Br. vet .
3. (1991) . 147, 469
HISTOLOGICAL CHANGES IN THE VAGINAL MUCOSA OF THE COW DURING THE OESTROUS CYCLE, AFTER OVARIECTOMY AND FOLLOWING EXOGENOUS OESTRADIOL BENZOATE AND PROGESTERONE TREATMENT
K . MIROUD* and D . E . NOAKESt Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA
SUMMARY The histological structure of the anterior vaginal mucosa was studied using sequential biopsies in five normal cyclical Friesian cows and in four of the same cows after ovariectomy and exogenous progesterone and oestradiol benzoate . During oestrus the epithelium comprised a single layer of tall columnar, highly active, mucus-secreting cells, whereas during the luteal phase of the cycle it comprised several layers of low columnar, cuboidal or flattened epithelial cells . Exogenous oestradiol benzoate and progesterone administered separately produced little change in the low columnar and cuboidal epithelium observed after ovariectomy .
INTRODUCTION Several studies have demonstrated the apparent inconsistency in the exfoliative vaginal cytology of the cow during the oestrous cycle (Cole, 1930 ; Hansel, 1950 ; Rao et al., 1979 ; Miroud & Noakes, 1990) . This may reflect a genuine inconsistency in the structure of the vaginal mucosa, or variations in the pattern of desquamation of the epithelial cells from the mucosa . However, studies on the histological structure of the vaginal mucosa, particularly the anterior vagina adjacent to the os uteri, have shown a considerable degree of consistency during the oestrous cycle (Hammond, 1927 ; Cole, 1930 ; Brown, 1944) . During oestrus and metoestrus the epithelium comprises non-cornified columnar, highly active mucussecreting cells, whilst after oestrus there was extensive sloughing of superficial epithelial cells with massive infiltration of leucocytes . Cupps et al. (1969) commented that the growth of the vaginal epithelium was an excellent indicator of oestrogen secretion . However, it should be noted that there are distinct variations in mucosal structure at different regions of the vagina (Hammond, 1927) . *Present address : 37 Rue Laboid . Alohamed S .A1 .K ., Constantine, Algeria . trot all correspondence .
470
BRITISH VETERINARY JOURNAL, 147, 5
Most studies have been performed on tissues obtained fresh from genital tracts removed at the abattoir . In this particular study sequential biopsies from the anterior vagina were obtained throughout the oestrous cycle in five normal cows so that comparisons could be made with the exfoliative smears collected from the same site a few minutes earlier and described previously (Miroud & Noakes, 1990) . Subsequently, similar studies were performed in the same cows after ovariectomy and exogenous oestradiol benzoate and progesterone .
MATERIALS AND METHODS Experimental animals
Five Friesian cows, aged between 2 .5 and 7 years, with normal breeding histories were used in the first experiment . They were housed in a conventional byre with water and hay ad libitum . When not on experiment the cows were kept at grass or, during the winter, loose-housed in a barn . Before starting the experiments, all cows were observed for at least three consecutive oestrous cycles . Four cows, 12, 17, 23 and 68, were subsequently ovariectomized and treated with slow-release progesterone and oestradiol benzoate as described previously (Miroud & Noakes, 1990) . Biopsy technique Biopsy instrument.
The biopsy instrument was similar to that described by Donker (1952) and used by Hartigan (1970) and Hartigan et al. (1974) to take bovine endometrial biopsies . The instrument was modified from that of Hartigan et al. (1974) in order to facilitate the interchange of the cutting blade, and was longer to improve the ease of manipulation during insertion and cutting . The basic design is that of a stainless steel tube with a free running, but close fitting, internal stainless steel rod running the length of the tube . A metal spike on the handle crossbar prevents the inner rod being pushed too far, thus damaging the cutting edge at the tip of the instrument . However, a hole in the handle allows the rod to pass over the spike and push the cutting blade out of the outer tube when the removable tip of the outer tube is unscrewed . Biopsy collection . After the vulva has been cleaned and the cow restrained with the tail held aside, the closed instrument was inserted into the vagina and guided per rectum by the left hand as far as the external os uteri. The cutting edge was drawn back, thus opening the window in the outer tube . The hand in the rectum was used to press the dorsal wall of the anterior vagina into the window of the instrument and the cutting edge pushed forward to remove a piece of tissue of about 1 cm long and 6 mm wide . The instrument was then withdrawn, and the piece of the vaginal mucosa was removed with a pair of fine pointed forceps and then placed in a 10 ml labelled plastic bottle containing Mirsky's fixative (National Diagnostic Ltd) where it remained for at least 24 h before processing . Tissue processing and staining
After fixation, processing and embedding in paraffin wax, several sections from the same block were cut at 5 um on a sledge microtome . Sections were stained using routine Harris's haematoxylin (BDH Ltd, Poole) .
EFFECT OF VARIOUS TREATMENTS ON BOVINE VAGINAL MUCOSA
471
Detection of oestrus In order to assist in the detection of oestrus, all five cows were synchronized with 500 µg cloprostenol (Estrumate, Coopers Animal Health) i .m . at an 11-day interval . From the time of the first injection, daily blood samples were obtained from the jugular vein by venepuncture so that plasma progesterone concentrations could be measured to monitor the stage of the oestrous cycle and predict the likely day of oestrus . Each cow was fitted with a KaMaR heat mount detector (KaMaR Inc ., Steamboat Springs, USA) at the time of the second cloprostenol injection, and from the day before the expected day of oestrus the cows were observed every 3 h so that the time of onset of oestrus could be determined . Collection of blood samples Cyclical cows. A 6 ml heparinized jugular blood sample was obtained by venepuncture daily except after the first sign of observed oestrus, i .e . activation of the KaMaR, when the frequency was increased to a 2-hourly interval on four occasions . The blood samples were centrifuged in a refrigerated centrifuge at 4°C for 10 min at 2000 r .p .m . The plasma was decanted and frozen at -20 ° C until assayed . Ovariectomized cows . A 6 ml heparinized jugular blood sample was obtained by venepuncture except when hourly or 4-hourly samples were required, when an indwelling catheter was inserted into one of the jugular veins . Following exogenous progesterone, blood samples were withdrawn twice daily in cows 12 and 23 (at 9 :00 and 21 :00) for 9 days, and in cows 17 and 68 every 4 h for 4 days and then twice daily for 5 days . All four cows were then sampled daily for 6 days . Following exogenous oestradiol-1713 (ODB) all four cows were sampled every hour for 5 h, then every 4 h for 36 h and then every 12 h for the next 72 h . Before the administration of exogenous hormones each cow was sampled daily for 5 days .
Assay of progesterone and oestradiol-1713 This has been described previously (Miroud & Noakes, 1990) . Biopsy frequency . Each cow was sampled eight times over four oestrous cycles to ensure that tissue was obtained at different stages of the oestrous cycle and so that at least 7 days elapsed between consecutive biopsies . Each cow was biopsied at least 3 weeks after ovariectomy and 48 h after the injection of the slow-release progesterone preparation and 24 h after oestradiol benzoate in oil . The rest periods between treatment regimens have been described previously (Miroud & Noakes, 1990).
RESULTS
Cyclical cows A total of 40 anterior vaginal mucosal biopsies were taken from the five cows at different stages of the oestrous cycle . During oestrus, the epithelium consisted of a single layer of very tall columnar, highly active mucus-secreting cells . The cells had thin elongated and compressed nuclei which stained intensely (Fig . 1) . In some sections there was a thin basal layer of polyhedral cells beneath, and in some other areas of the same section, the epithelium was composed of either one or two layers of flattened superficial
472
BRITISH
VETERINARY
JOURNAL,
147, 5
squamous epithelial cells or several layers of polyhedral cells. Leucocytes were absent or only occasionally seen, and no cornilication or desquamation was evident. The stroma was slightly oedematous and the blood vessels appeared slightly congested.
Fig. 1. Photomicrograph of transversesection of anterior vaginal mucosa taken at oestrus showing a single layer of tall, columnar mucus producing cells. (H & E X260).
At one day after oestrus, the epithelium was very similar to that of oestrus. The tall columnar cells were reduced in height and became low columnar but still secreted mucus. In two cows (12 and 23) the tall columnar, highly active mucus-secreting cells remained 2 days after oestrus. At this stage in some sections there was an increase in the number of layers of squamous or polyhedral cells (two to three layers). The degree of desquamation varied and leucocytes were occasionally observed (Fig. 2).
Photomicrograph of transversesection of anterior vaginal mucosa taken during metoestrus showing tall columnar mucus-secreting cells in one part of the section (M) and several layers of polyhedral cells in another(P). (H & E X130).
Fig. 2.
EFFECT OF VARIOUS
TREATMENTS
ON BOVINE
VAGINAL
MUCOSA
413
From day 2 after oestrus to the end of metoestrus, there was a further decrease in the height of the superficial cell layers and, at the same time, an increase in the number of layers. The superficial cells changed from low columnar to cuboidal, and sometimes to a polyhedral epithelial type. Leucocytes were present throughout this period. There was congestion of the blood vessels and the stroma was denser than that of oestrus. During dioestrus, some sections showed similar features to that of metoestrus, whilst in some others the superficial layers changed from cuboidal to flattened. The epithelium was usually six to eight layers thick (Fig. 3). Cell desquamation and leucocytes were also seen during this period.
Fig. 3. Photomicrograph of transversesection of anterior vaginal mucosa taken during dioestrus showing several layers of flattened epithelial cells in some parts of the section (F) and severallayers of polyhedral cells in others (P). (H & E X130). During pro-oestrus, the vaginal mucosa varied from two to seven layers of epithelial cells (Fig. 4). In early pro-oestrus, the superficial layer consisted of low columnar epithelial cells which rested on several layers of polyhedral cells. In mid pro-oestrus, the low columnar cells tended to increase in height and the number of polyhedral cells reduced greatly, so as in late pro-oestrus there were only one to two layers of epithelial cells. The superficial layer was formed by tall columnar mucus-secreting cells and the basal layer consisted of well stained polyhedral cells (Fig. 4). Leucocytes were present throughout this period. Ouariectomi
3. (1991) . 147, 469
HISTOLOGICAL CHANGES IN THE VAGINAL MUCOSA OF THE COW DURING THE OESTROUS CYCLE, AFTER OVARIECTOMY AND FOLLOWING EXOGENOUS OESTRADIOL BENZOATE AND PROGESTERONE TREATMENT
K . MIROUD* and D . E . NOAKESt Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA
SUMMARY The histological structure of the anterior vaginal mucosa was studied using sequential biopsies in five normal cyclical Friesian cows and in four of the same cows after ovariectomy and exogenous progesterone and oestradiol benzoate . During oestrus the epithelium comprised a single layer of tall columnar, highly active, mucus-secreting cells, whereas during the luteal phase of the cycle it comprised several layers of low columnar, cuboidal or flattened epithelial cells . Exogenous oestradiol benzoate and progesterone administered separately produced little change in the low columnar and cuboidal epithelium observed after ovariectomy .
INTRODUCTION Several studies have demonstrated the apparent inconsistency in the exfoliative vaginal cytology of the cow during the oestrous cycle (Cole, 1930 ; Hansel, 1950 ; Rao et al., 1979 ; Miroud & Noakes, 1990) . This may reflect a genuine inconsistency in the structure of the vaginal mucosa, or variations in the pattern of desquamation of the epithelial cells from the mucosa . However, studies on the histological structure of the vaginal mucosa, particularly the anterior vagina adjacent to the os uteri, have shown a considerable degree of consistency during the oestrous cycle (Hammond, 1927 ; Cole, 1930 ; Brown, 1944) . During oestrus and metoestrus the epithelium comprises non-cornified columnar, highly active mucussecreting cells, whilst after oestrus there was extensive sloughing of superficial epithelial cells with massive infiltration of leucocytes . Cupps et al. (1969) commented that the growth of the vaginal epithelium was an excellent indicator of oestrogen secretion . However, it should be noted that there are distinct variations in mucosal structure at different regions of the vagina (Hammond, 1927) . *Present address : 37 Rue Laboid . Alohamed S .A1 .K ., Constantine, Algeria . trot all correspondence .
470
BRITISH VETERINARY JOURNAL, 147, 5
Most studies have been performed on tissues obtained fresh from genital tracts removed at the abattoir . In this particular study sequential biopsies from the anterior vagina were obtained throughout the oestrous cycle in five normal cows so that comparisons could be made with the exfoliative smears collected from the same site a few minutes earlier and described previously (Miroud & Noakes, 1990) . Subsequently, similar studies were performed in the same cows after ovariectomy and exogenous oestradiol benzoate and progesterone .
MATERIALS AND METHODS Experimental animals
Five Friesian cows, aged between 2 .5 and 7 years, with normal breeding histories were used in the first experiment . They were housed in a conventional byre with water and hay ad libitum . When not on experiment the cows were kept at grass or, during the winter, loose-housed in a barn . Before starting the experiments, all cows were observed for at least three consecutive oestrous cycles . Four cows, 12, 17, 23 and 68, were subsequently ovariectomized and treated with slow-release progesterone and oestradiol benzoate as described previously (Miroud & Noakes, 1990) . Biopsy technique Biopsy instrument.
The biopsy instrument was similar to that described by Donker (1952) and used by Hartigan (1970) and Hartigan et al. (1974) to take bovine endometrial biopsies . The instrument was modified from that of Hartigan et al. (1974) in order to facilitate the interchange of the cutting blade, and was longer to improve the ease of manipulation during insertion and cutting . The basic design is that of a stainless steel tube with a free running, but close fitting, internal stainless steel rod running the length of the tube . A metal spike on the handle crossbar prevents the inner rod being pushed too far, thus damaging the cutting edge at the tip of the instrument . However, a hole in the handle allows the rod to pass over the spike and push the cutting blade out of the outer tube when the removable tip of the outer tube is unscrewed . Biopsy collection . After the vulva has been cleaned and the cow restrained with the tail held aside, the closed instrument was inserted into the vagina and guided per rectum by the left hand as far as the external os uteri. The cutting edge was drawn back, thus opening the window in the outer tube . The hand in the rectum was used to press the dorsal wall of the anterior vagina into the window of the instrument and the cutting edge pushed forward to remove a piece of tissue of about 1 cm long and 6 mm wide . The instrument was then withdrawn, and the piece of the vaginal mucosa was removed with a pair of fine pointed forceps and then placed in a 10 ml labelled plastic bottle containing Mirsky's fixative (National Diagnostic Ltd) where it remained for at least 24 h before processing . Tissue processing and staining
After fixation, processing and embedding in paraffin wax, several sections from the same block were cut at 5 um on a sledge microtome . Sections were stained using routine Harris's haematoxylin (BDH Ltd, Poole) .
EFFECT OF VARIOUS TREATMENTS ON BOVINE VAGINAL MUCOSA
471
Detection of oestrus In order to assist in the detection of oestrus, all five cows were synchronized with 500 µg cloprostenol (Estrumate, Coopers Animal Health) i .m . at an 11-day interval . From the time of the first injection, daily blood samples were obtained from the jugular vein by venepuncture so that plasma progesterone concentrations could be measured to monitor the stage of the oestrous cycle and predict the likely day of oestrus . Each cow was fitted with a KaMaR heat mount detector (KaMaR Inc ., Steamboat Springs, USA) at the time of the second cloprostenol injection, and from the day before the expected day of oestrus the cows were observed every 3 h so that the time of onset of oestrus could be determined . Collection of blood samples Cyclical cows. A 6 ml heparinized jugular blood sample was obtained by venepuncture daily except after the first sign of observed oestrus, i .e . activation of the KaMaR, when the frequency was increased to a 2-hourly interval on four occasions . The blood samples were centrifuged in a refrigerated centrifuge at 4°C for 10 min at 2000 r .p .m . The plasma was decanted and frozen at -20 ° C until assayed . Ovariectomized cows . A 6 ml heparinized jugular blood sample was obtained by venepuncture except when hourly or 4-hourly samples were required, when an indwelling catheter was inserted into one of the jugular veins . Following exogenous progesterone, blood samples were withdrawn twice daily in cows 12 and 23 (at 9 :00 and 21 :00) for 9 days, and in cows 17 and 68 every 4 h for 4 days and then twice daily for 5 days . All four cows were then sampled daily for 6 days . Following exogenous oestradiol-1713 (ODB) all four cows were sampled every hour for 5 h, then every 4 h for 36 h and then every 12 h for the next 72 h . Before the administration of exogenous hormones each cow was sampled daily for 5 days .
Assay of progesterone and oestradiol-1713 This has been described previously (Miroud & Noakes, 1990) . Biopsy frequency . Each cow was sampled eight times over four oestrous cycles to ensure that tissue was obtained at different stages of the oestrous cycle and so that at least 7 days elapsed between consecutive biopsies . Each cow was biopsied at least 3 weeks after ovariectomy and 48 h after the injection of the slow-release progesterone preparation and 24 h after oestradiol benzoate in oil . The rest periods between treatment regimens have been described previously (Miroud & Noakes, 1990).
RESULTS
Cyclical cows A total of 40 anterior vaginal mucosal biopsies were taken from the five cows at different stages of the oestrous cycle . During oestrus, the epithelium consisted of a single layer of very tall columnar, highly active mucus-secreting cells . The cells had thin elongated and compressed nuclei which stained intensely (Fig . 1) . In some sections there was a thin basal layer of polyhedral cells beneath, and in some other areas of the same section, the epithelium was composed of either one or two layers of flattened superficial
472
BRITISH
VETERINARY
JOURNAL,
147, 5
squamous epithelial cells or several layers of polyhedral cells. Leucocytes were absent or only occasionally seen, and no cornilication or desquamation was evident. The stroma was slightly oedematous and the blood vessels appeared slightly congested.
Fig. 1. Photomicrograph of transversesection of anterior vaginal mucosa taken at oestrus showing a single layer of tall, columnar mucus producing cells. (H & E X260).
At one day after oestrus, the epithelium was very similar to that of oestrus. The tall columnar cells were reduced in height and became low columnar but still secreted mucus. In two cows (12 and 23) the tall columnar, highly active mucus-secreting cells remained 2 days after oestrus. At this stage in some sections there was an increase in the number of layers of squamous or polyhedral cells (two to three layers). The degree of desquamation varied and leucocytes were occasionally observed (Fig. 2).
Photomicrograph of transversesection of anterior vaginal mucosa taken during metoestrus showing tall columnar mucus-secreting cells in one part of the section (M) and several layers of polyhedral cells in another(P). (H & E X130).
Fig. 2.
EFFECT OF VARIOUS
TREATMENTS
ON BOVINE
VAGINAL
MUCOSA
413
From day 2 after oestrus to the end of metoestrus, there was a further decrease in the height of the superficial cell layers and, at the same time, an increase in the number of layers. The superficial cells changed from low columnar to cuboidal, and sometimes to a polyhedral epithelial type. Leucocytes were present throughout this period. There was congestion of the blood vessels and the stroma was denser than that of oestrus. During dioestrus, some sections showed similar features to that of metoestrus, whilst in some others the superficial layers changed from cuboidal to flattened. The epithelium was usually six to eight layers thick (Fig. 3). Cell desquamation and leucocytes were also seen during this period.
Fig. 3. Photomicrograph of transversesection of anterior vaginal mucosa taken during dioestrus showing several layers of flattened epithelial cells in some parts of the section (F) and severallayers of polyhedral cells in others (P). (H & E X130). During pro-oestrus, the vaginal mucosa varied from two to seven layers of epithelial cells (Fig. 4). In early pro-oestrus, the superficial layer consisted of low columnar epithelial cells which rested on several layers of polyhedral cells. In mid pro-oestrus, the low columnar cells tended to increase in height and the number of polyhedral cells reduced greatly, so as in late pro-oestrus there were only one to two layers of epithelial cells. The superficial layer was formed by tall columnar mucus-secreting cells and the basal layer consisted of well stained polyhedral cells (Fig. 4). Leucocytes were present throughout this period. Ouariectomi
