Animal Reproduction Science 145 (2014) 8–14
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Associations between resumption of postpartum ovarian activity, uterine health and concentrations of metabolites and acute phase proteins during the transition period in Holstein cows Ana Rita T. Krause a,d , Luiz F.M. Pfeifer c,d,∗ , Paula Montagner a,d , Marina M. Weschenfelder a,d , Elizabeth Schwegler a,d , Márcio E. Lima a,d , Eduardo G. Xavier b , Cassio C. Brauner a,d , Eduardo Schmitt c,d , Francisco A.B. Del Pino a,d , Charles F. Martins a,d , Marcio N. Corrêa a,d , Augusto Schneider a,d a b c d
Universidade Federal de Pelotas, Pelotas, RS, Brazil Granjas 4 irmãos S.A., Rio Grande, RS, Brazil Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA Rondônia, Porto Velho, RO, Brazil Núcleo de Pesquisa, Ensino e Extensão em Pecuária (NUPEEC), Universidade Federal de Pelotas, Pelotas, RS, Brazil
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Article history: Received 31 July 2013 Received in revised form 19 December 2013 Accepted 21 December 2013 Available online 3 January 2014 Keywords: Albumin Haptoglobin Paraoxonase Uterus Cattle
a b s t r a c t The resumption of ovarian activity, uterine health, severity of the negative energy balance and the synthesis of inflammatory mediators during the transition period in dairy cows are interrelated. Therefore, the aim of this study was to evaluate the association between the resumption of postpartum ovarian activity and the percentage of polymorphonuclear (PMN) cells in endometrial cytology, lipid mobilization and the secretion of acute phase proteins. For this study, 20 multiparous Holstein cows were used. Blood samples that were collected from 21 d before calving to 44 d in milk (DIM) were analyzed for serum glucose, non-esterified fatty acids (NEFA), insulin, haptoglobin, albumin, paraoxonase and progesterone. Endometrial cytology was performed at 37 ± 2 DIM to evaluate the percentage of PMN cells in the uterine flushing. Cows were divided into two groups: (1) ovulatory cows (n = 12), which returned to ovarian activity by 44 ± 2 DIM; and (2) anovulatory cows (n = 8), which did not resume ovarian activity during this period. Ovulatory cows had a lower (P = 0.05) percentage of PMN cells in endometrial cytology than anovulatory cows (26.3 ± 8.3% vs. 53.4 ± 16.9%, respectively). Ovulatory cows had higher serum albumin during the pre- (P = 0.03) and postpartum periods (P = 0.01), and tended to have lower haptoglobin concentrations in the prepartum period (P = 0.07) and higher paraoxonase activity in the postpartum period (P = 0.09). In conclusion, cows that resumed ovarian activity early in the postpartum period had higher albumin concentrations in the peripartum period, which were associated with a lower percentage of uterine PMN cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Embrapa, Animal reproduction lab, Br 364 Km 5.5, 76815-800 Porto Velho, Rondonia, Brazil. Tel.: +55 69 3901 2526; fax: +55 69 3901 2510/+55 69 3222 0409. E-mail address: [email protected] (L.F.M. Pfeifer). 0378-4320/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.12.016
Only approximately 50% of healthy dairy cows ovulate the first dominant follicle within 3 weeks after calving (Kawashima et al., 2007). A healthy postpartum dairy cow can be defined as one that has resolved uterine involution,
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ovulated a dominant follicle early postpartum and continues to have a normal estrous cycle at regular intervals, coupled with homeostatic concentrations of insulin, IGF-I and glucose (Roche, 2006; Walsh et al., 2011). A delayed first postpartum ovulation is usually associated with a pronounced negative energy balance (NEB) in the early postpartum period (Butler, 2003), although other factors are also important (Santos et al., 2009). It is well known that cows that resume ovarian function early after calving are most likely to become pregnant in the first service (Staples et al., 1990). In dairy cows, to achieve a satisfactory reproductive performance during this critical period, it is necessary that the female ovulates a competent oocyte and provides a suitable uterine environment for fertilization and embryonic and fetal development (Pursley and Martins, 2011). Therefore, factors that affect the early resumption of ovarian cycles during the postpartum period have an impact on the fertility of dairy cows and should be further investigated. In addition to peripartum metabolic events that may affect reproductive performance, uterine contamination may also contribute to decreased conception rates in lactating dairy cows. In total, 80 to 100% of these cows have uterine bacterial contamination during the first weeks after calving (Sheldon et al., 2006). The presence of pathogenic bacteria in the uterus is associated with endometrial inflammation and purulent vaginal discharges (Sheldon et al., 2009), which increase the amount of polymorphonuclear (PMN) defense cells in the endometrium. Most cows recover naturally from these postpartum uterine infections, however, at least 20% of these cows have a persistent infection 3 weeks after calving and develop endometritis. Dairy cows that develop endometritis during the postpartum period have reduced conception at first insemination and increased days open (Fourichon et al., 2000). During the peripartum period, the increased release of inflammatory mediators, which are called acute phase proteins (APPs) has also been associated with decreased reproductive performance (Wira and Fahey, 2004). Some negative APPs synthesized in the liver, such as paraoxonase (PON) and albumin, have their serum concentrations reduced due to the typical inflammatory response, which is induced by parturition (Bionaz et al., 2007; Fleck, 1989). Conversely, the serum concentration of the positive APP haptoglobin (Hp) increases during the inflammatory process. Several studies have demonstrated increased haptoglobin and decreased albumin and paraoxonase concentrations in cows with postpartum uterine infections, even before calving (Sheldon et al., 2001; Turk et al., 2004, 2005; Huzzey et al., 2009; Burke et al., 2010; Schneider et al., 2013). Cows with bacterial infections in the reproductive tract have slower growth of the dominant follicle, lower plasma concentrations of estradiol and progesterone and are less likely to ovulate (Sheldon et al., 2002; Williams et al., 2007). Therefore, these results indicate that uterine health might be associated with the secretion of APPs and with the timing of the first postpartum ovulation. Based on these considerations, we hypothesized that cows resuming ovarian activity earlier during the postpartum period have a reduced percentage of PMN endometrial cells in the uterus, and that these conditions are reflected by
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the inflammatory status, which is indicated by the serum concentrations of APPs. Therefore, the aim of this study was to evaluate the association of the timing of the first postpartum ovulation with the following: (1) the percentage of PMN endometrial cells in the uterine flushing, (2) lipid mobilization, and (3) the secretion of APPs during the transition period of dairy cows that are maintained in a semi-extensive management system. 2. Materials and methods 2.1. Cows and experimental design The experiment was conducted between December 2011 and June 2012 in a commercial farm in Rio Grande, RS, Brazil (32◦ 16’ S, 52◦ 32’ W). The Ethics and Animal Experimentation Committee from the Federal University of Pelotas, under the registration number 4551, approved all procedures. The cows were selected for the study based on the number of lactations (≥3 lactations), the average production, which was adjusted for 305 days during the previous lactation (≥7.000 kg), and a negative history of any clinical disease during the last lactation. Initially, forty (n = 40) multiparous Holstein dairy cows (≥3 lactations), with an average body condition score (BCS) of 3.1 ± 0.4 and a body weight (BW) of 629.8 ± 42.7 kg, were enrolled in the study. Twenty (n = 20) cows were diagnosed with one or more pathological event, including dystocia, retained placenta, mastitis, laminitis and hypocalcemia, and were excluded from the experiment. The cows were maintained in a semi-extensive management system, which was based on pasture and concentrate supplementation after each milking. Milking was performed twice a day, at 12 h intervals. The cows were divided into two groups according to the return of luteal activity during the postpartum period as follows: (1) cows that resumed ovarian activity before 44 days in milk (DIM) (ovulatory cows), which had progesterone concentrations that were higher than 1 ng/mL in at least two consecutive blood samples that were collected between 16 and 44 DIM, and (2) cows that did not resume ovarian activity before 44 DIM (anovulatory cows), which had no increase in progesterone concentrations above 1 ng/mL during the same period (Stevenson and Britt, 1979). 2.2. Blood sampling and biochemical and hormonal analysis Blood sampling was performed on days −21, −14, −7, −3 before calving, at calving (0 DIM) and at 3, 6, 9, 16, 23, 30, 37 and 44 DIM. To evaluate the resumption of ovarian activity, blood sampling for the measurement of serum progesterone (P4) was performed at 16, 23, 30, 37 and 44 DIM. Blood was collected by coccygeal venipuncture using vaccutainer tubes. The blood samples were collected in tubes that contained clot activator (16 × 100 mm, 10 mL Vacuplast® , Shandong, China) and sodium fluoride (13 × 75 mm, 4 mL Vacuplast® , Zhejiang, China), centrifuged within 30 min after collection at 1.35 × g for 15 min. For all blood variables, except for progesterone, the
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serum was separated and frozen at −80 ◦ C for further analysis. The serum from the fluoride tube was harvested and cooled for the analysis of glucose concentration within 72 h. Blood samples for the analysis of progesterone were centrifuged (1.35 × g/15 min) after collection, and the serum was stored at −20 ◦ C. Serum samples were thawed at room temperature before each assay. Commercial kits (LabTest® , Lagoa Santa, Brazil) were used to evaluate serum albumin and glucose concentrations using colorimetric spectrophotometry (Biospectro® , SP-220, Curitiba, PR, Brazil); the intra- and inter-assay coefficients of variation (CVs) were 2.9% and 3.7%, respectively for albumin, and 1.9% and 13.9%, respectively for glucose. For the analysis of serum paraoxonase arylesterase activity, a commercial kit was used (ZeptoMetrix® Corporation, Buffalo, NY, USA), which was coupled to a kinetic spectrophotometry system (T80 UV/VIS Spectrometer, PG Instruments Ltd., PTC-2 Peltier Temperature Controller, software UVWin80 v5.0.5, Wibtoft, England), and the intra- and inter-assay CVs were 14.8% and 10.2%, respectively. Serum insulin concentrations were determined by a commercial ELISA assay according to Beitinger et al. (2012) (Ins-EASIA® , DIASource, Louvain-La-Neuve, Belgium), with a minimum detection limit of 1.13 IU/mL using a plate reader (Thermo Plate® , TP Reader, São Paulo, SP, Brazil) with intra- and inter-assay CVs of 5.4% and 6.2%, respectively. According to the manufacturer, the cross reactivity with bovine insulin is 100%. Non-esterified fatty acids (NEFA) were evaluated according to the micro-method that was previously described by Ballou et al. (2009) using a commercial kit (Wako NEFA-HR, Wako Chemicals® , Richmond, USA). Haptoglobin concentrations were measured according to the method that was previously described by Jones and Mould (1984). The intra- and inter-assay CVs for NEFA and Hp assays were 5% and 10.9%, respectively. The serum concentration of progesterone (P4) was determined using a commercial radioimmunoassay kit (Coat-A-Count® , Diagnostic Products Corporation, Los Angeles, USA) as previously described by Burke et al. (2003). All progesterone analyses were performed in a single batch and the intra-assay CV was 7.4%.
All statistical analyses were performed using the SAS 9.0 software (SAS Institute Inc., Cary, NC, USA). Variables for repeated measures (paraoxonase, haptoglobin, albumin, NEFA, glucose, insulin, milk yield, BCS and weight) were evaluated using an analysis of variance (ANOVA) with the MIXED procedure to test the main effects of the ovulatory group, days from calving and their interaction in two different models for the pre- and postpartum periods. The proportion of PMN cells at 37 ± 4 DIM was analyzed using a one-way ANOVA. Cows were also categorized by their subclinical endometritis (PMN cells ≥ 18%) status according to the method of Meira et al. (2012). Thus, the percentage of cows that had subclinical endometritis was compared between groups using the chi-square test. In all tests, the level of significance was set at P < 0.05, and P values between 0.05 and 0.1 were considered as a tendency.
2.3. Clinical examinations and uterine cytology
3. Results
Cows were examined at 18 ± 2, 25 ± 3 and 33 ± 2 DIM by vaginoscopy to evaluate and characterize the presence of vaginal discharge (Pleticha et al., 2009). For vaginoscopic examination, the vulva was cleaned with a paper towel, and a sanitized vaginal speculum was introduced into the vaginal canal. Using a light source, the cervix and the vaginal canal were inspected, and the presence of secretion was classified and registered. Vaginal discharge was graded on a scale from 0 to 3 (0 = mucus, 1 = mucus with flecks of pus, 2 ≥ 50% purulent exudate, 3 = hemorrhagic and/or purulent exudate), as adapted from Williams et al. (2005) and Sheldon et al. (2006). To count PMN cells on the uterus, uterine cytology was performed using low-volume uterine lavage (Gilbert et al., 2005) at 37 ± 4 DIM. For this procedure, 20 mL of isotonic saline solution was introduced into the uterus with a sterile syringe that was attached to a pipette for equine artificial
From calving to 44 ± 2 DIM, 12 cows (60%) ovulated, and 8 cows (40%) did not ovulate. The average interval from calving to the first ovulation for ovulatory cows was 29.8 ± 1.9 DIM. Among ovulatory cows, eight (67%) resumed cyclicity before 37 DIM, when the uterine aspirates were performed. Ovulatory cows had a lower PMN cell percentage (26.3 ± 8.3%) compared with anovulatory cows (53.4 ± 16.9%, P = 0.05). Despite this lower PMN cell percentage, the percentage of cows that were diagnosed with subclinical endometritis (PMN cells ≥ 18%; Meira et al., 2012) at 37 DIM was not different between ovulatory and anovulatory cows (58.3% vs. 62.5%, respectively; P > 0.05). No cows were observed with signs of clinical endometritis in the vaginoscopic examination at 18 ± 2, 25 ± 3 or 33 ± 2 DIM. Ovulatory cows had higher serum albumin concentrations during the pre- (P = 0.03) and postpartum periods
insemination. After a brief massage of the uterus by rectal palpation (10 s), the fluid was recovered into the same syringe, stored in a test tube, cooled and processed in a cytocentrifuge (14 TekLab Cytocentrifuge CT® , Camboriu, Brazil) within 6 h after collection. The amount of fluid that was recovered ranged from 2 to 6 mL. Slides were prepared with the recovered fluid and stained using a commercial kit (Quick Panoptic® , Laborclin, Pinhais, Brazil). Two hundred cells per slide were counted in an optical microscope (100 times magnification), including PMN, mononuclear and epithelial cells to calculate the proportion of PMN cells in the uterine lavage sample. 2.4. Milk production, body condition score and weight Individual milk yields (kg/cow) were measured daily (ALPRO, Tetra Laval Group® , Sweden) and averaged every 5 days from calving to 46 DIM. Body condition scores were measured weekly according to Wildman et al. (1982), and the body weight was measured using a digital scale (EziWeigh 5—TRU-TEST® , Farm Tech Group, Ljutomer, Slovenia). 2.5. Statistical analysis
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Fig. 1. Serum concentrations of albumin (A; g/dL), haptoglobin (B; g/L), and paraoxonase activity (C; kU/L) (mean ± SEM) during the transition period in ovulatory (n = 12) and anovulatory cows (n = 8) from −21 to 30 days in milk. Statistical analyses were performed separately for the pre- and postpartum periods for the effects of the ovulatory group, days from calving and the interaction between ovulatory group and days from calving.
(P = 0.01; Fig. 1A). Serum concentrations of haptoglobin during the prepartum period tended to be lower in ovulatory cows (P = 0.07); however, no difference was observed during the postpartum period (P > 0.10; Fig. 1B). Ovulatory cows also tended to have higher serum paraoxonase activity than anovulatory cows during the postpartum period (P = 0.09); however, no difference was observed during the prepartum period (P > 0.10; Fig. 1C). There were no differences in the serum concentrations of NEFA, glucose and insulin between ovulatory and anovulatory cows (P > 0.10; Fig. 2). No differences in BCS or body weight were detected between groups during the prepartum period (P > 0.05). However, ovulatory cows had a higher BCS (P = 0.001) than anovulatory cows (2.9 ± 0.06 vs. 2.6 ± 0.07, respectively) and tended (P = 0.06) to have a higher body weight (611.0 ± 15.9 vs. 560.9 ± 19.3 kg) during the postpartum
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Fig. 2. Serum concentrations of NEFA (A; mmol/L), glucose (B; mg/dL), and insulin (C; IU/mL) (mean ± SEM) during the transition period in ovulatory (n = 12) and anovulatory cows (n = 8) from −21 to 30 days in milk. Statistical analyses were performed separately for the pre- and postpartum periods for the effects of the ovulatory group, days from calving and the interaction between ovulatory group and days from calving.
period. Milk production was not different (P = 0.29) between ovulatory and anovulatory cows during the period from calving to 46 DIM (25.0 ± 1.5 kg vs. 27.0 ± 1.0 kg/day, respectively). 4. Discussion The aim of this study was to explore the associations between the percentage of PMN cells in the uterus, concentrations of inflammatory markers during the transition period, and resumption of postpartum ovarian activity in lactating dairy cows. The results indicated that the early resumption of postpartum ovarian activity was not associated with a lower incidence of subclinical endometritis, although it was associated with a lower percentage of uterine PMN cells and higher concentrations of albumin during the peripartum period. In addition, the energy status (NEFA, insulin and milk production) apparently was not associated
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with the postpartum resumption of ovarian activity in this group of cows that was studied. In the present study, we observed that anovulatory cows had a higher average percentage of PMN cells in the uterine lumen, which was associated with a reduced hepatic synthesis of albumin, although incidence of endometritis was not higher. Despite that, there was a tendency for higher serum concentrations of haptoglobin and lower serum concentrations of PON in anovulatory cows, with higher counts of PMN cells in the uterine lumen. Previous reports (Huzzey et al., 2009; Burke et al., 2010), have also indicated an association between APPs, uterine health and the resumption of postpartum ovarian activity. During the transition period dairy cows exhibit an exacerbated inflammatory response that is related to calving and to the onset of lactation (Sordillo et al., 2009), even when no signs of clinical disease are present (Bionaz et al., 2007), which may impair homeostasis and compromise the immune system of the cow (Castro et al., 2012). This observation is in agreement with our current observations because anovulatory cows had a higher uterine PMN cell count, which was associated with lower albumin concentrations and trends toward lower PON and higher haptoglobin, although the incidence of subclinical endometritis was not different between the groups. This result indicates that the severity of the uterine inflammatory response can potentially alter the inflammatory response of the cow, without any visible clinical alterations. In a similar study, Huzzey et al. (2009) demonstrated that cows with postpartum metritis had higher concentrations of haptoglobin than healthy cows from calving to 12 DIM, whereas the peak of haptoglobin for both healthy and unhealthy cows occurred between 3 and 6 DIM. An identical pattern of inflammatory response could be verified during the first week postpartum, particularly in anovulatory cows in the present study. In contrast, there was a trend for the reduced secretion of the negative inflammatory protein PON during the postpartum period. PON secretion is reduced in postpartum cows with uterine infection (Bionaz et al., 2007; Schneider et al., 2013). Nonetheless, to confirm this affirmation, more studies that address this topic during different situations of uterine contaminations and the inflammatory response are required. The effects of proinflammatory cytokines during the peripartum period may change according to their synthesis and release. These effects include impaired hepatic function, alterations in nutrient partitioning, reduced dry matter intake and decreased reproductive efficiency (Fleck, 1989). Lower serum albumin synthesis may indicate reduced liver function and a deviation of acute phase protein synthesis in the liver (Bertoni et al., 2008), as well as an increased catabolism of albumin because of an energy deficit (Bell et al., 2000). In the present study, the serum concentration of albumin was lower in anovulatory cows throughout the experiment. This observation suggests that albumin could represent an interesting biomarker for evaluating inflammatory/energetic status in association with the reproduction performance of postpartum cows. Because acute phase proteins are nonspecific markers of inflammation, the association of these proteins with uterine inflammation, which is diagnosed by endometrial cytology, becomes difficult to interpret. However, in an
attempt to avoid any misinterpretation of these results, this study involved only cows that remained clinically healthy throughout the experimental period. A specific designed experiment with many healthy cows should be performed to investigate the association between the profile of APP‘s secretion and reproduction performance in commercial herds. The early resumption of postpartum ovarian activity was associated with an increased serum albumin concentration and with a trend toward reduced PON and increased haptoglobin concentrations, which confirmed previous hypotheses that inflammatory phenomena associated with calving might have direct negative effects on the reproductive performance of dairy cows (Bertoni et al., 2008). It seems highly likely that within a herd, one of the factors that compromise fertility is the inflammatory status of individual cows during the peripartum period (Bertoni et al., 2008). The higher percentage of PMN cells in the uterine flushing, in anovulatory cows, although not related to a higher incidence of subclinical endometritis, was associated with a delayed first postpartum ovulation, which indicated that the severity of endometrium inflammation is detrimental to ovarian function. Conversely, the lack of postpartum ovarian activity can impair the removal of uterine pathogens and contribute to the persistence of uterine infection (Shrestha et al., 2004). The exacerbated release of proinflammatory cytokines by endometrial cells can affect follicular development, which may reduce steroidogenesis in granulosa cells (Spicer and Alpizar, 1994) and the growth of the first postpartum dominant follicle (Sheldon et al., 2002). The ovulation of these follicles creates smaller CL, which produces lower amounts of progesterone (Sheldon et al., 2002; Williams et al., 2007, 2008). Based on other studies, we can conclude that cows with higher uterine PMN cell counts were more likely to have a delayed resumption of ovarian activity, although not associated with a higher incidence of subclinical endometritis. Although postpartum subclinical endometritis is defined as an elevated percentage of inflammatory PMN cells in endometrial cytobrush or aspirate examinations, the threshold for determining endometritis is controversial. Apparently, there is no consensus regarding the percentage of PMN cells that are recovered from the uterus to classify a cow with subclinical endometritis. Moreover, it is clear that the earlier during postpartum period that the uterine examination is performed, the higher the percentage of PMN cells that are recovered. These factors cause the definition of subclinical endometritis to be challenging for researchers and clinical veterinaries. There are not only different methods to recover uterine cells, such as cytobrush (Barlund et al., 2008; Burke et al., 2010; Ghasemi et al., 2012; Meira et al., 2012), or low volume uterine lavage (Gilbert et al., 2005), but also different thresholds of PMN presence, e.g., 6% (Burke et al., 2010), 8% (Barlund et al., 2008) and 18% (Ghasemi et al., 2012; Meira et al., 2012), that are used to determine subclinical endometritis. In this study, which used the 18% threshold, there was no difference in the incidence of subclinical endometritis between groups; however, independent of the threshold that was used to consider a cow with or without subclinical endometritis, the data from our
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study demonstrated that cows with a delay in resuming postpartum ovulatory cycles had two-fold higher PMN cell count in endometrial aspirates than ovulatory cows. Several studies have associated NEB severity with the timing of the first postpartum ovulation (Wiltbank et al., 2006; Santos et al., 2009; Burke et al., 2010; Giuliodori et al., 2011; Castro et al., 2012). Although NEFA and insulin concentrations were strongly associated with the duration of the postpartum anovulatory interval previously (Butler, 2003; Wiltbank et al., 2006), in the present study there was no difference in the serum concentrations of NEFA and insulin between ovulatory and anovulatory cows. High yielding dairy cows are most likely to suffer the negative effects of a NEB: however, there is no consensus in the literature regarding whether milk yield is associated with a delayed first ovulation. Although some studies report that cows that are selected for high milk production postpone the first postpartum ovulation up to 14 days (Lucy, 2001; Gong et al., 2002), other studies have not found this correlation (Lopez et al., 2005). The latter result is in accordance with the current study, in which ovulatory cows showed similar levels of milk production compared with anovulatory cows. Bertoni et al. (2008) indicated that a close correlation between fertility and milk production is not obvious, although cows with high genetic merit for milk production had a decreased fertility, particularly when associated with exacerbated inflammatory conditions. Thus, a simple correlation between anovulation and NEB intensity apparently does not exist, and a more complex physiological model is required to fully explain the anovulatory conditions of dairy cows (Wiltbank et al., 2002; Gümen and Wiltbank, 2002). These records corroborate with the data obtained in the present study, bringing further evidence to the concept that the anovulatory conditions of postpartum dairy cows may be more related to inflammatory conditions than only strictly linked to nutritional stress conditions that are signaled by the levels of lipid mobilization and milk production. In summary, cows that resumed ovarian activity early during the postpartum period had higher albumin serum concentrations and lower counts of uterine PMN cells, which were not associated with higher subclinical endometritis incidence. Despite this observation, no changes in energy status markers or milk production were related to the anovulatory conditions. Therefore, this study suggests that the serum concentration of albumin may be an important biomarker of both the severity of uterine disease and the ovulatory potential in early postpartum lactating dairy cows. References Ballou, M.A., Gomes, R.C., Juchem, S.O., DePeters, E.J., 2009. Effects of dietary supplemental fish oil during the peripartum period on blood metabolites and hepatic fatty acid compositions and total triacylglycerol concentrations of multiparous Holstein cows. J. Dairy Sci. 92, 657–669. Barlund, C.S., Carruthers, T.D., Waldner, C.L., Palmer, C.W., 2008. A comparison of diagnostic techniques for postpartum endometritis in dairy cattle. Theriogenology 69, 714–723. Beitinger, P.A., Fulda, S., Dalal, M.A., Wehrle, R., Keckeis, M., Wetter, T.C., Han, F., Pollmächer, T., Schuld, A., 2012. Glucose tolerance in patients with narcolepsy. Sleep 35, 231–236.
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Sheldon, I.M., Noakes, D.M., Rycroft, A., Dobson, H., 2001. Acute phase protein responses to uterine bacterial contamination in cattle after calving. Vet. Rec. 148, 172–175. Sheldon, I.M., Noakes, D.E., Rycroft, A.N., Pfeiffer, D.U., Dobson, H., 2002. Influence of uterine bacterial contamination after parturition on ovarian dominant follicle selection and follicle growth and function in cattle. Reproduction 123, 837–845. Sheldon, I.M., Lewis, G.S., LeBlanc, S., Gilbert, R.O., 2006. Defining postpartum uterine disease in cattle. Theriogenology 65, 1516–1530. Sheldon, I.M., Price, S.B., Cronin, J., Gilbert, R.O., Gadsby, J.E., 2009. Mechanisms of infertility associated with clinical and subclinical endometritis in high producing dairy cattle. Reprod. Domest. Anim. 44, 1–9. Shrestha, H.K., Nakao, T., Higaki, T., Suzuki, T., Akita, M., 2004. Resumption of postpartum ovarian cyclicity in highproducing Holstein cows. Theriogenology 61, 637–649. Sordillo, L.M., Contreras, G.A., Aitken, S.L., 2009. Metabolic factors affecting the inflammatory response of periparturient dairy cows. Anim. Health Res. Rev. 10, 53–63. Spicer, L.J., Alpizar, E., 1994. Effects of cytokines on FSH-induced estradiol production by bovine granulosa cells in vitro: dependence on size of follicle. Domest. Anim. Endocrin. 11, 24–34. Staples, C.R., Thatcher, W.W., Clark, J.H., 1990. Relationship between ovarian activity and energy status during the early post-partum period of high producing dairy cows. J. Dairy Sci. 73, 938–947. Stevenson, J.S., Britt, J.H., 1979. Relationships among luteinizing hormone, estradiol, progesterone, glucocorticoids, milk yield, body weight and postpartum ovarian activity in Holstein cows. J. Anim. Sci. 48, 570–577.
Turk, R., Juretic, D., Geres, D., Turk, N., Rekic, B., Simeon-Rudolf, V., Svetina, A., 2004. Serum paraoxonase activity and lipid parameters in the early postpartum period of dairy cows. Res. Vet. Sci. 76, 57–61. Turk, R., Juretic, D., Geres, D., Turk, N., Rekic, B., Simeon- Rudolf, V., Robic, M., Svetina, A., 2005. Serum paraoxonase activity in dairy cows during pregnancy. Res. Vet. Sci. 79, 15–18. Walsh, S.W., Williams, E.J., Evans, A.C.O., 2011. A review of the causes of poor fertility in high milk producing dairy cows. Anim. Reprod. Sci. 123, 127–138. Wildman, E.E., Jones, G.M., Wagner, P.E., Bowman, R.L., 1982. A dairy cow body condition scoring system and its relationship to selected production characteristics. J. Dairy Sci. 65, 495–501. Williams, E.J., Fischer, D.P., Pfeiffer, D.U., England, G.E., Noakes, D.E., Dobson, H., Sheldon, I.M., 2005. Clinical evaluation of postpartum vaginal mucus reflects uterine bacterial infection and the immune response in cattle. Theriogenology 63, 102–117. Williams, E.J., Fischer, D.P., Noakes, D.E., England, G.E., Rycroft, A., Dobson, H., Sheldon, I.M., 2007. Uterine infection perturbs ovarian function in the postpartum dairy cow. Theriogenology 68, 549–559. Williams, E.J., Sibley, K., Miller, A.N., Lane, E.A., Fishwick, J., Nash, D.M., Herath, S., England, G.C.W., Dobson, H., Sheldon, I.M., 2008. The effect of Escherichia coli lipopolysaccharide and tumor necrosis factor alpha on ovarian function. Am. J. Reprod. Immunol. 60, 462–473. Wiltbank, M.C., Gümen, A., Sartori, R., 2002. Physiological classification of anovulatory conditions in cattle. Theriogenology 57, 21–52. Wiltbank, M., Lopez, H., Sartori, R., Sangsritavong, S., Gümen, A., 2006. Changes in reproductive physiology of lactating dairy cows due to elevated steroid metabolism. Theriogenology 65, 17–29. Wira, C.R., Fahey, J.V., 2004. The innate immune system: gatekeeper to the female reproductive tract. Immunology 111, 13–15.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Associations between resumption of postpartum ovarian activity, uterine health and concentrations of metabolites and acute phase proteins during the transition period in Holstein cows Ana Rita T. Krause a,d , Luiz F.M. Pfeifer c,d,∗ , Paula Montagner a,d , Marina M. Weschenfelder a,d , Elizabeth Schwegler a,d , Márcio E. Lima a,d , Eduardo G. Xavier b , Cassio C. Brauner a,d , Eduardo Schmitt c,d , Francisco A.B. Del Pino a,d , Charles F. Martins a,d , Marcio N. Corrêa a,d , Augusto Schneider a,d a b c d
Universidade Federal de Pelotas, Pelotas, RS, Brazil Granjas 4 irmãos S.A., Rio Grande, RS, Brazil Empresa Brasileira de Pesquisa Agropecuária, EMBRAPA Rondônia, Porto Velho, RO, Brazil Núcleo de Pesquisa, Ensino e Extensão em Pecuária (NUPEEC), Universidade Federal de Pelotas, Pelotas, RS, Brazil
a r t i c l e
i n f o
Article history: Received 31 July 2013 Received in revised form 19 December 2013 Accepted 21 December 2013 Available online 3 January 2014 Keywords: Albumin Haptoglobin Paraoxonase Uterus Cattle
a b s t r a c t The resumption of ovarian activity, uterine health, severity of the negative energy balance and the synthesis of inflammatory mediators during the transition period in dairy cows are interrelated. Therefore, the aim of this study was to evaluate the association between the resumption of postpartum ovarian activity and the percentage of polymorphonuclear (PMN) cells in endometrial cytology, lipid mobilization and the secretion of acute phase proteins. For this study, 20 multiparous Holstein cows were used. Blood samples that were collected from 21 d before calving to 44 d in milk (DIM) were analyzed for serum glucose, non-esterified fatty acids (NEFA), insulin, haptoglobin, albumin, paraoxonase and progesterone. Endometrial cytology was performed at 37 ± 2 DIM to evaluate the percentage of PMN cells in the uterine flushing. Cows were divided into two groups: (1) ovulatory cows (n = 12), which returned to ovarian activity by 44 ± 2 DIM; and (2) anovulatory cows (n = 8), which did not resume ovarian activity during this period. Ovulatory cows had a lower (P = 0.05) percentage of PMN cells in endometrial cytology than anovulatory cows (26.3 ± 8.3% vs. 53.4 ± 16.9%, respectively). Ovulatory cows had higher serum albumin during the pre- (P = 0.03) and postpartum periods (P = 0.01), and tended to have lower haptoglobin concentrations in the prepartum period (P = 0.07) and higher paraoxonase activity in the postpartum period (P = 0.09). In conclusion, cows that resumed ovarian activity early in the postpartum period had higher albumin concentrations in the peripartum period, which were associated with a lower percentage of uterine PMN cells. © 2014 Elsevier B.V. All rights reserved.
1. Introduction ∗ Corresponding author at: Embrapa, Animal reproduction lab, Br 364 Km 5.5, 76815-800 Porto Velho, Rondonia, Brazil. Tel.: +55 69 3901 2526; fax: +55 69 3901 2510/+55 69 3222 0409. E-mail address: [email protected] (L.F.M. Pfeifer). 0378-4320/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2013.12.016
Only approximately 50% of healthy dairy cows ovulate the first dominant follicle within 3 weeks after calving (Kawashima et al., 2007). A healthy postpartum dairy cow can be defined as one that has resolved uterine involution,
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ovulated a dominant follicle early postpartum and continues to have a normal estrous cycle at regular intervals, coupled with homeostatic concentrations of insulin, IGF-I and glucose (Roche, 2006; Walsh et al., 2011). A delayed first postpartum ovulation is usually associated with a pronounced negative energy balance (NEB) in the early postpartum period (Butler, 2003), although other factors are also important (Santos et al., 2009). It is well known that cows that resume ovarian function early after calving are most likely to become pregnant in the first service (Staples et al., 1990). In dairy cows, to achieve a satisfactory reproductive performance during this critical period, it is necessary that the female ovulates a competent oocyte and provides a suitable uterine environment for fertilization and embryonic and fetal development (Pursley and Martins, 2011). Therefore, factors that affect the early resumption of ovarian cycles during the postpartum period have an impact on the fertility of dairy cows and should be further investigated. In addition to peripartum metabolic events that may affect reproductive performance, uterine contamination may also contribute to decreased conception rates in lactating dairy cows. In total, 80 to 100% of these cows have uterine bacterial contamination during the first weeks after calving (Sheldon et al., 2006). The presence of pathogenic bacteria in the uterus is associated with endometrial inflammation and purulent vaginal discharges (Sheldon et al., 2009), which increase the amount of polymorphonuclear (PMN) defense cells in the endometrium. Most cows recover naturally from these postpartum uterine infections, however, at least 20% of these cows have a persistent infection 3 weeks after calving and develop endometritis. Dairy cows that develop endometritis during the postpartum period have reduced conception at first insemination and increased days open (Fourichon et al., 2000). During the peripartum period, the increased release of inflammatory mediators, which are called acute phase proteins (APPs) has also been associated with decreased reproductive performance (Wira and Fahey, 2004). Some negative APPs synthesized in the liver, such as paraoxonase (PON) and albumin, have their serum concentrations reduced due to the typical inflammatory response, which is induced by parturition (Bionaz et al., 2007; Fleck, 1989). Conversely, the serum concentration of the positive APP haptoglobin (Hp) increases during the inflammatory process. Several studies have demonstrated increased haptoglobin and decreased albumin and paraoxonase concentrations in cows with postpartum uterine infections, even before calving (Sheldon et al., 2001; Turk et al., 2004, 2005; Huzzey et al., 2009; Burke et al., 2010; Schneider et al., 2013). Cows with bacterial infections in the reproductive tract have slower growth of the dominant follicle, lower plasma concentrations of estradiol and progesterone and are less likely to ovulate (Sheldon et al., 2002; Williams et al., 2007). Therefore, these results indicate that uterine health might be associated with the secretion of APPs and with the timing of the first postpartum ovulation. Based on these considerations, we hypothesized that cows resuming ovarian activity earlier during the postpartum period have a reduced percentage of PMN endometrial cells in the uterus, and that these conditions are reflected by
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the inflammatory status, which is indicated by the serum concentrations of APPs. Therefore, the aim of this study was to evaluate the association of the timing of the first postpartum ovulation with the following: (1) the percentage of PMN endometrial cells in the uterine flushing, (2) lipid mobilization, and (3) the secretion of APPs during the transition period of dairy cows that are maintained in a semi-extensive management system. 2. Materials and methods 2.1. Cows and experimental design The experiment was conducted between December 2011 and June 2012 in a commercial farm in Rio Grande, RS, Brazil (32◦ 16’ S, 52◦ 32’ W). The Ethics and Animal Experimentation Committee from the Federal University of Pelotas, under the registration number 4551, approved all procedures. The cows were selected for the study based on the number of lactations (≥3 lactations), the average production, which was adjusted for 305 days during the previous lactation (≥7.000 kg), and a negative history of any clinical disease during the last lactation. Initially, forty (n = 40) multiparous Holstein dairy cows (≥3 lactations), with an average body condition score (BCS) of 3.1 ± 0.4 and a body weight (BW) of 629.8 ± 42.7 kg, were enrolled in the study. Twenty (n = 20) cows were diagnosed with one or more pathological event, including dystocia, retained placenta, mastitis, laminitis and hypocalcemia, and were excluded from the experiment. The cows were maintained in a semi-extensive management system, which was based on pasture and concentrate supplementation after each milking. Milking was performed twice a day, at 12 h intervals. The cows were divided into two groups according to the return of luteal activity during the postpartum period as follows: (1) cows that resumed ovarian activity before 44 days in milk (DIM) (ovulatory cows), which had progesterone concentrations that were higher than 1 ng/mL in at least two consecutive blood samples that were collected between 16 and 44 DIM, and (2) cows that did not resume ovarian activity before 44 DIM (anovulatory cows), which had no increase in progesterone concentrations above 1 ng/mL during the same period (Stevenson and Britt, 1979). 2.2. Blood sampling and biochemical and hormonal analysis Blood sampling was performed on days −21, −14, −7, −3 before calving, at calving (0 DIM) and at 3, 6, 9, 16, 23, 30, 37 and 44 DIM. To evaluate the resumption of ovarian activity, blood sampling for the measurement of serum progesterone (P4) was performed at 16, 23, 30, 37 and 44 DIM. Blood was collected by coccygeal venipuncture using vaccutainer tubes. The blood samples were collected in tubes that contained clot activator (16 × 100 mm, 10 mL Vacuplast® , Shandong, China) and sodium fluoride (13 × 75 mm, 4 mL Vacuplast® , Zhejiang, China), centrifuged within 30 min after collection at 1.35 × g for 15 min. For all blood variables, except for progesterone, the
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serum was separated and frozen at −80 ◦ C for further analysis. The serum from the fluoride tube was harvested and cooled for the analysis of glucose concentration within 72 h. Blood samples for the analysis of progesterone were centrifuged (1.35 × g/15 min) after collection, and the serum was stored at −20 ◦ C. Serum samples were thawed at room temperature before each assay. Commercial kits (LabTest® , Lagoa Santa, Brazil) were used to evaluate serum albumin and glucose concentrations using colorimetric spectrophotometry (Biospectro® , SP-220, Curitiba, PR, Brazil); the intra- and inter-assay coefficients of variation (CVs) were 2.9% and 3.7%, respectively for albumin, and 1.9% and 13.9%, respectively for glucose. For the analysis of serum paraoxonase arylesterase activity, a commercial kit was used (ZeptoMetrix® Corporation, Buffalo, NY, USA), which was coupled to a kinetic spectrophotometry system (T80 UV/VIS Spectrometer, PG Instruments Ltd., PTC-2 Peltier Temperature Controller, software UVWin80 v5.0.5, Wibtoft, England), and the intra- and inter-assay CVs were 14.8% and 10.2%, respectively. Serum insulin concentrations were determined by a commercial ELISA assay according to Beitinger et al. (2012) (Ins-EASIA® , DIASource, Louvain-La-Neuve, Belgium), with a minimum detection limit of 1.13 IU/mL using a plate reader (Thermo Plate® , TP Reader, São Paulo, SP, Brazil) with intra- and inter-assay CVs of 5.4% and 6.2%, respectively. According to the manufacturer, the cross reactivity with bovine insulin is 100%. Non-esterified fatty acids (NEFA) were evaluated according to the micro-method that was previously described by Ballou et al. (2009) using a commercial kit (Wako NEFA-HR, Wako Chemicals® , Richmond, USA). Haptoglobin concentrations were measured according to the method that was previously described by Jones and Mould (1984). The intra- and inter-assay CVs for NEFA and Hp assays were 5% and 10.9%, respectively. The serum concentration of progesterone (P4) was determined using a commercial radioimmunoassay kit (Coat-A-Count® , Diagnostic Products Corporation, Los Angeles, USA) as previously described by Burke et al. (2003). All progesterone analyses were performed in a single batch and the intra-assay CV was 7.4%.
All statistical analyses were performed using the SAS 9.0 software (SAS Institute Inc., Cary, NC, USA). Variables for repeated measures (paraoxonase, haptoglobin, albumin, NEFA, glucose, insulin, milk yield, BCS and weight) were evaluated using an analysis of variance (ANOVA) with the MIXED procedure to test the main effects of the ovulatory group, days from calving and their interaction in two different models for the pre- and postpartum periods. The proportion of PMN cells at 37 ± 4 DIM was analyzed using a one-way ANOVA. Cows were also categorized by their subclinical endometritis (PMN cells ≥ 18%) status according to the method of Meira et al. (2012). Thus, the percentage of cows that had subclinical endometritis was compared between groups using the chi-square test. In all tests, the level of significance was set at P < 0.05, and P values between 0.05 and 0.1 were considered as a tendency.
2.3. Clinical examinations and uterine cytology
3. Results
Cows were examined at 18 ± 2, 25 ± 3 and 33 ± 2 DIM by vaginoscopy to evaluate and characterize the presence of vaginal discharge (Pleticha et al., 2009). For vaginoscopic examination, the vulva was cleaned with a paper towel, and a sanitized vaginal speculum was introduced into the vaginal canal. Using a light source, the cervix and the vaginal canal were inspected, and the presence of secretion was classified and registered. Vaginal discharge was graded on a scale from 0 to 3 (0 = mucus, 1 = mucus with flecks of pus, 2 ≥ 50% purulent exudate, 3 = hemorrhagic and/or purulent exudate), as adapted from Williams et al. (2005) and Sheldon et al. (2006). To count PMN cells on the uterus, uterine cytology was performed using low-volume uterine lavage (Gilbert et al., 2005) at 37 ± 4 DIM. For this procedure, 20 mL of isotonic saline solution was introduced into the uterus with a sterile syringe that was attached to a pipette for equine artificial
From calving to 44 ± 2 DIM, 12 cows (60%) ovulated, and 8 cows (40%) did not ovulate. The average interval from calving to the first ovulation for ovulatory cows was 29.8 ± 1.9 DIM. Among ovulatory cows, eight (67%) resumed cyclicity before 37 DIM, when the uterine aspirates were performed. Ovulatory cows had a lower PMN cell percentage (26.3 ± 8.3%) compared with anovulatory cows (53.4 ± 16.9%, P = 0.05). Despite this lower PMN cell percentage, the percentage of cows that were diagnosed with subclinical endometritis (PMN cells ≥ 18%; Meira et al., 2012) at 37 DIM was not different between ovulatory and anovulatory cows (58.3% vs. 62.5%, respectively; P > 0.05). No cows were observed with signs of clinical endometritis in the vaginoscopic examination at 18 ± 2, 25 ± 3 or 33 ± 2 DIM. Ovulatory cows had higher serum albumin concentrations during the pre- (P = 0.03) and postpartum periods
insemination. After a brief massage of the uterus by rectal palpation (10 s), the fluid was recovered into the same syringe, stored in a test tube, cooled and processed in a cytocentrifuge (14 TekLab Cytocentrifuge CT® , Camboriu, Brazil) within 6 h after collection. The amount of fluid that was recovered ranged from 2 to 6 mL. Slides were prepared with the recovered fluid and stained using a commercial kit (Quick Panoptic® , Laborclin, Pinhais, Brazil). Two hundred cells per slide were counted in an optical microscope (100 times magnification), including PMN, mononuclear and epithelial cells to calculate the proportion of PMN cells in the uterine lavage sample. 2.4. Milk production, body condition score and weight Individual milk yields (kg/cow) were measured daily (ALPRO, Tetra Laval Group® , Sweden) and averaged every 5 days from calving to 46 DIM. Body condition scores were measured weekly according to Wildman et al. (1982), and the body weight was measured using a digital scale (EziWeigh 5—TRU-TEST® , Farm Tech Group, Ljutomer, Slovenia). 2.5. Statistical analysis
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Fig. 1. Serum concentrations of albumin (A; g/dL), haptoglobin (B; g/L), and paraoxonase activity (C; kU/L) (mean ± SEM) during the transition period in ovulatory (n = 12) and anovulatory cows (n = 8) from −21 to 30 days in milk. Statistical analyses were performed separately for the pre- and postpartum periods for the effects of the ovulatory group, days from calving and the interaction between ovulatory group and days from calving.
(P = 0.01; Fig. 1A). Serum concentrations of haptoglobin during the prepartum period tended to be lower in ovulatory cows (P = 0.07); however, no difference was observed during the postpartum period (P > 0.10; Fig. 1B). Ovulatory cows also tended to have higher serum paraoxonase activity than anovulatory cows during the postpartum period (P = 0.09); however, no difference was observed during the prepartum period (P > 0.10; Fig. 1C). There were no differences in the serum concentrations of NEFA, glucose and insulin between ovulatory and anovulatory cows (P > 0.10; Fig. 2). No differences in BCS or body weight were detected between groups during the prepartum period (P > 0.05). However, ovulatory cows had a higher BCS (P = 0.001) than anovulatory cows (2.9 ± 0.06 vs. 2.6 ± 0.07, respectively) and tended (P = 0.06) to have a higher body weight (611.0 ± 15.9 vs. 560.9 ± 19.3 kg) during the postpartum
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Fig. 2. Serum concentrations of NEFA (A; mmol/L), glucose (B; mg/dL), and insulin (C; IU/mL) (mean ± SEM) during the transition period in ovulatory (n = 12) and anovulatory cows (n = 8) from −21 to 30 days in milk. Statistical analyses were performed separately for the pre- and postpartum periods for the effects of the ovulatory group, days from calving and the interaction between ovulatory group and days from calving.
period. Milk production was not different (P = 0.29) between ovulatory and anovulatory cows during the period from calving to 46 DIM (25.0 ± 1.5 kg vs. 27.0 ± 1.0 kg/day, respectively). 4. Discussion The aim of this study was to explore the associations between the percentage of PMN cells in the uterus, concentrations of inflammatory markers during the transition period, and resumption of postpartum ovarian activity in lactating dairy cows. The results indicated that the early resumption of postpartum ovarian activity was not associated with a lower incidence of subclinical endometritis, although it was associated with a lower percentage of uterine PMN cells and higher concentrations of albumin during the peripartum period. In addition, the energy status (NEFA, insulin and milk production) apparently was not associated
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with the postpartum resumption of ovarian activity in this group of cows that was studied. In the present study, we observed that anovulatory cows had a higher average percentage of PMN cells in the uterine lumen, which was associated with a reduced hepatic synthesis of albumin, although incidence of endometritis was not higher. Despite that, there was a tendency for higher serum concentrations of haptoglobin and lower serum concentrations of PON in anovulatory cows, with higher counts of PMN cells in the uterine lumen. Previous reports (Huzzey et al., 2009; Burke et al., 2010), have also indicated an association between APPs, uterine health and the resumption of postpartum ovarian activity. During the transition period dairy cows exhibit an exacerbated inflammatory response that is related to calving and to the onset of lactation (Sordillo et al., 2009), even when no signs of clinical disease are present (Bionaz et al., 2007), which may impair homeostasis and compromise the immune system of the cow (Castro et al., 2012). This observation is in agreement with our current observations because anovulatory cows had a higher uterine PMN cell count, which was associated with lower albumin concentrations and trends toward lower PON and higher haptoglobin, although the incidence of subclinical endometritis was not different between the groups. This result indicates that the severity of the uterine inflammatory response can potentially alter the inflammatory response of the cow, without any visible clinical alterations. In a similar study, Huzzey et al. (2009) demonstrated that cows with postpartum metritis had higher concentrations of haptoglobin than healthy cows from calving to 12 DIM, whereas the peak of haptoglobin for both healthy and unhealthy cows occurred between 3 and 6 DIM. An identical pattern of inflammatory response could be verified during the first week postpartum, particularly in anovulatory cows in the present study. In contrast, there was a trend for the reduced secretion of the negative inflammatory protein PON during the postpartum period. PON secretion is reduced in postpartum cows with uterine infection (Bionaz et al., 2007; Schneider et al., 2013). Nonetheless, to confirm this affirmation, more studies that address this topic during different situations of uterine contaminations and the inflammatory response are required. The effects of proinflammatory cytokines during the peripartum period may change according to their synthesis and release. These effects include impaired hepatic function, alterations in nutrient partitioning, reduced dry matter intake and decreased reproductive efficiency (Fleck, 1989). Lower serum albumin synthesis may indicate reduced liver function and a deviation of acute phase protein synthesis in the liver (Bertoni et al., 2008), as well as an increased catabolism of albumin because of an energy deficit (Bell et al., 2000). In the present study, the serum concentration of albumin was lower in anovulatory cows throughout the experiment. This observation suggests that albumin could represent an interesting biomarker for evaluating inflammatory/energetic status in association with the reproduction performance of postpartum cows. Because acute phase proteins are nonspecific markers of inflammation, the association of these proteins with uterine inflammation, which is diagnosed by endometrial cytology, becomes difficult to interpret. However, in an
attempt to avoid any misinterpretation of these results, this study involved only cows that remained clinically healthy throughout the experimental period. A specific designed experiment with many healthy cows should be performed to investigate the association between the profile of APP‘s secretion and reproduction performance in commercial herds. The early resumption of postpartum ovarian activity was associated with an increased serum albumin concentration and with a trend toward reduced PON and increased haptoglobin concentrations, which confirmed previous hypotheses that inflammatory phenomena associated with calving might have direct negative effects on the reproductive performance of dairy cows (Bertoni et al., 2008). It seems highly likely that within a herd, one of the factors that compromise fertility is the inflammatory status of individual cows during the peripartum period (Bertoni et al., 2008). The higher percentage of PMN cells in the uterine flushing, in anovulatory cows, although not related to a higher incidence of subclinical endometritis, was associated with a delayed first postpartum ovulation, which indicated that the severity of endometrium inflammation is detrimental to ovarian function. Conversely, the lack of postpartum ovarian activity can impair the removal of uterine pathogens and contribute to the persistence of uterine infection (Shrestha et al., 2004). The exacerbated release of proinflammatory cytokines by endometrial cells can affect follicular development, which may reduce steroidogenesis in granulosa cells (Spicer and Alpizar, 1994) and the growth of the first postpartum dominant follicle (Sheldon et al., 2002). The ovulation of these follicles creates smaller CL, which produces lower amounts of progesterone (Sheldon et al., 2002; Williams et al., 2007, 2008). Based on other studies, we can conclude that cows with higher uterine PMN cell counts were more likely to have a delayed resumption of ovarian activity, although not associated with a higher incidence of subclinical endometritis. Although postpartum subclinical endometritis is defined as an elevated percentage of inflammatory PMN cells in endometrial cytobrush or aspirate examinations, the threshold for determining endometritis is controversial. Apparently, there is no consensus regarding the percentage of PMN cells that are recovered from the uterus to classify a cow with subclinical endometritis. Moreover, it is clear that the earlier during postpartum period that the uterine examination is performed, the higher the percentage of PMN cells that are recovered. These factors cause the definition of subclinical endometritis to be challenging for researchers and clinical veterinaries. There are not only different methods to recover uterine cells, such as cytobrush (Barlund et al., 2008; Burke et al., 2010; Ghasemi et al., 2012; Meira et al., 2012), or low volume uterine lavage (Gilbert et al., 2005), but also different thresholds of PMN presence, e.g., 6% (Burke et al., 2010), 8% (Barlund et al., 2008) and 18% (Ghasemi et al., 2012; Meira et al., 2012), that are used to determine subclinical endometritis. In this study, which used the 18% threshold, there was no difference in the incidence of subclinical endometritis between groups; however, independent of the threshold that was used to consider a cow with or without subclinical endometritis, the data from our
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study demonstrated that cows with a delay in resuming postpartum ovulatory cycles had two-fold higher PMN cell count in endometrial aspirates than ovulatory cows. Several studies have associated NEB severity with the timing of the first postpartum ovulation (Wiltbank et al., 2006; Santos et al., 2009; Burke et al., 2010; Giuliodori et al., 2011; Castro et al., 2012). Although NEFA and insulin concentrations were strongly associated with the duration of the postpartum anovulatory interval previously (Butler, 2003; Wiltbank et al., 2006), in the present study there was no difference in the serum concentrations of NEFA and insulin between ovulatory and anovulatory cows. High yielding dairy cows are most likely to suffer the negative effects of a NEB: however, there is no consensus in the literature regarding whether milk yield is associated with a delayed first ovulation. Although some studies report that cows that are selected for high milk production postpone the first postpartum ovulation up to 14 days (Lucy, 2001; Gong et al., 2002), other studies have not found this correlation (Lopez et al., 2005). The latter result is in accordance with the current study, in which ovulatory cows showed similar levels of milk production compared with anovulatory cows. Bertoni et al. (2008) indicated that a close correlation between fertility and milk production is not obvious, although cows with high genetic merit for milk production had a decreased fertility, particularly when associated with exacerbated inflammatory conditions. Thus, a simple correlation between anovulation and NEB intensity apparently does not exist, and a more complex physiological model is required to fully explain the anovulatory conditions of dairy cows (Wiltbank et al., 2002; Gümen and Wiltbank, 2002). These records corroborate with the data obtained in the present study, bringing further evidence to the concept that the anovulatory conditions of postpartum dairy cows may be more related to inflammatory conditions than only strictly linked to nutritional stress conditions that are signaled by the levels of lipid mobilization and milk production. In summary, cows that resumed ovarian activity early during the postpartum period had higher albumin serum concentrations and lower counts of uterine PMN cells, which were not associated with higher subclinical endometritis incidence. Despite this observation, no changes in energy status markers or milk production were related to the anovulatory conditions. Therefore, this study suggests that the serum concentration of albumin may be an important biomarker of both the severity of uterine disease and the ovulatory potential in early postpartum lactating dairy cows. References Ballou, M.A., Gomes, R.C., Juchem, S.O., DePeters, E.J., 2009. Effects of dietary supplemental fish oil during the peripartum period on blood metabolites and hepatic fatty acid compositions and total triacylglycerol concentrations of multiparous Holstein cows. J. Dairy Sci. 92, 657–669. Barlund, C.S., Carruthers, T.D., Waldner, C.L., Palmer, C.W., 2008. A comparison of diagnostic techniques for postpartum endometritis in dairy cattle. Theriogenology 69, 714–723. Beitinger, P.A., Fulda, S., Dalal, M.A., Wehrle, R., Keckeis, M., Wetter, T.C., Han, F., Pollmächer, T., Schuld, A., 2012. Glucose tolerance in patients with narcolepsy. Sleep 35, 231–236.
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