Investigations into the puerperium of Beagle-breed female dogs
Authors: Ορφανού Διονυσία 
Published: August 2021

The present thesis focusses on the study of the puerperium of female dogs, with the general objective to increase available knowledge regarding the puerperium of that species and to elucidate the process of involution of the uterus and the mammary glands in female dogs. Specific objectives of the thesis were as follows: (i) a proposal of ‘reference’ values for haematological and blood biochemical parametres during the puerperium, (ii) the description of anatomical details and their progressive changes in the uterus and the mammary glands during the puerperium, (iii) the identification of time periods, during which the uterus and the mammary glands are more susceptible to infection, (iv) the identification of cells present in the uterine content and in the milk and (v) the study of sucking behaviour during lactation and the identification of potential risk factors for mammary infections.Initially (1st Chapter), the relevant literature is reviewed. The Chapter is subdivided into three Parts. In Part A, principles of anatomy of the reproductive system and of physiology of the puerperium of female dogs are briefly reviewed. In Part B, the literature on pathological conditions of female dogs during the puerperium (systemic post-partum pathological conditions, post-partum pathological conditions of the uterus, post-partum pathological conditions of the mammary glands) is reviewed. In Part C, the ovario-hysterectomy during the puerperium is presented.Materials and methods used are described in detail in the 2nd Chapter. In total, 12 primiparous Beagle-breed female dogs were used in the study. Animals whelped normally. Subsequently, all animals were monitored until the 84th day post-partum. The following examinations were carried out: (i) general clinical examination (female dogs and puppies), (ii) clinical examination of the genital system, (iii) ultrasonographic examination of the uterus and the ovaries, (iv) clinical examination of the mammary glands, (v) ultrasonographic examination of the mammary glands, (vi) measurement of haematological and blood biochemical parametres, (vii) measurement of blood serum progesterone concentration, (viii) bacteriological and cytological examination of vaginal swab samples, (ix) bacteriological examination and cytological examination of teat duct material and milk samples, (x) behavioural observations. Dogs were subjected to ovario-hysterectomy and partial mastectomy on each of 4th, 7th, 10th, 14th, 21st, 28th, 35th, 42nd, 56th, 70th and 84th day after whelping. Appropriate tissue samples were collected for detailed examinations and the following were studied: (i) gross appearance of the uterus and the ovaries, (ii) bacteriological and cytological examination of uterine content samples, (iii) histological, histometric and ultrastructural examination of uterus and ovary tissue samples, (iv) bacteriological examination of mammary parenchyma samples, (v) histological, histometric and ultrastructural examination of mammary parenchyma tissue samples. Standard clinical, paraclinical, laboratory, behavioural and data analysis techniques were employed. For the purposes of analysis of results of the study, the puerperium was divided into four stages: L1 included samples collected from D0 (i.e., day of whelping) to D7 (7 days after whelping) (n=59), L2 included samples collected from D8 to D21 (n=27), L3 included samples collected from D22 to D42 (n=18) and L4 included samples collected from D43 to D84 (n=18). Specifically for analysis of haematological and blood biochemical parametres, samples collected during pregnancy were also taken into account.The results of the study are presented in the 3rd Chapter and are summarised herebelow.•Animals were clinically healthy throughout the study.•A significant effect of the peri-parturient period (last week of pregnancy and first week of the puerperium) was observed for the following parametres, rendering proposed ‘reference’ values during that period as follows: haematocrit 24.1-35.3%, leucocyte counts 13,200-31,400 μL-1, thrombocyte counts 502,000-912,000 μL-1, haemoglobin concentration 7.7-10.6 g dL-1, mature neutrophil counts 8,500-25,200 μL-1, lymphocyte counts 2,200-7,500 μL-1, total protein concentration 4.3-6.4 g dL-1, albumin concentration 1.7-2.6 g dL-1 and C-reactive protein concentration 30.0-180.0 mg L-1. Other parametres (alkaline phosphatase activity, calcium-ion concentration and total calcium concentration) were significantly affected during the peri-parturient period, but remained within the established ‘reference’ values. Finally, other parametres (mean corpuscular haemoglobin concentration, fibrinogen concentration, mature neutrophils proportion, immature neutrophils counts and proportion, lymphocytes proportion, monocyte counts and proportion, eosinophil counts, basophil counts, globulin concentration, glucose concentration, magnesium concentration) were not affected.•Median value of blood serum progesterone concentration was 0.43 ng mL-1; it was higher in [L1+L2] compared to [L3+L4] (0.48 ng mL-1 versus 0.32 ng mL-1, P=0.013).•Vaginal discharge was observed in 58% of sampling occasions, continuously up to D21 and intermittently up to D77, in ‘scanty’ to ‘small’ amount. Median diametre of the body of the uterus, as estimated during palpation through the abdominal wall, was progressively reduced during the puerperium; it was estimated at ~3.0 cm on D0 to <1.0 cm on D28 and subsequently (P=0.01). Median value of dimensions of the vulva during the puerperium were as follows: total horizontal width × total vertical length = 2.7 cm × 3.4 cm; median value of distance from anus to upper vulval commisure was 8.0 cm. Dimensions of the external genitalia progressively decreased during the study.•Up to D21, the uterus appeared with a heterogeneous echopattern; it was easy to recognize and image the organ, because width of the uterine horns was estimated to be ≥1.0 cm. After that day, it was more difficult to image it, as their width was progressively decreasing and, on D70, was estimated to be 0.6 to 0.7 cm (P<0.001). In longitudinal images, the uterus appeared consistently cylindrical in shape. In transverse images taken up to D21, the sections of the organ appeared polygonal to compressed circular to circular; in transverse images taken after D21, they appeared consistently circular. There was evidence of a hypoechoic rim, with a more echogenic inner architecture. In some images, the anechoic lumen was barely visible, because of the more echogenic content, whilst in others, it was observed greatly contrasting to the hyperechoic endometrium. The placental sites appeared as granulated structures of medium echogenicity, with small anechoic areas. Initially, the uterine wall was imaged as a multi-layer structure, but, subsequently, number of layers imaged was reduced. Thickness of myometrium and endometrium was found to progressively decrease as the puerperium advanced (P<0.001), as was diametre of the lumen of uterine horns (P=0.003).•In total, 51 of 122 vaginal samples examined bacteriologically, yielded bacteria. Frequency of bacterial isolation from vaginal samples was 0.525 during L1, 0.333 during L2, 0.333 during L3 and 0.278 during L4 (P=0.094). Median time to first infection after whelping was 0.25 days. Estimated median duration of infection (all infections taken into account) was 5.25 days. Most of the organisms recovered were Escherichia coli and Trueperella (Arcanobacterium) pyogenes: 22 and 16, respectively, of the 55 isolates recovered.•In smears from swab samples from the anterior part of the vagina, the majority of cells observed were epithelial cells. Uterine epithelial cells, normal or degenerated, were observed characteristically clustered, whilst vaginal cells observed were primarily parabasal or small intermediate cells. Leucocytes were also observed in these smears; neutrophils predominated. Trophoblast-like cells were also evident in L1 and L2. There was a progressive increase in epithelial cell counting scores and progressive decrease in leucocyte counting scores observed during the puerperium (P<0.04). Cell counting scores for samples which had yielded bacteria were significantly higher for leucocytes (P=0.045), but not for for epithelial cell counting scores (P=0.383).•During the study, the mammary glands were soft and with homogeneous consistency. On D1, the mammary secretion was thick and yellowish, thereafter (occasionally on D2, but always on D4) becoming ‘milky’.•The mammary parenchyma showed, in general, a homogeneous consistency and medium echogenicity. As the puerperium advanced, a less homogeneous echopattern was seen and (compared to previous periods) lower echogenicity was recorded; increased amount of connective tissue was also evident in ultrasonograms during that period. Occasionally, it was possible to image different lobules within the same mammary gland, separated by connective interlobular tissue.•Frequency of bacterial isolation from teat ducts was 0.068 during L1, 0.130 during L2, 0.0 during L3 and 0.028 during L4; frequency of bacterial isolation from mammary glands was 0.091, 0.104, 0.122 and 0.017, respectively. There was a greater probability of bacterial isolation from Rc1 (right most caudal gland) than [Rc3 or Rc4] glands, as well as from Rc2 than Rc4 glands (P<0.02); there was also a greater probability of bacterial isolation from [Rc1+Rc2] than from [Rc3+Rc4] glands (P<0.001). Median time to first infection after whelping was significantly (P=0.044) shorter for Rc1 than for Rc2, Rc3 or Rc4 (median time: 3 days for Rc1, >7 days for Rc2, Rc3 or Rc4). Most of the organisms recovered were staphylococci: 14 of the 17 isolates (82%) from teat duct material and 45 of the 53 isolates (85%) from milk; Staphylococcus pseudintermedius was the most frequently recovered species (7 and 24 isolates, respectively).•Mean (median) Whiteside test (WST) score of bacteriologically negative milk samples was 1.78 (2.0) and that of bacteriologically positive milk samples was 2.20 (2.5) [in a 0 to 5 scale corresponding to scores ‘-’ to ‘3+’]. There was a significant association between increased WST scores and bacteriologically positive results in milk samples. Overall sensitivity of the test was 74% for ‘high’ (i.e., ≥’1+’) WST scores to identifying bacterial isolation from milk samples. Mean (median) WST scores of milk samples collected during each of the four puerperium stages were as follows: 1.73 (2.0) in L1, 1.68 (2.0) in L2, 1.58 (2.0) in L3 and 3.12 (4.0) in L4; scores in samples collected during L4 were significantly (P<0.001) greater. Scores in samples collected from ‘Rc1’ gland were significantly (P<0.015) greater than in samples from the other mammary glands. The majority of cells observed in milk films were macrophages, although lymphocytes and neutrophils were also present therein.•As the puerperium advanced, there was a significant progressive increase in mobility of the bitches (‘Inside whelping box’: P≤0.001, ‘Outside whelping box’: P<0.001), as well as a significant progressive decrease of interaction with puppies (‘Grooming puppy’: P<0.001, ‘Contact with puppies’: P<0.001). Moreover, there was a significant increase in mobility (‘Inside whelping box’: P≤0.001, ‘Outside whelping box’: P<0.001) and activity (‘Lie down’: P<0.001, ‘Playing’: P<0.001) of the puppies during L4. Sucking behaviours decreased progressively as the puerperium advanced (‘Successful suck’: P<0.09, ‘Sucking bout’: P<0.08). There was no significant difference in the frequency of successful sucks of puppies between the right and the left mammary glands (P=0.973), but there was a significant difference in the frequency of successful sucks of puppies between the more caudal (i.e., [Rc1+Rc2]) and more cranial (i.e., [Rc3+Rc4+Rc5]) mammary glands (P<0.001). Tendency lines drawn for frequency of ‘Successful sucks’ of puppies and risk of infection for five mammary glands (Rc1, Rc2, Rc3, Rc4 and Lc2) and two teat ducts (Rd2, Ld2) of their dams were associated throughout the study.•Externally, the uterus was pink, with longitudinal folds and mild vascularization. The two horns were symmetrical between them and laterally oblate; macroscopically, the placental sites could be appreciated even up to D84. Mean widths of the uterine horns were different across the various time-points: 2.8 / 2.7 cm (placental sites / interplacental areas) on D7, 1.7 / 1.2 cm (placental sites / interplacental areas) on D35 and 0.9 / 0.8 cm (placental sites / interplacental areas) on D84 (P≤0.001). There was a small amount of viscous to mucous, red to dark-brownish fluid inside the uterus of seven bitches up to D42 of the puerperium. The placental sites were initially of dark green to grey colour, whilst later they were dark brown; these were easily recognized, thickened and with ‘foamy’-like surface; their length progressively decreased. The interplacental areas were white to pink colour with mild longitudinal wrinkles thereon; their length progressively increased.•Bacteria were isolated from swabs cultured from the uterine content of the bitch operated on D4 and from that of the bitch operated on D7; E. coli and T. pyogenes were isolated, respectively. In smears from swab samples from the uterine content, the majority of cells observed were initially leucocytes, with neutrophils predominating. Uterine epithelial cells predominated from D56 of the puerperium onwards.•During L1, the myometrium was very thickened (1887 μm on D7). The thickened endometrium showed folding and increased vascularisation in the lamina propria. High numbers of inflammatory cells were observed subepithelially. A simple, cuboidal to columnar epithelium was seen; the epithelial cells were enlarged, clustered and with a ‘foamy’ cytoplasm. Clusters of ‘foamy’ cells were evident along the entire epithelial lining. Increased desquamation of epithelial cells was seen into the lumen. During L2, the intensity of the above findings progressively decreased. During L3, thickness of all layers of the myometrium was reduced. Inflammatory cells were first evident in the inner layer of the myometrium. A simple, cuboidal to columnar epithelium was seen; ‘foamy’ cells were also observed, but only at the part of the epithelium lining corresponding to the placental sites. During L4, thickness of the myometrium was further decreased (1255 μm of D70). Uterine glands were evident, although their diametre had decreased. A simple, cuboidal epithelium was seen; some ‘foamy’ cells were also observed at the part of the epithelium lining corresponding to the placental sites. Median diametre of the uterine glands' transverse sections was 79 μm on D7, 53 μm on D56 and 52 μm on D84 (P=0.032); moreover, significant differences were also seen in the height of glandular epithelial cells: 9.4 μm on D4, 6.2 μm on D35, 6.4 μm on D70 (P=0.035).•The mammary glands appeared rectangular to almost square, with pink to mildly red colour; from D56 of the puerperium, pale yellow colouration was also evident in some areas of the mammary glands. There was no association between the number of teat orifices and the risk of infection of the respective mammary gland. Dimensions of the mammary glands were greatest during D14 to D42, then, progressively, decreased. From D4 to D42, dimensions of Rc2 were greater than those of Rc1, a pattern which changed from D56 onwards; in general, dimensions of Rc1 and Rc2 were greater than those of Rc3. The decrease of dimensions of the mammary glands observed during the study was significant (P<0.001).•No bacteria were isolated from any mammary parenchyma tissue sample of any bitch.•In all mammary glands studied, all lobes and lobules within the same sample had similar histological features. Similarities were evident in the mammary glands of the same animal: up to D42, all mammary glands of the same animal were in full lactation, whilst on D70 and thereafter, all mammary glands of the same animal showed evidence of involution. Specifically on D56, differences were observed among the mammary glands of the animal, i.e. three mammary glands showed findings of being in full lactation, whilst a fourth showed evidence of involution. Inflammatory cells (predominantly macrophages and lymphocytes) were observed in the inter-alveolar space of all mammary glands. Until D42, mammary lobules were separated with a scant amount of connective tissue. Alveoli were well developed and distended; they appeared to have a spherical to slightly ovoid structure, with myoepithelial cells grasping them around. The epithelial cells were uniform, cubical to slightly parallelepiped in shape. From D70, the between-lobules connective tissue was abundant and dense, occupying a major part of each optical field; increased numbers of fibroblasts were observed; the stroma of connective tissue within the lobules contained inflammatory cells; alveoli appeared irregularly-shaped and collapsing, shrunken or fully collapsed; the few epithelial cells in each alveolus were flattened and slender. Median number of alveoli per lobule decreased as the puerperium progressed (P<0.001); median number of epithelial cells per alveolus also decreased (P=0.005). Rc3 mammary glands had a significantly smaller number of alveoli than Rc1 or Rc2 mammary glands. No significant differences were evident between the number of epithelial cells per alveolus between the mammary glands. Median diametre of alveoli and median height of epithelial cells decreased significantly in L4 (P<0.045).The conclusions from the results of the present thesis are summarised herebelow.(a) ‘Reference’ values for haematological and blood biochemical values are proposed for samples collected from bitches during the peri-parturient period. New values are proposed for haematocrit, leucocyte counts, thrombocyte counts, haemoglobin concentration, mature neutrophil counts, lymphocyte counts, total protein concentration, albumin concentration and C-reactive protein concentration.(b) The present ultrasonographic findings of the uterus and the mammary glands can be used as further reference data for imaging standards in bitches during the puerperium.(c) Current concepts about the length of post-partum involution of the genital system of bitches might need to be re-addressed. In primiparous animals, one may propose that involution could normally take longer than in multiparous bitches. The significance of ‘foamy’ cells in the uterus could be re-assessed and their presence could be considered as a normal feature. Perhaps, the quantity of such cells observed in samples from the animals, as well as other abnormal findings that might indicate subinvolution of placental sites, should be taken into account.(d) Despite a very high infection rate of the genital system in the immediately post-partum period, effective cellular (neutrophils) defences of the animal contribute to protection from development of metritis.(e) Involution of the mammary glands starts around the end of the 2nd month after whelping. By the end of the 3rd month after whelping (in cases the dam is still suckling puppies), the process is almost complete.(f) Staphylococci are the primary bacteria isolated from milk samples of healthy female dogs during lactation. The organisms likely originate from the skin of the animal. Infection risk of the caudal mammary glands is increased compared to that of the cranial glands. Infection risk during the early post-partum period is increased compared to that in later stages.(g) Macrophages and lymphocytes constitute the main cells present in mammary glands. The Whiteside test appears to be useful for detecting increased cellular content in the milk of female dogs.(h) Ethological observations indicate an association of normal behaviour with potential health problems. Sucking is a factor contributing to the increased infection risk of the caudal glands and of the early post-partum period. No adverse behaviours were observed in the dams. In general, litter size did not affect behaviours of female animals and their puppies.