Placental and fetal development during multiple bovine pregnancy. Anatomical and physiological studiesкод для вставкиСкачать
Placental and Fetal Development during Multiple Bovine Pregnancy ANATOMICAL AND PHYSIOLOGICAL STUDIES 1n2r3 E. S. E. HAFEZ AND E. FAJAKOSKI* Department of Animal Sciences, Washington State University, Pullman, Washington ABSTRACT Cattle were injected with PMS, bred and slaughtered 3, 30, 45, 60 and 90 days after ovulation and the genital tracts dissected. From 30 to 90 days of pregnancy the percentage increase in the weight of single embryos was 85 times as much as the increase in crown-rump length. The volume of amniotic fluid increased 580 times in the second month of pregnancy and increased twice during the third month of pregnancy. The allantochorion did not lengthen between 60 and 90 days of pregnancy. At 30 days of pregnancy there was no significant difference in the crown-rump length or the weight of embryos of singles, twins, triplets, quadruplets or quintuplets. The volume of placental fluid per embryo was higher in singles than in twins and triplets; the volume of placental fluids per embryo decreased further in quadruplets and quintuplets. The weight of the placental membranes per embryo was heavier in singles than in twins, triplets and quadruplets; the weight of membranes was markedly decreased in quintuplets. The effects of overcrowding in utero on the conceptus was more pronounced at 60 days than at 30 days and in quintuplets than in triplets and quadruplets. Anastomosis of vascular supply of allantochorions occurred as early as 30 days of pregnancy. Extensive morphological studies have and Reece, '53). Interlocking between the provided information on the development chorion and the maternal cotyledons does of the trophoblast and the early embryol- not begin until the fortieth to fiftieth day ogy of the cow during single pregnancy (Chang, '52). Before that time, the rela(Hammond, '27; Winters et id.,'42; Boyd tionship between the chorion and the endoet al., '44; and Hamilton and Laing, '46). metrium is more or less of a diffused epiWinters et al. ('42) described the first theliochorial placenta; during the second stages of chorionic elongation in a 13-day month this begins to change to a cotyledonembryo. Chang ('52), studied embryos of ary placenta. The development of the allantochorion unknown age and observed an extremely rapid growth of the trophoblast prior to in the cow is of particular interest. Essenan estimated age of 18 days. Such findings tially, the chorion is formed by elongation were augmented by Greenstein, et al. ('58) of the trophoblast, which becomes invested who attempted to define daily standards of with a layer of endodenn followed by the normal variation and development for lateral development of the mesoderm. The accurately timed embryos recovered 16 to allantois develops as an outgrowth of the hind end of the gut, and rapidly elongates 26 days post coitum. Other investigations have contributed and closely invests the chorion. The vasdata relative to the mode of placentation cularized allantois brings its blood vesseIs in the cow. The data of Green ('46) may in close opposition with the chorion, so indicate that the site of implantation of the bovine blastocysts is determined at the eleventh day. The allantochorion becomes attached to the endometrial caruncles at some 33 days of gestation. At 35 days this attachment is firm enough for the embryo to receive part of its nourishment through the cotyledons (Melton et al., '51; Foley ANAT. REC., 1 5 0 : 303-316. 303 304 E. S. E. HAFEZ AND E. RAJAKOSKI forming the vascular allantochorion. Fusion is practically complete by the twentyeighth day (Amoroso, '58). In multiple pregnancies the allantoida of embryos elongate within the common trophoblast and they commonly anastomose where they meet. The body proportions of the fetus change markedly as gestation advances; some approximate the corresponding proportion of the adult at one age, some at another. The stage of gestation at which the bovine fetus most nearly approximates the adult in body proportions is 2 to 3 months for ratio of height at withers to depth and width of chest and 6 to 7 months for ratio of height at withers to length of head (Swett et al., '48). Conspicuously absent from the literature ture has been any detailed studies on the developmental morphology of the placenta and fetus of a monotocous species during induced multiple pregnancy. Such a study is obviously vital to an understanding of the nature of the high incidence of embryonic mortality during multiple pregnancy in cattle. This investigation was made to study the effect of crowding in utero on the developmental morphology of the conceptus during early stages of multiple pregnancy with special emphasis given on the development of fetus, its internal organs and ossification centers. dissected, and their lengths measured (fig. 1 ) . The lengths and diameters of the gravid and non-gravid horns were recorded. The uterus was cut open from the right uterotubal junction and the fetuses were recovered and identified. The length of allantochorion and necrotic tips, dimensions of amniotic vesicle, volume of amniotic and allantoic fluids, and weight of amniotic membranes were measured. The crown-rump length, umbilical cord length and weight of fetuses were recorded. The fetuses were immersed in warm saline to judge their viability by heart beat. Degenerating and non-viable fetuses were not considered and were excluded from the data on fetal development. The fetuses were cut open and the following glands and organs were dissected from the adjacent connective tissues and weighed: gonads, digestive tract (full), liver, kidneys, adrenals, lungs and heart. Tissue clearing techniques Eviscerated fetuses were fixed in 10% formalin for at least 48 hours, cleared in 2% KOH solution for 7 to 10 days and stained for two hours with Alizarin Red S (1: 10,000 in 2% KOH). The specimens were rinsed in several changes of distilled water over a period of two hours and gradually dehydrated through a series of increasing concentrations of glycerol ( 1/4 glycerol: 3/4 H20: 1/2 glycerol; 1/2 H,O; 3/4 glycerol; 1/4 HzO: 1 glycerol) and MATERIALS AND METHODS then stored in a second or even third Fifty nulliparous beef cattle heifers of change of glycerol. This gradual dehydraHereford breed aged one and one-half to tion was necessary to prevent shrinkage two years were used. The heifers were and deformity of body shape. The specichecked twice daily for the incidence of mens were turned every few hours to inestrus with a vasectomized bull. On day 16 sure even clearing on all sides. Great care (the day of estrus = day 1 ) each animal was taken to handle the fragile jelly-like was injected with 1,500 to 2,000 i.u. of fetuses by supporting both the head and PMS (Equinex, Ayerst) subcutaneously. At the body with rubber-gloved hands. the following estrus the heifers were bred Microscopic examination of to a fertile bull and artificially inseminated ossification centers with frozen semen. Pregnancy was diagnosed by palpation per rectum. Sixteen 60-day and six 90-day fetuses were used to study the effect of sex and Autopsy litter size on ossification pattern. The The animals were slaughtered 3, 30, 45, length and thickness of the following ossi60 and 90 days after ovulation ( f 12 hrs). fied bones were measured microscopically, The genital tract was removed after using a calibrated ocular or calipers: manslaughter. The corpora lutea on each dible, scapula, tenth rib, ilium, femur, ovary were counted, the uterine tubes were tibia, fibula, humerus, ulna and radius. MULTIPLE PREGNANCY IN CATTLE ?I 305 TI I I I I I I I I I I I 1I e I I I I I I I I I I I I I 5 I I V 3 _ _ _ _h r _-_-_-_-4 ----- ----- +---- - ----- ----- , Y Fig. 1 Technique used to measure the dimensions of the reproductive organs and conceptus of experimental animals. a,b, length of gravid and non-gravid uterine horns; c,d, width of uterine horns; e, length of uterine tubes; f, length and width of fimbriae; g, largest dimension of ovary, length and width of corpus luteum; h, interamnion spaces; i, length of allantochorion; j, length and width of amnion. RESULTS Morphology of reproductive organs and conceptus during single pregnancy ovaries. The dimension, volume and weight of ovaries at 30 days of pregnancy were higher than that at three days after ovulation. There were no significant changes in these values from 30 to 90 days of pregnancy (table 1). In general, ovary weight was sigmficantly related to number of corpora lutea (fig. 2). The number of unruptured follicles, over 10 mm, at three days after ovulation ranged from nil to 16 with an average of 3.4. Such unruptured follicles seemed to regress throughout later stages of pregnancy. The diameter of corpus luteum at three days after ovulation ranged from 11 to 14 mm and the average was 12.3 mm. From 30 to 90 days of pregnancy the diameter of corpus luteum vaned from 14 to 22 mm with an overall average of 19.7 mm. At three days after ovulation the weight of corpus luteum, during early pregnancy, fluctuated between 1,813 and 5,240 mg. The stage of pregnancy from 30 to 90 days had no significant effect on the diameter or weight of the corpus luteum. Reproductive tract. In all experimental animals the length of the fimbriae ranged from 44 to 84 mm with an overall average of 68 mm. The width of fimbriae ranged from 23 to 38 mm and averaged 30 mm. The length of the uterine tube ranged from 15 to 25 cm and averaged 21 cm. The stage of pregnancy had no significant effect on the size of fimbriae or length of uterine tube. The length of the uterine horn during non-pregnancy varied from 22 to 34 cm with a mean of 27.0 cm. The gravid horn lengthend progressively throughout pregnancy and reached an Stage of pregnancy (days) 3 453-820 Weight of corpus luteum ( m g ) 657 12.3 3.4 1.3 4.65 4.95 28.4 Mean 1813-5240 14-23 0-2 1-3 7.0-13.8 4.0-15.5 21-36 283 224-304 283 2.2 0-5 224-304 6-10 2 14 88-232 94-364 0-10 0-5 27-34 1.1 27 0-2 22-32 + 28-38 27.0 30 Range 22-34 Mean Length of gravid horn (cm) D B . in length of both horns ( c m ) Diameter of gravid horn ( m m ) DifF. in diameter of both horns ( m m ) Diff. in number of visible caruncIes in both horns Weight of uterus conceptus +cervix (gm) Weight of empty uterus cervix ( g m ) 3 Range Stage of pregnancy (days) Measurements + 30 Range TABLE 2 3605 19.1 0.7 1.5 9.19 9.83 29.7 Mean 45 23284686 17-21 0-5 1 4.6-6.6 3.5-9.0 23-35 Range 3470 19.3 19.8 2.0 1.5 10.5 13.0 35.7 Mean 2550-4970 3416 17-22 14 1-3 0.8 5.3-19.3 60 1.o 8-18 25-48 Range 5.91 6.11 29.5 Mean 45 181 203 385 - 8.8 1-16 7.0 244 11 6-16 10.3 37 40 8 ~ Mean 7-13 32-41 35-46.5 Range 2.3 31 33.4 Mean 60 259-452 655-1092 3-9 10-31 1-9 60-75 4225 Range 343 829 2.5 20 6.0 67 43.7 Mean 90 19-22 0 1 6.7-10.2 8.5 2642 Range 20.5 0 1.o 8.25 8.5 34.1 Mean 417-640 1200-1820 4-12 35-85 8-18 90-135 37-50 Range 90 566 1600 7.2 62 13.0 115 45.0 Mean 3117-4570 3839 Differential developments in the morphology of the gravid and n o n gravid horns during single pregnancy 11-14 0-16 Diameter of corpus luteum ( m m ) Number of unruptured follicles (over 10 m m ) 3.3-6.4 Weight ( g m ) 1-2 2.8-6.5 Volume ( m l ) Number of corpora lutea 22-35 Range Largest diameter ( m m ) Measurements TABLE 1 Developmental changes i n morphology of ovaries duTing single pregnancy 307 MULTIPLE PREGNANCY IN CATTLE 80 - 70 - 60 - E50 - 6 v t c rn 40 - 3 2 ? 30 - 3 0 Days P r e g n a n c y 0 20 - 10 - a;:@ : 01 @ ' I I I I 0 2 4 6 8 I 10 I I 12 14 I 16 18 20 I 22 Number Of Ovulation I n O v a r y Fig. 2 Relationship between ovarian weight and number of ovulations at 30 days of pregnancy. average of 45.0 cm at 90 days of pregnancy (table 2). The average difference in length of non-gravid and gravid uterine horns was 2.3 cm at 30 days of pregnancy and 13.0 cm at 90 days. The diameter of the uterine horn during non pregnancy ranged from 22 to 32 mm with an average of 27 mm. The diameter of the gravid uterine horn increased progressively throughout pregnancy and reached an average of 115 mm at 90 days of pregnancy. Difference in the diameter of gravid and non-gravid horns was first noted at 30 days of pregnancy. Each uterine horn during non pregnancy had 43 to 72 visible caruncles; the gravid horn had a few more visible caruncles. The percentage increase in weight of uterus and conceptus was greater in the second month of pregnancy than in the first and third. Embryos. The crown-rump length of 30-day embryos varied from 8.1 to 12.5 mm with an average of 10.0 mm. The average crown-rump lengths of 45-, 60-, and 90-day fetuses were 28.0, 74.3 and 159.3 mm respectively. The weight of 30-day embryos ranged from 69 to 178 mg with an average of 114 mg. The average weights of 45-, 60-, and 90-day fetuses were 2.0, 16.8 and 156.8 gm respectively. From 30 to 90 days of pregnancy the percentage increase in the weight of embryo was on an average 85 times as much as the increase in crownrump length. The average length of umbilical cord was 2.5 cm at 60 days of pregnancy and 6.5 cm at 90 days. Placenta. The average length of amniotic vesicle at 45, 60 and 90 days was 3.9, 9.4 and 32.7 cm respectively. At 30 days of pregnancy, the volume of amniotic fluid was 0.1 to 0.2 ml; this fluid increased 570 times in the second month of pregnancy and increased twice during the third month of pregnancy. The percentage increase in the volume of allantoic fluid was much slower than in the amniotic fluid. The ratio between the volume of amniotic fluid: volume of allantoic fluid at 45, 60 and 90 days of pregnancy was 1:16, 1:3 and 1:4 respectively. 308 E. S. E. HAFEZ AND E. RAJAKOSKI 60 DAY - 90 DAY I . I . I . 1 40 0 --- . 1 80 1 . I 120 Lenqfh of ollontochorion fcm) Embryo or fetus -. Allontrochorion Necrotic tip Fig. 3 Diagram of the pattern of spacing of embryos along the allantochorion during multiple pregnancy at 60 and 90 days of gestation in cattle. The length of allantochorion is drawn to scale and the width of the amnion is not considered. Note the uneven spacing of the fetuses along the allantochorion and the shorter placenta at 90 days as compared to that at 60 days. There was no increase in the length of the allantochorion between 60 and 90 days of pregnancy (fig. 3). At 30 days of pregnancy the weight of the amnion and allantochorion ranged from 0.59 to 3.77 gm with an average of 1.81 gm. The relative increase in the weight of placental membranes was much slower in the third month of pregnancy than in the second month, whereas the relative increase in fetal weight was much higher in the third month (fig. 4). ERect of litter size on weight of embryos a n d placenta At 30 days of pregnancy there was no significant difference in the crown-rump lengths or the weights of embryos of singles, twins, triplets, quadruplets or quintuplets (table 4). The volume of placental fluid per embryo was significantly higher in singles than in twins and triplets; the volume of placental fluids was decreased further in quadruplets and quintuplets. The weight of the placental membranes per embryo was heavier in singles than in twins, triplets and quadruplets; the weight of membranes was markedly decreased in quintuplets. At 60 days of pregnancy there was a slight increase in the crown-rump length and weight of embryos of singles as compared to twins and triplets. Assuming that sex differences in fetal weight exist at 60 days of pregnancy, there is evidence to indicate that overcrowding in utero reduced weight of viable fetuses (fig. 5). Unlike the results at 30 days of pregnancy there were no significant differences between the volumes of placental fluids at 90 days in singles, twins and triplets. Litter size exerted a reducing effect on the weight of placental membranes. The effects of overcrowding in utero on the components of the conceptus was more pronounced at 90 days than at 60 days of MULTIPLE PREGNANCY 309 IN CATTLE 140,000 I20,OOO a 0 100,000 m Ea 0 M t 0 Em 60,000 .- P 0 t Q) 40,000 ? 0 .-r 20,000 Membranes 0 30 60 90 Stage of Pregnancy (days) Fig. 4 Relative increase in components of conceptus (fetus and placenta) at 45 to 90 days of pregnancy as compared to that at 30 days in single pregnancy. Note the high rate of increase in fetal weight and low rate of increase in placental weight during the third month of gestation. gestation and in quintuplets than in triplets and quadruplets (fig. 6). At 30 days of pregnancy, the adjacent placentae were either separated by necrotic tips or were completely or partially envaginated (fig. 7). Necrotic tips between adjacent allantochorions were found in some twin and triple pregnancy (fig. 8). Anastomosis of vascular supply of allantochorions occurred as early as 30 days of pregnancy. E. S. E. H A F E Z AND E. RAJAKOSKI 0 rl 0 8 rl I rl Q) al 0) m rl rl 0) I m B J I n a, M 9 1 Fr; 9 i I 5 8 9 Fr; I Effect o f litter size and sex of fetus on development o f organs and ossification centers In 60-day fetuses the weight of major internal organs was not significantly affected by litter size (table 5). There were no significant differences in organ weights of males and females in 60- and 90-day fetuses. The weight of a single fetus at 90 days of pregnancy was approximately 11 times its weight at 60 days and it seemed that the internal organs had developed at a similar rate from 60 to 90 days of single pregnancy. I n 60-day fetuses, the following bones were ossified and readily stained by the Alizarin Red S: mandible, scapula, ribs, ilium, femur, tibia, fibula and metacarpus. The ischium, however, was not well ossified to be stained with Alizarin Red. The dimension of the ossified bones did not differ significantly between singles, twins and triplets of 60-day fetuses (table 6). Also, there were no significant differences between dimensions of ossified bones of males and females in 60- and 90-day fetuses. There was no evidence to show that the growth rate, between 60 and 90 days of gestation, is higher for some bones than for others (fig. 9). Fig. 5 Gravid uteri cut open to show the degree of development of endometrial caruncles in relation to number of implantations at 60 days of gestation. Note the effect of crowding in utero on fetal weight. a (102) Triplets: Two implantations, evenIy spaced in the right uterine horn and one in the left horn. The two fetuses in the right horn weighed 14.21 (d) and 14.88 gm ( 8 ) whereas the single fetus in left horn weighed 15.38 gm ( 9 ) . Note a similar degree of development of caruncles in both horns. b (103) Twins: Two implantations (see two arrows) in the right horn; the fetuses weighed 14.58 ( 9 ) and 15.48 gm ( 8 ) . There were n o implantations in the left horn but the allantochorion migrated from the right horn to half the length of the left horn (see arrow). The caruncles and the fetal-maternal attachments were well developed in the right horn whereas the caruncles were only eroded in the left horn. c (97) Twins: Two implantations in the right horn, the fetuses weighing 12.45 ( 0 ) and 13.49 gm (d). Note the lack of development of caruncles in the left side which was a blind uterine horn; the allantochorion was confined to the right horn only. The fetuses in this case were lighter than in "b" (cow with a double cervix). (All implantations were represented by viable fetuses.) MULTIPLE PREGNANCY IN CATTLE Figure 5 311 E. S . E. H A F E Z A N D E. RAJAKOSKI DISCUSSION m w e m m c - e c o w e 126.96.36.199.3. 0 0 0 0 0 & & i d & w m o o w 99??9 0 0 0 0 0 E v1 +lo O R 02 Z 8 aJw u g;. I4 0 co The relative growth of the different components of the gravid uterus during early gestation is correlated with functional necessity. After implantation, the allantochorion develops faster than other parts of the conceptus : this is achieved by "creep" growth through the non-gravid horn. Following the rapid growth of the placental membranes, the fetal fluids increase rapidly; the distension of the blastocyst presses the trophoblast cells firmly against the uterine mucosa and so allows the intimate contact between fetal and maternal cotelydons. The fetal weight increases very slowly during early stages of gestation, but rapidly late in gestation. Similar patterns in the differential growth of the conceptus take place in other species (Malan and Curson, '37; Wallace, '48; Huggett and Hammond, '58). The periods of morphological changes of the developing embryo are preceded by developmentally adaptive stages aimed to supply the developing organism with nutritional requirements including oxygen. Smidt ('51 ) characterized six prenatal stages in cattle : ( 1) intracellular nutrition and anaerobic respiration 1 to 12 days after fertilization: ( 2 ) nutrition through the trophoblast, 12 to 15 days: ( 3 ) nutrition through the trophoblast and yolk sac, 16 to 2 1 days: ( 4 ) the allantois, in close association with the chorion, 22 to 27 days: (5) the allantochorion with its primitive cotyledons 27 to 50 days: and ( 6 ) the complex cotyledons of the placenta, 51 to 280 days. During multiple pregnancy the number and size of eroded caruncles are greater than in single pregnancy. The surface area of eroded caruncles corresponds to litter size. It is not known whether the surface area of fetal-maternal attachment, the eroded caruncles, is correlated with fetal weight. The morphological changes during the pre-fetal period are very extensive. The occipital and the parietal protuberances of the head decrease, the facial parts elongate, the neck and the extremities lengthen, the umbilical hernia disappears, the abdominal wall strengthens, and the head is inflexed away from the chest. The prefetal stage may be divided into two stages: 313 MULTIPLE PREGNANCY IN CATTLE ( a ) the early stage of development (30 to 45 days) when the embryo enlarges from 100 mg to 2 gm and ( b ) the late stage of development (45 to 60 days) when the embryo enlarges from 2 to 17 gm. Over- crowding in utero did not seem to affect the size of the embryo in the pre-fetal stages; the retarding effect was shown at the early fetal stages. In superovulated rabbits overcrowding in utero had no TABLE 5 Effect of litter size on organ development in 60-day f e t u s e s Singles Measurements Triplets Twin Range Mean Range Mean Range Mean Liver weight (gm) 0.74-0.98 0.86 0.55-0.79 0.67 0.63-0.82 0.71 Digestive tract weight (gm) 0.43-0.77 0.60 0.38-0.56 0.47 0.33-0.53 0.45 Adrenals and kidney weight * 95-148 (mg) Lungs weight (gm) 0.42-0.74 97-178 Heart weight (mg) % eviserated fetus weight to fetal weight - 66-106 113 0.58 0.27-0.44 137.5 76 100-119 - 91 0.36 57-132 0.32-0.45 96 0.37 109.5 110-124 116.8 77 - 77 Skull length ( m m ) 22-26 24 20-22 21 21-32 24 Tail length ( m m ) 16-17 17 14-20 17 16-20 17 1 Both right and left. Singles Trip Ie t s Twins 20 - 15 E 0, z L 10 01 r r 0 f 5 Fetus w Membranes 0 Fluids \ 400 c c 0 0) 0 Cr 200 r 0 f Fig. 6 Effect of litter size on components of conceptus. Note the relative effect of overcrowding in utero on weight of fetus and placenta at 60 and 90 days of gestation. 314 E. S . E. HAFEZ A N D E . RAJAKOSKI B A 1 I I I I I , of the placenta before it shows its retarding effect on the size of the fetus. From 60 to 90 days of single pregnancy there was no evidence of differential growth in the internal organs: i.e. no organs grew faster than others. There is no data to illustrate the effect of litter size on differential growth at that stage of pregnancy. Further research is needed to study the effects of litter size on the prenatal development of different organs. The differential growth rate throughout prenatal development has been studied in sheep, by Wallace ('48), who found that organs such as kidney, liver, lungs, bladder and brain make a larger proportion of their fetal growth I 1 I I I !~=;d \ I I I I I I ' ' I I I II I I I 1 I Chori onic ----- A l la n t o i c Fig. 7 Diagram showing patterns of placentation in multiple bovine pregnancy at 30 days. A. Well developed necrotic tips and no attachment between adjacent allantochorions. B. Partial attachment of adjacent chorions but the allantoida not attached. C . Complete attachment of allantoida and anastomosis of the adjacent vascular supplies. was affected. It is known that the sex differentiation of the embryonic gonads starts at 45 days and is well defined histologically at 60 days, Meanwhile, there were no significant differences in the weight or dimenOf Ossified bones in both 90-day fetuses. Of TABLE 6 Development of ossification centers in 60-day-old fetuses (measurements i n rnm):' Measurements Mandible Scapula 10th Rib Ilium Femur Tibia Fibula Metatarsus Humerus Radius Ulna Metacarpus Eye ball Triplets Twins Singles Range Mean 11.8 -13.0 4.25- 5.16 7.04- 8.43 1.78- 2.59 2.54- 3.03 2.88- 3.54 2.34- 2.91 1.30- 1.79 2.78- 3.43 2.84- 3.76 3.37- 3.85 1.50- 1.92 4.27- 5.11 12.2 4.47 7.95 2.07 2.78 3.15 2.55 1.54 3.15 3.29 3.57 1.70 4.65 Range Mean 11.9 11.0 -12.5 3.89 3.58- 4.32 6.63 5.52- 8.15 1.70 1.27- 2.08 2.47 2.05- 2.90 2.67 2.19- 3.38 2.15 1.67- 2.86 1.05 0.64- 1.42 2.74 2.39- 3.13 2.84 2.47- 3.35 3.18 2.75- 3.70 1.27 1.02- 1.55 4.53 4.29- 4.76 Range Mean 11.0 -12.9 3.70- 4.39 6.30- 8.50 1.35- 2.25 2.29- 2.96 2.46- 3.15 1.94- 3.33 0.87- 1.58 2.61- 3.24 2.65- 3.15 2.90- 3.43 1.05- 1.69 3.91- 5.09 12.3 4.02 7.34 1.79 2.62 2.84 2.41 1.30 2.98 2.97 3.25 1.42 4.48 Effect of multiple pregnancy 1 0.81 11.41 12.20 15.46 8.27 12.38 10.59 23.38 9.21 11.55 9.80 20.59 3.01 3 Difference between measuremeiits of single versus multiple fetuses as a percentage of measurements of single fetus. MULTIPLE PREGNANCY IN CATTLE 315 TWINS TRIPLETS I QUADRUPLETS Fig. 8 Diagram showing degree of attachment between adjacent allantochorions during multiple pregnancy. A. No attachment between allantoida. B,C. Partial attachment of allantoida. D. Complete attachment of allantoida i n both horns. E,G,H. Envagination of allantoida in one uterine horn. F. Envagination of allantoida in both uterine horns. 1,J. Complete attachment of allantoida i n one uterine horn. Fig. 9 Ossification centers in 60- and 90-day fetuses. Note the proportional increase during that stage of fetal development. 316 E. S . E. HAFEZ AND E. RAJAKOSKI LITERATURE CITED Amoroso, E. C. 1958 Placentation, Chapter 15 In: Marshall’s Physiology of Reproduction, A. S. Parkes, edit. Vol. 11. Longmans, London. Boyd, J. D., W. J. Hamilton and J. Hammond 1944 Transuterine (“internal”) migration of the ovum i n sheep and other mammals. J. Anat., 78: 5-14. Chang, M. C. 1952 Development of bovine blastocyst with a note on implantation. Anat. Rec., 113: 143-161. Foley, R. C., and R. P. Reece 1953 Histological studies of the bovine uterus, placenta, and corpus luteum. Mass Agr. Exp. Sta. Bull. no. 468. Green, W. W. 1946 Comparative growth of the sheep and bovine animal during prenatal life. Amer. J. Vet. Res., 7: 395402. Greenstein, J. S., R. W. Murray and R. C. Foley 1958 Observations on the morphogenesis and histochemistry of the bovine preattachment placenta between 16 and 33 days of gestation. Anat. Rec., 232: 321-341. Hafez, E. S. E., and E. Rajakoski 1964 Growth and survival of blastocysts in the domestic rabbit. I. Effect of maternal factors. J. Reprod. Fertil., 7: 229-240. Hamilton, W. J., and J. A. Laing 1946 Development of the egg of the cow up to the stage of the blastocyst formation. J. Anat., 80: 194-204. Hammond, J. 1927 The Physiology of Reproduction in the Cow. Cambridge University Press, Cambridge. Huggett, A. St. G., and J. 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