Equine fetal kinetics: Allantoic-fluid shifts and uterine-horn closures

Uterine filling by the conceptus and uterine constrictions and allantoic-fluid shifts after filling were studied by daily ultrasound examinations on Days 40 to 74 in 10 mares. Allantoic fluid was first detected at the extremities of the uterus on the following days: 1) tip of umbilical-cord horn, Day 60.0 +/-1.9 (mean +/-SEM); 2) tip of noncord horn, Day 64.8 +/-1.3; and 3) near cervix, Day 48.5 +/-1.3. Transient uterine constrictions with expulsion of allantoic fluid were detected in 37% of 271 examinations. Other end points were studied by weekly ultrasound examinations from Day 40 to term. Height of cross-sections of the fetal body increased (P<0.01) progressively over Monthes 2 to 8, whereas the height of placental fluid minus fetal body decreased (P<0.05) after Month 3. An increasing (P<0.05) frequency of examinations with a reclosed uterine horn occurred between Months 3 and 4. The frequency of reclosed noncord horns continued to increase (P<0.05) after Month 4 and reached 96% at Month 7. The frequency of reclosure of cord horns plateaued (no significant differences) at 54% over Months 4 to 7 and then rapidly decreased (P<0.05). Both uterine horns were closed during the examination that preceded the permanent entry of the fetal hind limbs into the cord horn in all of 9 mares in which this temporal relationship was studied. After entry of the fetal hind limbs, the cord horn remained constricted upon the limbs until at least Month 10. The results provide rationale for the following hypotheses: 1) The gradual decrease in fetal mobility (location and presentation changes) previously reported for Months 4 to 8 is associated with growth of the fetus and a concomitant decrease in volume of allantoic fluid and number of open uterine segments; 2) Closure of both uterine horns near mid-pregnancy plays a role in final selection of cranial fetal presentation followed by entry of the fetal hind limbs into the cord horn.     

Equine fetal kinetics: entry and retention of fetal hind limbs in a uterine horn

Transrectal ultrasonic examinations were made in 31 pregnant pony mares once a week during Months 6 to 11. Each uterine horn was divided into 3 approximately equal segments (caudal, middle, cranial). The percentage of examinations with cranial fetal presentation increased (P<0.05) progressively from 58% at Month 6 to 99% at Month 9 and was followed in all mares by entry of the fetal hind limbs into one uterine horn. The mean number of uterinehorn segments with limb parts increased (P<0.05) between each set of consecutive months from Month 6 to Month 10. Initially (Months 7 and 8), retraction of limbs after entry into the caudal and middle segments of the horn was common (31% incidence). The mean day of final entry of the limbs without detection of subsequent retraction was Day 230 +/- 2.2. Both uterine horns were closed during the examination preceding final entry of the hind limbs into one horn in 25 of 29 (86%) mares. The limbs reached the cranial segment in most examinations by Months 9 and 10 (73% and 98%). The cross-sectional height, as seen on the ultrasound screen, of both uterine horns increased (P<0.05) progressively during Months 7 to 10. Between Months 10 and 11, the height of the horn containing the limbs decreased (P<0.05); this result was attributable to a flattening of the horn and thinning (P<0.05) of the horn wall above a hoof and metatarsal bone. Apparent placental fluid (nonechogenic areas >5 mm in height) between the hind limbs and the cornual wall was detected in 0, 10, 25, and 63% (P<0.01) of examinations for Months 8 to 11, respectively. Results indicated that entry of the fetal hind limbs into a uterine horn was initially tentative and became final during a mean of Month 8. The apposition between the cornual wall and limbs was close during Months 7 to 11. By Month 11, the fetal-limb horn became flatter and thin-walled and usually contained placental fluid.     

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA)

In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.     

Equine fetal gender determination from mid- to advanced-gestation by ultrasound

Equine fetal gender can be easily determined by ultrasound between Days 120 and 210. A combination of transrectal and transabdominal ultrasound examinations can visualise fetal sex organs up to 8 months of gestation. Early equine fetal sexing techniques, performed between approximately Days 58 and 70, are aimed at identifying the position of the genital tubercle. A wider diagnostic window, a diagnosis based on several parameters, and the ease of identification of fully developed fetal primary sex organs make gender diagnosis in mid- to advanced-gestation a preferable technique.     

Growth kinetics of hemopoietic fetal stem cells

Fetal spleen stem cells have growth characteristics similar to those of normal adult spleen stem cells. On the contrary there is an early fetal liver stem cell population which possesses a lag time longer than that of adult stem cells. The duration of the lag time is controlled by a built-in biological timer which seems to regulate some proliferative functions of the primitive liver stem cell.     

Temporal kinetics of fluoride accumulation: from fetal to adult deer

In June 2011, a volcano deposited about 100 million tons of tephra over parts of Chile and over 36 million ha of Argentina. Initially, fluoride was considered irrelevant; however, recently wild deer exhibited strong fluorosis, with fluoride level increasing 38-fold among severely affected deer. Whereas mothers averaged 2,151 ppm, their late-term fetuses had only 19.8 ppm, indicating a barrier to fluoride transport in utero. Levels among four age classes increased significantly, at a rate of about 1,000 ppm/year. The temporal kinetics of accumulation suggests that sources of available fluoride are highly effective. Thus, compared to prior background levels (63 ppm in adults) and to fetuses starting at about 20 ppm, 1-year-old calves averaged 1,035 ppm (maximum 1,830 ppm), 2-year olds averaged 2,151 ppm (maximum 2,513 ppm), and older deer averaged 2,806 ppm (maximum 5,175 ppm). As osteofluorosis occurs in deer with >4,000 ppm, accumulation of 1,000 ppm/year would result in adults reaching levels causing osteopathology in 1–2 years. Importantly, impacts may be further exacerbated by regional iodine and selenium deficiencies. Iodine deficiency may increase incidences of dental fluorosis and severity of damages, while selenium deficiency impacts iodine metabolism. Fluorosis will affect population dynamics, morbidity, predation susceptibility, and other ecosystem components like scavenger and plant communities.     

Equine fetal sex determination using a single ultrasonic examination under farm conditions

The aims of this study were to evaluate the reliability, under general farm conditions, of the use of a single transrectal sonogram in pregnant mares to determine fetal sex by locating the genital tubercle, and the feasibility of extending the period of gestation in which this examination can be carried out. This research was done during routine reproductive examinations on three different stud farms. Forty mares between the 54th and the 89th day of gestation were examined once; gestation was calculated by identifying the last day of mating as Day 0. In order to verify the precision of the determination, data regarding the sex of the foals at birth were collected. This information was not available for two of the 40 mares examined. The diagnosis was made without any certainty level or any other methods which might quantify or increase the reliability of the examination. Of 40 mares, a diagnosis was possible in 35 (87%); in five mares (12%), it was not possible due to insufficient data. Twenty-six of 40 sex determinations (65%) were correct, 9 of 40 (22%) were incorrect. The comparison of the percentages obtained using the chi2-test was P = 0.0011. In spite of positive statistical analysis, the results of this study are not sufficient to allow systematic use of this approach due to the high percentage of error.     

DNA-damage checkpoints: location, location, location

The DNA-damage response (DDR) is an evolutionarily conserved signaling cascade crucial for sensing DNA damage and activating cellular responses such as cell-cycle arrest, DNA repair, senescence and apoptosis. Excitingly, two recent studies describe activation of this checkpoint in the absence of DNA damage. These studies support the idea that accumulation of checkpoint proteins and changes in global-chromatin structure are important signaling intermediates for the activation of the DDR.     

Planar polarity: location,location, location

Recent studies have shown that many proteins that regulate planar polarity in the fly eye are organized into discrete membrane sites which may be crucial for coordinating groups of cells.     

Cell kinetics in the fetal mouse thymus: precursor cell input, proliferation, and emigration

The entry and differentiation of lymphoid precursor cells (LPC) in grafted mouse fetal thymuses and the emigration of explants thymocytes has been followed in a system in which donor and host lymphocytes could be distinguished on the basis of Thy-1 expression. It appears that LPC that invade the fetal mouse thymus between 10 and 13 days rapidly differentiate into Thy-1 positive thymocytes, giving rise to all of the lymphoid populations of both cortical and medullary locations until approximately the end of the first week after birth. Lymphoid precursor cells that enter the fetal thymus after 13 days of fetal life only differentiate into Thy-1 positive lymphocytes 6 or 7 days after birth, when they give rise to a second generation of thymocytes that grows exponentially and completely replaces the first generation in approximately 8 days. All cells leaving the thymus during the first 2 wk of life appear to be derived from the first wave of precursors.     

Equine clinical cytogenetics: the past and future

Cytogenetic analyses of horses have benefited the horse industry by identifying chromosomal aberrations causing congenital abnormalities, embryonic loss and infertility. Technical advances in cytogenetics enabled the identification of chromosome specific aberrations. More recently, advances in genomic tools have been used to more precisely define chromosome abnormalities. In this report we review the history of equine clinical cytogenetics, identify historical landmarks for equine clinical cytogenetics, discuss how the current use of genomic tools has benefited this area, and how future genomics tools may enhance clinical cytogenetic studies in the horse.     

Equine viral arteritis: current status and prevention

Recently, there has been increased interest in equine viral arteritis (EVA) among veterinarians and horse owners. Outbreaks of the disease were identified initially in New Mexico, USA in 2006, and in the Normandy region of France in the summer of 2007. Both occurrences were associated with AI of cool-shipped semen. Each was linked to respiratory illness, neonatal death, abortion, development of carrier stallions, and cancellation of equestrian events. In light of the increased interest, this paper will present a brief case history, followed by a review addressing common concerns regarding EVA, current status, and control and prevention strategies, including vaccination, and recommended bio-security measures.     

Growth factor receptor signalling: location, location, location

Ligand binding to plasma membrane receptors initiates a series of events culminating in a variety of changes in cellular phenotypes. Although numerous publications have documented the activation/inactivation of signalling molecules following receptor binding, relatively few investigations have focused on the cellular compartment responsible for either initiating or selecting the particular pathway that mediates the response. Specifically, does receptor signalling occur only at the plasma membrane; is signalling dependent upon the location of defined endosome populations; or are components of both plasma membrane and endosomal activity operative depending upon the particular signalling pathway or cell type? This review addresses aspects of these questions by discussing the evidence supporting or contrasting the interplay between the endocytic and signalling systems for a subset of tyrosine kinase, serine/threonine kinase and G-protein-coupled receptors.     

Equine frozen semen: freezability and fertility field results

The freezability of stallion semen defined as the number of selected ejaculates/total number of ejaculates frozen from 161 different stallions was analyzed. Of the stallions, 19, 30, 27 and 24% had a freezability of 0%, 0 to 33%, 33 to 66%, over 66%, respectively In 85 different stallions, the correlation of freezability between first and second year was 0.60 (P < 0.001). The relationship between fertility with fresh and frozen semen and freezability was analyzed in 40 stallions whose freezability and fertility information was recorded during 5 years. There was a strong relationship between fertility of fresh semen and semen freezability (P < 0.001). However, the relationship between fertility of frozen semen and freezability was not as marked (P < 0.05). Analysis of the field fertility per cycle results when mares were bred with 300 or 150 x 10(6) total spermatozoa at different frequencies until ovulation indicated that mares that were inseminated 2 times or more per estrus show an improved fertility in comparison with mares inseminated only once (34%, n = 1576 vs 26%, n = 626; P < 0.001). Foaling rate when mares were inseminated with frozen semen (1858 mares during 8 breeding seasons) was mainly influenced by mare age (< 16 years: 54% vs >/= 16 years 42% p < 0.001). Date of first insemination (before May 15: 58% vs after May 15: 37%) also had a significant effect on foaling rate (P < 0.001).     

Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart.

The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.     

Linoleic acid kinetics and conversion to arachidonic acid in the pregnant and fetal baboon.

Linoleic acid plasma kinetics in pregnant baboons and its conversion to long chain polyunsaturates (LCP) in fetal organs is characterized over a 29-day period using stable isotope tracers. Pregnant baboons consumed an LCP-free diet and received [U-13C]linoleic acid (18:2*) in their third trimester of gestation. In maternal plasma, 18:2* dropped to near baseline by 14 days post-dose, while labeled arachidonic acid (20:4*) plateaued at 10 days at about 70% of total labeled fatty acids. After 2;-5 days, total tracer fatty acids decreased in visceral organs, but increased in the fetal brain. Maximal fetal incorporation of 18:2* was 1;-2 days post-dose; thereafter it dropped while 20:4* increased reciprocally. Labeled 20:4 replaced 18:2* in neural tissues by 5 days post-dose. In liver, kidney, and lung, 20:4* became dominant by 12 days, but in heart the crossover was >29 days. Fetal brain 20:4* plateaued by 21 days at 0. 025% of dose, while fetal liver 20:4* was constant from 1 to 29 days at 0.006% of dose. Under these dietary conditions we estimate that the fetus derives about 50% its 20:4 requirement from conversion of dietary 18:2, with the balance from maternal stores, and conclude that 1) fetal organs accumulate 18:2 within a day of a maternal dose and convert much of it to 20:4 within weeks, 2) modest dietary 18:2 levels may support fetal brain requirements for 20:4, and 3) the brain retains n;-6 fatty acids uniquely compared with major visceral organs.