stallions at 82 estrous periods. All the stallions had, after thawing of a frozen semen sample, > 40% progressively motile spermatozoa. The spermatozoa concentration before freezing varied from 50xl06/ml to 500x 106/ ml and the inseminating volume varied from 0.5 ml to 4 ml to give a final insemination dose of
Seminal fluid contains the secretion of the testis, epididymis and sex accessory glands. Its components can influence the motility and the metabolism of spermatozoa with opposite effects: either a positive or a negative influence on the fertilizing capacity of the sperm has been reported. The presence of membrane particles in the seminal plasma of rabbit, ram, bull and man has been already documented. The vesicles isolated from horse seminal plasma present on their surface enzyme activities which, according to literature, are considered to be markers of human prostasomes. In addition, the anatomy of the horse prostate closely resembles the human gland; therefore, these equine particles might be, unless further investigation will prove otherwise, of prostatic derivation. The particles in rabbit seminal fluid originate from epididymis and cause sperm decapacitation and inhibit the fertility whereas human prostasomes are involved in stimulation of forward motility of the spermatozoa. However, the functional importance of the particles in seminal
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fluid are contradictory since these vescicles differ in their detailed biochemical composition. Electron microscopy of particles present in horse seminal plasma shows vesicular structures (100m diameter) surrounded by a bilaminar membrane containing unevenly distributed floccular material. Our analysis of the motility of horse spermatozoa with and without added prostasomes do not reveal any dramatic change. Preliminary investigation carried out with frozen semen samples seem to lead to different results. In fact, when frozen spermatozoa are thawed in the presence of prostasomes, there is a major change in the sperm motility pattern indicating an induction of the forward motility. Further studies will be needed to assess this preliminary positive effect on the fertilizing capacity of the horse frozen semen. PREGNANCIES AFTER EMBRYO T R A N S F E R IN A N E S T R U S R E C I P I E N T MARES: N A T U R A L AND A R T I F I C I A L M O D I F I C A T I O N S OF OVARIAN AND H O R M O N A L ACTIVITIES D. Lagneaux, E. Palmer Haras Nationaux and INRA Mares in anestrus were used as a recipient for non-surgical embryo transfers. They were treated with 25 mg estradiol-benzoate daily, during three days up to the day of ovulation of the donor, then with 44 mg Altrenogest daily from the day after the ovulation of the donor mare: up to Day 100 in EXP 1 or up to the first day endogenous progesterone levels were analyzed and ovarian activity, follicular growth and luteinized unruptured follicles (LUF) were detected by ultrasound echography. In EXP 1, two groups of pregnant anestrus recipient mares were (I-A): stimulated with lighting procedure (16 hours daily) (N=4) or (I-B): control, under natural lighting conditions (N=7). In EXP 2, mares were allotted in two groups: (2-A): treated daily with eLH, 25 mg CEG (i.m.) (Crude Equine Gonadotrophin) up to day 70 or when progesterone level was higher than 4 ng/ml (N=5), or (2B): control without treatment (N=7). In EXP 3, mares were also allotted in two groups, (3-A): treated two times (Day 40 and Day 47) with 4.5 mg (6 times more than usual) (i.m.) e FSH (purified Crude Equine Gonadotrophin) (N=10), or (3-B) control, without treatment (N=9). Results of these three experiments are showed in this table: groups
I-A
I-B
2-A
2-B
3-A I 3-B
pregnaneie~ I 4 I 7 I s I 7 I 1~ 9 Day 1st ov ]73_+24154_+6158_+16168_+22155_+12170+16 1st Day progt 175-+21158+6163-+21/71_+201 60_+14172_+13 Day 55* 2/4 2/7 4/5 2/7 7/10 I 2/9 Day 70* 2/4 / 7/7 / 4/5 4/7 I 9/10 4/9 Day 85* 2/4 7/7 4/5 5/7 9/10 4/9
Treatmentl-A= 16 h light Treatment2-A=eLH daily Treatment3-A=2X6 eFSH TreatmentsB= controls #= progesterone>4ngper ml *ovulationor LUF Mean of the first days of ovulation and endogenous progesterone level in control groups (1, 2 & 3 - B) suggested that ovarian activity is different from one mare to the other during early
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pregnancy. Stimulated groups (1, 2 & 3 -A) were provided to stimulate ovarian activity and induce ovulation or luteinization. Neither lighting nor long period eLH treatment seem efficient. This suggests that hormonal environment and the response to stimulation of pregnant anestrus mares are different than of normal anestrus mares in which light or eLH are efficient to induce ovulations. The high dose of eFSH (3-A) seems efficient to induce early ovulations or LUF in anestrus recipient mares compared to control group (3-B). Treatment of eFSH stimulates follicular growth and when follicles larger than 25 m m diameter are obtained, eCG induces their luteinization or ovulation (before days 55 or 70 in 7 or 9 cases out of 10). Efficiency of such a treatment is only 90% at day 70 which doesn't allow interruption of exogenous Altrenogest treatment except if previous results showing that exogenous supplementation is not useful up to day 70 are confinned. PROBLEMS ASSOCIATED WITH O V A R I A N ACTIVITY IN B A R R E N AND P O S T P A R T U M M A R E S E A R L Y IN THE BREEDING SEASON R Nagy 1, Gy- Huszenicza1, J. Jnhfisz2 L. Solti 1, M. Kulcsfir1 1Department at'Obstetricsand Reproduction,2Research Insititute, University of Veterinary Science, Budapest, Hungary In two studies ovarian activity was monitored with regular teasing, ultrasonography and blood sampling for progesterone in the winter and spring period. Of the 50 barren mares studied 41 (82%) stopped cycling during the autumn and winter period. The anovulatory season started before December 1 in 61% of the mares (25 animals). In 16 mares (39%) cyclic ovarian activity ceased after December 1. Of these 16 mares 5 (12.2%) stoppedcycling after January 1 (Jan. 4, Jan. 10, Jan. 11, Feb. 7). The lengths of the anovulatory season varied between 20 to 172 days. There was a strong correlation between the time of cessation and the time of resumption of cyclic ovarian activity (P <0.01). Mares that stopped cycling before December 1 resumed cyclic ovarian activity one month later than mares in which the anovulatory season began after December 1 (98.6+-30.5 days and 67.9+-35.0 days after January 1 for mares that stopped cycling before and after December 1, respectively). The first ovulation was not preceded by overt estrous (silent heat) in 64.7% of the cases (22 of 34 mares). Overt estrous was observed without ovulation in 8 mares (22.9%). Thirty-eight mares were followed in the postpartum period. Foalings occurred in the first three months of the year. The season of foaling strongly influenced ovarian activity and sexual behavior. Only 71.1% of the mares (27 animals) ovulated within 20 days post partum. Foal heat was observed in 17 mares (51.5%). The mean intervals from parturition to the 1st and 2nd ovulation and to 1st overt estrous were 25.4+-25.74 days; 52.6+-26.42 days and 35.6_+34.38 days from f o a l i n g . The
interovulatory interval was also longer then expected (32.2+ 19.44 days). In some cases the lutelysis of the first corpus lutenm was followed by delayed follicular growth resulting in a prolonged follicula phase (17.8 +/- 18.06 days). It is concluded that seasonal reproductive activity is one of the most important research and practical problems in equine reproduction. Management of both barren and postpartum mares early in the breeding season gives alot of problems to veterinarians each year. The use of mare-side progesterone assays (like the semiquantitative tube test used by us) can provide a powerful mean to determine the background of anestrous early in the breeding season both in barren and postpartum mares. H A P T O G L O B I N IN T H O R O U G H B R E D MARES ANTE AND POST P A R T U M C. Gothe,A. Vandenhouten,C. Lippeheide, H. Sommer InstituteofAnatomy,Physiologyand Hygieneof Domestic Animals, Dept. Hygiene, Bonn, Germany Parturation is always a critical stage for both mares and foals. Therefore blood parameters are discussed to indicate possible difficulties in time. Concentration levels of Acute-Phase-Proteins change rapidly in case of inflammations, infections or tissue damages. For this reason they are used as inflammation markers to determine the status of health in animals. The aim of the study was to observe the physiological course of haptoglobin around the time of foaling. Furthermore, we investigated whether haptoglobin (Hp) is a suitable parameter in preventive health control in mares during this time. Blood samples were taken from 8 clinically healthy thoroughbred mares four and two weeks ante partum and at day 1,3, 5, 7 and 14 post parturation between 8-9 a.m. For the determination of Hp in the plasma the automated nephelometer analyser 100 (Behring Diagnostics, Germany) was used. Our results show that haptoglobin always increased immediately after foaling. Hp-values at day 1 and 3 p.p. were significantly higher than a.p. and decreased slowly during the following 14 days to their fundamental basis. Values ranged between 9 and 97.4 mg/dl in mares with "normal" parturation and healthy foals (n=6). Hp-values increased up to 130mg/dl in mares with still birth (n= 1) and retained placenta (n= 1). No correlation between haptoglobin and other biochemical parameters (aspartatamino-transferase, lactate-dehydrogenase, creatinkinase, total protein, albumin, globulin, urea, bilirubin, gamma-GT, cholesterol, phosphor, magnesium, calcium) could be found. So, it seems to be useful to investigate haptoglobin as a parameter for ante partum health control in further studies. E F F E C T S OF A N T I S P E R M ANTIBODIES ON THE F U N C T I O N A L I N T E G R I T Y OF S T A L L I O N SPERMATOZOA C. Lee, H.S. Joe, H.W. Moment I, B.G. Crabo,
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SCIENCE
Hunter University of Minnesota, 1University of Wisconsin A.G.
Antisperm antibody is one of the factors associated with infertility or subfertility in domestic animals. Antisperm antibody interferes with the reproduction in various ways including sperm motility, binding, capacitation, penetration, and zygote development, etc. Antibody to nine major polypeptide groups (85, 51-59, 34-37, 26, 2324,16-19, 15, 10, and 5-6 kDa) of stallion sperm membrane protein were produced in rabbits. ELISA, indirect immunofluorescent antibody test, sperm motility, Glass wool/Sephadex filtration, zone-free hamster oocytes test were used to characterize and determine the effects of the antisperm antibodies on stallion spermatozoa. Antiserum to 85 kDa polypeptide was localized in the acrosome, to 51-59 kDa in the sharp line on the tail, to 34-37 kDa in the tail midpiece, to 26 kDa in the head, midpiece, and neck, to 23-24 kDa in the head and midpiece, to 16-19 kDa in the head and midpiece, to 15 kDa in the head and midpiece, to 10 kDa in the equatorial and tail, and to 5-6 kDa in the head and tail. Motility data were collected during three periods, before, during and after in vitro capacitation process. In glass wool/Sephadex filtration, the antisera to 85, 34-37, 23-24 kDa increased the filtered stallion spermatozoa, and the antisera to 16-19 and 5-6 kDa decrease the filtered spermatozoa comparing with the control serum produced against PB S. In zone-free hamster oocytes test, the antisera to 23- 24, 16-19, 15, 10, 5-6 kDa showed a decreased rate in the penetration test when spermatozoa were exposed to antibodies before in vitro capacitation. When stallion spermatozoa were exposed to antibodies after in vitro capacitation, the antiserum to 23-24 kDa only decreased the result of penetration. More detail data was shown at the presentation. F E R T I L I T Y AT T H E F I R S T POST P A R T U M ESTRUS C O M P A R E D W I T H F E R T I L I T Y AT T H E F O L L O W I N G E S T R O U S CYCLES IN F O A L I N G M A R E S AND W I T H F E R T I L I T Y IN NON F O A L I N G M A R E S F. Camillo, P.Marmorini,S. Romagnoli,L. Vannozzi, M. Bagliacca Istituto di Patologia Speciale e Clinica Chirurgica Veterinaria, Universith di Pisa, Italia The aim of this study was to compare conception rate (CR) (pregnant mares/inseminated mares) and foaling rate (FR) (foaling mares/ pregnant mares) at the first post partum estrus with fertility at the following estrous cycles in foaling mares and with fertility of non foaling mares (maiden+barren). Estrous Trotter mares were inseminated, through > 6 consecutive breeding seasons, with fresh semen of 3 fertile stallions on alternate days between the ultrasound detection of a follicle 2 35 m m and ovulation. Pregnancy diagnosis was performed with ultrasound at day 14 post ovulation and, when possible, confirmed once a week until about day 35. Parturition was checked by consulting the official Italian Stud Book of Trotters. Results are reported in the following table.
V o l u m e 17, N u m b e r 9, 1997
Cycle
Inseminated
Pregnant mares mares Post Not Post Not partum foa}ing partum foaling % %
3st
251
123
2rid
113
31
3rd
41
13
>3rd
15
12
Foaling mares Post Not partum foalinc % %
78/251 96/123" 97/132" 58/79' (71)a (78) (73) (73) 75/113 19/31 40/56* 12/16' (66) (61) (71) (75) 27/41 8/13 17/19" 4/8 (66) (61) (89) (50) 9/15 3/12 6/8* 3/3 (60) (25)b (75) (100)
* The occurrence of foaling during 1996 has not yet been recorded for some mares a > b (P< 0.01).
This study indicates that CR and FR of Trotter mares inseminated with fresh semen at the first post partum estrus is not different from CR and FR achievable in any other estrous cycles of the mare. THE FUNCTIONAL COMPETENCE E V A L U A T I O N OF P O L Y M O R P H O N U C L E A R N E U T R O P H I L S IN M A R E S R E S I S T A N T AND S U S C E P T I B L E T O CHRONIC ENDOMETRITIS C.M.Q. Barros 1., M.D. Lopes 1, N.P. Rocha 2, H. Langoni 3 1Department of Animal Reproduction and Veterinary Radiology, *MS student sponsored by FAPESP; 2Department of Pharmacology, IB UNESP; 3 D e p a r t m e n t of Public Health, Botucanu, Sao Paulo Chronic uterine infection represents a major cause of infertility in mares. Most younger mares are capable of spontaneously clearing and induced uterine infection within a few days and are classified as resistant to chronic endometritis. In contrast, mares referred to as susceptible lack the ability to spontaneously clear bacterial contamination of the uterus, resulting in a persistent inflammation that interferes with fertility. It has been suggested that susceptibility to chronic endometritis is associated with an impaired local uterine immune response and other causes. The functional competence evaluation of uterine and blood derived polimorphonuclear neutrophils (PMN) from resistant and susceptible mares to uterine infection were measured for migration responsiveness by use of chamber (filter) assay. Uterine infection was induced with 1x 109 of Streptococcus zooepidemicus in mares during estrus. Six (6) hours after inoculation uterine flush was performed and blood samples were collected in order to obtain PMNs and protein concentration was determined. For a sequential analysis of concentration of MMNs and number of bacteria, uterine flushes were obtained 6,30 and 102 hours after inoculation. The chemotactic responsiveness of the uterine PMNs of susceptible mares (12.75+0.96) was lower than resistant mares (61.64+3.45) for p< 0.01. Pheripheral blood PMNs obtained from mares experimentally inoculated (13.23+11.62) and control mares (11.08_+6.53) had similar chemo-
tactic responsiveness for p > 0.50. protein concentration was similar among groups. In the sequential analysis of the concentration of uterine PMNs and number of bacteria from the uterine flushings, differences among groups at fixed moments were observed. Six hours after inoculation there was an increase in the concentration of PMNs and number of bacteria in both groups. Thirty (30) hours after inoculation, there was a decline in the concentration of PMNs in both groups, but a different response with regard to the number of bacteria. At 102 hours after inoculation, the number of bacteria decreased in resistant mares and increased in susceptible mares. The PMNs concentration decreased in resistant mares and remained elevated in susceptible mares. The result of this study suggested that uterine derived PMN obtained from mares susceptible to endometritis have a compromised ability to migrate. L A R Y N G E A L D Y S F U N C T I O N IN HORSES: E F F E C T S ON RESPIRATORY FUNCTION DURING EXERCISE R. Rose, R. Christley, D. Evans, D. Hodgson Equine Performance Laboratory, University of Sydney, Rural Veterinary Centre, Camden, Australia The relationship between different grades of laryngeal function, as assessed by endoscopy at rest, and the measurements of indices of gas exchange and exercise capacity was assessed during a standardized treadmill exercise test in 149 horses. Horses underwent a rapid incremental exercise test with the treadmill set at a slope of+10 per cent. The test commenced at 4 m/s (3 min) with subsequent 60 s steps at each of 6, 8, 10, 11 and 12 m/s or until horses were unable to keep pace with the treadmill. The duration of the run was recorded as an objective measure of exercise capacity. Horses with abnormalities other than idiopathic laryngeal hemiplegia (ILH) were excluded from the study and laryngeal function was graded according to the description given by Lane (1995). There was no significant difference in age, weight, maximum oxygen uptake, maximum carbon dioxide production, maximum respiratory exchange ratio, maximum oxygen pulse, and run time between the different grades of ILH. Blood lactate concentration at 10 m/s was greater in horses with grade 5 laryngeal function than in horses with lesser grades of laryngeal function. Minimum PaO2 and SaO2 w e r e lower, and maximum PaCO2, higher in horses with grade 4 and 5 laryngeal function than other grades. Horses with grade 4 function had a lower minimum CaO 2 than horses with other grades. Minimum PaO2 decreased from grades 1 and 2 to grades 4 and 5. The minimum alveolar ventilation was lower in horses with grades 4 and 5 laryngeal function compared to other grades. There is some difficulty in comparisons across groups of horses with differing abilities and levels of fitness. However, the results of this study indicate that endoscopic assessment of laryngeal function at rest using a simple grading system provides an indication of dynamic changes in ventilation and the effects on gas
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exchange during exercise. From the data we suggest that horses that have some movement of the left arytenoid cartilage but are unable to achieve full abduction have similar ventilatory effects and blood gas responses during maximal exercise to those with complete paralysis. Some horses with grade 3 laryngeal function had blood gas results similar to those of horses with grades 4 and 5 laryngeal function, indicating that discrepancies may occur between the resting assessment of laryngeal function and laryngeal function during strenuous exercise. Arterial blood gas analyses provides functional physiological information that can be useful to supplement or complement resting and exercising upper respiratory tract endoscopy.
PHYSIOLOGICAL PARAMETERS IN O V E R T R A I N E D S T A N D A R D B R E D PACERS M.J. Hamlin, W.G. Hopkins Departmentof Physiology,University of Otago, Dunedin NZ A 34-week longitudinal study was conducted to investigate the effects of overtraining on physiological parameters in 10 standardbred pacers. The first 24 weeks involved jogging, conventional training, and taper training. For the next eight weeks the horses received up to a 61% greater weekly workload, followed by two weeks of reduced training. Horses performed a 2400 m time trial every second week, in which they covered the first 1200 m in 105 seconds and the final 1200 m as fast as possible. Every other week horses performed a sub maximal exercise test, in which they exercised for 3 min at each of three velocities; recordings of heart rate were used to interpolate velocities at l g0 and 200 min-1 (VI80 and V200). Venous blood was sampled at rest, and at 1 and 4 rain after exercise tests. Significant increases in final 1200 m time (p = 0.03- 0.5) and decreases in peak velocity (p = 0.02 - 0.04) by the end of the period of heavy training suggested overtraining had occurred. Body weight showed a trend downward as the study progressed, but during overtraining weight showed a trend downward as the study progressed, but during overtraining weight dropped significantly below the trend line (p=0.03 - 0.05). Other changes in physiological parameters during overtraining included significant increases in blood lactate concentration after the time trial and submaximal test, venous P02, and venous HC03 - (p = 0.001 - 0.05). Significant decreases were found in mean velocity, V 180, V200, plasma cortisol concentration after the time trial, post-exercise hematocrit, and total C02 (p= 0.001 - 0.05). Some of these measures may be useful for detecting overtraining in standardbred pacers. Measures that showed no change with overtraining or were otherwise unreliable included maximal and recovery heart rate, basal plasma cortisol, plasma and red cell volume, creatine kinase, aspartate aminotransferase, and venous
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Na+, K+, pH and PC02. The changes in physiological parameters during this study occurred only after unusually large increases in training. It is not clear whether these markers would be useful in detecting overtraining in more realistic training situations. EXTRAVASCULAR LUNG WATER IN T H E HORSE AT REST A N D EXERCISE P.A. Wilkins, R.D. Gleed, A. Dobson DepartmentsofClinicalSciencesand Physiology,College of Veterinary Medicine, CorneUUniversity, Ithaca, NY, USA Unlike most other athletic species, the maximally exercising horse experiences both pulmonary hypertension and arterial hypoxemia. These findings suggest that the pulmonary vascular bed is at the limit of its ability to accommodate increased cardiac output and oxygen exchange in the maximally exercising horse. We have demonstrated that capillary pressure also increases with exercise in the horse, most closely following pulmonary wedge pressure. The measured values of pulmonary capillary pressure are not as great as those estimated by the midpoint between the pulmonary artery and wedge pressures. Nevertheless, capillary pressure is high enough to cause the formation of interstitial extravascular lung water. Should this accumulate in the lung, it might disrupt blood oxygenation by inhibiting diffusion. We measured extravascular lung water, as the extravascular thermal volume, by a dual indicator dilution technique using electrical conductivity as the intravascular marker and heat as the total water marker in 6 Standardbred horses on a treadmill. Thermal volume determinations were made at rest, 1.8 m/s, 75% heart rate maximum and 100% heart rate maximum. Extravascular thermal volume increased from ~5.9 ml/kg at rest to ~7.7 ml/kg at 75% HRmax, parallel to the increase in pulmonary artery pressure, but was measured at ~5.4 ml/kg at 100% HRmax. We conclude that extravascular thermal volume, a measure of extravascular lung water, increases under conditions of maximal exercise and may contribute to the arterial hypoxemia seen in exercising horses. Supported by the Grayson Jockey Club Research Foundation and Harry M. Zweig Memorial Foundation. T H E USE OF STICK D I A G R A M S IN THE K I N E M A T I C S T U D Y OF LAME HORSES E Audigir, E Pourcelot, C. Deguerce, M.J. Denoix, E.A. Biomrcanique du Cheval EcoleVEtrrinaired"A1fort, Maisons-Alfort Cedex, France Visual examination of the lame horse in motion is difficult because locomotion is a complex phenomenon requiring simultaneous assessment of many events. The Equine Kinematic Analysis System (E.K.A.S.) developed at the veterinary school of Alfort allows to record in outdoor conditions the 3D motion of both sides of the horse. Kinematics
of sound and lame horses were obtained while trotting guided by hand in the conditions of the clinical lameness examination. Each horse was fitted with skin markers. The motion of the markers were collected using specific softwares and presented as stick diagrams. The stick diagrams of a sound trotting horse are shown. Differences are found in the trajectories of the proximal markers between non painful and painful lamenesses. Stick diagrams allow also to quantify the decrease of protection in the lame hind limb, changes in the hoof trajectories and changes with the lameness degree. Providing a general view of limb movement patterns stick diagrams could help to understand the way horses adapt their movements during lameness. SOME E F F E C T S OF E Q U I N E S O M A T O T R O P I N IN HORSES R. Rose, L. Creis, A. Dart, D. Hodgson Rural Veterinary Centre, University of Sydney, PMB, Australia Equine somatotropin or growth hormone has only been studied relatively recently in the horse. A number of experiments have shown that the pattern of secretion of growth hormone is pulsatile in nature. Growth hormone has a range of important physiological effects and studies in many species, including humans, have indicated exogenous administration of growth hormone can have beneficial effects on immune function and body condition. Equine somatotropin (eST) has been shown to be effective in improving the body condition of mares by anticatabolic actions. Somatotropin and associated growth factors such as the insulin like growth factor family of proteins are major determinants of tissue growth and repair. There is evidence in the literature to suggest that somatotropin can increase collagen deposition and maturation rate. Administration of somatotropin releasing factor has been shown to increase cutaneous wound strength and collagen maturation in granulation tissue. Two major studies were undertaken in our laboratory to evaluate possible adverse effects of eST and to assess any beneficial effects in a model of wound healing. Because it is possible that higher doses than those recommended may be inadvertently administered in therapeutic situations, clinical, hematological and biochemical indices were evaluated in Thoroughbred mares administered 2.5 and 5 times the recommended normal dose rate. The highest dose rate of 100 ug/kg was selected because for a 500 kg horse, this dose rate would represent a full vial of the eST, which is the likely maximum that would be inadvertently administered. The only significant effects associated with the eST administration were on plasma glucose and heart rate. However, with both these measurements all values fell within the normal ranges and there was no obvious hyperglycemia or tachycardia following administration of eST. The wound healing study utilized a model of full thickness skin wounds 2.5cm x 2.5 cm (area 6.25cm2), created on the dorsomedial aspect of either the left metacarpus and metarsus or right
JOURNAL OF EQUINE VETERINARY SCIENCE