Embryonic Loss in Mares - PDF

P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come Chapter 239 Embryonic Loss...

P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come Chapter 239 Embryonic Loss Barry A. Ball Introduction aged mares survive after embryo transfer to healthy recipient mares.7 Because the equine embryo enters In contrast to other domestic livestock, the horse is the uterus at Day 6 to 6.5, it was unclear from these maintained as part of the breeding population for a observations whether the decline in number and ap- relatively longer period of time. In many situations, parent viability of blastocysts from aged mares was older mares may be more valuable because of their related to an abnormal uterine environment, abnor- proven produce record, and it is common to find malities of the oviductal environment, or abnormali- mares that are bred at greater than 20 years of age. For ties inherent in the embryo. these reasons, age-related reproductive failure has re- Several studies have characterized the early ceived considerable study. This chapter will summa- equine embryo during oviductal passage in mares of rize our current understanding of reproductive fail- different ages and fertility status. As in other species, ure in mares, particularly as it relates to embryonic the overall fertilization rate in young mares appears loss. high (> 90%) based upon the cleavage rate of ova re- Foaling rates decline with maternal age after 14 to covered 2 days after fertilization whereas the fertil- 16 years.1–3 Although pregnancy losses during late ization rate in aged mares was 80 to 90%. Estimates of gestation increase in older mares, it appears that fail- the embryonic loss rate between fertilization and Day ures of early pregnancy account for the majority of 10 were 9% for young mares compared to 60 to 70% the reduced fertility in aged mares. The widespread for aged mares.8–10 Although pregnancy rates at Day application of transrectal ultrasonography for preg- 2 were similar in young and aged mares, by 4 days nancy detection in mares at Days 10 to 14 after ovu- after fertilization, there was a significant reduction lation led to evaluation of the incidence of embryonic in pregnancy rates in aged mares. This finding sug- loss in mares between Days 14 and 40, and these stud- gested that the interval between Day 2 and 4 might ies indicate that overall pregnancy rates declined and represent a critical period in pregnancy failure in that detected embryonic loss rates increased with in- aged mares. These studies confirmed the importance creasing mare age.1, 4 A recent review evaluated the of very early embryonic loss in the reduced fertility overall incidence of detected embryonic loss in mares of aged mares; however, such studies do not provide as approximately 7.7%.5 Data from field studies such information as to the causation of these losses. as these provided a strong impetus to examine very early embryonic mortality in mares. Factors in embryonic loss Incidence of early embryonic loss Pre-uterine events associated with Prior to Day 10, ultrasonographic evaluation can- embryonic loss in mares not accurately detect the early equine conceptus; however, several reports based upon embryo collec- Events that occur prior to Day 6 to 6.5 of pregnancy tion and transfer provide information regarding very in the mare within the oviduct have received limited early embryonic loss in mares. From these studies, it study due to the limited accessibility to the oviduct. appears that there is a sharp decline in the recovery However, as noted above, a relatively high rate of em- rate of blastocysts from the uterus of aged mares.6, 7 bryonic loss appears to occur prior to entry of the em- In addition, blastocysts from aged mares have more bryo into the uterus, and therefore these pre-uterine morphological defects,6 and fewer blastocysts from events are of particular importance in understanding Equine Reproduction, Second Edition. Edited by Angus O. McKinnon, Edward L. Squires, Wendy E. Vaala and Dickson D. Varner
c 2011 Blackwell Publishing Ltd. P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come 2328 Mare: Problems of Pregnancy early embryonic loss in mares. The role of pre-uterine mares has not been established, it is possible that events in embryonic wastage in aged mares was delayed embryonic development in these mares is examined by collection of embryos from the uter- also associated with delayed or abnormal transit ine tube of aged and young mares at 4 days after through the oviduct. fertilization.9 The viability of these embryos was assessed after transfer to the uterus of young recip- Oviductal (uterine tubal) factors ient mares. The survival of embryos from aged mares Because the period of oviductal transit of the em- was significantly lower than those from young mares bryo appears to be critical to early embryonic de- after placement into a normal uterine environment, velopment and loss, two studies were conducted to which indicates a role for either the oviductal envi- evaluate the role of the uterine tubal environment ronment or defects inherent in the early embryo for on early embryonic loss in aged mares. In the first the high rate of early losses in aged mares. study, histopathological evaluation of both the am- The equine embryo resides in the oviduct for a rel- pulla and isthmic regions of the uterine tube was atively long time (6 days) and reaches a late stage conducted on aged and young mares. In contrast to of development (morula or early blastocyst) before the findings associated with the endometrium, there entering the uterus. There are, consequently, a num- was no association between mare age and the inci- ber of important events that occur during oviductal dence of salpingitis.16 In a second study, differences transit that could affect embryonic survival. During in proteins synthesized and secreted in vitro by ex- this time, the embryonic genome is activated, and planted oviductal tissue was examined by incorpo- the transition from maternal to embryonic control of ration of 35 S-methionine, two-dimensional polyacry- development has been proposed as a critical junc- lamide gel electrophoresis, and fluorography. Both ture in embryonic development.11 Early blastomere qualitative and quantitative differences in the pat- cleavage progresses to further differentiation includ- terns of proteins from oviductal epithelium were de- ing compaction, blastulation, and initial formation of tected between young and aged mares; however, the the inner-cell mass and trophoblast. In order to inves- importance of these differences relative to embryonic tigate developmental capacity of early embryos, we development remains to be determined.17 examined the in vitro development of early, Day-4 Another, unique, feature of the equine oviduct is embryos from both aged and young mares during the presence of oviductal masses (Figure 239.1) or co-culture with oviductal epithelial cells. There was ‘plugs’ that have been characterized by several au- no catastrophic failure of development of embryos thors.12, 18, 19 The exact origin of these masses has been from aged mares during in vitro culture as the de- debated, but it appears likely that these aggregates velopmental rate of embryos from young and aged of collagen and fibroblasts originate from connec- mares to blastocysts was similar. However, embryos tive tissue debris that is carried into the oviduct at from aged mares had fewer blastomeres and poorer the time of ovulation.18 Irrespective to their origin, quality scores than did embryos from young mares.10 these oviductal plugs are observed with consider- This study supported a reduction in embryo quality able frequency in mares, and it has been proposed in embryos from aged mares during the preblastocyst that they may act as physiological ‘ball’ valves which period. allow the passage of sperm up the oviduct but Oviductal transit of the embryo in the mare is prevent the larger embryo from passing through the unique among the domestic animals because the early embryo actively regulates transit from the oviduct into the uterus.12, 13 Regulation of embryo transit through the isthmic portion of the oviduct is dependent upon normal development of the embryo14 and is regulated through secretion of prostaglandin E2 in part by the early conceptus.15 Therefore, if embryonic development is abnormal during the early cleavage stages, the equine blasto- cyst will not passage through the oviduct into the uterus as expected at Day 6. Anecdotally, embryo transit into the uterus of aged mares appears to be delayed and many embryo transfer practitioners delay embryo recovery attempts from such mares until Day 8 after ovulation. Although the relationship Figure 239.1 Low-magnification micrograph demonstrating between delayed rates of embryonic development an equine oviductal ‘plug’ recovered after lavage of the oviduct and embryo transit through the oviduct of older of a mare. P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come Embryonic Loss 2329 oviduct and into the uterus.19 In support of this hy- strated more ultrastructural abnormalities. Because pothesis, Zent et al. demonstrated that lavage of the the events of oocyte maturation and fertilization oviduct to remove these oviductal plugs improved are highly energy-dependent processes, and because fertility in three of five mares with long-standing sub- there are major changes in distribution of mitochon- fertility.20 More recently, Allen and co-workers19 used dria during oocyte maturation in the horse,25 these the novel approach of application of PGE2 gel (Pre- findings suggest that alterations in mitochondria in podil) to the serosal surface of the oviduct in 15 mares oocytes from aged mares may be an important under- with long-standing subfertility. Laparoscopic appli- lying factor associated with reduced oocyte quality in cation of PGE2 to the oviduct was theorized to in- these mares. duce relaxation of the oviductal smooth muscle with Delayed ovulation and preovulatory aging of subsequent passage of these oviductal plugs into the oocytes has been proposed as another factor associ- uterus, thereby relieving the oviductal obstruction. ated with abnormal oocytes in aged females. Aged Although the study was not controlled, 14 of these 15 mares appear to undergo a reproductive senescence mares established detectable pregnancies subsequent characterized by lengthening of the follicular phase, to this treatment.19 irregular ovulations, and eventually a cessation of follicular activity.23, 26, 27 The onset of these changes appears to occur over a relatively broad age range Oocyte abnormalities but appears most commonly in mares > 20 years of Deterioration of oocytes associated with increased age. Prolongation of the follicular phase appears to maternal age has been well described in other be associated with an elevation of both FSH and LH species.21 Unfortunately, few studies have addressed in aged mares.27 The relationship of declining follicu- this issue in the horse. In one study, we examined the lar populations, altered gonadotropin levels, and pro- in vitro maturation of oocytes from young and aged longed follicular development in aged mares with an mares as well as the rate of aneuploidy in oocytes increased incidence of abnormal oocytes remains to from each group. Unfortunately, inadequate num- be explored as an explanation for age-related infer- bers of chromosomal spreads were available to as- tility in mares. In cattle, a decreased concentration sess the rate of aneuploidy in oocytes from these of circulating progesterone in diestrus prior to the two groups of mares, and the relative incidence of subsequent estrus influences LH pulse frequency and aneuploidy in oocytes from aged mares remains to may lead to prolonged follicular development and be defined.22 However, oocytes from aged mares premature resumption of meiosis.28 Oocytes derived reached metaphase II at a much lower rate than did from these large persistent follicles are able to initiate oocytes from younger mares, with more oocytes from fertilization but have a high rate of embryonic death older mares arresting at metaphase I, which suggests that occurs by the 2- to 16-cell stage.28 Although simi- that meiotic division of oocytes from older mares lar data are lacking for the mare, it appears likely that is more likely to be abnormal.22 In another report, prolonged follicular development in older mares may Carnevale and Ginther reported the results of ga- contribute to increased abnormalities in the oocyte mete intrafallopian transfer studies in which oocytes with a subsequent increase in embryonic loss. were collected from both young and aged mares and Aging of the oocyte after ovulation with subse- transferred into the uterine tube of young recipient quent fertilization may also result in abnormalities of mares.23 Oocytes from aged mares resulted in sig- oocyte function. In mares, aging of the oocyte subse- nificantly fewer pregnancies than those from young quent to ovulation results in a progressive decline in mares after transfer to the uterine tube of a young pregnancy rates,29, 30 a delayed embryonic develop- recipient mare. This study provides the most con- ment, and possibly an increased rate of subsequent vincing evidence to date of an age-related decline in embryonic loss. Although the mechanism for the re- oocyte quality in mares as a major factor in the re- duced developmental capacity of embryos derived duced fertility of these mares. It appears likely that from oocytes that have undergone post-ovulatory ag- this reduction is secondary to abnormalities during ing in mares is not known, information from other meiosis in oocytes from aged mares possibly result- species suggests that breakdown of the meiotic spin- ing in an increased incidence of aneuploidy; however, dle or reduced efficacy of the cortical reaction with this remains to be established for the horse. subsequent polyspermic fertilization are likely fac- Although the mechanism responsible for reduced tors to consider. oocyte quality from aged mares remains undefined, a recent study indicated that there is a quantita- Genetic factors tive reduction in mitochondria in oocytes from aged compared to young mares.24 Mitochondria from in Although infrequently reported, autosomal translo- vitro matured oocytes from aged mares also demon- cations have been described in phenotypically P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come 2330 Mare: Problems of Pregnancy normal mares that were associated with reduced corpus luteum with ultrasonography of the ovaries fertility.31, 32 More recently, a high rate of recur- might all represent situations in which determining rent early embryonic loss was reported in three serum progesterone concentrations would be useful. Thoroughbred mares that had reciprocal autosomal Minimal accepted concentrations of serum proges- translocations detected by karyotyping.33 These terone vary according to the laboratory or study re- findings suggest that mares with repeated early ported; however, values between 2.0 and 4.0 ng/mL embryonic losses may be considered as candidates are typically used as the minimal serum progesterone for karyotypic investigation in the absence of other concentration during early gestation.34, 43 Because abnormal clinical findings. serum progesterone concentrations vary over time, it is typically recommended that a minimum of two daily samples be evaluated to more accurately assess Uterine period the progestational status of the pregnant mare. It has become common for practitioners to deter- Endocrine factors mine serum progesterone concentrations in embryo- Progesterone is critical for the continued mainte- recipient mares at the time of embryo transfer and at nance of pregnancy in mares throughout gesta- the first pregnancy determination.44 In mares used tion and applications for progestin administration in for embryo recipients, there appears to be a rela- pregnant mares have been previously reviewed.34, 35 tionship between circulating progesterone concentra- During the embryonic period (through Day 40) pro- tions at 7 days after transfer and subsequent survival gesterone is produced solely by the primary corpus of transferred embryos; however, there was no rela- luteum that is formed at the time of the initial ovu- tionship between progesterone concentrations at the lation. Lysis of the corpus luteum associated with time of transfer and subsequent embryo survival.44 failure of maternal recognition of pregnancy36–38 can These results suggest that non-surgical embryo trans- lead to early embryonic death. Failure of the embryo fer techniques may reduce serum progesterone con- to block luteolysis has been identified in mares with centrations, possibly associated with a partial lute- embryonic loss prior to day 20 and was character- olysis initiated at the time of non-surgical embryo ized by the presence of embryonic vesicles that were transfer. too small for days of age and by failure of fixation.39 Hypothyroidism has also been cited as a potential In these mares, serum progesterone concentrations factor contributing to reduced fertility or early em- were lower at days 12, 15, and 18 as was the diame- bryonic loss in mares. Two recent reports have ex- ter of the corpus luteum. Although the role of abnor- amined the relationship between thyroid status and mal luteal function and deficiency of progesterone se- early pregnancy in mares. There was no associa- cretion during early pregnancy is cited as a cause of tion between basal serum T4 concentrations or thy- early embryonic loss in mares,39 there is little, if any, roid supplementation and early pregnancy rates in scientific evidence to support such a cause of em- mares.45 There was also no association between thy- bryonic loss in mares.40–42 However, in cattle, circu- roid function as determined by TRH stimulation test- lating progesterone concentrations in the late embry- ing.46 Together these studies suggest that thyroid onic period (5 weeks) were positively associated with supplementation is unlikely to affect early embryonic embryo survival over the next several weeks. Cows loss rates in mares. with low progesterone at 5 weeks had a higher rate of late embryonic death.28 The frequent use of exoge- Endometrial factors nous progestins to prevent early embryonic loss in mares is not well justified based upon available sci- Clinically, abnormalities of the endometrium have entific evidence. This said, the veterinary practitioner been considered an important factor in the reduced is frequently faced with clients who demand the use fertility of aged mares. A number of studies have of exogenous progestins in pregnant mares. The prac- characterized degenerative changes in the equine en- titioner is trapped in a ‘Catch 22’ in such situations dometrium as a function of increased mare age, and and frequently resorts to the use of such therapy as a the decline in mare fertility with increased age has means to satisfy client demands. been frequently attributed to these changes. These Interpretation of circulating progesterone concen- changes include endometrosis, and endometritis as trations in the early pregnant mare can be useful to well as vascular changes. We examined the impact assess luteal function in situations where inadequate of uterine environment on embryo survival in young progestational support is suspected clinically. Mares and aged mares in which endometrial histopathol- that have poor uterine or cervical tone, increased ogy was evaluated.47 As expected, aged mares had uterine edema, failure of fixation of the conceptus at a higher incidence of degenerative and inflammatory Day 16 post-ovulation, or do not have a detectable changes in the endometrium. When morphologically P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come Embryonic Loss 2331 normal, Day-7 or -8 blastocysts were transferred into Two general categories of uterine cysts have been de- the uterus of young and aged recipient mares, em- scribed including either glandular cysts or lymphatic bryo survival rates at Days 12 and 28 were not dif- cysts.52 However, most macroscopically evident uter- ferent between young and aged mares (55% vs 45% ine cysts are lymphatic in origin because glandular at Day 12). In another report comparing the effect cysts tend to remain microscopic in size.53 of recipient mare age on subsequent embryonic sur- Lymphatic cysts occur with increasing frequency vival and embryonic loss rates, there were no differ- in mares after 10 years of age.37, 54 Although the ences in survival of equine embryos transferred into pathogenesis of lymphatic cysts remains unclear, it is mares that were 2 to 9 years of age compared to 10 to likely that these distended lymphatics originate as a 18 years of age based upon embryo survival rates at consequence of vascular degenerative changes and fi- Day 12.48 There tended, however, to be more embry- brosis in the endometrium that occur with increased onic losses between Day 12 and Day 50 in the older age and parity in mares.55 mares that received embryos. These two studies sug- The effect of lymphatic cysts on fertility in mares gest that uterine effects did not play a major role in remains controversial. Many studies which have the high incidence of early (prior to Day 12) embry- examined the relationship between the presence onic loss that had been previously detected in aged of lymphatic cysts and fertility in mares are con- mares. The impact of uterine abnormalities may be founded by mare age because the presence of cysts more likely during later periods in gestation in these is strongly age-associated and because fertility is also mares and this conclusion is supported by the higher age-dependent. The frequency of lymphatic cysts in rate of embryonic loss in older mares between Days mares over 10 years of age varies between 13% and 12 and 50.48 27% of mares examined54,56–58 with most cysts being The uterine environment clearly plays a role in located near the base of the uterine horn (near the site embryonic losses detected by ultrasound in subfertile of embryo fixation). Eilts et al.56 found no relationship mares and many of these losses are detected in the between the presence or absence of lymphatic cysts period between the first detection of the embryonic on either pregnancy rates or pregnancy loss rates in vesicle with ultrasound at Day 11 and Day 15.37 274 Thoroughbred mares. In contrast, several other Beyond Day 20, embryo loss rates in subfertile mares studies suggest that the presence of lymphatic cysts appeared no higher than those detected in normal had a negative impact on fertility54, 57 although other populations of mares. Endometritis has long been effects such as mare age were not considered in these held as an important factor in embryo losses in sub- studies. fertile mares, and this is supported by the finding that Irrespective of the effect of lymphatic cysts on fer- the rate of embryo loss in mares with intrauterine tility, the presence of single or multiple lymphatic collections of fluid during early pregnancy are associ- cysts can make early pregnancy diagnosis and twin ated with higher rates of embryo loss, lower proges- pregnancy diagnosis based upon transrectal ultra- terone concentrations, and more evidence of inflam- sonography in mares much more difficult. Although mation based upon endometrial histopathology.37, 49 cysts can often be distinguished from the embry- A negative impact of foal-heat breeding on em- onic vesicle based upon their appearance (lymphatic bryonic loss has been reported3 but has not been a cysts are often irregular and multiloculated), soli- consistent finding across studies.1 In some cases it is tary cysts can be confused with the early conceptus difficult to differentiate the effect of the postpartum and the presence of multiple cysts can greatly ham- uterine environment and other effects such as lac- per early pregnancy diagnosis. Therefore, removal of tation or nutritional status.50 Interestingly, there ap- lymphatic cysts has become a more common proce- pears to be an effect of side of fixation of the con- dure in barren mares. ceptus relative to the prior pregnancy in postpartum Ablation of lymphatic cysts can be conducted us- mares. The rate of pregnancy loss (as determined by ing either laser photoablation or loop electrocautery palpation per rectum) was higher when the concep- depending upon the preference of the veterinarian tus was located in the previously gravid uterine horn. and the availability of equipment. Photoablation is 51 These observations await confirming studies based based upon neodymium : yttrium (Nd:YAG) laser ir- upon transrectal ultrasonography, but suggest that ridation.59–61 Loop electrocautery is an alternative for the local uterine environment may indeed play a role ablation of lymphatic cysts that has advantages of re- in survival of the conceptus in the postpartum mare. duced equipment costs and reduced thermal trauma to the endometrium.62 There are no controlled studies which have exam- Lymphatic cysts ined the effect of lymphatic cyst ablation on subse- Among the most common pathology identified by quent mare fertility. Several clinical reports, however, hysteroscopy in mares is the presence of uterine cysts. suggest that subfertile mares that underwent cyst P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come 2332 Mare: Problems of Pregnancy ablation were able to establish pregnancy after the the resulting embryo undergoes embryonic loss at a procedure. Griffin and Bennett60 presented follow- much higher rate as demonstrated by experimental up information on 39 mares that had undergone studies in mice and in cattle.72, 73 laser cyst ablation. These mares were barren with a mean age of 18 years. Of these mares, 26 of 39 (66%) Immune-mediated effects on pregnancy loss conceived the following season. Unfortunately, this study was not controlled and no further information Although stallion ‘incompatibilities’ have been sug- (such as endometrial biopsy) was available to char- gested to cause pregnancy loss in mares (see above), acterize these mares. Other clinical reports have also there is little documented evidence for such causes. suggested that ablation of lymphatic cysts can im- In horses, immune recognition of paternal major his- prove mare fertility59, 63 but appropriately controlled tocompatability antigens appears to occur early in studies are needed to accurately assess the benefits of pregnancy,74 and these antigens are expressed by cyst ablation on fertility of mares. the invasive trophoblast of the chorionic girdle as soon as 32 to 36 days of gestation.74 In extraspe- cific pregnancies in equids (donkey embryos trans- Effect of sire on embryonic loss ferred to horse recipient mares) a high proportion of Some3, 4, 64, 65 but not all66 field studies have described donkey embryos are lost by Days 85 to 90 of ges- an effect of sire on the detected incidence of early tation, and these losses are characterized by a fail- pregnancy loss in mares. In two studies in Thorough- ure of the invasive trophoblast of the donkey con- breds, there appeared to be a subpopulation of stal- ceptus to form endometrial cups within the uterus.76 lions with increased rates of early pregnancy loss These pregnancy losses are associated with little to rates in mares.3, 4 In the group of 49 Thoroughbred no eCG present in the recipient mare and a failure stallions which had mated > 30 mares, 10 stallions of formation of normal allantochorionic placenta.77 had a higher rate of early embryonic loss (13–23%).4 Although the basis for the high rate of early fetal Interestingly, increasing sire age was not associated loss in these extraspecific pregnancies remains to be with the rate of early pregnancy loss in mares bred determined, it has been postulated that these early to those sires in this study,4 contrary to observations fetal losses have an immunological basis.77 This pos- from other species. tulate was supported by the observation that immu- The influence of stallion on embryonic loss rate in nization of pregnant mares with donkey peripheral mares could be related to genetic factors, infectious lymphocytes during early gestation (between Days diseases, or damaged sperm chromatin among oth- 20–50) increased the survival of donkey embryos in ers. There is relatively little information concerning horse recipient mares. However, subsequent attempts possible genetic influence on embryonic loss in to reduce the incidence of recurrent early pregnancy horses. In cattle, a 10% inbreeding coefficient of the loss in mares based upon immunization with pater- embryo was associated with a 1% decline in fertility nal lymphocytes had no effect on the incidence of rates.67 Similar studies have not been conducted for early pregnancy loss.78 the horse; however, increased inbreeding is likely to increase the incidence of early pregnancy loss associ- Effect of heat stress on embryonic loss ated with recessive lethal traits. Early pregnancy loss can also be associated with structural chromosomal Heat stress has long been defined as an important fac- abnormalities in cattle such as the 1/29 Robertsonian tor in early embryonic loss in ruminants; however, translocation.68 An increased rate of early pregnancy there has been little information available concerning loss was present in mares bred to a Thoroughbred the effect of heat stress on early pregnancy in mares. stallion with an autosomal translocation.69 These In a recent abstract, exercise-induced hyperthermia in studies suggest that genetic factors should be consid- mares was associated with a reduced embryo recov- ered as a possible cause of embryonic loss in mares ery rate at Day 7, possibly implying a similar effect bred to individual stallions with unexplained high of heat stress on early pregnancy in mares.79 Future rates of embryonic loss in their mares. studies should examine this possibility in the horse Damage to sperm chromatin is another potentially and address management issues to control heat stress important stallion factor that may influence early em- in mares in hot climates. bryonic loss rates in mares. Sperm chromatin damage may increase during sperm storage70 or after cryop- Effect of nutrition on embryonic loss reservation.71 Spermatozoa have no ability to repair this damage although some DNA repair may occur Adverse affects of poor nutrition and low body in the oocyte after fertilization. If damage to sperm condition scores on fertility in mares have been DNA is extensive, fertilization may occur; however, described,80, 81 and mares that experience increased P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come Embryonic Loss 2333 nutritional demands during lactation appear to be at risk of higher early pregnancy loss rates if under- fed.50, 80 In another trial, mares fed a low-protein diet had a higher rate of early pregnancy loss than did mares fed a high-quality diet (36% vs 7%, respec- tively).82 Lactating mares that experienced embryo loss had weight loss (25 kg) which was not experi- enced by mares fed a higher-quality ration. Further observations on this group of mares suggested that there were changes in serum progestagen concen- trations associated with dietary restriction although mares on the low-protein diet had higher serum progesterone concentrations than did mares fed normally.83 Figure 239.2 Photomicrograph of an equine oocyte demon- strating large number of lipid droplets (L) along with polar dis- Ultrasonographic indicators of tribution of mitochondria (arrows). impending embryonic loss A number of ultrasonographic findings during early tected as early as Days 17 to 20 under experimental pregnancy in mares may suggest impending em- conditions.89 bryonic loss.84 Embryos detected by transrectal ul- trasonography that are significantly retarded in de- Treatment/management of velopment at Days 14 to 21 have a higher rate of subsequent embryonic loss than do embryos that embryonic loss are normally sized for days of age.41,85–87 Although Embryonic loss, particularly when it occurs over the causal relationship between delayed embryonic multiple cycles in a mare, is a particularly challeng- development and subsequent embryo loss is not ing and frustrating reproductive problem to manage. known, two possible mechanisms may account for In many cases, diagnostic approaches may be limited these failures. If the early embryo growth is retarded to ultrasonographic imaging or measurement of cir- due to an embryonic defect, the embryo may not sig- culating progesterone, and these may be undertaken nal appropriately to block luteolysis in the mare, with subsequent embryonic loss. Similarly, the early em- bryo with retarded development may be inherently defective with subsequent failure associated with those defects. Such embryos have been described in some cases as trophoblastic vesicles in which the embryonic trophoblast continues to develop without subsequent development of the inner-cell mass and embryo proper. These pregnancies are typically lost by approximately Day 30 of gestation.88 The presence of intraluminal fluid accumulations during Days 11 to 15 is also associated with a high rate of embryonic loss, and many of these losses appear related to en- dometritis.84 There is also a higher rate of embryonic loss if the embryonic vesicle fails to undergo fixation at the base of the uterus horn around Day 16 or if an embryonic vesicle that was previously fixed moves caudally into the uterine body.84 After the appearance of the embryo proper, detec- tion of embryonic cardiac motion beginning around Day 20–22 of gestation is a useful indicator of em- bryonic viability (Figures 239.2–239.5). The more widespread availability of color Doppler ultrasonog- Figure 239.3 Power Doppler ultrasound scan of a Day raphy provides a useful adjunct for determina- 28 equine conceptus demonstrating blood flow within the tion of embryonic blood flow which may be de- conceptus. (See color plate 119). P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come 2334 Mare: Problems of Pregnancy order to begin the breeding season as early as possi- ble. An early start to the breeding season will provide as many cycles as possible to establish pregnancy. Likewise, frequent pregnancy examination with ul- trasound will allow the early detection of embryonic loss and allow the mare to be re-bred as soon as pos- sible, thus allowing more attempts per season to es- tablish pregnancy. In older mares, it is also prudent to utilize ovulation induction with preparations such as human chorionic gonadotropin (hCG) or GnRH analogs in order to limit the duration of the follicular phase and thereby reduce the prospects of oocyte ag- ing that may contribute to abnormalities of the oocyte in these animals. Because this group of mares is also more prone to post-mating endometritis, aggressive breeding management utilizing uterine lavage and administration of uterine ecbolics should help avoid problems attributed to uterine inflammation that per- sists into diestrus. Insemination or breeding once per cycle is beneficial in older mares and will further min- imize post-mating endometritis. As noted above, supplementation with exoge- nous progesterone has been commonly used in at- tempts to prevent embryonic loss although the effi- cacy of such treatment has yet to be established. In Figure 239.4 Ultrasonographic image of a Day 32 concep- situations where administration of exogenous pro- tus undergoing embryonic death. The conceptus demonstrates gesterone is selected as a means to treat or pre- general disorganization, excess embryonic membranes, and loss of fetal heartbeat. vent embryonic loss in mares, it seems prudent to choose a progestin and a treatment protocol that has been demonstrated to maintain pregnancy in after the loss has occurred. Nonetheless, there are the absence of any exogenous progesterone. A num- several approaches which may improve the chance ber of progestins have been shown to be ineffec- of successfully re-establishing pregnancy in mares tive in maintaining early pregnancy in the absence that have undergone embryonic loss. of endogenous progesterone including medroxypro- In older, problem-breeding mares, it will be useful gesterone acetate, hydroxyprogesterone caproate or to place these animals under artificial photoperiod in hexanoate, norgestomet, and megesterol acetate.90, 91 Preparations which have been shown to be effective in maintaining pregnancy include daily altrenogest (0.44 mg/kg; orally), daily injectable progesterone in oil (150–300 mg, i.m.) or various preparations of controlled-release progesterone which allow longer intervals between administration.92–95 Although the recommended duration of progesterone supplemen- tation varies, in most cases it seems prudent to continue administration of supplemental progestins through 100–120 days of gestation.34 This includes the period of time in which placental production of progestins is adequate to maintain pregnancy as well as the period of time in which much of placental for- mation is complete in mares. If progestin therapy is discontinued prior to 60 days of gestation, it may be prudent to assay serum progesterone concentra- Figure 239.5 Color Doppler ultrasonographic image of a Day 45 equine conceptus with displacement of the fetus toward the tions in the mare before discontinuing progesterone cervix and loss of cardiac motion. Blood flow is detectable in the administration. This is possible in those mares being caudal vaginal artery but not within the conceptus. (See color supplemented with altrenogest which does not cross- plate 120). react in most progesterone assays. P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come Embryonic Loss 2335 Although no long-term deleterious effects on the is unclear, these studies suggest that in some mares conceptus have been established for the administra- with persistent endometritis, the administration of tion of supplemental progestins in mares, care should immunostimulants should be considered as a part of be used in supplementation with progestins. Mares the therapeutic regimen. should be monitored regularly for continued devel- Likewise, investigators have suggested that ad- opment of normal pregnancy because death and re- ministration of either corticosteroids or NSAIDs may tention of the conceptus may occur in later gesta- also improve fertility or reduce early pregnancy loss tion in mares receiving progesterone.96 Mares that are in mares. Acute administration of flunixin meglu- given supplemental progestins soon after mating and mine at the time of nonsurgical embryo transfer has ovulation should be monitored closely for develop- been suggested as a means to improve pregnancy ment of post-mating endometritis, which may be ex- rates44 although controlled studies are lacking to acerbated by exogenous progestin administration. support a positive effect of NSAID administration In a series of field trials, mares were given the on embryo survival. Another study demonstrated GnRH analog, buserelin (20 or 40 µg) between days an increased early pregnancy rate in barren mares 8 to 12 after mating and ovulation in mares.97 Over- that received prednisolone (0.1 mg/kg, twice daily) all pregnancy rates were significantly increased by for 4 days before and at the time of artificial in- approximately 10% in buserelin-treated mares com- semination with frozen semen compared to control pared to controls. The authors of this study specu- mares.104 Wilsher et al. demonstrated that admin- lated that differences in early pregnancy rates in their istration of NSAIDs (meclofenamic acid) increased study may have been due to a luteotrophic effect of the range of synchrony between donor and recipient administration of GnRH possibly through the induc- mares that resulted in successful embryo transfer.105 tion of additional corpora lutea formation;97 how- Administration of meclofenamic acid to recipient ever, a subsequent study was unable to demonstrate mares beginning at 9 days after ovulation supported either luteinization or ovulation of diestrous follicles the establishment of pregnancy in recipient mares in mares after administration of another GnRH ana- that ovulated up to 3 days prior to the donor mare log, deslorelin, at days 8 or 12.98 More recently, mares although the mechanism of action did not appear to that received 40 µg of buserelin at day 10 after ar- act via suppression of prostaglandin F2α -mediated tificial insemination and ovulation had an increase luteolysis.105 Together, these studies suggest that in circulating LH concentrations but no change in the immune response around the time of mating circulating progesterone concentrations.99 Pregnancy may alter the subsequent uterine environment and rates, although numerically higher in buserelin- embryo survival, and future studies should be treated mares, were not significantly different from directed towards a better understanding of the mech- controls in this particular study.99 Overall, these anisms involved as they relate to embryonic loss in studies suggest that administration of potent GnRH mares. analogs during early diestrus in mares may posi- Because of the potential adverse effect of chronic tively influence pregnancy rates although the mech- or persistent endometritis on survival of the early anism of action of such treatment remains to be de- embryo in mares, some authors have advocated ad- termined. In other species, multiple studies suggest ministration of systemic antibiotics to mares with re- a positive effect of administration of GnRH analogs current early embryonic loss.35 In situations where during early diestrous on fertility100 although this re- chronic endometritis has been documented or is sus- sult has not been supported in other reports.101 pected, administration of broad-spectrum antibiotics In addition to administration of exogenous pro- during early gestation may be indicated. No con- gestins to mares in attempts to prevent embryonic trolled studies exist to support this application; how- loss, a number of studies have examined the ad- ever, anecdotal information suggests that treatment ministration of corticosteroids, non-steroidal anti- with systemic antibiotics may be helpful in selected inflammatory drugs (NSAIDs), and immunomodu- cases. lators to the mare to improve early pregnancy rates Assisted reproductive techniques may offer some or prevent embryonic loss. The administration of im- benefit to improve the prospects of establishing preg- munomodulators or immunostimulants have been nancy from older, subfertile mares. Unfortunately, used in attempts to improve fertility in mares with embryo recovery from uterine flushes is often re- persistent endometritis.102, 103 In a recent report, fer- duced, and survival of embryos from such mares tility of mares with persistent endometritis was im- is lower after embryo transfer. Likewise, the ability proved after administration of Propionibacterium ac- to establish pregnancies from older, subfertile mares nes (EqStimTM ) compared to control mares, and mares also appears to be somewhat reduced after proce- treated with P. acnes tended to have a lower rate of dures such as oocyte transfer, and more attempts pregnancy loss.102 Although the mechanism of action will often be necessary to successfully establish P1: SFK/UKS P2: SFK Color: 1C c239 BLBK232-McKinnon February 11, 2011 18:54 Trim: 279mm X 216mm Printer Name: Yet to Come 2336 Mare: Problems of Pregnancy pregnancy from these mares. Carnevale et al.106 re- 15. Weber JA, Freeman DA, Vanderwall DK, Woods GL. ported that Day 50 pregnancy rates in recipient mares Prostaglandin E2 hastens oviductal transport of equine embryos. Biol Reprod 1991;45:544–6. that received oocytes tended to be lower from donor 16. Ball BA, Brinsko SP, Schlafer DH. Histopathologic exam- mares over 20 years of age than from donor mares ination of the oviduct and endometrium of fertile and less than 20 years of age. When mares at the ex- subfertile mares. Pferdeheilkunde 1997;13:548–9. treme of age ranges were compared, mares less than 17. Brinsko SP, Ignotz GG, Ball BA, Thomas PGA, Currie 15 years of age established pregnancies from 50% of WB, Ellington JE. Characterization of polypeptides syn- oocytes compared to 16% of oocytes from mares over thesized and secreted by oviductal epithelial cell explants obtained from young, fertile mares and aged, subfertile 23 years of age.106 mares. Am J Vet Res 1996;57:1346–58. 18. Lantz K, Enders A, Liu I. Possible significance of cells within intraluminal collagen masses in equine oviducts. Anat Rec 1998;252:568–79. References 19. Allen WR, Wilsher S, Morris L, Crowhurst JS, Hillyer 1. Woods GL, Baker CB, Baldwin JO, Ball BA, Bilinski JL, MH, Neal HN. Laparoscopic application of PGE2 to re- Cooper WL, Ley WB, Mank EC, Erb HN. Early preg- establish oviducal patency and fertility in infertile mares: nancy loss in broodmares. J Reprod Fertil Suppl 1987;35: a preliminary study. Equine Vet J 2006;38:454–9. 455–9. 20. Zent WW, Liu IKM, Spirito MA. Oviduct flushing as a 2. McDowell KJ, Powell DG, Baker CB. Effect of book size treatment for infertility in the mare. Equine Vet J Suppl and age of mare and stallion on foaling rate in Thorough- 1993;15:47–8. bred horses. J Equine Vet Sci 1992;12:364–7. 21. Armstrong DT. Effects of maternal age on oocyte devel- 3. Morris LHA, Allen WR. Reproductive efficiency of in- opmental competence. Theriogenology 2001;55:1303–22. tensively managed Thoroughbred mares in Newmarket. 22. Brinsko SP, Ball BA, Ellington JE. In vitro maturation of Equine Vet J 2002;34:51–60. equine oocytes obtained from different age groups of sex- 4. Allen WR, Brown L, Wright M, Wilsher S. Reproduc- ually mature mares. Theriogenology 1995;44:461–9. tive efficiency of Flatrace and National Hunt Thorough- 23. Carnevale EM, Ginther OJ. Defective oocytes as a cause bred mares and stallions in England. Equine Vet J 2007; of subfertility in old mares. Biol Reprod 1995; Mono. 1: 39:438–45. 209–14. 5. Vanderwall DK, Newcombe JR. Early embryonic loss. In: 24. Rambags BPB, van Boxtel DCJ, Tharasanit T, Lenstra Samper JC, Pycock JF, McKinnon AO (eds) Current Ther- JA, Colenbrander B, Stout TAE. Maturation in vitro apy in Equine Reproduction. St Louis: Saunders Elsevier, leads to mitochondrial degeneration in oocytes recovered 2007; pp. 374–83. from aged but not young mares. Anim Reprod Sci 2006; 6. Woods GL, Hillman RB, Schlafer DH. Recovery and eval- 94:359–61. uation of embryos from normal and infertile mares. Cor- 25. Torner H, Alm H, Kanitz W, Goellnitz K, Becker F, nell Vet 1986;76:386–94. Poehland R, Bruessow KP, Tuchscherer A. Effect of initial 7. Vogelsang SG, Vogelsang MM. Influence of donor par- cumulus morphology on meiotic dynamic and status of ity and age on the success of commercial equine embryo mitochondria in horse oocytes during IVM. Reprod Dom transfer. Equine Vet J Suppl 1989;8:71–2. Anim 2007;42:176–83. 8. Ball BA, Little TV, Hillman RB, Woods GL. Pregnancy 26. Carnevale EM, Bergfelt DR, Ginther OJ. Aging effects rates at Days 2 and 14 and estimated embryonic loss rates on follicular activity and concentrations of FSH,LH, and prior to Day 14 in normal and subfertile mares. Theri- progesterone in mares. Anim Reprod Sci 1993;31:287–99. ogenology 1986;26:611–19. 27. Carnevale EM, Bergfelt DR, Ginther OJ. Follicular activ- 9. Ball BA, Little TV, Weber JA, Woods GL. Viability of Day- ity and concentrations of FSH and LH associated with 4 embryos from young, normal mares and aged, subfer- senescence in mares. Anim Reprod Sci 1994;35:231–46. tile mares after transfer to normal recipient mares. J Re- 28. Inskeep EK. Preovulatory, postovulatory, and postma- prod Fertil 1989;85:187–94. ternal recognition effects of concentrations of proges- 10. Brinsko SP, Ball BA, Miller PG, Thomas PGA, Ellington terone on embryonic survival in the cow. J Anim Sci 2004; JE. In vitro development of day two embryos obtained 82:E24–39. from young, fertile mares and aged, subfertile mares. 29. Woods JA, Bergfelt DR, Ginther OJ. Effects of time of in- J Reprod Fertil 1994;102:371–8. semination relative to ovulation on pregnancy rate and 11. Brinsko SP, Ball BA, Ignotz GG, Thomas PGA, Currie embryonic loss rate in mares. Equine Vet J 1990;22:410–15. WB, Ellington JE. Initiation of transcription and nucle- 30. Huhtinen M, Koskinen E, Skidmore JA, Allen WR. Re- ologenesis in equine embryos. Mol Reprod Dev 1995;42: covery rate and quality of embryos from mares insemi- 298–302. nated after ovulation. Theriogenology 1996;45:719–26. 12. Van Niekerk CH, Gerneke WH. Persistence and 31. Power MM. The first description of a balanced reciprocal parthenogenetic cleavage of tubal ova in the mare. translocation [t(1q;3q)] and its clinical effects in a mare. Onderstepoort J Vet Res 1966;31:195–232. Equine Vet J 1991;23:146–9. 13. Betteridge KJ, Mitchell D. Direct evidence of retention of 32. Lear TL, Layton G. Use of zoo-FISH to characterise a unfertilized ova in the oviduct of the mare. J Reprod Fertil reciprocal translocation in a thoroughbred mare: t(1;1 1974;39:145–8. 6)(q16;q21.3). Equine Vet J 2002;34:207–9. 14. Betteridge KJ, Eaglesome MD, Flood PF. Embryo trans- 33. Lear TL, Lundquist J, Zent WW, Fishback WD, Clark A. port through the mare’s oviduct depends on cleavage Three autosomal chromosome translocations associated and is independent of the ipsilateral corpus luteum. J Re- with repeated early embryonic loss in the domestic horse prod Fertil Suppl 1979;27:387–94. (Equus caballus). Cytogenet Genome Res 2008;120:117–22. P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 1 (Figure 4.5 – Volume 1) B cells CD4+ T cells CD8+ T cells equine fetus equine neonate Plate 2 (Figure 32.3 – Volume 1) Plate 4 (Figure 40.2 – Volume 1) Plate 3 (Figure 37.1a-b – Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) (c) Plate 5 (Figure 56.2a-c – Volume 1) Plate 6 (Figure 57.1 – Volume 1) Plate 7 (Figure 59.1 – Volume 1) Plate 8 (Figure 59.2 – Plate 9 (Figure 59.3 – Volume 1) Plate 10 (Figure 59.4 – Volume 1) Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 11 (Figure 59.5 – Volume 1) Plate 12 (Figure 59.6 – Volume 1) Plate 13 (Figure 59.7 – Volume 1) Plate 14 (Figure 59.8 – Volume 1) Plate 15 (Figure 59.9 – Volume 1) Plate 16 (Figure 59.10 – Volume 1) Plate 17 (Figure 59.11 – Volume 1) Plate 18 (Figure 59.12 – Volume 1) Plate 19 (Figure 59.13 – Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) (c) Plate 20 (Figure 59.14a-c – Volume 1) Plate 21 (Figure 59.15 – Volume 1) Plate 22 (Figure 59.17 – Volume 1) Plate 23 (Figure 59.18 – Volume 1) Plate 24 (Figure 59.19 – Volume 1) Plate 25 (Figure 59.20 – Volume 1) Plate 26 (Figure 59.21 – Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 27 (Figure 59.22 – Volume 1) Plate 28 (Figure 59.23 – Volume 1) Plate 29 (Figure 59.24 – Volume 1) (a) (b) Plate 30 (Figure 59.25a-b – Volume 1) Plate 31 (Figure 59.26 – Volume 1) Plate 32 (Figure 59.27 – Volume 1) Plate 33 (Figure 59.28 – Volume 1) Plate 34 (Figure 59.29 – Volume 1) Plate 35 (Figure 59.31 – Volume 1) Plate 36 (Figure 73.4 – Volume 1) Plate 37 (Figure 74.1 – Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 39 (Figure 74.3 – Volume 1) Plate 38 (Figure 74.2 – Volume 1) Satge V Plate 41 (Figure 124.7 – Volume 1) Spermatogonia Plate 40 (Figure 97.21 – Volume 1) Plate 42 (Figure 124.8 – Volume 1) (a) (b) Plate 43 (Figure 126.7a-b – Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) (c) (d) Plate 44 (Figure 126.9a-d – Volume 1) (a) (b) (c) (d) Plate 45 (Figure 126.10a-d – Volume 1) (a) (b) Plate 46 (Figure 150.2a-b – Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come T T T T T (a) (b) Plate 47 (Figure 151.11a-b – Volume 1) Plate 48 (Figure 152.3 – Volume 1) Plate 49 (Figure 152.4 – Volume 1) Plate 50 (Figure 153.3 – Volume 1) (a) (b) (c) Plate 51 (Figure 153.4 – Volume 1) Plate 52 (Figure 153.6a-c – Volume 1) Plate 53 (Figure 153.7 – Plate 54 (Figure 155.1 – Volume 1) Plate 55 (Figure 155.2 – Volume 1) Volume 1) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 56 (Figure 156.6 – Volume 1) Plate 57 (Figure 156.7 – Volume 1) Plate 58 (Figure 156.8 – Volume 1) Plate 59 (Figure 156.10 – Volume 1) (a) (b) (c) (d) Plate 60 (Figure 172.4a-d – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) (c) (d) (e) (f) Plate 61 (Figure 172.5a-f – Volume 2) Plate 62 (Figure 203.8 – Volume 2) Plate 63 (Figure 204.1 – Volume 2) Plate 64 (Figure 204.2 – Volume 2) Plate 65 (Figure 204.3 – Volume 2) Plate 66 (Figure 204.4 – Volume 2) Plate 67 (Figure 204.5 – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 68 (Figure 204.6 – Volume 2) Plate 69 (Figure 204.7 – Volume 2) Plate 70 (Figure 204.8 – Volume 2) Plate 71 (Figure 204.9 – Volume 2) Plate 72 (Figure 204.10 – Volume 2) Plate 73 (Figure 204.11 – Volume 2) (a) (b) Plate 74 (Case 205-1a-b – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 75 (Case 205-2 – Volume 2) Plate 76 (Case 205-3 – Volume 2) Plate 78 (Case 205-5 – Volume 2) Plate 77 (Case 205-4 – Volume 2) Plate 79 (Case 205-6 – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come Plate 80 (Case 205-7 – Volume 2) Plate 81 (Case 205-8 – Volume 2) Plate 82 (Figure 207.6 – Volume 2) Plate 83 (Figure 208.6 – Volume 2) Plate 84 (Figure 208.8 – Volume 2) Plate 85 (Figure 208.9 – Volume 2) Plate 86 (Figure 208.12 – Volume 2) Plate 88 (Figure 209.1 – Volume 2) Plate 87 (Figure 208.13 – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) (e) (c) (d) Plate 89 (Figure 209.2 a-e – Volume 2) (a) (b) (c) (d) Plate 90 (Figure 209.3 a-d – Volume 2) (a) (b) Plate 91 (Figure 209.4 a-b – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) Plate 92 (Figure 209.5 a-b – Volume 2) Plate 93 (Figure 209.7 – Volume 2) (a) (b) (c) (d) Plate 94 (Figure 209.8 a-d – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (b) Plate 95 (Figure 209.9 a-b – Volume 2) Plate 97 (Figure 213.1 – Volume 2) (a) (a) (b) Plate 96 (Figure 209.10a-b – Volume 2) (b) (a) (b) (c) Plate 98 (Figure 215.3 a-b – Volume 2) Plate 99 (Figure 215.6 a-c – Volume 2) P1: SFK/UKS P2: SFK/UKS QC: SFK/UKS T1: SFK Color: 1C cp-final BLBK232-McKinnon October 27, 2010 17:6 Trim: 279mm X 216mm Printer Name: Yet to Come (a) (d) Maximum Blood Flow Serration Minimum Apex (b) (e) ) Blood Flow ( Ovulation Maximum Minimum Blood Flow Serration Apex ? Septated Evacuation, HAF, Atresia -24 -12 -6 -5 -4 -3 -2 -1 (c) (f) Hours before expected evacuation Plate 100 (Figure 215.7 a-f – Volume 2) Plate 101 (Figure 215.10 – Volume 2) (a) (b) 9 12 6 3 (c) 12 100 Clock-face 11 1 positions Apical 80 % of mares (n=21) area 10 2 60 Preovulatory 9 Follicle 3 40 20 8 Basal 4 Serration area Blood-flow 0 7 5 1 2 3 4 5 6 7 8 9 10 11 12 Serrated granulosa 6 Clock-face position Vascularized theca (d)