Molecular typing of enteroviruses: comparing 5'UTR, VP1 and whole genome sequencing methods.

Enteroviruses (EV) commonly cause hand, foot and mouth disease (HFMD), and can also cause potentially fatal neurological and systemic complications. In our laboratory, sequencing 5' untranslated region (UTR) of the viral genome has been the routine method of genotyping EVs. During a recent localised outbreak of aseptic meningitis, sequencing the 5'UTR identified the causative virus as EV-A71, which did not fit with the clinical syndrome or illness severity. When genotyped using a different target gene, VP1, the result was different. This led us to evaluate the accuracy of the two different target genome regions and compare them against whole genome sequencing (WGS). We aimed to optimise the algorithm for detection and characterisation of EVs in the diagnostic laboratory. We hypothesised that VP1 and WGS genotyping would provide different results than 5'UTR in a subset of samples. Clinical samples from around New South Wales which were positive for EV by commercial polymerase chain reaction (PCR) assays were genotyped by targeting three different viral genome regions: the 5'UTR, VP1 and WGS. Sequencing was performed by Sanger and next generation sequencing. The subtyping results were compared. Of the 74/118 (63%) samples that were successfully typed using both the 5'UTR and the VP1 method, the EV typing result was identical for 46/74 (62%) samples compared to WGS as the gold standard. The same EV group but different EV types were found in 22/74 (30%) samples, and 6/74 (8%) samples belonged to different EV groups depending on typing method used. Genotyping with WGS and VP1 is more accurate than 5'UTR. Genotyping by the 5'UTR method is very sensitive, but less specific.

The immunolocalization of Galectin-1 and Progesterone-Induced Blocking Factor (PIBF) in equine trophoblast: Possible roles in trophoblast invasion and the immunological protection of pregnancy.

INTRODUCTION: The proteins galectin-1 and Progesterone Induced Blocking Factor (PIBF) are present on human and murine trophoblast and are thought to influence both immunomodulation and trophoblast invasion. In equids, the invasive component of the placenta, the endometrial cups, stimulate maternal cell-mediated and humoral immune responses. It was therefore of interest to know if galectin-1 or PIBF could be immunolocalised to the invasive and/or non-invasive components of the equine placenta. MATERIALS: Horse and mule (♀ horse X ♂ donkey) embryos and placental tissues between Days 12 and 124 of gestation were stained immunohistochemically with antibodies raised against galectin-1 and PIBF. RESULTS: Galectin-1 stained the non-invasive trophoblast between Days 15 and 20 but thereafter stained only the invasive trophoblast cells of the chorionic girdle, both before and after they invaded the endometrium to form the endometrial cups. PIBF, on the other hand, stained both the invasive and non-invasive trophoblast throughout the period of gestation studied. Of particular interest was the relative lack of staining of the endometrial cup cells in mule compared to horse pregnancies for galectin-1 and PIBF prior to the earlier and more rapid death and desquamation of the mule cup cells. DISCUSSION: The expression of galectin-1 and PIBF proteins in equine trophoblast and the marked difference in lifespan between the endometrial cups in intraspecies horse versus interspecies mule pregnancies support a likely role for these two proteins protecting the fetal trophoblast from maternal immune attack and/or modulation of the invasiveness of endometrial cup cells.

Effect of exogenous progesterone administration on luteal sensitivity to PGF during the early development of the corpus luteum in mares and cows.

The objective of this study was to determine the effect of exogenous progesterone administration at ovulation and during the early development of the CL, on its future sensitivity to a single administration of PGF2a in mares and cows. Horse Retrospective reproductive data from an equine clinic in the UK during three breeding seasons were used. Mares were divided into: control group, cycles with single ovulations; double ovulation group cycles with asynchronous double ovulations; and PRID group: cycles with single ovulations and treatment with intravaginal progesterone device (CIDR) immediately after the ovulation. All mares were treated with d-cloprostenol (PGF) at either: (i) 88 hr; (ii) 96 hr; (iii) 104 hr; or (iv) 112 hr after the last ovulation. Cattle A total of nine non-lactating Holstein cows were used. All cows were administered PGF14 d apart and allocated to one of two groups control group GnRH was administered 56 hr after the second PGF administration. CIDR group CIDR was inserted at the same time of GnRH administration. All cows were administered PGF at 120 hr post-ovulation. The complete luteolysis rate of mares with double ovulation (66.7%) and those treated with exogenous progesterone (68.4%) was significantly higher than the rate of mares with single ovulation (35.6%) at 104 hr. In the cow, however, the treatment with CIDR did not increase the luteolytic response in cows treated at 120 hr post-ovulation. In conclusion, the degree of complete luteolysis can be influenced by increasing the concentration of progesterone during the early luteal development in mares.

The period of the follicular phase during which the uterus of mares shows estrus-like echotexture influences the subsequent pregnancy rate.

The interval from both spontaneous and prostaglandin (PGF)-induced luteolysis to ovulation is greatly variable in mares. Several reports have shown a positive association between the length of the interval from PGF treatment to ovulation (ITO) and the subsequent pregnancy rate (PR). However, it is not known whether this association also occurs in estrous cycles with spontaneous luteolysis. The main objective of this study was to determine the effect of the duration of estrus-like echotexture of the uterus during the follicular phase on the subsequent PR in both spontaneous and PGF-induced cycles. A total of 768 estrous cycles from 325 thoroughbred mares were analyzed (401 estruses were induced with exogenous PGF and 367 cycles were not treated with PGF). The following factors were taken into account to determine the effect on PR: age of the mare, stallion, year of breeding, month of season, reproductive status of the mare, use of PGF treatment, duration of follicular phase with estrus-like echotexture, interovulatory interval (IOI; in spontaneous cycles), and ITO (in PGF-induced cycles). The age of the mare (P = 0.017), mare status (P = 0.031), the ITO (P = 0.041), and the duration of the follicular phase with estrus-like echotexture (P < 0.001) influenced the PR. The PR increased with the duration of estrus and of endometrial edema in both PGF-induced and spontaneous cycles. The correlation between the duration of endometrial edema and the IOI and ITO was positive (r = 0.5) and significant (P < 0.05).

The effect of the interval from PGF treatment to ovulation on embryo recovery and pregnancy rate in the mare.

The objective of this study was to determine the effect of the interval from induced luteolysis to ovulation on fertility of mares from two different farms. At farm 1, 215 mares were inseminated with frozen/thawed semen during 513 estrous cycles over seven consecutive breeding seasons. Estrus was induced with analogues of PGF2α in 179 cycles. At farm 2, 375 embryo flushings were performed in 65 donor mares inseminated with fresh semen; of which, 327 were performed following artificial insemination after PGF-induced luteolysis. In both farms, the intervals from PGF treatment to ovulation (ITO) data were divided into three interval groups: less than 6 days, 6 to 8 days, and greater than 8 days. A mixed regression model was created to determine the effect of different factors on the pregnancy rate (PR) and embryo recovery rate (ERR). Of all factors analyzed, the ITO was the only one that significantly influenced the PR and ERR (P < 0.05). In farm 1, the PR of mares with an ITO of less than 6 days, 6 to 8 days, and greater than 8 days was 26.6%, 39.4%, and 55.9%, respectively (P = 0.01). The PR for mares inseminated after spontaneous luteolysis (without PGF) was 42.5%. In farm 2, the ERR of donor mares for the same ITO groups was 55.0%, 62.6%, and 73.7%, respectively (P = 0.02). The ERR for mares flushed after a previous spontaneous estrus was 75.0%. In conclusion, the ITO had a significant effect on the PR and ERR in the mare. Fertility was reduced as the ITO became shorter.

Asynchronous ovulation in mares: seasonal variations in frequency.

The mare's reproductive system is rarely capable of sustaining multiple pregnancies to term. Multiple pregnancies largely derive from multiple ovulations, most commonly double ovulations, hence, double ovulations are of significant concern to breeders/veterinarians. Double ovulations may be synchronous or asynchronous. Asynchrony of up to 96 hours may result in two embryonic vesicles of very different sizes, which are challenging to detect at early ultrasonic pregnancy detection. This study aims to (1) document the incidence of synchronous and asynchronous double ovulation and (2) determine whether this varies with month/season. 506 cycles from double ovulating mares were monitored at 8±1 hour intervals before expected ovulation until 96 hours post initial ovulation. Mares were grouped according to eight-hour ovulation intervals and month/season. When asynchrony was classed as greater than or equal to eight hours or greater than or equal to 24 hours apart, respectively, a significant (P<0.001) difference existed between the number of mares demonstrating asynchronous double ovulations (65.8 and 28.5 per cent) and synchronous double ovulations (34.2 and 71.5 per cent). Significantly (P<0.05), more asynchronous ovulations occurred in the seasonal transition periods. This study demonstrates that asynchronous double ovulation is common and emphasises the importance of closely monitoring mares, particularly at the extremes of the breeding season, for double ovulation up to or at 96 hours postinitial ovulation in order to minimise the chances of missing multiple pregnancy.

Ultrasound confirmation of ovulation in mares: a normal corpus luteum or a haemorrhagic anovulatory follicle?

The most common pathological anovulatory condition that occurs spontaneously during the breeding season in the mare is the haemorrhagic anovulatory follicle (HAF). A relatively high proportion of mares, soon after ovulation, develop a corpus haemorrhagicum (CH) with a central lacuna. This type of corpora lutea may resemble an HAF, which may complicate the accurate diagnosis of ovulation. The main objective of this study was to compare the ultrasound data of mares examined frequently with HAFs and CHs to elucidate whether it is possible to distinguish them from each other. A total of 135 ovulating mares were classified according to the morphology of the corpus luteum (CL) in mares with: a solid CL, a CH with small or with large central cavities. Ultrasound characteristics of the development of 11 HAF and 13 CHs with a large central cavity were compared. The pre-ovulatory follicular diameter of ovulatory mares was significantly correlated with the diameter of CH with large central cavities. The percentage of mares with post-ovulatory areas eligible to be mistaken with a CH was <25%. Although a predictive diagnosis of an HAF/CH can be made on the basis of several ultrasonographic endpoints, the only parameter that allows a definitive diagnosis is the thickness of the luteal border. This is <3 mm in HAFs in contrast to >5 mm in CHs. However, this only applies when the unidentified structure has non-organized contents.

Ultrasound characteristics of experimentally induced luteinized unruptured follicles (LUF) and naturally occurring hemorrhagic anovulatory follicles (HAF) in the mare.

The development of hemorrhagic anovulatory follicles (HAF) involves luteinization and hemorrhage of the follicle. This is observed on ultrasound as an increase in the echogenicity of the granulosa layer and formation of echoic particles in the antrum. The inhibition of prostaglandin synthesis with flunixin meglumine (FM) during the periovulatory period induces ovulatory failure with development of luteinized unruptured follicles (LUF). These two types of anovulatory follicles appear to share similar ultrasound features but they have not been compared critically. The following endpoints: follicle diameter, follicular contents score, interval from hCG administration to beginning of follicular hemorrhage, interval from hemorrhage to organization of follicular contents, and cycle length were studied and compared in mares with HAF (n = 11) and LUF (n = 13). The objective of this study was to elucidate whether these two unruptured follicles have a consistent clinical pattern of development and therefore can be considered as part of the same anovulatory syndrome. None of the endpoints analyzed differed significantly between HAF and LUF. However, there was a greater individual variation in HAF as compared with LUF in regards to interval from hCG to hemorrhage, follicular diameter at the administration of hCG, and beginning of hemorrhage. In conclusion, HAF share a similar cascade of ultrasound characteristics with the experimentally induced LUF. This finding may provide new insights in elucidating the pathogenesis of HAF.

Relationship between dose of cloprostenol and age of corpus luteum on the luteolytic response of early dioestrous mares: a field study.

The objective of this study was to establish and characterize the relationship between the dose of cloprostenol (37.5, 250, 500 and 750 µg) and the age of the early corpus luteum (CL) (80, 88, 96, 104 and 112 h) on the luteolytic response of mares. Behavioural oestrus and ultrasonographic signs of return to oestrus were considered as the occurrence of full luteolysis. A total of 298 mares were divided into groups according to dose of cloprostenol and CL age. There was an effect of dose of cloprostenol (p < 0.001) and age of the CL at the time of treatment (p < 0.001) on the percentage of mares with full luteolysis. The efficacy of 37.5 µg of d-cloprostenol was similar to that of 250 µg of d,l-cloprostenol (p > 0.05); and that of 500 similar to that of 750 µg (p > 0.05). The higher dose groups (500 and 750 µg) induced full luteolysis more frequently than the lower dose groups (37.5 and 250 µg) 96-104 h post-ovulation. There was no effect of CL age or cloprostenol dose on the interovulatory interval (p > 0.05). In conclusion, the effect of cloprostenol on the percentage of mares undergoing full luteolysis is dose-dependent. However, this effect is only evident in mares with CLs aged between 96 and 104 h. There is no advantage of administering more than 500 µg of d,l-cloprostenol (Estrumate(®)), to obtain a higher percentage of mares with full luteolysis in mares with CLs aged 80-112 h.

Manual reduction of multiple embryos in the mare: the effect on subsequent pregnancy outcome.

To determine whether manually reduced multiple pregnancies (MPs) are at a greater risk of pregnancy loss than single pregnancy (SP) in mares, and to examine if a difference exists in the timing of pregnancy loss between manually reduced MPs and SPs, 1916 Thoroughbred mares were ultrasonically monitored every 2 days during oestrus to confirm ovulation, and up to Day 40 post-ovulation to confirm pregnancy. Ultimate pregnancy outcome was ascertained from the General Thoroughbred Stud book and classified as live foal (LF), early abortion (EA; Days 40-150), slipped foal (SF; Days 150-term), and barren (B; Time of pregnancy loss unknown). Significantly (P<0.05) more SPs failed (17.23%; 226/1312) than manually reduced MPs (13.41%; 81/604). Both SPs and MPs were at greatest risk of being lost as EA (72.16%; 127/176 and 61.67%; 37/60, respectively) compared to SF (27.84%; 49/176 and 38.33%; 23/60). There was no significant difference in the time of greatest risk of pregnancy loss between manually reduced MPs and SPs. It was concluded that owners and veterinarians can be assured that manual reduction of MPs does not increase the risk of pregnancy loss above those associated with SPs.

The effect of time of insemination with fresh cooled transported semen and natural mating relative to ovulation on pregnancy and embryo loss rates in the mare.

One hundred and fifty-four mares were inseminated with fresh semen either during the pre- or post-ovulatory periods at different intervals relative to ovulation: 36-24 h (n = 17) and 24-0 h (n = 30) before ovulation; 0-8 h (n = 21), 8-16 h (n = 24), 16-24 h (n = 48) and 24-32 h (n = 14) h after ovulation. All mares received the same routine post-mating treatment consisting of an intrauterine infusion with 1 litre of saline and antibiotics followed 8 h later by an intravenous administration of oxytocin. Artificial inseminations (AI) from 36 h before ovulation up to 16 h post-ovulation were performed with transported cooled semen. While there was no data available for inseminations later than 16 h, data from natural mating after 16 h post-ovulation were included. Pregnancy rate (PR) of mares inseminated 36-24 h (29.4%) was significantly lower (p < 0.05) than mares inseminated 24-0 h before ovulation (60%), 0-8 h (66.7%) and 8-16 h (70.1%) post-ovulation. Embryo loss rate (ELR) was highest in mares mated 24-32 h after ovulation (75%). PR of mares mated 16-24 h post-ovulation (54.1%) did not differ significantly from any other group (p > 0.05); however, the ELR did increased markedly (34.6%) compared with inseminations before 16 h post-ovulation (<12%). At ≥ 30 days post-ovulation, PR of mares mated 16-24 h after ovulation (35.4%) was significantly lower than mares mated 0-16 h after ovulation (62%). Good PR with acceptable ELR can result from inseminations within 16 h of ovulation, at least with this specific post-mating routine treatment.

Reducing the examination interval to detect ovulation below 12h does not improve pregnancy rates after postovulatory insemination with frozen/thawed semen in mares.

Data were analysed retrospectively from fourteen breeding seasons at an Equine Fertility Clinic for the effect of interval between pre- and postovulatory examinations for immediate postovulatory insemination on pregnancy rate (PR) and embryo loss rate (ELR). Mares of various breeds and ages were examined at intervals which varied from 0.5 to 15h between the pre- and postovulatory period over 867 cycles. When ovulation was detected they were inseminated with a single dose of commercial frozen-thawed semen. All mares were treated in the post-insemination period with intrauterine antibiotics and then with oxytocin. Pregnancy diagnoses were made at 12-17 days post-ovulation and at intervals up to 40 days. The overall PR was 47.9%. The data were pooled into 3h examination intervals. In the first interval, mares were inseminated at the time of ovulation to 3h post-ovulation (n=44) with a PR of 43.2%. Results of insemination to consecutive 3h intervals gave PR of 44.7% (3-6h, n=150), 45.1% (6-9h, n=432), 55.8% (9-12h, n=190) and 54.9% (12-15h, n=51). ELR was 10.5%, 11.9%, 5.6%, 7.5% and 3.6% respectively for the same intervals. There was no statistical difference in either the PR or ELR. It is concluded that in a postovulatory insemination regime with routine post-insemination treatment as described, examination of mares at intervals of any less than 12-15h does not improve pregnancy or embryo loss rates.

Factors affecting pre-ovulatory follicle diameter in the mare: the effect of mare age, season and presence of other ovulatory follicles (multiple ovulation).

The importance of elucidating factors affecting reproductive performance and efficiency is of paramount concern to the equine industry. Oocyte viability is known to be one of the determinants of reproductive success and evidence suggests that it may be linked to follicle size. The aims of this study were, therefore, to ascertain: i) the average diameter and range of pre-ovulatory follicles in Thoroughbred mares; ii) whether this is affected by either mare age, time within the breeding season, or the presence of multiple pre-ovulatory follicles (MO). One thousand, four hundred and ninety two Thoroughbred mares, aged 2-26 years, were examined with ultrasound to ascertain ovulation date to within 24h, and pre-ovulatory follicle(s) (F1) diameter. Mares were divided into groups according to age (7 groups, 2-4 yr, 5-7 yr, 8-10 yr, 11-13 yr, 14-16 yr, 17-19 yr, >19 yr), time within the season (16 half-month groups, from Feb 1(st) to Sept 30(th)), and pre-ovulatory follicles (single, {SO} or multiple {MO}). Overall average F1 diameter was 39.95 ± 4.84 mm (range 22-50 mm). Mare age had a significant (P < 0.001) negative effect on F1 diameter (largest F1 38.95 ± 5.61 mm, mares 2-4 yrs; smallest F1 33.30 ± 4.66 mm, mares >19 yrs) as did season (largest F1 44.20 ± 3.95 mm, Feb 1(st)-14(th); smallest F1 33.74 ± 4.87 mm, Aug 15(th)-31(st)) and the presence of more than one pre-ovulatory follicle (MO F1 35.45 ± 4.53 mm; SO F1 37.44 ± 4.84 mm). In conclusion older mares, bred towards the end of the breeding season, especially if MO were present, were more likely to ovulate from smaller follicles. If, as suggested, small pre-ovulatory follicle size is associated with low oocyte viability, then this may account, at least in part, for the poor fertility rates characteristic of older MO mares, bred later in the season and so justify increased monitoring and careful reproductive management of such mares.

Risk factors for the development of haemorrhagic anovulatory follicles in the mare.

Haemorrhage into the dominant follicle during the reproductive season is a subtle but definitive cause of infertility in the mare population. This condition however can be of high relevance for an individual in which its incidence is abnormally high. Little is known about the nature and factors affecting the incidence of haemorrhagic anovulatory follicles (HAFs) in the mare. The objectives of the study were to define and characterize the ultrasonographic development and incidence of HAFs and to investigate possible risk factors influencing its occurrence. Detailed reproductive and ultrasound records of seven mares studied during their entire reproductive lives (>10 years and 612 oestrous cycles) were analysed retrospectively and computed into a statistical mixed model. Of all animal studied, two mares were found to have an unusually high incidence of HAFs of approximately 25%. Time of season and use of induction treatments (Cloprostenol) were found to influence its incidence. It appears that early-enhanced stimulatory effect of LH on an ovary with the presence of small and immature follicles might increase the risk of ovulatory failure of those follicles later in the cycle. Mares during the months of highest follicular activity (May to August) and after treatment with hormones to induce oestrus and ovulation are at greater risk to develop HAFs. The potential relevance of this study is two folds: clinical relevance for the practitioner to better understand this condition and so improve reproductive management of mares with abnormally high incidence; and to provide useful insights for researchers willing to further investigate the nature of this phenomenon.

Cloprostenol in equine reproductive practice: something more than a luteolytic drug.

Prostaglandin F(2α) and its analogues (PGF) are widely used in equine reproductive practice. The interval from PGF treatment to ovulation (ITO) varies greatly with a range from 2 to 16 days. Clinical observation suggests that mares mated and ovulated soon after PGF treatment may have poor fertility. Reproductive records of 329 cyclic Thoroughbred mares were analysed retrospectively. The following parameters were analysed: (i) use of cloprostenol; (ii) ITO and (iii) number of ovulations per cycle. According to these parameters, mares were classified into four groups. (i) mares with spontaneous ovulations, n = 57; (ii) mares induced with cloprostenol and ITO = 4-7 days, n = 77; (iii) ITO = 8-10 days, n = 89 and (iv) ITO = ≥ 11 days, n = 106. Differences in pregnancy (PR) and multiple ovulation (MO) rates among groups were tested using chi-squared test. PR rates for groups 1-4 were: 73.7%, 46.7%, 64% and 71.7% respectively (p < 0.05). Groups 1 and 2 had lower (p < 0.05) MO rate (24.6% and 20.8%) than groups 3 and 4 (40.4% and 44.3%). It appears that ovulation soon after PGF-induced luteolysis is detrimental to PR rates. It was found highly significant that in cloprostenol-treated mares, the MO rate was enhanced without subsequent increase in multiple pregnancies.

The effect of hormone treatments (hCG and cloprostenol) and season on the incidence of hemorrhagic anovulatory follicles in the mare: a field study.

The association between use of hormone treatments to induce estrus and ovulation and the incidence of hemorrhagic anovulatory follicles (HAFs) was studied in a mixed population of mares (Equus caballus) during two breeding seasons in a commercial breeding clinic. Mares treated with cloprostenol (CLO) were more likely to develop HAFs than were mares with spontaneous cycles (P<0.001) or those treated with human chorionic gonadotropin alone (P=0.08). There was no significant effect of season on the incidence of HAFs. The mean (+/-SEM) interval from CLO treatment to beginning of HAF development was 6.1+/-0.5 d. Age of mares with HAF cycles was not different (12+/-1.3 yr; P>0.05) from that of mares with ovulatory cycles (10.5+/-1.5 yr).

The relationship between consecutive pregnancies in Thoroughbred mares. Does the location of one pregnancy affect the location of the next, is this affected by mare age and foal heat to conception interval or related to pregnancy success.

Delayed uterine involution is a major cause of early reproductive failure in mares. Involution is affected by mare age, and foaling to covering interval. Involution rates vary between the previously non-gravid horn (PNGH), which recovers the quicker, and the previously gravid horn (PGH). Location of a pregnancy and its likely success may, therefore, be affected by its location relative to the previous pregnancy. This study aimed to determine: (i) the location of concepti in consecutive pregnancies; (ii) whether this varies with mare age or foaling to conception interval; (iii) whether location in relation to the previous pregnancy affects success. 1383 Thoroughbred mares were monitored by ultrasonic scanning during oestrus and early pregnancy. Significantly (p<0.01) more pregnancies were located in the PNGH (79.2%) than the PGH (20.8%). The number of pregnancies in PGH significantly increased with mare age (p<0.01) and foaling to conception interval (p<0.05). Significantly (p<0.001) more pregnancies located in the PGH (16.5%) failed, than those in the PNGH (4.6%). It can be concluded that most pregnancies locate in the PNGH where their chances of success are greatest. The larger number of pregnancies locating in the PGH in older mares and those with shorter foaling to conception intervals may in part account for the higher conceptus mortality rates in such mares. Hence breeding older mares on alternate years and maximising foaling to conception interval may improve reproductive success. Alternatively termination of pregnancies located in the PGH followed by timely recovering may be justifiable as might ET in older mares covered close to foaling.

Effect of type of semen, time of insemination relative to ovulation and embryo transfer on early equine embryonic vesicle growth as determined by ultrasound.

Embryonic vesicle growth in the mare is easily monitored by ultrasound. Apart from pregnancy diagnosis, assessment of the embryonic vesicle in practice is also useful to evaluate its viability. Although subject to individual variation, embryo growth rate follows a constant pattern in the early stages of development in relation to embryonic age. Previous studies have shown a significant effect of some factors routinely used in practice, such as post-ovulation insemination and embryo transfer, on embryonic growth and the time in which the vesicle is first detected. This study attempts to confirm previous results in different settings and characterise the causes for this delay in growth. A total of 159 pregnancies from different mating protocols: (1) pre-ovulation natural mating, (2) pre-ovulation natural mating and transfer into recipient mares, (3) post-ovulation natural mating, and (4) post-ovulation AI with frozen/thaw spermatozoa were evaluated ultrasonographically from day 12 to 19 of pregnancy and vesicle diameters recorded. Regression analysis between embryonic vesicle diameters and embryonic ages was performed for each group and mean vesicle diameter at different age periods among groups were tested for statistical difference with a general linear model of variance. There was no significant difference between groups 1 and 2 (P=0.73) or between groups 3 and 4 (P=0.71). However both pre-ovulation groups (1 and 2) had larger vesicle diameters (P<0.000) at any embryonic age analysed than either of the post-ovulation groups (3 and 4). In conclusion, post-ovulation inseminations produced pregnancies with smaller vesicle diameters equivalent to approximately 1 day's growth.