Follicular development in primiparous lactating sows

Animal Reproduction Science, 5 (1982) 47--56 47 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands FOLLICULAR DEVELOPMENT IN PRIMIPAROUS LACTATING SOWS A. K U N A V O N G K R I T , S. E I N A R S S O N and I. S E T T E R G R E N Department of Obstetricsand Gynaecology and Department of ClinicalChemistry, College of Veterinary Medicine, Swedish Universityof AgriculturalSciences, S-750 07 Uppsala (Sweden) (Accepted ~ February 1982) ABSTRACT Kunavongkrit, A., Einarsson, S. and Settergren, I., 1982. Follicular development in primiparous lactating sows. Anita. Reprod. Sci., 5 : 47--56. The object of this investigation was to study ovarian activity with particular reference to follicular development in primiparous sows during the lactation period. Thirteen primiparous sows were slaughtered at day 4, 5, 14, 21, 28, 42 or 56 of lactation. The ovaries were examined macroscopically, serially sectioned and photographed. Follicles ~ 1 mm were counted and classified as normal or atretic. Corpora lutea of pregnancy were identified and the size measured. The peripheral plasma progesterone levels were determined throughout the lactation period. There was a tendency towards increasing numbers of normal and atretic follicles of 2.00--4.99 m m size during later stages of lactation. During the first week of lactation there was an accumulation of atretic follicles. About equal numbers of normal and atretic follicles were recognized at days 14, 21 and 28 of lactation. There were more normal than atretic follicles at days 42 and 56. The peripheral plasma progesterone levels were constantly low in all sows, indicating that no ovulation occurred during lactation. It can be concluded that the ovaries had some development in follicular growth during lactation but they did not reach the ovulatory size. INTRODUCTION During the lactation period sows do not normally show oestrus (Burger, 1952). Some lactating sows, however, may exhibit signs of oestrus within a few days after parturition but without ovulating (Warnick et al., 1950). After a lactation length of 4--8 weeks, oestrus usually occurs within a few days after weaning (Self and Grummer, 1958; Svajgr et al., 1974). The interval is longer in primiparous sows (Rasbech, 1969; Dyck, 1971; Einarsson and Settergren, 197~; Karlberg, 1980). Several factors, such as feeding (Dyck, 1972; Karlberg, 1980), management (Allrich et al., 1979) and season of the year (Hurtgen and Leman, 1978; Fahmy et al., 1979; Hurtgen et al., 1980; Karlberg, 1980) influence the length of the interval between weaning and the first observed oestrus. 0378-4320/82/0000---0000/$02.75 © 1982 Elsevier Scientific Publishing Company 48 Very few studies cover the ovarian morphology in sows during lactation. Palmer et al. (1965), studying the ovarian morphology of sows during second lactation, found no ovulation in any of 67 sows slaughtered during different stages of the lactation period. Atretic follicles appeared more numerous in the ovaries during the immediate post-partum period than later during lactation. Crighton and Lamming (1969) and Edqvist et al. (1974), measuring the progesterone level in the peripheral blood during lactation, found no increased levels indicating formation of corpora lutea. The objective of this investigation was to study the ovarian activity of primiparous sows during the lactation period with particular reference to number and quality of follicles ~ 1 mm in size. MATERIALS AND METHODS Thirteen primiparous sows (nos. 1--13, Table I) of Swedish Landrace or crosses between Landrace and Yorkshire were used in this study. They were purchased from commercial herds and brought to the Department of Obstetrics and Gynaecology 3--4 weeks before expected farrowing. The sows were housed in individual pens and kept there throughout the experimental period. They were fed a commercial feed containing all necessary nutrients (Eriksson et al., 1972). The dally ration per sow was approximately 3 kg during late pregnancy. The ration was slowly increased after farrowing and reached a daily amount of 2 kg plus 0.4 kg per piglet 2 weeks after farrowing. Testing for oestrus was performed once daily in the presence of a boar throughout the lactation period. Blood samples were drawn from an ear vein once a week in sows nos. 1--4 and 8--13 for progesterone determination. In sows nos. 5--7 silastic collecting tubes were inserted into the cephalic vein under general anaesthesia (Karlbom et al., 1982). This operation was done a week before expected farrowing and blood collection was done once daily (9 a.m.) until slaughter. The blood samples were collected into heparinized tubes and centrifuged immediately at 3000 r.p.m. Plasma was removed and stored at - 2 0 ° C until assayed. The levels of peripheral plasma progesterone of the sows nos. 1--4 and 8--13 were determined by the rapid competitive protein-binding m e t h o d (Edqvist et al., 1970). This assay has a relatively low sensitivity and a relatively high blank. From the assay of plasma pools from ovariectomized pigs a practical detection limit of 2.5 ng/ml (mean + 2 S.D.) was established. Later this technique was replaced by a radioimmunoassay (Bosu et al., 1976) and the samples from sows nos. 5--7 were analyzed by this technique. This assay is much more sensitive and also has a lower blank. The practical detection limit determined as above for this technique was 0.25 ng/ml. The sows were slaughtered from approximately 1 to 8 weeks of lactation (Table I). The reproductive organs were removed and examined macroscopically within 1 h after slaughter. The ovaries were fixed in Bouin's fluid (about 50 ml for each ovary) for 30 h. They were dehydrated according to standard methods and 49 embedded in histowax at 56°C. The tissues were serially sectioned in 10 ~m thick sections. Every twentieth section was stained with hemalum eosin. The stained sections were projected on photographic paper with 5X magnification. In the photographs, the numbers and sizes of the follicles 1 mm were determined using t h e same methods as described by Rajakoski (1960) for heifers. The follicles were divided into the following classes according to the diameter: 1.00--1.99, 2.00--2.99, 3.00--3.99 and 4.00-4.99 ram. The mean diameter of the follicles (d) was obtained from the formula: d = dl + d2 + d3/3 in which dl = the greatest diameter of the follicle d~ = the diameter at right angles to d~ d3 = the diameter perpendicular to the plane of sectioning obtained by counting the total number of sections in which the follicle was seen. The identification of normal and atretic follicles was carried out microscopically. The attetic follicles were characterized by degeneration of the granulosa cells with pycnosis of their nuclei. During early stages degenerated granulosa cells became detached from the follicular wall and pycnotic nuclei floated in the follicular cavity (Fig. 1). During later stages of atresia the Fig. 1. Early stage of follicular atresia in the pig ovary showing pycnotic nuclei of granulosa cell in the lumen (750x). 50 Fig. 2. Late stage of atresia in the pig ovary with disappearance of the granulosa layer and ingrowth of connective tissue (500x). granulosa cells disappeared completely and the thecal connective tissue started to grow and began to fill up the lumen (Fig. 2). The number of corpora lutea of pregnancy was counted and the largest corpus luteum in each sow was measured. Conventional statistical methods were used for analyses of the results (Snedecor, 1956). RESULTS The gestation length and the litter sizes at birth and at slaughter are given in Table I. Two sows (nos. 1 and 11) showed weak signs of oestrus 3 days after parturition. No signs of oestrus were seen in the other sows. At slaughter, the ovaries showed only small follicles (< 5 mm). In all ovaries, including those of sow no. 1 which was slaughtered 1 day after showing signs of heat, no newly formed corpora lutea or corpora hemorrhagica were found. Microscopically, regressing corpora lutea of pregnancy or corpora albicans were seen in all sows (Table II). In sows nos. 1, 2 and 3 they were also seen macroscopically. No pathological lesions were found macroscopically or microscopically in oviducts, uterus, cervix or vagina. The number of corpora lutea in the right and the left ovaries is given in 51 io Clinical data of sows slaughtered at different stages of lactation SOW n o . 1 2 3 4 5 6 7 8 9 10 11 12 13 Gestation (days) 117 114 115 115 114 116 115 115 118 116 114 114 114 Lactation (days) 4 5 14 14 21 21 21 28 28 42 42 56 56 Litter size At birth At slaughter Heat symptoms after parturition (day) 9 11 12 4 9 12 13 12 5 15 11 10 7 9 11 11 3 9 11 11 11 4 11 11 10 5 3 nil nil nil nil nil nil nil nil nil 3 nil nil TABLE II Numbers and largest sizes of old corpora lutea (CL) (1--8 weeks) in sows slaughtered during different stages of lactation Sow no. 1 2 3 4 5 6 7 8 9 10 11 12 13 Slaughtered on day 4 5 14 14 21 21 21 28 28 42 42 56 56 No. of CL Right Left 4 9 7 6 6 7 7 3 6 10 7 6 4 7 6 6 ] 7 7 7 9 2 5 5 4 3 Total no. Size of the largest corpus luteum (mm) 11 15 13 7 13 14 14 12 8 15 12 10 7 6.5 6.1 4.3 2.8 2.9 2.8 2.8 3.1 2.5 2.4 2.5 2.3 2.1 T a b l e II. T h e r e w e r e m o r e i n t h e r i g h t o v a r y t h a n i n t h e l e f t b u t t h e d i f f e r e n c e is n o t s t a t i s t i c a l l y s i g n i f i c a n t ( P > 0 . 0 5 ) . T h e sizes o f t h e l a r g e s t c o r p o r a l u t e a i n d i c a t e t h a t t h e y d e c r e a s e i n size w i t h i n t h e f i r s t w e e k a f t e r p a r t u r i t i o n . The total n u m b e r of n o r m a l follicles per sow a n d the percentage in differe n t size classes a r e g i v e n i n T a b l e I I I f o r d i f f e r e n t l a c t a t i o n a l stages. T h e findings indicate a m a r k e d v a r i a t i o n in the t o t a l n u m b e r of follicles b e t w e e n 52 TABLE III Number of normal follicles of different sizes during lactation per sow Week of lactation No. of sows Number of normal follicles of different sizes (mm) 1.00--1.99 2.00--2.99 mean and (range) % mean and (range) % 3.00--3.99 4.00--4.99 mean and (range) mean and (range) % 1 2 108.0 (108) 93.9 7.0 (0--14) 6.1 2 2 116.5 (72--161) 74.4 38.0 (23--53) 24.3 2.0 (0--4) 1.3 3 3 66.0 (51--74) 79.9 15.6 (0--25) 18.9 1.0 (0--3) 1.2 4 2 111.0 (34--188) 65.3 53.0 (33--73) 31.2 6.0 (3--9) 3.5 6 2 82.0 (52--112) 68.9 27.5 (0--55) 23.1 9.5 (0--19) 8.0 8 2 66.0 (44--88) 50.6 43.5 (25--62) 33.3 19.5 (6--33) % -- -- 14.9 1.5 (0--3) 1.2 TABLE IV Number of atretic follicles of different sizes during lactation per sow Week of lactation No. of sows Number of atretic follicles of different sizes (ram) 1.00--1.99 mean and (range) 2.00--2.99 % mean and (range) % 3.00--3.99 4.00--4.99 mean and (range) mean and (range) -- % % 1 2 183.0 89.7 (161--205) 21.0 (0--42) 10.3 -- 2 2 126.0 76.4 (103--149) 38.0 (1--75) 23.0 1.0 (0--2) 0.6 3 3 50.7 (28--65) 78.4 12.3 (0--22) 19.0 1.7 (0--5) 2.6 4 2 122.5 (14--231) 64.3 62.5 (38--87) 32.8 5.5 (4--7) 2.9 6 2 40.5 (10--71) 60.4 17.0 (0--34) 25.4 9.0 13.4 0.5 (0--18) (0--1) 0.8 8 2 54.0 (44--64) 59.7 23.5 (22--25) 26.0 12.5 (0--25) 0.5 13.8 0.5 (o-1) 53 individuals. T h e r e is a clear t e n d e n c y t o w a r d s a n increasing n u m b e r o f follicles 2 . 0 0 - - 3 . 9 9 m m in size d u r i n g l a t e r stages o f l a c t a t i o n . Follicles >i 4 . 0 0 m m in size o c c u r r e d o n l y in o n e a n i m a l in t h e last g r o u p w h e n t h e length o f l a c t a t i o n was 8 weeks. N o follicles o f o v u l a t o r y size w e r e f o u n d in a n y o f t h e animals. T h e n u m b e r a n d p e r c e n t a g e o f a t r e t i c follicles d u r i n g d i f f e r e n t l a c t a t i o n a l stages are s h o w n in T a b l e IV. T h e findings s h o w a n increased average size o f a t r e t i c follicles w i t h increasing length o f l a c t a t i o n in t h e s a m e w a y as f o r n o r m a l follicles. A c o m p a r i s o n o f t h e n o r m a l a n d a t r e t i c follicles ( T a b l e V) s h o w s a relatively high n u m b e r o f a t r e t i c follicles d u r i n g early l a c t a t i o n w i t h a b o u t t w o - t h i r d s o f all follicles atretic d u r i n g t h e first w e e k . D u r i n g w e e k s 2 - - 4 o f l a c t a t i o n t h e r e w e r e a b o u t e q u a l n u m b e r s o f n o r m a l a n d a t r e t i c follicles, whereas later on during weeks 6--8 of lactation there were m o r e normal t h a n a t r e t i c follicles. T h e p e r i p h e r a l p l a s m a p r o g e s t e r o n e levels a f t e r p a r t u r i t i o n are s h o w n in TABLE V Total numbers of normal and atreticfollicles> 1 m m Week of lactation 1 2 3 4 6 8 Normal during lactation per sow Atretic Total no. No. % No. % 115.0 156.5 82.7 170.0 119.0 130.5 36.0 48.6 56.1 47.2 64.0 59.0 204.0 165.0 64.6 190.5 67.0 90.5 64.0 51.4 43.9 52.8 36.0 41.0 319.0 321.5 147.3 360.5 186.0 221.0 TABLE V I a Peripheral plasma progesterone levels in Day after parturition 1--7 8--14 15--21 22--28 29--35 36--42 43--57 No. of animals 10 6 6 6 3 4 2 sows nos. No. of samples 34 6 7 7 3 4 4 1--4 and 8--13 a ng/ml Mean Range 1.6 1.5 1.3 1.1 1.3 1.5 1.3 < 2.5--4.5 < 2.5 < 2.5 < 2.5 < 2.5 < 2.5 < 2.5 aAnalysed by competitive protein binding, the practical detection limit being 2.5 ng/ml. 54 TABLE VI b Peripheral plasma progesterone levels in sows nos. 5, 6 and 7a Days after parturition 1--7 8--14 15--21 No. of animals 3 3 3 No. of samples 19 21 21 ng/ml Mean Range 0.22 0.11 0.11 <0.25--0.35 <0.25 <0.25 aDetermined by radioimmunoassay, the practical detection limit being 0.25 ng/ml. Table Via and b according to m e t h o d of analysis. The plasma of sows nos. 1--4 and 8--13 was examined by competitive protein binding and showed a mean progesterone value of about 1.5 ng/ml (range < 2.5--4.5). The peripheral plasma levels of progesterone in sows nos. 5--7, where a radioimmunoassay system was used, were very low during the first 3 weeks of lactation. DISCUSSION The lengths of gestation and litter sizes were within the normal range for primiparous sows of the breeds to which they belonged. Two of the thirteen sows showed signs of oestrus at day 3 after parturition. This is in agreement with previous reports (Warnick et al., 1950; Baker et al., 1953). No newly formed corpora lutea or corpora hemorrhagica were found in the ovaries of any of the sows. The constantly low levels of peripheral plasma progesterone in all sows confirmed that no ovulation occurred during the lactation period. These results are in conformity with earlier presented results by Palmer et al. (1965) and Crighton and Lamming (1969). Regressing old corpora lutea were f o u n d microscopically in all ovaries examined. These corpora lutea decreased rapidly in size after parturition, which was also shown by Palmer et al. (1965). In all animals slaughtered more than 2 weeks after parturition the old corpora lutea were discovered only microscopically. There was a marked variation in the total number of follicles between individuals. With the limited number of sows available no statistical analysis was made on variation in the total number of follicles between different lactational stages. There was, however, a clear tendency towards an increasing number of follicles 2.00--4.99 mm in size during later stages of lactation both for normal and atretic follicles. No follicles of ovulatory size were found in any sow. Palmer et al. (1965) found that the follicular size decreased during the first week and thereafter gradually increased as the lactation period progressed. Follicles of 1> 5 m m size were f o u n d by Palmer et al. during most of the lactational stages but in no case did they find follicles of ovulatory size. No follicles ~> 5 mm size were found in any of the sows in the present study. One reason might be that only primiparous sows were 55 used in the present study while Palmer et al. used sows in their second lactation. The reason for the suppression of follicular growth during lactation is not fully known. Lactation or the suckling stimulus, or both, might be responsibl, for insufficient gonadotropic stimulation of the ovaries. It has been shown that the plasma levels of LH and FSH are low during the first 3 weeks of of lactation and then begin to rise (Stevenson et al., 1981). Peripheral plasma levels of prolactin are high during lactation in the sow (Bevers et al., 1978). A relationship might exist between the release of the gonadotropins and prolactin (Rothchild, 1966). The suckling stimulus may promote prolactin release (Smith and Wagner, 1980) and thereby inhibit the release of gonadotropins. Under normal conditions milk production increases to a m a x i m u m level during the third week of lactation and thereafter decreases as the piglets start consuming creep feed (Elsley, 1970). This might explain why there are a higher number of atretic follicles in the beginning of lactation while the reverse is the situation during late lactation. The increase in follicular size during the later stages of lactation, demonstrated by the present study, might be due to the same condition. It can be concluded from the present study that no ovulation occurred during the lactation period in primiparous sows. There was, however, a clear tendency towards increasing numbers of normal follicles 2.00--4.99 m m in size during the later stages of the lactation period. ACKNOWLEDGEMENT We would like to t h a n k Mrs Kerstin Lindblad and Miss Catharina Falkenberg for excellent histological and technical assistance. This work was supported by the Swedish Council for Forestry and Agricultural Research. A scholarship awarded to A. Kunavongkrit by Chulalongkorn University, Bangkok, is acknowledged. REFERENCES Allrich, R.D., Tilton, J.E., Johnson, J.N., Slanger, W.D. and Marchello, M.J., 1979. Effect of lactation length and fasting on various reproductive phenomena of sows. J. Anim. Sci., 48: 359--362. Baker, L.N., Woehling, H.L., Casida, L.E. and Grummer, R.H., 1953. Occurrence of estrus in sows following parturition. J. Anim. Sci., 12: 33--38. Bevers, M.M., Willemse, A.H. and Kruip, Th.A.M., 1978. Plasma prolactin levels in the sow during lactation and the postweaning period as measured by radioimmunoassay. Biol. Reprod., 19: 628--634. Bosu, W.T., Edqvist, L.E., Lindberg, P., Martinsson, K. and Johansson, E.D.B., 1976. 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