THE ANATOMICAL RECORD 293:1873–1879 (2010)

Morphometric Analysis of YakPlacentomes During Gestation

BEN LIU,1 YAN CUI,1 BO YANG,2 JIANGFENG FAN,1 ZHIWEN ZHAO,1

AND SIJIU YU1*

1Gansu Province Livestock Embryo Engineering Research Center, Department of ClinicalVeterinary Medicine, Faculty of Veterinary Medicine, Gansu Agricultural University,

Lanzhou, China2Laboratory of Animal Anatomy & Tissue Embryology, Department of Basic Veterinary

Medicine, Faculty of Veterinary Medicine, Gansu Agricultural University, Lanzhou, China

ABSTRACTTo study the morphological characteristics of placentomes during ges-

tation, thirty-one yaks were used to determine anatomical and histologicaldata of placentomes by morphometric method. The results showed thatthere were no significant changes in the total number of yak placentomesduring gestation, but remarkably fewer placentomes in the non-pregnanthorn compared with the pregnant horn. The mean size of placentomesincreased significantly from <61 days to 181–210 days and decreased in211þ days. The development of placentomes in the pregnant horn wereobviously different from that of the non-pregnant horn except <61 days,which were longer, wider, and thicker. The volume density of fetal villiincreased during gestation, with a concomitant reduction in the volumedensity of caruncular endometrium. However, there was no significantchange in the surface density of fetal villi. The volume density of binu-cleate cell increased in prophase and then decreased. No apparent changesin the volume densities of maternal and fetal pyknotic cell occurredthroughout gestation. There was a significant increase in the average totalvolume of yak placentomes from 61–90 days to 181–210 days and a signifi-cant decrease in 211þ days. The same patterns were showed in the aver-age total volumes of caruncular endometrium and binucleate cell as wellas the mean total surface area of fetal villi. However, the mean totalvolumes of fetal villi, maternal and fetal pyknotic cell increased constantlywith increasing gestation time. Three types of placentome shape were iden-tified in yak. The results showed that the maternal-fetal contact area ofyak was enlarged by increasing the placentome size as well as the totalvolume and surface area of placentome component during pregnancy, soplacental function could be enhanced further to meet the needs of fetaldevelopment. Anat Rec, 293:1873–1879, 2010. VVC 2010 Wiley-Liss, Inc.

Keywords: yak; gestation period; placentome; morphometry;stereology

In the cow, the allantochorionic membrane and endome-trium become modified for exchange at specialized pointof contact, the placentome. This structure, composed ofmaternal caruncle and foetal cotyledon, is the organthrough which respiratory gases, nutrients, and wastesare transported. Normal placentome growth and develop-ment is essential for foetal growth and development(Laven and Peters, 2001). After transfer of in vitro pro-duced embryos, developmental problems of the fetus, the

Grant sponsor: National Natural Science Foundation ofChina; Grant number: 30871903.

*Correspondence to: Sijiu Yu, Faculty of Veterinary Medicine,Gansu Agricultural University, Lanzhou, China. Fax: 86-931-7631220. E-mail: sjyu@163.com

Received 14 December 2009; Accepted 16 June 2010

DOI 10.1002/ar.21231Published online 9 September 2010 in Wiley Online Library(wileyonlinelibrary.com).

VVC 2010 WILEY-LISS, INC.

placenta, or both occurred in cattle, including increasedpregnancy loss, oversized or malformed fetuses and calves,increased size and decreased number of placentomes, andother abnormalities of placental development (Farin et al.,2001; Bertolini et al., 2002; Miglino et al., 2007).

The yak (Bos grunniens) reproductive efficiency is low. Itreach puberty late, at a mean age of 33.4 � 6.0 months,and most yak cows calve only once every 2 years (Yu et al.,1993). To improve the reproductive efficiency, Cui and Yu(1999) studied the anatomical characteristics of internalgenital organs and growth of various parameters of non-pregnant uterus in different ages yaks. Besides, the previ-ous research also studied on the apoptosis and its pathwayduring implantation and placentation in yaks (Ju, 2006;Yong, 2006). There is, however, very little informationavailable on the normal variability of the yak placentomeduring gestation. Present study was undertaken to investi-gate the changes in individual placentome during preg-nancy, particularly parameters in their anatomy andhistology, to provide a basic knowledge of yak reproductivephysiology and a normal referenced database of placentalabnormality as a contribution to the improvement of yakreproduction.

MATERIALS AND METHODSCollection and Recording

Thirty-one female yaks from 6 to 9 years of age, with thebreeding season range from July to October, were includedin the investigation. They were purchased from the abat-toir in Xining City of Qinghai Province, but there were fouryaks whose gestation stage were 181–210 days and 211þdays were purchased from small holders in Tianzhu Auton-omous County of Gansu. Gestational age was estimatedaccording to the mating record provided by the host and fe-tal crown-rump length after slaughter (Wang, 2008), andthe uterus categorized into one of seven groups depending

on gestational age (Table 1). Experimental yaks wereslaughtered at an abattoir, immediately after collection,placental samples were chosen and fixed first (see detailsbelow). The organs were then removed and brought to theplace of examination immediately to be examined, meas-ured, and weighed. The fetuses were weighed, and fetalcrown-rump length was measured, in addition, fetaldevelopmental detail was recorded. All placentomes weredissected away from the uterine mucosa and counted, andthe dimensions (length, width, and depth) of all the placen-tomes were measured (Table 2), and the placentome shapewas also recorded (Fig. 1).

Histology

Four placentomes from the same area of the pregnanthorn (close to the fetus) from each yak were randomlyselected and further processed for the stereological analy-sis. About 1 cm3 tissue blocks were obtained from each pla-centome, then fixed into 4% neutral paraformaldehydephosphate buffer (PH 7.4) for 24 h, dehydrated in gradedalcohol, and embedded in paraffin wax. Vertical sections 4lm thick were cut and stained using a standard

TABLE 1. Number of yak uteri collected and fetal developmentin each stage during gestation

Days ofgestation(day)

Number ofuteri

collectedCrown-rumplength (cm)

Range ofcrown-rumplength (cm)

Fetalweight (g)

<61 7 2.8 � 0.7 1.2–5.8 1.9 � 0.561–90 7 12.6 � 1.1 7.3–15.6 100.0 � 22.091–120 5 23.3 � 1.2 21.0–27.0 462.0 � 52.4121–150 4 33.9 � 2.0 28.0–36.5 1637.5 � 293.9151–180 4 44.5 � 2.5 42.0–47.0 3310.0 � 560.0181–210 2 60.0 � 1.0 59.0–61.0 9445.0 � 105.0211þ 2 74.0 � 5.0 69.0–79.0 11295.0 � 295.0

TABLE 2. Number and size of yak placentomes in each stage during gestation

Days ofgestation(day)

Pregnant horn Non-pregnant horn

Number Length (cm) Width (cm) Depth (cm) Number Length (cm) Width (cm) Depth (cm)

<61 36.90 � 1.70 0.42 � 0.02 0.31 � 0.01 0.22 � 0.02 32.60 � 1.50 0.37 � 0.01 0.30 � 0.01 0.15 � 0.0161–90 46.00 � 4.30 1.85 � 0.06 1.31 � 0.04 0.60 � 0.03 41.00 � 2.90 1.19 � 0.04 0.82 � 0.03 0.48 � 0.0291–120 41.00 � 2.10 2.58 � 0.07 1.94 � 0.06 0.84 � 0.02 34.20 � 2.50 1.72 � 0.07 1.27 � 0.05 0.50 � 0.03121–150 41.50 � 4.50 4.19 � 0.11 2.90 � 0.08 1.04 � 0.02 27.30 � 3.30 2.97 � 0.12 2.11 � 0.10 0.78 � 0.03151–180 43.00 � 3.00 4.65 � 0.19 3.36 � 0.14 1.52 �0.04 29.00 � 7.00 2.70 � 0.17 1.83 � 0.12 0.97 � 0.06181–210 44.50 � 2.50 6.56 � 0.22 4.09 � 0.16 1.70 � 0.06 34.00 � 1.00 5.27 � 0.26 3.33 � 0.19 1.40 � 0.07211þ 54.50 � 0.50 5.84 � 0.12 4.17 � 0.09 1.08 � 0.03 35.50 � 5.50 4.37 � 0.19 2.98 � 0.13 0.81 � 0.04

Fig. 1. Graphic illustrations of yak placentome shapes found in thisstudy (black area: caruncular tissue). (a) The flat placentome is slightlyconvex and non-stalked. (b) The convex placentome is mushroomshaped due to a distinct caruncular stalk formation. (c) The concaveplacentome is cup shaped similar to the sheep.

1874 LIU ET AL.

hematoxylin and eosin (HE) protocol. Light microscopicobservations and section images acquirement were carriedout using Olympus DP71 Light Microscope (including DPcontrol and Image-Pro Express, Japan).

Stereological Estimations

Each placentome was cut transversely and serially sec-tioned to generate a certain number of slices. Then, thelength of the placentome was divided by the number of sli-ces to obtain a slab thickness. Subsequently, a test-pointsystem was placed over each slab to estimate its area. TheCavalieri principle was applied to reach a volume estimate(Gundersen and Jensen, 1987; Michel and Cruz-Orive,1988): Vplac ¼ d � (a/p) � RP, where d was the average slicethickness, (a/p) was the area associated with each testpoint and RP was the total number of points hitting the cutsurfaces of slabs. To obtain the total volume, all placentomevolumes in a given placenta were summed, and this value,Vref, was represented as the total placentome volume(Ribeiro et al., 2008).

One section from each of four different blocks of thesame placenta was used for further analysis. Systematicrandom sampling was used to select fields for stereologicalanalysis, including the volume densities of fetal villi, car-uncular endometrium, fetal binucleate cells, fetal pyknoticcells, and maternal pyknotic cells, were evaluated by pointcount methods using computer-assisted image analysis(Image-Pro Express). For analysis of fetal villi and carun-cular endometrium, 10 fields of view representing a total of1.090 mm2 of each placentome tissue were examined usinga 256-point grid system. For analysis of fetal binucleatecells, fetal and maternal pyknotic cells, 10 fields of viewrepresenting a total of 0.275 mm2 of each placentome tis-sue were examined using a 256-point grid system. In addi-tion, the surface density of fetal villi was estimated byusing a test system of lines superimposed on images pro-jected onto a computer screen, for analysis of it, 10 fields ofview representing a total of 0.275 mm2 of each placentometissue were examined.

The volume densities (Vv) of fetal villi, caruncular endo-metrium, fetal binucleate cells, fetal pyknotic cells, andmaternal pyknotic cells were calculated using the formulaVv ¼ Pa/Pt, where Pa was the total number of points fallingon the given tissue, and Pt was the total number of pointsfalling within placentomes. The total volume of placentalcomponent in total placentome volume was estimated indi-

rectly by multiplying the total placentome volume (Vref) byvolume density for each component. The surface density offetal villi (Sv) was measured by counting the line interceptswith the same grid in the same fields and was calculatedusing the formula Sv ¼ 2 � Ia/Lt, where Ia was the numberof intercepts of fetal villi with the line, and Lt was the totallength of the lines applied. The total surface area of fetalvilli (Sfv) in the placentomes was estimated by multiplyingthe surface density (Sv) by the total placentome volume(Baur, 1973; Miles et al., 2004; Ribeiro et al., 2008).

Statistical Analysis

Data were analyzed using the SPSS software, version13.0. First, comparisons between the different gestationalages were drawn by means of one-way analysis of variancewhen two conditions were simultaneously fulfilled, that is,data distributions were normal and variances were homo-geneous. Second, differences for placentome number andsize were compared between the pregnant horn and thenon-pregnant horn using the paired Student t test. Differ-ences were considered significant below the 5% level ofprobability. Mean data were expressed � SEM.

RESULTS

The average crown-rump length and weight of thefoetuses of 31 yaks in different gestation ages were givenin Table 1. As could be seen from the table, the length andweight of the foetuses became larger and heavier, withincreasing gestation age (P < 0.05). They were 2.8 � 0.7cm and 1.9 � 0.5 g in <61 days foetuses, and increased to74.0 � 5.0 cm and 11,295.0 � 295.0 g in 211þ days,respectively.

Placentome Number

The placentomes were arranged in three or four rows oneach side of the horns during gestation, but diminished intwo or three rows to the apex of the horns. The mean totalnumber of placentomes per uterus at each stage of preg-nancy had been shown in Fig. 2. There was no significantchange in the total number of placentomes during gesta-tion (P > 0.05). As could be seen from Table 2, the meantotal number of placentomes in the pregnant horn showedno significant differences at each stage of pregnancy (P >0.05). The similar results were also presented in the non-pregnant horn. However, the number of placentomes in the

Fig. 3. The mean size of yak placentomes in each stage duringgestation.

Fig. 2. The total number of yak placentomes in each stage duringgestation.

MORPHOMETRY OF YAK PLACENTOMES 1875

non-pregnant horn was significantly fewer than that in thepregnant horn at all stages of pregnancy (P < 0.05).

Placentome Size

The average size of placentomes in both horns for eachstage of gestation was presented in Fig. 3. The size of pla-centomes increased significantly from 0.39 � 0.30 � 0.19cm3 at <61 days to 6.00 � 3.77 � 1.57 cm3 at 181–210 daysbut decreased to 5.21 � 3.66 � 0.97 cm3 at 211þ days (P <0.05). As shown in the Table 2, the mean size of placen-tomes in each horn increased significantly during gestation(P < 0.05). At all gestation stages, the average size of pla-centomes in the pregnant horn were significantly greaterthan that in the non-pregnant horn (P < 0.05), except atgestation of <61 days.

The present study found that, in two of the 31 uteri, alarge mauve diffuse area of villous attachment between thematernal and foetal epithelia occurred in the pregnanthorn. This structure developed around the normal placen-tomes, spreading along endometrium between them. It wasprominent in the area of the largest placentomes (thosenearest the foetus). However, there was no evidence of thistype of placental structure in non-pregnant horn. In yak,otherwise, placentomes with length <1 cm were found inboth horns during late gestation, distributing aroundlarger placentomes, but the number of them in each hornwas very little.

Placentome Shape

There were three significantly different placentomeshapes in yak (Fig. 1). The majority of them were flat(non-pedunculated), and very few were mushroom-

shape (pedunculated). These two types of placentomeshape remained with the foetus-folding-mother type.Fifty percent of all uteri of greater than 121 days gesta-tion had at least one mushroom-shape placentome.There was no evidence that the proportion of mushroom-shape placentomes changed with the stage of pregnancy(P > 0.05) (see Table 3). The average sizes of mushroom-shape placentomes were presented in Table 3 andshowed no significant differences with that of flatplacentome at each stage of pregnancy (P > 0.05). Onlythree concave placentomes appeared calyx-shape in onepregnant horn between days 121–150, belonging to themother-folding-foetus type. The proportion and averagesize of this type of placentome were 4.48% and 1.87 �1.07 � 0.57 cm3, respectively. The size of concave placen-tome was similar to that of mushroom-shape placentome(P > 0.05), but significantly smaller than that of flatplacentome between days 121–150 (P < 0.05).

Relative Vv and Sv of Specific Tissues

Figure 4 showed a section of placentome demonstrat-ing both maternal and fetal components. Results of thestereological analysis of the placentomes at each stage ofpregnancy, except <61 days, were presented in Table 4.Based on morphometric analysis, the placentomes exhib-ited a significant increase in the volume density of fetalvilli (from 61 to 90 days, 36.75% � 1.05%, to 211þ days,54.04% � 2.12%; P < 0.05), and a significant reductionin the volume density of caruncular endometrium (from63.25% � 1.05% to 45.96% � 2.12%; P < 0.05).

The volume density of binucleate cells increased from9.78% � 0.30% at 61–90 days to 11.01% � 0.39% at 91–120 days (P < 0.05), after that significant decrease took

TABLE 3. The proportion and average size of mushroom-shapeplacentome during gestation

Days ofgestation(day)

Proportion ofplacentae withat least one

mushroom-shapeplacentome

Proportion ofmushroom-shapeplacentome (%)

Average size ofmushroom-shapeplacentome (cm)

121–150 2/4 2.10 2.30 � 1.60 � 0.63151–180 1/4 2.44 2.70 � 1.90 � 1.00181–210 1/2 1.33 5.00 � 2.30 � 1.30211þ 2/2 4.44 4.28 � 3.23 � 0.88

Fig. 4. Light micrograph of the developing fetal villi (FV) within thecaruncular endometrium (CE) of a yak placentome during gestation,crown-rump length 42.0 cm, stained using a standard hematoxylinand eosin (HE) protocol. Note that the fetal villi contain binucleate cells(arrows), and there are maternal and fetal pyknotic cells (arrowhead) in

the fetal villi and caruncular endometrium. (a) A point grid projectedon the image that is used for assessment of volume densities of pla-centome component. (b) A test system of lines projected on the imagethat is used to count the number of intersections with the fetal villi (Ia)and estimate the surface density of fetal villi.

1876 LIU ET AL.

place, the lowest value of the volume density was obtainedin 211þ days (8.16% � 0.47%).The results indicated thatthe volume densities of maternal pyknotic cells variedbetween 0.63% � 0.05% and 0.82% � 0.11%, with no sig-nificant changes during pregnancy (P > 0.05). There werealso no significant variations in the volume densities of fe-tal pyknotic cells during pregnancy (P > 0.05), the highestvalue was found in 151–180 days (0.50% � 0.07%) and thelowest was in 61–90 days (0.26% � 0.05%). No apparentchanges in the surface density values of fetal villi occurredthroughout gestation in either group (P > 0.05).

Total Volumes and Surface Areas of SpecificTissues and Vref

In Table 5, the average total volumes of placentomes,relational tissues and cells as well as the mean totalsurface area of fetal villi in different gestation stageswere given. As could be seen from the table, there was astatistically sharp increase in the average total volume ofyak placentomes from 61–90 days to 181–210 days (from204.64 � 43.72 cm3 to 2,255.36 � 138.79; P < 0.05) and asignificant decrease at 211þ days (2,206.96 � 81.42 cm3).The average total volumes of caruncular endometriumand binucleate cell as well as the mean total surface areaof fetal villi shared the same pattern as the average totalvolumes of placentomes, increasing from 61–90 days to181–210 days and declining in 211þ days. However, themean total volumes of fetal villi, maternal and fetalpyknotic cell exhibited a different pattern and increasedconstantly with increasing gestation time.

DISCUSSIONPlacentome Number

The present study found that neither the total numberof placentomes nor the number in each horn varied withstage of gestation, similar to the results in cattle (Laven

and Peters, 2001). These changes reflected that no com-pensatory mechanism existed in the yak to satisfy fetalincreasing demands by forming more placentomes (Doizeet al., 1997). In yak, the number of placentomes in thenon-pregnant horn was significantly less than that inthe pregnant horn. Laven and Peters (2001) reportedthe similar results in cattle and indicated that this maybe a diffusion effect, with the concentrations of growthfactors being highest in the placentomes nearest theconceptus and declining with distance from the foetus.

Placentome Size

In cattle, the placentome length increased significantlyduring gestation (Laven and Peters, 2001). The presentstudy showed that there was a significant increase inthe average size of placentomes, but slightly decreasedat 211þ days, the reasons for the decrease were notclear. The increased size of placentome enhanced therate of physiological exchange between the fetal andmaternal systems (Reynolds and Ferrell, 1987). Atall stages, the average size of placentomes in the non-pregnant horn were significantly less than that of thepregnant horn, except at gestation age of <60 days. Theresults were similar to those in cattle (Laven and Peters,2001). However, the placentome length of yak was lessthan that of cattle.

In two of the 31 yak uteri, a large mauve diffuse areaoccurred in the pregnant horn. Otherwise, placentomeswith length <1 cm were found in both horns during lategestation. The two types of placental structure, referredto by Bjorkman (1954), were accessory placentation.According to Hill et al. (2001), a similar number ofsmall, nonfunctional placentomes were present in clonedcattle uterus, and the abnormal placentation wasregarded as a pathologic situation. However, Kohan-Ghadr et al. (2008) carried out a study on clone placen-tae in dairy cattle and found that these mini-

TABLE 4. Stereological parameters of yak placentomes in each stage during gestation

Days ofgestation(day)

Volume density (%)Fetal villous

surfacedensity (cm�1)

Fetalvilli

Caruncularendometrium

Binucleatecell

Maternalpyknotic cell

Fetal pyknoticcell

61–90 36.75 � 1.05 63.25 � 1.05 9.78 � 0.30 0.63 � 0.05 0.26 � 0.05 310.65 � 9.3691–120 36.80 � 0.51 63.20 � 0.51 11.01 � 0.39 0.73 � 0.05 0.34 � 0.05 328.67 � 11.95121–150 43.20 � 1.97 56.80 � 1.97 10.19 � 0.72 0.75 � 0.07 0.29 � 0.03 350.18 � 13.96151–180 45.92 � 1.39 54.08 � 1.39 10.14 � 0.14 0.82 � 0.11 0.50 � 0.07 320.76 � 23.84181–210 49.68 � 0.46 50.32 � 0.46 9.09 � 0.17 0.67 � 0.03 0.38 � 0.01 311.10 � 3.32211þ 54.04 � 2.12 45.96 � 2.12 8.16 � 0.47 0.76 � 0.18 0.43 � 0.12 294.76 � 9.40

TABLE 5. Volume and surface area of relevant tissues and cells of yak placentomesin each stage during gestation

Days ofgestation(day)

The total volume (cm3)The total

surface areaof fetalvilli (m2)Placentomes

Fetalvilli

Caruncularendometrium

Binucleatecell

Maternalpyknotic

cell

Fetalpyknotic

cell

61–90 204.64 � 43.72 75.96 � 16.95 128.32 � 26.93 20.53 � 4.56 1.19 � 0.25 0.53 � 0.09 6.93 � 0.8491–120 278.59 � 56.74 101.19 � 18.73 177.09 � 37.90 29.61 � 5.08 2.11 � 0.61 0.93 � 0.31 9.07 � 1.88121–150 795.11 � 118.70 339.11 � 45.69 455.57 � 75.00 82.74 � 17.18 5.93 � 1.10 2.42 � 0.51 27.90 � 4.59151–180 1325.65 � 53.43 607.97 � 6.15 716.39 � 42.11 134.40 � 3.50 10.90 � 1.96 6.55 � 0.63 42.39 � 1.45181–210 2255.36 � 138.79 1118.10 � 21.03 1137.26 � 159.81 204.33 � 26.89 14.98 � 1.50 8.48 � 1.00 70.28 � 11.29211þ 2206.96 � 81.42 1190.97 � 2.98 1016.83 � 81.59 180.56 � 16.99 16.67 � 1.26 9.37 � 0.22 65.13 � 4.47

MORPHOMETRY OF YAK PLACENTOMES 1877

placentomes appeared normal, except for their size. Inprevious studies of cloned pregnancies (Miglino et al.,2007), many well developed tiny placentomes (<1.0 cm)were reported, they apparently had normal function,based on the presence of the PAS reaction for glyco-proteins in the trophoblastic giant cells and intense sub-epithelial capillary organization. Further evidence froma stereological study of cloned pregnancies suggestedthat they were fully functional (Constant et al., 2006),reflecting an adaptation that contributed to the continu-ation of these pregnancies to term.

Placentome Shape

According to Laven and Peters (2001), two morphologi-cally different types of placentomes, normal and flat,appeared in cattle. In their study, a flat placentome wasdefined as a placentome, which placed on a level surface,rested mainly on caruncular rather than cotyledonarytissue. In yak, there were three significantly differentplacentome shapes. The majority of placentomes wereflat and very few were mushroom-shape with a distinctcaruncular stalk, which were similar to those in cattleand African buffalo (Schmidt et al., 2006). In contrast tothe yak, the most placentomes were mushroom-shape incattle. However, the African buffalo placentomes remaineddome-shaped (non-pedunculated) in all three groups.Only three concave placentomes which resembled thatin sheep (Vatnick et al., 1991) were found in yak. Thepresent investigation did not determine the importanceof placentome shape and further research is needed.

Placentome Stereology

Compared with placentas from the artificially insemi-nated (AI) group, the in vitro fertilization (IVF) grouphad decreased feto-maternal contact area by decliningthe placentome surface area and volume density of fetalvilli (Miles et al., 2004). The mean volume density of thefetal component was higher in the clone group than thatin control group (the AI and IVF groups) because of anincrease in mean volume density of the fetal connectivetissue and a decrease in mean volume density of mater-nal epithelium (Constant et al., 2006). Our researchdata of stereologic analysis included only placentomes ofmore than 61 days gestation, as prior to this stage itwas the period of implantation and placentation (Ju,2006; Yong, 2006). In yak, there was a significantincrease in the mean total volume of yak placentomes.Our results indicated that the placentomes exhibited asignificant increase in the volume density of fetal villi, asignificant increase in the mean total volume of fetalvilli, and a significant increase in the mean total surfacearea of fetal villi, suggesting that the proportion of fetaltissue increased and the villi exhibited extensive growthand development as pregnancy advanced. So the surfacearea at the feto-maternal junction could be enlarged tomeet the needs of conceptus development duringpregnancy.

Fetal binucleate cells produced a variety of hormones,such as placental lactogen and pregnancy-associatedglycoproteins, which played an essential role in mainte-nance of normal pregnancy (Wooding, 1992; Guillomot,1995). The increased mean total volume of binucleate

cells in yak implied that placentomes might have anincreased availability of placental related hormones,suggesting enhanced placental development. The presentwork found that there were no significant changes inthe volume density of maternal and fetal pyknotic cellduring pregnancy. However, the average total volume ofmaternal and fetal pyknotic cell existed a significantincrease, impling that there was an increased cell deathwithin the fetal villi and caruncular endometrium, eitherby apoptosis or necrosis (Van Cruchten and Van DenBroeck, 2002). According to previous reports (Rileyet al., 2000; Boos et al., 2003), apoptosis might contrib-ute to placental remodeling and reflect the continuinghistiotrophic nutrition to the fetus, which consisted inphagocytosis of maternal apoptotic epithelial cells. Suchvariations were considered part of the placental matura-tion process, which could be a requirement to maternal-fetal detachment and fetal membranes release.

In summary, this study had shown that these dramati-cally macroscopical and cytomorphometric variations ofplacentomes in yak could be related to the placentalregeneration and maturation, which was most vital tofetal survival and development during gestation.

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