Effect of uterine lavage on neutrophil counts in postpartum dairy cows

Uterine disease in cattle causes economic losses due to reduction of pregnancies per insemination, extended calving to conception intervals and increased culling rates(Gilbert et al., 2005). Postpartum uterine diseases can be classified as puerperal metritis, clinical metritis, clinical endometritis and subclinical endometritis (SCE; Sheldon et al., 2006). Subclinical endometritis is a post partumuterine disease characterized by inflammation of the endometrium in the absence of clinical signs of the dis-ease (Sheldon et al., 2006). According to a review (Galvão,2012), there is general agreement that SCE is highly prevalent affecting approximately 30% of lactating dairy cows with a within herd prevalence ranging from 11% to 70%. In animals without signs of clinical endometritis, SCE is diagnosed by measuring the proportion of neutrophils present in a sample collected by a small-volume lavage of the uterine lumen or by means of a cytobrush (Gilbert et al., 2005).Parturition, expulsion of fetal membranes and uterine involution all involve an inflammatory process (Sheldon et al.,2008). This uterine inflammation normally decreases with time in healthy cows. The proportion of cows with uterine inflammation diagnosed by cytology decreased from100% at 2 weeks postpartum to 89%, 58%, and 41% at 4, 6,and 8 weeks postpartum, respectively (Gilbert et al., 2005).However, in cows with SCE, the inflammatory process in the uterus remains for a longer time interval postpartum(Gilbert et al., 2005). Subclinical endometritis is defined by the presence of more than 18% PMNs in an uterine cytology sample collected between 21 and 33 days in milk (DIM)or more than 10% PMNs between 34 and 47 DIM (Sheldon al., 2006).

 

It has been postulated in sub-fertile cows, that more cows were diagnosed with uterine inflammation than uterine infection (McDougall et al., 2011; Bara´nski et al., 2012;Madoz et al., 2014; Sens, 2014). Factors responsible for failure to resolve the postpartum endometrial inflammation at the beginning of the breeding period remain to be elucidated (Gilbert et al., 2005).

 

Inflammation of the uterus (SCE) leads to adverse effects on reproductive performance, and also interferes with proper fertility (Gilbert, 2012). PMNs and inflammatory mediators such as cytokines, chemokines, eicosanoids, nitric oxide and oxidative stress, are characteristically associated with SCE and are shown to have negative effects on sperm, endometrium and embryos (Gilbert, 2012).

 

Currently, several controversial reports on the efficiency of treatment protocols for SCE are available in the literature(Kasimanickam et al., 2005b; Galvão et al., 2009; Dubucet al., 2011; Lima et al., 2013b). There is little evidence that treatments based on intra-uterine infusions are beneficial, except for the infusion of cephapirin which was reported to improve reproductive performance in cows suffering from SCE (Kasimanickam et al., 2005b). Because the use of cephalosporins in food producing animals, and in veterinary medicine as a whole, is currently under pressure there is an urgent need to find other, non-antibiotic based therapies. Administration of PGF2 _ was believed to improvere productive performances due to mechanical expulsion of inflammatory products by eliciting estrus and concomitant uterine contractions but field studies failed to show beneficial effects of PGF2 _ injections on the incidence of SCE(Kasimanickam et al., 2005b; Galvão et al., 2009; Dubucet al., 2011). Taken together, the absence of a rapid, reliable “cow side” diagnostic test and the lack of insight into the pathogenesis of this disease are main reasons why an effective treatment for SCE is not yet available.

 

Removing the inflammatory content from the uterus might be the key for improving later reproductive function and pregnancy outcome. Uterine lavage is an important therapeutic tool for treatment of uterine inflammation in equine medicine (Hurtgen, 2006; Liu and Troedsson,2008). It was proposed that it removes non-functional neutrophils and other inflammatory products and causes uterine contractions which aid in a physical clearance of uterine contents (Brinsko et al., 2011). Although the etiology and pathology of uterine inflammation are different in cattle compared to horses, anecdotal reports of beneficial impacts exist from practitioners who use uterine lavage to improve fertility in cows suspected to suffer from SCE.

 

In the present study, the hypothesis was tested that rinsing the uterus with sterile saline reduces the number of PMNs, an indicator of intra-uterine inflammation. If the present hypothesis can be accepted, this would suggest that uterine lavage using physiological saline can be pro-posed to be tested on a larger scale as a therapeutic method for treatment of SCE in the postpartum period and in repeat breeder cows.

 

The present study was conducted in two commercial dairy herds (Herd 1: 2100 dairy cows; Herd 2: 800 dairy cows) located in the Tehran Province, Iran (35?39_35__N,51?03_33__E and 35?56_13__N, 50?38_38__E, respectively).Selection of the herds was based on willingness of the herd manager to participate in the study and on the availability of facilities to efficiently conduct the study. Briefly, cows were housed in open-shed barns and milked three times daily. The average 305 days milk production was 10,223and 9562 kg in Herds 1 and 2, respectively. The reported voluntary waiting period before breeding was 60 days for primiparous and 55 days for multiparous cows. Cows were feed twice daily, with a total mixed ration including corn silage, alfalfa hay, corn meal, barley, and a protein and mineral supplement.

 

Samples were taken in January and February 2013. Cows that had no degree of dystocia, retained placenta, abnormal uterine or vaginal discharge (defined as purulent or mucopurulent discharge), were eligible for the experiment. Cows that had a history of fever, mastitis, laminitis or any clinical disease after calving and that had received ecbolic drugs or hormones after calving were not included in the study.

 

Holstein Friesian cows (n = 50), in second to fourth lactation and with a body condition score >2.5 and <3.25 (scales1–5 with 0.25-point increments) were randomly assigned at 30 ± 3 DIM to a control or treatment group (Edmonsonet al., 1989).

 

At 30 and 40 DIM, a small-volume uterine lavage was performed to harvest samples for endometrial cytology analysis for all cows (Gilbert et al., 2005; Kasimanickam et al., 2005a). Briefly, 30 mL sterile 0.9% saline solution (Daropakhsh Co., Tehran, Iran) was infused into the uterus using a 40-cm long, sterile 18Fr Foley catheter, with a30-cm3balloon and a protection sheet (Rusch Co., Waiblingen, Germany). To minimize contamination, the vulva and perineum were thoroughly cleaned with water. The catheter inside the protection sheet was passed through the vagina using a stainless steel stylet. Only the catheter (without protection sheet) was manipulated through the cervix. Once the catheter was passed through the cervix and placed in the body of the uterus, the balloon was inflated. After catheter fixation, 30 mL of sterile saline solution 0.9%(35–40?C) was infused in the uterus using a 60-mL syringe. Subsequently, the uterus was gently massaged for about10 s, and some of the infused fluid was harvested in a 15 mL falcon tube by means of gravity without aspiration. The samples were transported to the laboratory within 4 h.

 

Samples were centrifuged for 5 min at 700 × g, after which a drop of the sediment was placed on a micro-scope slide and spread on the glass slide with the tip of the sampler. Air-dried cytology smears were stained with modified Wright-Giemsa stain (Diff-Quik; Dade Behring) (Kasimanickam et al., 2005a). All slides were examined using light microscopy at a magnification of 400× and1000× to identify individual cell types, including endometrial epithelial cells and PMNs (Gilbert et al., 2005). Per slide, a total of 300 cells were counted. Polymorpho nuclear cell counts were expressed as a percentage and was equal to the number of PMNs divided by the number of PMNs and epithelial cells combined.

 

In the treatment group, the uterine lavage was per-formed immediately following the cytology sampling at 30DIM. With the Foley catheter still in place, 60 mL of normal saline at 35–40?C was infused in the uterus and recovered in a similar manner as for the collection of the samples for cytology. The procedure was repeated 8–10 times consecutively aiming for a total flush volume of 500–600 mL.

 

Coccygeal blood samples were collected at each uterine lavage (30 and 40 DIM) into Vacutest standard tubes with-out anticoagulant (Vacutest Kima srl, Arzergrande (PD),Italy). Samples were transported to the laboratory within4 h. Serum was separated by centrifugation at 2000 × g for15 min and frozen at −20?C until assayed for progesterone(P4) using a progesterone ELISA kit (Demeditec Diagnostic Company, Kiel, Germany) with a sensitivity, inter- andintra-assay and coefficients of variation of 0.2 ng/mL, 10.6%and 7.4%, respectively. Progesterone concentrations were categorized into HIGH (>1 ng/mL) and LOW (<1 ng/mL)(Stevenson et al., 2006; Dubuc et al., 2011).

 

Data from individual cows were exported from the farm managing software program to an Excel spreadsheet(Microsoft Corporation, Seattle, WA, USA). All statistical analyses were performed using the SAS software (SAS Institute Inc., Cary, NC, USA, 2010). Descriptive statistics were done using PROC MEANS of SAS (SAS Institute Inc., Cary, NC,USA, 2010) (Table 1). The distribution of PMNs% was evaluated to approximate the normal Gaussian distribution. Because  PMNs% was LOG-normally distributed, a generalized linear mixed model was designed using the PROCGLIMMIX procedure from SAS version 9.2 for Windows(SAS Institute Inc., Cary, NC, USA, 2010), to detect the factors that are significantly associated with the PMNs% at 40 DIM. Least square means (LSM; Table 2) were calculated after back transformation using the LSMEANS option. To con-firm the present hypothesis that uterine lavage at 30 DIM decreases the PMNs% the non-parametric Mann–Whitney test was used to compare the PMNs% at 30 DIM compared with 40 DIM both in the control as well as in the treatment group (Fig. 1). P-values <0.05 were considered statistically significant.

 

Independent variables withheld in the final model(Table 2) were: herd, lactation number, treatment, P4 at40 DIM and the first order interaction between the latter two. Herd and PMNs% at 30 DIM were tested but not with-held in the final model because these were not  significant. The latter indicates that flushing the uterus with saline at30 DIM did decrease the percentage of PMNs irrespective of the percentage of PMNs present at the moment of treatment. The percentage of PMNs at 40 DIM was estimated to be greater in cytology samples of cows in the second compared with those in the third or fourth lactation. Cows with lesser concentrations of progesterone (P4 < 1 ng/mL)at 40 DIM were estimated to have a greater percentage of PMNs in endometrial smears compared with cows having progesterone concentrations of ≥1 ng/mL (Table 2).

 

The principal aim of the present study was to determinate the effect of uterine lavage on percentage of PMNs in uterine cytology samples which is considered an indicator of SCE.

 

Small-volume uterine lavage was used to obtain acytological sample. Cytology can be performed using the cytobrush or the uterine lavage technique (Kasimanickam et al., 2004; Gilbert et al., 2005; Kasimanickam et al.,2005a). Harvesting of endometrial cells by cytobrush is convenient and practical (Kasimanickam et al., 2005a;Overbeck et al., 2013), whereas, the major disadvantage is that only a small surface area of the uterine body is sampled (LeBlanc, 2011). However, by using the uterine lavage technique, one collects cells over a larger surface area pro-viding for a more representative uterine content sample(LeBlanc, 2011). More studies are needed to determine the distribution of inflammation in the uterus of cattle, and to ascertain that cytobrush samples are as reliable as a small volume lavage.

 

Uterine lavage using sterile saline is a well-accepted method to treat persistent endometritis and post-breeding endometritis in mares (Troedsson, 1999). The efficacy of this treatment is based upon the removal of exudate from the uterine lumen and the reduction of the uterine bacterial population (Brinsko et al., 1990). Uterine lavage may also exert beneficial effects by stimulating uterine contractions, there by aiding in the expulsion of debris out of the uterus(Brinsko et al., 1990). Results of the present study suggest that flushing the uterus with 500–600 mL of physiological saline around 30 DIM decreases the number of PMNs in the uterus of dairy cows. Uterine lavage might help the uterus in the latter stages of debris clearance, thereby, positively contributing to fertility. Flushing might, however, be considered problematic because the procedure may provoke endometrial and cervical damage due to manipulation of the genital tract which might negatively affect subsequent reproductive performance. The latter possibility was proposed by Kasimanickam et al. (2005a) who described the failure to recover fluids from the uterus and the trauma caused to the endometrium during attempts to recover the infused fluids indicating that the results from using the lavage technique might be inconsistent and technique might possibly be detrimental in some cases. However, the aim of the latter study was to lavage the uterus to obtain a sample for cytological evaluation using only a small volume of fluid (Kasimanickam et al.,2005a). Remarkably, in the present study, the PMNs% in the untreated cows was numerically increased from 30to 40 DIM. It has been hypothesized that uterine lavage might trigger an irritation of the endometrium, inducing the migration of neutrophils into the uterine lumen or stimulation of serum opsonins (Asbury, 1984; Le Blancand Asbury, 1987). The replacement of nonfunctional neutrophils with active neutrophils can be considered a helpful phenomenon which may aid in killing and removing of bacteria located in the uterus (Brinsko et al., 1990). Recent findings, however, question the association of SCE with bacterial infection because no bacteria could be isolated from the uteri of grazing dairy cows suffering from SCE(Madoz et al., 2014). The latter had also been concluded by Bara´nski et al. (2012) where SCE was thought to be associated to a greater extent with the postpartum recovery of the endometrium than with bacterial infection. In summary, recruitment of new neutrophils, following uterine lavage, might not be beneficial in terms of bacterial killing due to the absence of germs in cows suffering from post-partum SCE. The results of the present study indicate that rinsing the uterus of cattle with normal saline 0.9% reduces the percentage of PMNs in cytology samples taken 10 days later. However, differences between different properties of neutrophils were not ascertained in the present study. 

 

The type of liquid used for uterine lavage may influence the potential for benefit (Blanchard et al., 1989).Many fluids have been tested to perform uterine lavage inmares such as hypertonic 5% NaCl (Webb et al., 1983; Lenz,1986; Blanchard et al., 1989), saline solutions diluted with antiseptics (e.g., povidoneiodine solutions at 0.02% or less) (Watson, 1987), Lugol’s solution and others (Threlfall,1980; Bennett et al., 1981). Isotonic saline or other balanced salt solutions such as lactated Ringer’s solution are generally preferred because these are considered to not be or tobe only mildly irritating, cause a cellular response, and are translucent which allows easy inspection of the recovered material (Brinsko et al., 2011). Sterile physiological saline would appear to be a logical choice due its neutral, low cost feasibility and overall availability.

 

As described in several previous studies (LeBlanc et al.,2002; Gilbert et al., 2005; Barlund et al., 2008; Bara´nskiet al., 2012), SCE should be considered a management dis-ease, being associated with nutritional practices during the transition period, the presence/absence of stress and good obstetrical and gynecological practices (even artificial insemination). In the present study, herd, however, was not found to be a factor associated with the prevalence of SCE which might be due to the relatively small number of cows included in the present study.

 

Moreover, parity had an impact on the percentage of PMNs in uterine cytology samples in the present study. Cows in their second lactation had a greater percentage of PMNs in the uteri in comparison to cows in third and fourth lactation. In a previous study (Kaufmann et al., 2009) found that primiparous cows suffered from greater numbers of uterine PMNs at 4 h after insemination. Cheonget al. (2011) and Dourey et al. (2011) revealed that high producing primiparous cows had an increased probability of suffering from SCE in comparison to multiparous cows. However, Gilbert et al. (2005) found no significant effect of parity on the prevalence of endometritis diagnosed by cytology. In contrast to these findings, LeBlanc et al.(2002) found clinical endometritis to be more prevalent in cows in third or greater lactation (21% in third and greater lactations compared with 16% in second and 12% in first lactation cows. The latter finding was in agreement with the previous report of Gröhn et al. (1990) that indicated the incidence of most peri-parturient diseases in dairy cows increases with increasing parity. Negative energy balance and insulin resistance have a role in the immune regulation and defense mechanism of the reproductive tract of postpartum dairy cows (LeBlanc, 2014). The fact that primiparous in comparison to multiparous cows differentially undergo the metabolic adaptation to the establishment of milk production (Wathes et al., 2007) led Kaufmann et al.(2009) to suggest the former cows had a greater number of PMNs at 4 h after insemination. However, in that previous study, there was no conclusion as to whether greater numbers of PMNs should be regarded as a hallmark for a more active immunity in younger animals or whether younger cows were simply more at risk to suffer from postpartum SCE.

 

At each sampling in the present study, blood samples were taken to measure serum progesterone concentrations as an indicator of ovarian cycles. Madoz et al. (2013)had previously showed in grazing dairy cows that there is no significant correlation between the endometrial PMN% harvested by cytobrush and the stage of the estrus cycle(Madoz et al., 2013). However, the present results indicate that at 30 and 40 DIM progesterone concentrations have aninverse effect on the amount of neutrophils harvested from the uterus.

 

Secretions from uterine glands and contractions of the uterine musculature during the estrous phase are beneficial for uterine clearance. Thus, cows that had greater progesterone concentrations at the time of sampling had already resumed ovarian cycles and had expressed at least one period of behavioral estrus consequently having lesser amounts of PMNs in the uterus. Furthermore, there may also be a direct effect of progesterone and estrogen on the PMN population present in the uterus (Subandrio et al.,2000).

 

Although several alternatives for the treatment of SCE have been suggested, an ideal and generally accepted therapy is not yet available and the topic is still controversial. Preventive PGF2 _treatment at 21, 35 and 49 DIM did not decrease the prevalence of SCE at 35 and 49 DIM (Galvãoet al., 2009). Treatment with 1 or 2 injections of PGF2 _inthe early lactation period before cows were subjected to a resynchronized timed AI protocol was unable to improve uterine health and subsequent fertility indices in lactating dairy cows (Lima et al., 2013a). Although post-partum administration of NSAIDs improved liver function in dairy cows this treatment did not reduce local inflammation inthe uterus as represented by the percentage of PMNs in the endometrium (McDougall et al., 2013). Kasimanickam et al.(2005b) found that the usage of cephapirin or cloprostenolat 20–33 DIM improved reproductive performance of cows suffering from SCE although effects on PMN percentage was not reported. To the ves of our knowledge, the present paper is the first to report data of a study regarding the treatment of SCE by measuring its efficacy at reducing the percentage of PMNs in uterine cytology samples.

 

In summary, it is concluded that rinsing the uterus of dairy cows at 30 DIM decreases the number of PMNs in cytology samples taken 10 days later. The latter suggests that this treatment might contribute to the prevention of cows suffering from postpartum SCE. To propose this method as a treatment protocol for SCE it should be tested on a larger scale including evaluations of subsequent reproductive performance of the treated cows.

 

None.

 

The authors thank the managers and staffs of Yoosh dairy farm and Taliseh Nemoone dairy farm for their kind collaboration, and the technicians of the Mabna veterinary laboratory for their excellent technical support. Authors are also grateful to Prof. Dr. Richard Ducatelle, Prof. Dr. PeterDaels and Prof. Dr. Bahram Salasel for their constructive remarks and critical reading of the manuscript.

 

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