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Embryonic development, conception rate, ovarian function and structure in pregnant rabbits heat-stressed before or during implantation
Animal Reproduction Science, 17 (1988} 259-270
259
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Embryonic Development, Conception Rate, Ovarian Function and Structure in Pregnant Rabbits Heat-Stressed Before or During Implantation D. WOLFENSON ~and ORLY BLUM
Department of Animal Science, Faculty o[ Agriculture, Hebrew University, Rehovot 76100 (Israel) 1 To whom reprint requests should be addressed. (Accepted 25 March 1988 )
ABSTRACT Wolfenson, D. and Blum, Orly, 1988. Embryonic development, conception rate, ovarian function and structure in pregnant rabbits heat-stressed before or during implantation. Anim. Reprod. Sci., 17: 259-270. Rabbits were heat-stressed during two definite stages of pregnancy: 1. during early stages of blastocyst formation and rapid growth of the corpus luteum (Days 3-5), and 2. during implantation period (Days 6-8). Hyperthermia was created ( + 1.2 ° C ) in which food intake was reduced to 1/3 of control. Both treatments, H(3-5) and H(6-8), reduced conception rate to 59% from 74% of the control. The development of the remaining live embryos was retarded in the H (3-5) group only. Plasma progesterone concentration in the H (3-5) group was significantly lower than in the control, immediately following termination of heat exposure. In the H (6-8) group, progesterone was only slightly changed, as compared to control. Histological examination of the ovarian corpus luteum indicated that size and number of the large luteal cells were reduced in both heat-stressed groups, as compared with the control. These results indicate that: (a) rabbit embryos are more susceptible to heat stress during blastocyst formation than during implantation, (b) ovarian function impairment is more pronounced during the rapid linear growth stage of the corpus luteum (Days 3-5), and (c) a possible link between impaired ovarian function and reduced reproductive performance can be suggested.
INTRODUCTION
Exposure of domestic animals to naturally high environmental temperatures, or laboratory animals to a controlled heat stress, impairs reproductive performance and embryonic development. Many studies have documented a pronounced decline in conception rate, the occurrence of embryonic death, 0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
260 retardation of embryonic growth and teratogenic symptoms due to hyperthermia (Edwards, 1978; Johnson, 1985). The duration of heat stress and the time of its occurrence during pregnancy determine the resulting symptoms; early stages of pregnancy are more sensitive than later ones (Edwards, 1978). Increased embryo mortality was noted in does that were inseminated with heated sperm cells (Burfening and Ulberg, 1968) or, when zygotes of mouse, rabbits and sheep were heated during the division of the first cells (Alliston et al., 1965). In agreement with these studies, hyperthermia was considered as most critical to conception in cows when it occurred on the day following insemination (Badinga et al., 1985 ). In rabbits also, embryo losses occurred when animals were exposed to severe heat stress during the first 6 days of pregnancy (Shah, 1956). A functional corpus luteum is essential for successful conception. The effect of heat stress on progesterone (Pt) concentration is controversial. In long term experiments (several weeks) in cattle, thermal stress was associated with decreased (Rosenberg et al., 1977; Folman et al., 1983; Wolfenson et al., 1988) or increased P4 concentrations (Abilay et al., 1975; Roman-Ponce et al., 1981). Rats, acclimated to heat before mating, and heat-stressed during pregnancy, had lower Pt levels than control (Bedrak et al., 1979). A possible association between impaired ovarian endocrine function and fertility in heat-stressed animals needs further examination. Histological changes in the ovary following heat exposure have been examined, to the best of our knowledge, in only one study in the ewe (Thwaites, 1970). No morphological change was found in the five different cell types classified in that study. The present study aimed to examine, in the rabbit, the effect of heat stress on embryonic death, embryonic development, ovarian endocrine function and morphological structure. Animals were exposed to heat during two definite stages of pregnancy: 1. during early blastocyst development in the uterus, which coincides with the period of rapid linear growth of the corpora lutea (CL), and 2. during implantation. MATERIALSAND METHODS
Animals Mature virgin does (Oryctolagus cuniculus), aged 4-6 months and weighing 3.2 kg, were held in a temperature controlled room at 21°C and 14 h of light per day. They were fed a commercial rabbit food mixture (Ambar Ind., Hedera, Israel) containing at least 16% protein, 10.5% cellulose, 0.85% calcium, 0.5% phosphate, vitamins and micro-elements. Females were transferred to the male cage for a day and mating was confirmed; that day was defined as Day 0. The
261 following morning, animals were randomly assigned to the control or one of the two heat-stressed groups.
Experimental procedure Control group animals (C; n = 11) were held for the experimental period (Days 1-14 ) at 21 ° C. Another group [H (3-5); n = 8] was transferred for Days 3-5 to a hot chamber at 33.6°C air temperature (T a), 34% relative humidity. On the morning of Day 6, the animals were returned to the 21°C room to stay there until Day 14. A third group [H(6-8); n--8], was similarly exposed to heat stress during Days 6-8 post-mating.
Measurements Body weights were recorded on Day 1 and Day 14, and food intake was recorded daily. Body temperature (Tb), ear temperature and respiratory rate were recorded at thermoneutral and hot conditions. Temperatures were measured using thermistor probes calibrated to the nearest 0.1 ° C (Model 46 TUC, Yellow-Springs Instruments, Yellow Springs, OH 45387). Blood samples (4 ml) were collected daily from the marginal ear vein when rabbits were in the hot chamber and every other day when they were in the control room. Heparinized blood was centrifuged and the plasma samples stored at - 20 ° C. Progesterone concentration was assayed in plasma by a dextran-coated charcoal radioimmunoassay procedure as previously described and validated (Wolfenson et al., 1988). Duplicate samples (25/ll) were extracted with 2 ml distilled petroleum ether. Average extraction efficiency (88 % ) was determined for each assay by a set of external standards. Sensitivity of the assay was 10 pg/tube. Antiserum (Bio-Yeda, Rehovot, Israel; 1:3000 dilution in 0.05M Tris buffer, pH 8, containing 0.1% gelatin) cross-reactivity with related compounds was: 5a-pregnene-3-20-dione, 6%; lift-OH-P4, 1.8%; 17a-OH-P4, 2.5%; 20aOH-P4, 0.3%; 20fl-OH-P4, 1.2%; 17fl-estradiol, 0.1%; and corticosterone, 0.3%. Intra- and interassay coefficients of variation were 11% and 13 %, respectively. On Day 14 of pregnancy, the rabbits were euthanized by an overdose of sodium pentobarbital. At autopsy, the following were determined: weight of ovaries, number of CL, number and weights of gestation sacs and embryos, crown to rump length and transumbilical distance of embryos. Ovaries were placed in Bouin-Hollande fixative and slides were stained with haematoxylin-eosin. Characteristics of two types of luteal cells, large and small, were examined (Niswender et al., 1985). The population of small cells is heterogeneous and may include stromal, endothelial and small epithelial cells (Osteen et al., 1986) in addition to the steroidogenic small luteal cells. A population of small non-steroidogenic cells was identified in Day 9 pseudopregnant rabbits (Hoyer et al., 1986), as well as in sheep (Rodgers and O'Shea,
262 1982). Identification of the sub-population of small cells with steroidogenic capacity is difficult using standard staining. For the above reasons, the histological analysis in this study is limited to the large luteal cells, since the large cells are easily identified among other cells by their size and shape. For each animal an average of five CL were examined; in each CL three fields were chosen at random, and, in each field, the following were recorded using a calibrated micrometer scale: 1. the number of large luteal cells along vertical and horizontal 250 pm long scale lines, and, 2. the average diameter of five large luteal cells. About 2000 cells were measured for size analysis, and 405 measurements (2000 cells) were carried out for analysis of the number of large cells along the scale line. A similar degree of shrinkage, as well as similar cell shape and cell shape variability was assumed for the experimental groups.
Data analyses Results were analyzed by least squares analysis of variance according to the methods presented in the General Linear Models procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC 27511 ). For progesterone profiles, a split-plot analysis, suitable for repeated measures within the animal, was employed. Treatment effect was tested using rabbits nested within treatment as an error term. To examine whether the shape and trends of P4 curves differ among treatments, a heterogeneity of regression test was performed using the day post-mating as a continuous independent variable. The number of embryos in a uterine horn served as a covariate in analyses of variance of embryo parameters. Implantation rate (number of gestation sacs per CL), conception rate (number of embryos per CL ) and post implantation survival rate (number of embryos per gestation sacs), were calculated 'for each animal and the effect of treatments was examined by analysis of variance. RESULTS
Thermal responses and food intake Thermal responses, food intake of heat-stressed rabbits while in the hot room and body weight measurements are summarized in Table 1. Responses of the two heat-stressed groups were grouped together, as their responses did not differ. Body temperature of the heat-stressed groups increased by 1.2 °C above that at thermal neutrality (P < 0.01 ). Such increments in body temperature reflect a mild hyperthermia. A typical vasodilatory response was indicated by an increase in the ear skin temperature, and a rise in the average respiratory rate above 200 respirations per minute ( P < 0.01). Average daily food intake of the two heat-stressed groups was 30% of C level ( P < 0.01 ) and
263 TABLE 1 Means and SEM of daffy measurements of thermal responses and food intake at thermoneutral and hot conditions 1, and body weight gain during 14-day experimental period Parameter
Control
Heat stress
Number of rabbits Body temperature ( oC ) Ear temperature ( ° C) Respiratory rate (rain -1 ) Food intake (g/day) Body weight2 (kg) Body weight gain (kg)
11 38.4 _+0.1 25.9 _+0.4 149 _+8 161 _+15 3.16 _+0.09 0.25 _+0.05
16 39.6 _+0.1" 31.5 _+0.6* 215 _+8* 48 _+8* 3.22 _+0.10 0.13 _+0.06
1Means of H(3-5) and H(6-8) groups. 2Initial body weight on day of mating. *P < 0.01, from Control. TABLE 2 Reproductive performance and embryo development in rabbits heat-stressed during Days 3-5 [H (3-5) ] or Days 6-8 [H (6-8) ] of pregnancy 1 Parameter
Control
H (3 -5 )
H (6-8)
Implantation rate (To) Conception rate (%) Survival rate (%) Gestation sac weight (g) Embryo weight (g) Crown-rump length (ram) Transumbilical distance (ram)
80___3 74 ___4b 92_+3 4.31 _+0.07a 0.24 _+0.01a 14.02+0.15 a 5.59 -+0.10
71+4 59 _+5a 81_+4 3.47 _+0.09c 0.16 _+0.01b 13.8 +0.19 b 5.23 -+0.13
67_+4 59 _ 5a 90_+4 4.07 _+0.08b 0.22 _+0.01~ 13.79+0.18 ~ 5.55 _+0.12
iValues are means_ SEM. a'b'¢Differentletters denote statistically significant differences at the P < 0.05 level. a v e r a g e b o d y w e i g h t g a i n for t h e i n t e r v a l b e t w e e n D a y I a n d D a y 14 was 0.25 kg a n d 0.13 kg for t h e c o n t r o l a n d h e a t - s t r e s s e d g r o u p s ( N S ) , respectively.
Reproductiveperformance and embryonic development R e p r o d u c t i v e p e r f o r m a n c e a n d e m b r y o n i c d e v e l o p m e n t are p r e s e n t e d in T a ble 2. I m p l a n t a t i o n r a t e s in H ( 3 - 5 ) a n d H ( 6 - 8 ) g r o u p s w e r e lower b y 10% u n i t s t h a n in t h e c o n t r o l g r o u p ( N S ) . S i m i l a r l y , p o s t i m p l a n t a t i o n s u r v i v a l r a t e s o f b o t h h e a t - s t r e s s e d g r o u p s did n o t differ f r o m t h e control. C o n c e p t i o n r a t e for D a y 14 o f p r e g n a n c y , w h i c h t a k e s i n t o a c c o u n t all e m b r y o n i c losses before, d u r i n g a n d a f t e r i m p l a n t a t i o n , was lower in t h e t w o h e a t - s t r e s s e d groups, as c o m p a r e d w i t h c o n t r o l (74% vs. 5 9 % ; P < 0.05 ).
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Number of embryos in a uterine horn had an effect on embryonic development when a single embryo was present, or when more than six embryos were present in a horn. No effect of the number of embryos in a uterine horn was detected if two to six embryos were present per horn, the situation found in more than 80% of all embryo populations. Mean weight of embryos of the H (35) group was two-thirds that of the control group (0.24 g vs. 0.16 g; P < 0.05). Heating on Days 6-8 did not affect embryo weight. The weight of gestation sacs containing embryos in the H(3-5) group was 80% that of the C group ( P < 0.05), while, in the H (6-8) group, it was only slightly reduced. Crownrump length was smaller in H(3-5) embryos (P<0.05) and that of H(6-8) was the same, compared with controls. Transumbilical distance was similar in all three groups.
Progesterone concentration Ovarian measurements for Day 14 and mean P4 concentrations in plasma for the period from Day 1 to Day 14 are presented in Table 3. Mean P4 level was lowest in the H (3-5) group and highest in the H (6-8) group. In another study in our laboratory (A. Lublin, unpublished data, 1986), a linear relationship between P4 concentration and CL number was found. As P4 concentration is influenced by the number of CL in the ovary, and as a coincidental difference was noted in the number of CL between experimental groups, P4 concentration in plasma per number of CL (P4/CL) was calculated to allow for comparison of heat stress effect among treatments. P4/CL of the C group was similar to that of the H (6-8) group (0.89 and 0.86 ng/ml, respectively). That of the H (35) group was significantly lower, by 30%, than that of the other groups (0.62 ng/ml; P < 0.02 ). The pattern of P4/CL concentrations over all experimental periods was best described by a second order polynomial regression (Fig. 1 ). The C group curve differed from that of both treatment curves: it was higher and the curve did not decline on Day 14 (P<0.01). The curve of the H(3-5) TABLE3 Ovarian weight, number of CL per animal, concentration of progesterone in plasma (P4), and concentration of progesterone in plasma per number of CL (P4/CL), during Days 1-14 of pregnancy, in control and heat-stressed rabbits ~ Parameter
Control
H (3-5)
H (6-8)
Ovarian weight (g) Corporalutea (n) P4 (ng/ml) P a / C L (ng/ml)
0.30 +_0.01 9.6 +0.4 b 8.40 _+0.43 b 0.89 _+0.04 a
0.35 +_0.02 11.1 +0.5 ab 6.80 +_0.46 ¢ 0.62 _+0.04 b
0.36 _+0.02 11.9 +0.5 a 10.22 +_0.46 ~ 0.86-2-_0.04 a
1Values are means +_SEM. ~'b'¢Different letters denote statistically significant differences at the P < 0.05 level.
265 1.6
~ J 1.2 •
j
0.0 2
~ 4-
6
~ 8
~ 10
J
J
t2
14
DAYS
Fig. 1. Concentration of progesteronein plasma per number of CL (P4/CL; ng/ml), as described by secondorder polynomialregressionsof control and heat-stressed rabbits. Control, Q; H (3-5), A; H(6-8), V1.SEM of the three groups: 0.05, 0.04 and 0.05, respectively. group was affected more t h a n that of the H (6-8) group ( P < 0.05). It is noteworthy that the difference between curves of H (3-5) and C groups was detected right at the end of the hot period, on Day 6. On the other hand, the curve of the H ( 6 - 8 ) group declined only on Day 12-Day 14, 4-5 days following the termination of heat exposure. Mean plasma Pt concentrations of the control and heat-stressed groups did not statistically differ ( P > 0.20) on Day 3, before the start of exposure to heat. Neither were differences between groups observed during the periods of exposure to heat.
Histology of the corpus luteum
The number of large luteal cells and their size are presented in Table 4. A typical photograph of a luteal tissue of a control animal is presented in Fig. 2. The number of large cells, along a scale line, was significantly smaller in both heat-stressed groups (82% of C group; P < 0 . 0 5 ) . The mean diameter of the large luteal cells was smaller by about 10% in the two heat-stressed groups, as compared with the C group ( P < 0.05 ).
266 TABLE 4 Number of large luteal cells along a 250/1m scale line and its average diameter (/~m), in control and heat-stressed rabbits 1 Parameter
Control
H (3-5 )
H (6-8)
Number Diameter
6.7 ___0.1a 36.0 _+0.3a
5.3 _+0.25 32.3 _+0.35
5.6 _+0.25 32.7 _+0.35
1Valuesare means _+SEM. a'5Different letters denote statistically significant differences at the P < 0.05 level.
Fig. 2. Luteal tissue of Day 14 control pregnant rabbit, × 750. L, large luteal cell. DISCUSSION The thermal stress elevated body temperature by only 1.2 °C above normal Tb. Such an increment in Tb is experienced by many species of animals during the hot hours of summer days (Berman et al., 1985 ). During the three-day heat stress, food intake was about one-third of control, but body weight gain was only slightly reduced (Table 1 ). Low level of nutrition, over a few estrous cycles, decreased conception rate in cattle (Folman et al., 1973). Low intake during the first 11 or 21 days of pregnancy in sheep was associated with high peripheral P4 and impaired embryonic development (Parr et al., 1982). Undernutrition also increased P4 in late pregnant cows (Gauthier et al., 1983). However, Cartwright and Thwaites (1976), in a pair-feeding experiment in pregnant sheep, did not observe effects of a low nutrition level on embryonic develop-
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ment. In the present experiment, relatively short term heat stress was imposed, in contrast to the forementioned studies. Though possible nutritional effects can not be discarded, it is unlikely that low food intake for a three-day period might significantly affect reproductive and endocrine responses. In support of this presumption, Lublin et al. (1984), in our laboratory, found that reducing feed intake during Days 3-5 of pregnancy in rabbits did not significantly affect P4 concentration or embryonic development. Shah (1956) also claimed that a high rate of embryo mortality in rabbits, in response to a severe 6-day heat exposure, was unrelated to the lower food intake during heat exposure. There are various stages between mating and implantation which are critical for the success of pregnancy. The H (3-5) treatment heat stressed the animals during the period of blastocyst development and rapid CL growth. Conception rate of the H (3-5) group was significantly lower than control. Implantation and post-implantation survival rate suggested that 41% of the potential embryos of that group were lost before and after implantation, as compared with 26% in the C group. On a longer and more severe pre-implantation heat stress (Days 0-6, Ta=36°C, Tb=40.6°C; Shah, 1956), most rabbit embryos were resorbed by Days 10-14, i.e. after implantation. In rats, 1-day heat stress was found to increase embryo mortality when it was applied during blastocyst development (Arora et al., 1979). In contrast, the sheep zygote was found to be most sensitive to thermal stress during the initial stages of cleavage (Days 15 ) and not during the initial blastocyst stage (Day 8; Dutt, 1964). The H (6-8) treatment (33.6 ° C, 3 days) coincided with early implantation. Conception rate was, as in the H (3-5) group, significantly lower than in C group. Only few studies have examined specifically the effect of heat stress during implantation. Heat stressing pregnant rats around implantation time (Ta 43-44°C, Day 6), did not affect reproductive performance (Arora et al., 1979). Gilts were most susceptible to thermal stress during implantation in one study (Ta 37.8°C for 17 h and 32.2°C for rest of day, between Days 8-16; Omtvedt et al., 1971 ), but not in another at slightly lower heat stress ( Ta 35 ° C and 32 ° C, each for 12 h, on Days 8-16; Hoagland and Wettemann, 1984). These studies, as a whole, demonstrate species differences in the susceptibility periods of pregnancy to thermal stress. They also emphasize difficulties encountered in comparing experiments where severity of thermal stress or its duration differ. Unlike percentage of embryonic losses, which was similar in rabbits exposed to heat between Days 3-5 or Days 6-8 post-mating, development of the surviving embryos was impaired in the H(3-5) but not in the H(6-8) group. This reflected itself in smaller gestation sacs, embryo weight and length. The above agrees with the teratogenic effects and retarded embryonic development induced by hyperthermia pre-implantation (Edwards, 1978 ). Most studies in the rabbit examined embryo mortality rate, but not alterations of embryo development, resulting of thermal stress around fertilization (Ulberg and Sheean,
268 1973), during the first 6 days post-mating (Shah, 1956) or during 21 days of pregnancy (Trammel et al., 1986). The specific contribution of this study, in this respect, is that, in the rabbit, a significant retardation of embryo development occurred following thermal stress on Days 3-5 of pregnancy. It does not indicate, however, whether embryos found retarded on Day 14 would have been carried to term. Lublin et al. (1984) suggested that an early heat stress (Days 3-5 ) might cause embryonic death extending until close to term. Progesterone levels were reduced by thermal stress, more so for the H (3-5) group than for the H (6-8) group. In the H (3-5) group, it coincided with the period of CL linear growth. The lesser reduction in the H (6-8) group could be detected only by 4-5 days following cessation of heat stress. Similar results were obtained in studies in dairy cows and gilts. In dairy cows exposed to environmental heat, P4 was lower than in cows cooled for the first 10 days after insemination (Gauthier, 1983). Biggers et al. (1987) did not find changes in P4 level in beef cows following later thermal stress on Days 8-16 post insemination. Also, heat stressing pregnant gilts during Days 8-16 post-mating, had no effect on post-exposure P4 (Hoagland and Wettemann, 1984). These studies suggest that progesterone secretion is more sensitive to heat stress when applied during the period of rapid CL growth. However, heat stressing gilts for the first 8 days post-mating increased P4 concentrations following heat exposure (Wettemann et al., 1984). In the rabbit, as in other species, two steroidogenic cell types are found small and large - which originate from granulosa and theca cells, respectively (Niswender et al., 1985). The regulation of progesterone secretion by the two cell types differs as does their sensitivity to various mediators or hormones (Hoyer et al., 1986). In the present study, the large cells (Fig. 1) which were easily identified among other cells, had a diameter similar to that found by Hoyer et al. (1986) and Osteen et al. (1986). In these studies, rabbit luteal cells were dispersed, their steroidogenic capacity examined by histochemical technique and their size visually determined. Similarity between large cells in the present study and large cells in the other studies is suggested. The histology of the ovaries in this study indicated that heating induced some morphological changes in the population of the CL cells, in contrast to a study in sheep (Thwaites, 1970). A small but significant reduction in size and number of large cells was noted in both H (3-5) and H (6-8) treatments. As the size of the large cells declined following heat stress, and less cells were counted along a scale line, this may indicate an increase in the proportion of other cell types (stromal or vascular cells ) following heat stress. The relationship of endocrine capacity to changes in ovarian morphology of the various cell populations needs further study. The present study suggests a relationship between the reduction in reproductive performance and embryo development following heat stress and some impairment of ovarian structure and function. Along the same line in an ex-
269
periment performed in our laboratory (Blum et al., unpublished results), exogenous progesterone administration improved reproductive performance and embryonic development in pregnant rabbits heat-stressed on Days 3-5 postcoitus. In conclusion, heat stress-induced impairment and retardation of development of rabbit embryos was more pronounced during the period of blastocyst formation. This coincided with heat stress-induced reduction of ovarian function, which was larger at this period than during implantation. Possible involvement of a sub-functional CL in reduced reproductive performance is suggested. ACKNOWLEDGEMENT
The assistance of Mrs. Y. Graber, Mrs. R. Steinberg, Mr. A. Lublin and Mr. M. Maman in carrying out various parts of this research, is much appreciated.
REFERENCES Abilay, T.A., Johnson, H.D. and Madan, M., 1975. Influence of environmental heat on peripheral plasma progesterone and cortisol during the bovine estrous cycle. J. Dairy Sci., 58: 1836-1840. Alliston, C.W., Howarth, B. and Ulberg, L.C., 1965. Embryonic mortality following culture in vitro of one- and two-cell rabbit eggs at elevated temperatures. J. Reprod. Fertil., 9: 337-341. Arora, K.L., Cohen, B.J. and Beaudoin, A.R., 1979. Fetal and placental responses to artificially induced hyperthermia in rats. Teratology, 19: 251-260. Badinga, L., Collier, R.J., Thatcher, W.W. and Wilcox, C.J., 1985. Effects of climatic and management factors on conception rate of dairy cattle in subtropical environments. J. Dairy Sci., 68: 78-85. Bedrak, E., Gueron, E. and Ladany, S., 1979. Progesterone concentration and reproduction: an evaluation in pregnant heat-acclimated white rats. J. Therm. Biol., 4: 311-315. Berman, A., Folman, Y., Kaim, M., Mamen, M., Hertz, Z., Wolfenson, D., Arieli, A. and Graber, Y., 1985. Upper critical temperature and forced ventilation effects for high-yielding dairy cows in a subtropical climate. J. Dairy Sci., 68: 1488-1495. Biggers, B.G., Geisert, R.D., Wettemann, R.P. and Buchanan, D.S., 1987. Effect of heat stress on early embryonic development in the beef cow. J. Anita. Sci., 64:1512-1518. Burfening, P.J. and Ulberg, L.C., 1968. Embryonic survival subsequent to culture of rabbit spermatozoa at 38°C and 40°C. J. Reprod. Fertil., 15: 87-92. Cartwright, G.A. and Thwaites, C.J., 1976. Foetal shunting in sheep. 1. The influence of maternal nutrition and high ambient temperature on the growth and proportions of Merino foetuses. J. Agric. Sci., 86: 573-580. Dutt, R.H., 1964. Detrimental effects of high ambient temperature on fertility and early embryo survival in sheep. Int. J. Biometeorol., 8: 47-56. Edwards, M.J., 1978. Congenital defects due to hyperthermia. Adv. Vet. Sci. Comp. Med., 22: 2952. Folman, Y., Resenberg, M., Herz, Z. and Davidson, M., 1973. The relationship between plasma
270 progesterone concentration and conception in post-partum dairy cows maintained on two levels of nutrition. J. Reprod. Fertil.,34: 267-278. Folman, Y., Rosenberg, M., AscareUi, I.,Kaim, M. and Herz, Z., 1983. The effect of dietary and climatic factors on fertility,and on plasma progesterone and oestradiol-17plevels in dairy cows. J. Steroid Biochem., 19: 863-868. Gauthier, D., 1983. The influence of season and shade on oestrous behaviour, timing of preovulatory LH surge and the pattern of progesterone secretion in FFPN and Creole heifers in tropical climate. Reprod. Nutr. Develop., 26: 767-775. Gauthier, D., Terqui, M. and Mauleon, P., 1983. Influence of nutrition on pre-partum plasma levels of progesterone and total oestrogens and post-partum plasma levels of luteinizing hormone and folliclestimulating hormone in suckling cows. Anim. Prod., 37: 89-96. Hoagland, T.A. and Wettemann, R.P., 1984. Influence of elevated ambient temperature after breeding on plasma corticoids,estradiol and progesterone in gilts.Theriogenology, 22" 15-24. Hoyer, P.B., Keyes, P.L. and Niswender, G.D., 1986. Size distribution and hormonal responsiveness of dispersed rabbit lutealcellsduring pseudopregnancy. Biol. Reprod., 34: 905-910. Johnson, H.D., 1985. Physiological responses and productivity in cattle.In: M.K, Yousef (Editor), Stress Physiology in Livestock, Vol. 2. CRC Press, Boca-Raton FL, pp. 3-23. Lublin, A., Wolfenson, D. and Berman, A., 1984. The rabbit as a polyovulating animal model for the study of heat stress effects on fertilityduring early pregnancy. Q. Isr.Zootech. Assoc., 13: 9-17. Niswender, G.D., Schwall, R.H., Fitz, T.A., Farin, C.A. and Sawyer, H.R., 1985. Regulation of luteal function in domestic ruminants: new concepts. Recent Prog. Horm. Res., 41: 101-151. Omtvedt, I.T.,Nelson, R.E., Edwards, R.L., Stephens, D.F. and Turman, E.J., 1971. Influence of heat stress during early, mid and late pregnancy of gilts.J. Anita. Sci.,32: 312-317. Osteen, K.G., Thompson, E.B., Gorsein, F. and Hoffman, L.H., 1986. Enhancement of steroidogenesis in separated luteal cellpopulation in the rabbit using collagen substrate. Biol. Reprod. (Suppl.), 34: 128, Abst. Parr, R.A., Cumming, I.A. and Clarke, I.J.,1982. Effects of maternal nutrition and plasma progesterone concentrations on survival and growth of the sheep embryo in early gestation. J. Agric. Sci.,98: 39-46. Rodgers, R.J. and O'Shea, J.D., 1982. Purification, morphology, and progesterone production and content of three celltypes isolated from the corpus luteum of the sheep. Aust. J. Biol. Sci.,35: 441-445. Roman-Ponce, H., Thatcher, W.W. and Wilcox, C.J., 1981. Hormonal interrelationships and physiological responses of lactating dairy cows to a shade management system in a subtropical environment. Theriogenology, 16: 139-154. Rosenberg, M., Herz, Z., Davidson, M. and Folman, Y., 1977. Seasonal variations in post-partum plasma progesterone levels and conception in primiparous and multiparous dairy cows. J. Reprod. Fertil., 51: 363-367. Shah, M.K., 1956. Reciprocal egg transplantations to study the embryo-uterine relationship in heat-induced failure of pregnancy in rabbits. Nature, 177: 1134-1135. Thwaites, C.J., 1970. Embryo mortality in the heat stressed ewe. 3. The role of the corpus luteum, thyroid and adrenal glands. J. Reprod. Fertil., 21: 95-107. Trammel, T., Stallcup, O.T., Rakes, J.M. and Daniels, L.B., 1986. Influence of high ambient temperature and humidity on feed intake, weight gains and reproduction in rabbit does. J. Anita. Sci. (Suppl.), 63:176-177 (abstract). Ulberg, L.C. and Sheean, L.A., 1973. Early development of mammalian embryos in elevated ambient temperatures. J. Reprod. Fertil. (Suppl.), 19" 155-161. Wettemann, R.P., Bazer, F.W., Thatcher, W.W. and Hoagland, T.A., 1984. Environmental influence on embryonic mortality. 10th Inter. Congr. Anita. Reprod. A.I., Urbana, IL, Vol. 4, Syrup. 10. pp. 26-32. Wolfenson, D., Flamenbaum, I. and Berman, A., 1988. Dry period heat stress relief effects on prepartum progesterone, calf birth weight and milk production. J. Dairy Sci., 71: 809-818.
259
Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands
Embryonic Development, Conception Rate, Ovarian Function and Structure in Pregnant Rabbits Heat-Stressed Before or During Implantation D. WOLFENSON ~and ORLY BLUM
Department of Animal Science, Faculty o[ Agriculture, Hebrew University, Rehovot 76100 (Israel) 1 To whom reprint requests should be addressed. (Accepted 25 March 1988 )
ABSTRACT Wolfenson, D. and Blum, Orly, 1988. Embryonic development, conception rate, ovarian function and structure in pregnant rabbits heat-stressed before or during implantation. Anim. Reprod. Sci., 17: 259-270. Rabbits were heat-stressed during two definite stages of pregnancy: 1. during early stages of blastocyst formation and rapid growth of the corpus luteum (Days 3-5), and 2. during implantation period (Days 6-8). Hyperthermia was created ( + 1.2 ° C ) in which food intake was reduced to 1/3 of control. Both treatments, H(3-5) and H(6-8), reduced conception rate to 59% from 74% of the control. The development of the remaining live embryos was retarded in the H (3-5) group only. Plasma progesterone concentration in the H (3-5) group was significantly lower than in the control, immediately following termination of heat exposure. In the H (6-8) group, progesterone was only slightly changed, as compared to control. Histological examination of the ovarian corpus luteum indicated that size and number of the large luteal cells were reduced in both heat-stressed groups, as compared with the control. These results indicate that: (a) rabbit embryos are more susceptible to heat stress during blastocyst formation than during implantation, (b) ovarian function impairment is more pronounced during the rapid linear growth stage of the corpus luteum (Days 3-5), and (c) a possible link between impaired ovarian function and reduced reproductive performance can be suggested.
INTRODUCTION
Exposure of domestic animals to naturally high environmental temperatures, or laboratory animals to a controlled heat stress, impairs reproductive performance and embryonic development. Many studies have documented a pronounced decline in conception rate, the occurrence of embryonic death, 0378-4320/88/$03.50
© 1988 Elsevier Science Publishers B.V.
260 retardation of embryonic growth and teratogenic symptoms due to hyperthermia (Edwards, 1978; Johnson, 1985). The duration of heat stress and the time of its occurrence during pregnancy determine the resulting symptoms; early stages of pregnancy are more sensitive than later ones (Edwards, 1978). Increased embryo mortality was noted in does that were inseminated with heated sperm cells (Burfening and Ulberg, 1968) or, when zygotes of mouse, rabbits and sheep were heated during the division of the first cells (Alliston et al., 1965). In agreement with these studies, hyperthermia was considered as most critical to conception in cows when it occurred on the day following insemination (Badinga et al., 1985 ). In rabbits also, embryo losses occurred when animals were exposed to severe heat stress during the first 6 days of pregnancy (Shah, 1956). A functional corpus luteum is essential for successful conception. The effect of heat stress on progesterone (Pt) concentration is controversial. In long term experiments (several weeks) in cattle, thermal stress was associated with decreased (Rosenberg et al., 1977; Folman et al., 1983; Wolfenson et al., 1988) or increased P4 concentrations (Abilay et al., 1975; Roman-Ponce et al., 1981). Rats, acclimated to heat before mating, and heat-stressed during pregnancy, had lower Pt levels than control (Bedrak et al., 1979). A possible association between impaired ovarian endocrine function and fertility in heat-stressed animals needs further examination. Histological changes in the ovary following heat exposure have been examined, to the best of our knowledge, in only one study in the ewe (Thwaites, 1970). No morphological change was found in the five different cell types classified in that study. The present study aimed to examine, in the rabbit, the effect of heat stress on embryonic death, embryonic development, ovarian endocrine function and morphological structure. Animals were exposed to heat during two definite stages of pregnancy: 1. during early blastocyst development in the uterus, which coincides with the period of rapid linear growth of the corpora lutea (CL), and 2. during implantation. MATERIALSAND METHODS
Animals Mature virgin does (Oryctolagus cuniculus), aged 4-6 months and weighing 3.2 kg, were held in a temperature controlled room at 21°C and 14 h of light per day. They were fed a commercial rabbit food mixture (Ambar Ind., Hedera, Israel) containing at least 16% protein, 10.5% cellulose, 0.85% calcium, 0.5% phosphate, vitamins and micro-elements. Females were transferred to the male cage for a day and mating was confirmed; that day was defined as Day 0. The
261 following morning, animals were randomly assigned to the control or one of the two heat-stressed groups.
Experimental procedure Control group animals (C; n = 11) were held for the experimental period (Days 1-14 ) at 21 ° C. Another group [H (3-5); n = 8] was transferred for Days 3-5 to a hot chamber at 33.6°C air temperature (T a), 34% relative humidity. On the morning of Day 6, the animals were returned to the 21°C room to stay there until Day 14. A third group [H(6-8); n--8], was similarly exposed to heat stress during Days 6-8 post-mating.
Measurements Body weights were recorded on Day 1 and Day 14, and food intake was recorded daily. Body temperature (Tb), ear temperature and respiratory rate were recorded at thermoneutral and hot conditions. Temperatures were measured using thermistor probes calibrated to the nearest 0.1 ° C (Model 46 TUC, Yellow-Springs Instruments, Yellow Springs, OH 45387). Blood samples (4 ml) were collected daily from the marginal ear vein when rabbits were in the hot chamber and every other day when they were in the control room. Heparinized blood was centrifuged and the plasma samples stored at - 20 ° C. Progesterone concentration was assayed in plasma by a dextran-coated charcoal radioimmunoassay procedure as previously described and validated (Wolfenson et al., 1988). Duplicate samples (25/ll) were extracted with 2 ml distilled petroleum ether. Average extraction efficiency (88 % ) was determined for each assay by a set of external standards. Sensitivity of the assay was 10 pg/tube. Antiserum (Bio-Yeda, Rehovot, Israel; 1:3000 dilution in 0.05M Tris buffer, pH 8, containing 0.1% gelatin) cross-reactivity with related compounds was: 5a-pregnene-3-20-dione, 6%; lift-OH-P4, 1.8%; 17a-OH-P4, 2.5%; 20aOH-P4, 0.3%; 20fl-OH-P4, 1.2%; 17fl-estradiol, 0.1%; and corticosterone, 0.3%. Intra- and interassay coefficients of variation were 11% and 13 %, respectively. On Day 14 of pregnancy, the rabbits were euthanized by an overdose of sodium pentobarbital. At autopsy, the following were determined: weight of ovaries, number of CL, number and weights of gestation sacs and embryos, crown to rump length and transumbilical distance of embryos. Ovaries were placed in Bouin-Hollande fixative and slides were stained with haematoxylin-eosin. Characteristics of two types of luteal cells, large and small, were examined (Niswender et al., 1985). The population of small cells is heterogeneous and may include stromal, endothelial and small epithelial cells (Osteen et al., 1986) in addition to the steroidogenic small luteal cells. A population of small non-steroidogenic cells was identified in Day 9 pseudopregnant rabbits (Hoyer et al., 1986), as well as in sheep (Rodgers and O'Shea,
262 1982). Identification of the sub-population of small cells with steroidogenic capacity is difficult using standard staining. For the above reasons, the histological analysis in this study is limited to the large luteal cells, since the large cells are easily identified among other cells by their size and shape. For each animal an average of five CL were examined; in each CL three fields were chosen at random, and, in each field, the following were recorded using a calibrated micrometer scale: 1. the number of large luteal cells along vertical and horizontal 250 pm long scale lines, and, 2. the average diameter of five large luteal cells. About 2000 cells were measured for size analysis, and 405 measurements (2000 cells) were carried out for analysis of the number of large cells along the scale line. A similar degree of shrinkage, as well as similar cell shape and cell shape variability was assumed for the experimental groups.
Data analyses Results were analyzed by least squares analysis of variance according to the methods presented in the General Linear Models procedure of the Statistical Analysis System (SAS Institute Inc., Cary, NC 27511 ). For progesterone profiles, a split-plot analysis, suitable for repeated measures within the animal, was employed. Treatment effect was tested using rabbits nested within treatment as an error term. To examine whether the shape and trends of P4 curves differ among treatments, a heterogeneity of regression test was performed using the day post-mating as a continuous independent variable. The number of embryos in a uterine horn served as a covariate in analyses of variance of embryo parameters. Implantation rate (number of gestation sacs per CL), conception rate (number of embryos per CL ) and post implantation survival rate (number of embryos per gestation sacs), were calculated 'for each animal and the effect of treatments was examined by analysis of variance. RESULTS
Thermal responses and food intake Thermal responses, food intake of heat-stressed rabbits while in the hot room and body weight measurements are summarized in Table 1. Responses of the two heat-stressed groups were grouped together, as their responses did not differ. Body temperature of the heat-stressed groups increased by 1.2 °C above that at thermal neutrality (P < 0.01 ). Such increments in body temperature reflect a mild hyperthermia. A typical vasodilatory response was indicated by an increase in the ear skin temperature, and a rise in the average respiratory rate above 200 respirations per minute ( P < 0.01). Average daily food intake of the two heat-stressed groups was 30% of C level ( P < 0.01 ) and
263 TABLE 1 Means and SEM of daffy measurements of thermal responses and food intake at thermoneutral and hot conditions 1, and body weight gain during 14-day experimental period Parameter
Control
Heat stress
Number of rabbits Body temperature ( oC ) Ear temperature ( ° C) Respiratory rate (rain -1 ) Food intake (g/day) Body weight2 (kg) Body weight gain (kg)
11 38.4 _+0.1 25.9 _+0.4 149 _+8 161 _+15 3.16 _+0.09 0.25 _+0.05
16 39.6 _+0.1" 31.5 _+0.6* 215 _+8* 48 _+8* 3.22 _+0.10 0.13 _+0.06
1Means of H(3-5) and H(6-8) groups. 2Initial body weight on day of mating. *P < 0.01, from Control. TABLE 2 Reproductive performance and embryo development in rabbits heat-stressed during Days 3-5 [H (3-5) ] or Days 6-8 [H (6-8) ] of pregnancy 1 Parameter
Control
H (3 -5 )
H (6-8)
Implantation rate (To) Conception rate (%) Survival rate (%) Gestation sac weight (g) Embryo weight (g) Crown-rump length (ram) Transumbilical distance (ram)
80___3 74 ___4b 92_+3 4.31 _+0.07a 0.24 _+0.01a 14.02+0.15 a 5.59 -+0.10
71+4 59 _+5a 81_+4 3.47 _+0.09c 0.16 _+0.01b 13.8 +0.19 b 5.23 -+0.13
67_+4 59 _ 5a 90_+4 4.07 _+0.08b 0.22 _+0.01~ 13.79+0.18 ~ 5.55 _+0.12
iValues are means_ SEM. a'b'¢Differentletters denote statistically significant differences at the P < 0.05 level. a v e r a g e b o d y w e i g h t g a i n for t h e i n t e r v a l b e t w e e n D a y I a n d D a y 14 was 0.25 kg a n d 0.13 kg for t h e c o n t r o l a n d h e a t - s t r e s s e d g r o u p s ( N S ) , respectively.
Reproductiveperformance and embryonic development R e p r o d u c t i v e p e r f o r m a n c e a n d e m b r y o n i c d e v e l o p m e n t are p r e s e n t e d in T a ble 2. I m p l a n t a t i o n r a t e s in H ( 3 - 5 ) a n d H ( 6 - 8 ) g r o u p s w e r e lower b y 10% u n i t s t h a n in t h e c o n t r o l g r o u p ( N S ) . S i m i l a r l y , p o s t i m p l a n t a t i o n s u r v i v a l r a t e s o f b o t h h e a t - s t r e s s e d g r o u p s did n o t differ f r o m t h e control. C o n c e p t i o n r a t e for D a y 14 o f p r e g n a n c y , w h i c h t a k e s i n t o a c c o u n t all e m b r y o n i c losses before, d u r i n g a n d a f t e r i m p l a n t a t i o n , was lower in t h e t w o h e a t - s t r e s s e d groups, as c o m p a r e d w i t h c o n t r o l (74% vs. 5 9 % ; P < 0.05 ).
264
Number of embryos in a uterine horn had an effect on embryonic development when a single embryo was present, or when more than six embryos were present in a horn. No effect of the number of embryos in a uterine horn was detected if two to six embryos were present per horn, the situation found in more than 80% of all embryo populations. Mean weight of embryos of the H (35) group was two-thirds that of the control group (0.24 g vs. 0.16 g; P < 0.05). Heating on Days 6-8 did not affect embryo weight. The weight of gestation sacs containing embryos in the H(3-5) group was 80% that of the C group ( P < 0.05), while, in the H (6-8) group, it was only slightly reduced. Crownrump length was smaller in H(3-5) embryos (P<0.05) and that of H(6-8) was the same, compared with controls. Transumbilical distance was similar in all three groups.
Progesterone concentration Ovarian measurements for Day 14 and mean P4 concentrations in plasma for the period from Day 1 to Day 14 are presented in Table 3. Mean P4 level was lowest in the H (3-5) group and highest in the H (6-8) group. In another study in our laboratory (A. Lublin, unpublished data, 1986), a linear relationship between P4 concentration and CL number was found. As P4 concentration is influenced by the number of CL in the ovary, and as a coincidental difference was noted in the number of CL between experimental groups, P4 concentration in plasma per number of CL (P4/CL) was calculated to allow for comparison of heat stress effect among treatments. P4/CL of the C group was similar to that of the H (6-8) group (0.89 and 0.86 ng/ml, respectively). That of the H (35) group was significantly lower, by 30%, than that of the other groups (0.62 ng/ml; P < 0.02 ). The pattern of P4/CL concentrations over all experimental periods was best described by a second order polynomial regression (Fig. 1 ). The C group curve differed from that of both treatment curves: it was higher and the curve did not decline on Day 14 (P<0.01). The curve of the H(3-5) TABLE3 Ovarian weight, number of CL per animal, concentration of progesterone in plasma (P4), and concentration of progesterone in plasma per number of CL (P4/CL), during Days 1-14 of pregnancy, in control and heat-stressed rabbits ~ Parameter
Control
H (3-5)
H (6-8)
Ovarian weight (g) Corporalutea (n) P4 (ng/ml) P a / C L (ng/ml)
0.30 +_0.01 9.6 +0.4 b 8.40 _+0.43 b 0.89 _+0.04 a
0.35 +_0.02 11.1 +0.5 ab 6.80 +_0.46 ¢ 0.62 _+0.04 b
0.36 _+0.02 11.9 +0.5 a 10.22 +_0.46 ~ 0.86-2-_0.04 a
1Values are means +_SEM. ~'b'¢Different letters denote statistically significant differences at the P < 0.05 level.
265 1.6
~ J 1.2 •
j
0.0 2
~ 4-
6
~ 8
~ 10
J
J
t2
14
DAYS
Fig. 1. Concentration of progesteronein plasma per number of CL (P4/CL; ng/ml), as described by secondorder polynomialregressionsof control and heat-stressed rabbits. Control, Q; H (3-5), A; H(6-8), V1.SEM of the three groups: 0.05, 0.04 and 0.05, respectively. group was affected more t h a n that of the H (6-8) group ( P < 0.05). It is noteworthy that the difference between curves of H (3-5) and C groups was detected right at the end of the hot period, on Day 6. On the other hand, the curve of the H ( 6 - 8 ) group declined only on Day 12-Day 14, 4-5 days following the termination of heat exposure. Mean plasma Pt concentrations of the control and heat-stressed groups did not statistically differ ( P > 0.20) on Day 3, before the start of exposure to heat. Neither were differences between groups observed during the periods of exposure to heat.
Histology of the corpus luteum
The number of large luteal cells and their size are presented in Table 4. A typical photograph of a luteal tissue of a control animal is presented in Fig. 2. The number of large cells, along a scale line, was significantly smaller in both heat-stressed groups (82% of C group; P < 0 . 0 5 ) . The mean diameter of the large luteal cells was smaller by about 10% in the two heat-stressed groups, as compared with the C group ( P < 0.05 ).
266 TABLE 4 Number of large luteal cells along a 250/1m scale line and its average diameter (/~m), in control and heat-stressed rabbits 1 Parameter
Control
H (3-5 )
H (6-8)
Number Diameter
6.7 ___0.1a 36.0 _+0.3a
5.3 _+0.25 32.3 _+0.35
5.6 _+0.25 32.7 _+0.35
1Valuesare means _+SEM. a'5Different letters denote statistically significant differences at the P < 0.05 level.
Fig. 2. Luteal tissue of Day 14 control pregnant rabbit, × 750. L, large luteal cell. DISCUSSION The thermal stress elevated body temperature by only 1.2 °C above normal Tb. Such an increment in Tb is experienced by many species of animals during the hot hours of summer days (Berman et al., 1985 ). During the three-day heat stress, food intake was about one-third of control, but body weight gain was only slightly reduced (Table 1 ). Low level of nutrition, over a few estrous cycles, decreased conception rate in cattle (Folman et al., 1973). Low intake during the first 11 or 21 days of pregnancy in sheep was associated with high peripheral P4 and impaired embryonic development (Parr et al., 1982). Undernutrition also increased P4 in late pregnant cows (Gauthier et al., 1983). However, Cartwright and Thwaites (1976), in a pair-feeding experiment in pregnant sheep, did not observe effects of a low nutrition level on embryonic develop-
267
ment. In the present experiment, relatively short term heat stress was imposed, in contrast to the forementioned studies. Though possible nutritional effects can not be discarded, it is unlikely that low food intake for a three-day period might significantly affect reproductive and endocrine responses. In support of this presumption, Lublin et al. (1984), in our laboratory, found that reducing feed intake during Days 3-5 of pregnancy in rabbits did not significantly affect P4 concentration or embryonic development. Shah (1956) also claimed that a high rate of embryo mortality in rabbits, in response to a severe 6-day heat exposure, was unrelated to the lower food intake during heat exposure. There are various stages between mating and implantation which are critical for the success of pregnancy. The H (3-5) treatment heat stressed the animals during the period of blastocyst development and rapid CL growth. Conception rate of the H (3-5) group was significantly lower than control. Implantation and post-implantation survival rate suggested that 41% of the potential embryos of that group were lost before and after implantation, as compared with 26% in the C group. On a longer and more severe pre-implantation heat stress (Days 0-6, Ta=36°C, Tb=40.6°C; Shah, 1956), most rabbit embryos were resorbed by Days 10-14, i.e. after implantation. In rats, 1-day heat stress was found to increase embryo mortality when it was applied during blastocyst development (Arora et al., 1979). In contrast, the sheep zygote was found to be most sensitive to thermal stress during the initial stages of cleavage (Days 15 ) and not during the initial blastocyst stage (Day 8; Dutt, 1964). The H (6-8) treatment (33.6 ° C, 3 days) coincided with early implantation. Conception rate was, as in the H (3-5) group, significantly lower than in C group. Only few studies have examined specifically the effect of heat stress during implantation. Heat stressing pregnant rats around implantation time (Ta 43-44°C, Day 6), did not affect reproductive performance (Arora et al., 1979). Gilts were most susceptible to thermal stress during implantation in one study (Ta 37.8°C for 17 h and 32.2°C for rest of day, between Days 8-16; Omtvedt et al., 1971 ), but not in another at slightly lower heat stress ( Ta 35 ° C and 32 ° C, each for 12 h, on Days 8-16; Hoagland and Wettemann, 1984). These studies, as a whole, demonstrate species differences in the susceptibility periods of pregnancy to thermal stress. They also emphasize difficulties encountered in comparing experiments where severity of thermal stress or its duration differ. Unlike percentage of embryonic losses, which was similar in rabbits exposed to heat between Days 3-5 or Days 6-8 post-mating, development of the surviving embryos was impaired in the H(3-5) but not in the H(6-8) group. This reflected itself in smaller gestation sacs, embryo weight and length. The above agrees with the teratogenic effects and retarded embryonic development induced by hyperthermia pre-implantation (Edwards, 1978 ). Most studies in the rabbit examined embryo mortality rate, but not alterations of embryo development, resulting of thermal stress around fertilization (Ulberg and Sheean,
268 1973), during the first 6 days post-mating (Shah, 1956) or during 21 days of pregnancy (Trammel et al., 1986). The specific contribution of this study, in this respect, is that, in the rabbit, a significant retardation of embryo development occurred following thermal stress on Days 3-5 of pregnancy. It does not indicate, however, whether embryos found retarded on Day 14 would have been carried to term. Lublin et al. (1984) suggested that an early heat stress (Days 3-5 ) might cause embryonic death extending until close to term. Progesterone levels were reduced by thermal stress, more so for the H (3-5) group than for the H (6-8) group. In the H (3-5) group, it coincided with the period of CL linear growth. The lesser reduction in the H (6-8) group could be detected only by 4-5 days following cessation of heat stress. Similar results were obtained in studies in dairy cows and gilts. In dairy cows exposed to environmental heat, P4 was lower than in cows cooled for the first 10 days after insemination (Gauthier, 1983). Biggers et al. (1987) did not find changes in P4 level in beef cows following later thermal stress on Days 8-16 post insemination. Also, heat stressing pregnant gilts during Days 8-16 post-mating, had no effect on post-exposure P4 (Hoagland and Wettemann, 1984). These studies suggest that progesterone secretion is more sensitive to heat stress when applied during the period of rapid CL growth. However, heat stressing gilts for the first 8 days post-mating increased P4 concentrations following heat exposure (Wettemann et al., 1984). In the rabbit, as in other species, two steroidogenic cell types are found small and large - which originate from granulosa and theca cells, respectively (Niswender et al., 1985). The regulation of progesterone secretion by the two cell types differs as does their sensitivity to various mediators or hormones (Hoyer et al., 1986). In the present study, the large cells (Fig. 1) which were easily identified among other cells, had a diameter similar to that found by Hoyer et al. (1986) and Osteen et al. (1986). In these studies, rabbit luteal cells were dispersed, their steroidogenic capacity examined by histochemical technique and their size visually determined. Similarity between large cells in the present study and large cells in the other studies is suggested. The histology of the ovaries in this study indicated that heating induced some morphological changes in the population of the CL cells, in contrast to a study in sheep (Thwaites, 1970). A small but significant reduction in size and number of large cells was noted in both H (3-5) and H (6-8) treatments. As the size of the large cells declined following heat stress, and less cells were counted along a scale line, this may indicate an increase in the proportion of other cell types (stromal or vascular cells ) following heat stress. The relationship of endocrine capacity to changes in ovarian morphology of the various cell populations needs further study. The present study suggests a relationship between the reduction in reproductive performance and embryo development following heat stress and some impairment of ovarian structure and function. Along the same line in an ex-
269
periment performed in our laboratory (Blum et al., unpublished results), exogenous progesterone administration improved reproductive performance and embryonic development in pregnant rabbits heat-stressed on Days 3-5 postcoitus. In conclusion, heat stress-induced impairment and retardation of development of rabbit embryos was more pronounced during the period of blastocyst formation. This coincided with heat stress-induced reduction of ovarian function, which was larger at this period than during implantation. Possible involvement of a sub-functional CL in reduced reproductive performance is suggested. ACKNOWLEDGEMENT
The assistance of Mrs. Y. Graber, Mrs. R. Steinberg, Mr. A. Lublin and Mr. M. Maman in carrying out various parts of this research, is much appreciated.
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