{"paper_id":"e885b11c-88df-4131-ba2a-34a88e4760fb","body_text":"Polish  Journal of Veterinary Sciences  Vol. 23, No. 4 (2020), 619–626\nDOI 10.24425/pjvs.2020.135810\nOriginal article\nCorrespondence to: \nV . Juodžentis, e-mail: vilmantas.juodzentis@lsmuni.lt\nInfluence of puerperal metritis on the first \novulation after calving in dairy cows\nV . Juodžentis1, J. Šiugždaitė2, G. Palubinskas3, D. Juodžentė4, A. Rekešiūtė1,  \nH. Žilinskas1\n1 Large Animal Clinic, Faculty of Veterinary, Veterinary Academy,  \nLithuanian University of Health Sciences, Tilžės 18, LT-47181, Kaunas, Lithuania   \n2 Department of Veterinary Pathobiology, Veterinary Faculty,  \nVeterinary Academy, Lithuanian University of Health Sciences, Tilžės 18, LT-47181, Kaunas, Lithuania  \n3 Animal Breeding Department, Faculty of Animal Sciences,  \nVeterinary Academy, Lithuanian University of Health Sciences, Tilžės 18, LT-47181, Kaunas, Lithuania  \n4 Dr. L. Kriaučeliūnas Small Animal Clinic, Veterinary Faculty,  \nVeterinary Academy, Lithuanian University of Health Sciences, Tilžės 18, LT-47181, Kaunas, Lithuania\nAbstract\nThe aim of this study was to determine how puerperal metritis influences the resumption  \nof estrous cycle in dairy cows. The ovaries of 72 multiparous Holstein cows (38 healthy and  \n34 metritic – after treatment) were ultrasonographically scanned until the first ovulation post-  \npartum and 7 days after to confirm the ovulation. All 72 cows were divided in to 4 groups:  \nHSO (healthy with single ovulation) (n=29), MSO (metritic with single ovulation) (n=21),  \nHDO (healthy with double ovulation) (n=9), and MDO (metritic with double ovulation) (n=13). \nThe proportion of cows that had DO in the first ovulation postpartum was similar between M  \nand H groups, 38.2% and 23.6%, respectively (p>0.05). There was a difference between HDO \nand MDO groups comparing the first dominant follicle ovulation postpartum (11.4±2.7 and  \n20±1 days, respectively p <0.05) and the diameter of the ovulatory follicles (15.3±1.9 mm  \nand 17.3±1.7 mm, respectively p <0.05). The percentage of cows that had double follicle domi -\nnance in the first follicular wave after first ovulation was higher in the M groups (33.3% (MSO) \nvs. 6.9% (HSO) (p<0.05) and (69.2% (MDO) vs. 22.2% (HDO) (p<0.05)). The MSO group  \ndominant follicle diameter was bigger for cows which had one dominant follicle (p<0.05).  \nIt might be concluded that dairy cows after puerperal metritis need more time until the first ovu-\nlation. Also, metritic cows have a higher risk for double dominance in the first follicular wave, \nafter the first ovulation.\nKey words: corpus luteum, dominant follicle, ovulation, metritis, progesterone\n\n620\nV . Juodžentis et al.\nIntroduction\nFast recovery of ovarian activity postpartum is rele-\nvant for high yielding dairy cows. It greatly influences \nthe risk of successful insemination in early lactation. \nHowever, in modern dairy cows, slow resumption  \nof the estrous cycle postpartum leads to the negative \neffects which compromise fertility. The occurrence  \nof early ovulation is associated with the probability that \na cow will have frequent and normal estrous cycles, \nwhich will increase the reproductive performance  \nof the uterine environment (Thatcher et al. 2005).\nIn postpartum dairy cows, metritis is a common  \ndisease. It leads to milk production losses and also  \nto reduced fertility compared with healthy herd mates \n(Fourichon et al. 2000, Bell and Roberts 2007, Wittrock \net al. 2011, Giuliodori et al. 2013). The definitions  \nof metritis have been standardized by Sheldon et al. \n(2009). According to these authors, cows which have  \nan abnormally enlarged uterus and a fetid, reddish \nbrown, watery uterine discharge detectable in the vagi-\nna with the presence of pyrexia (≥39.5°C) are defined \nas having puerperal metritis. Puerperal metritis can be \nhighly prevalent in some dairy farms and can vary from \n7% to 20% for multiparous dairy cows (Benzaquen et \nal. 2007, Giuliodori et al. 2013, Armengol and Fraile \n2015). Furthermore, multiparous cows have a higher \nrisk of puerperal metritis than primiparous cows \n(Kawashima et al. 2006, Wittrock et al. 2011). However, \nthere are numerous studies investigating the resumption \nof ovarian activity in the postpartum period (Peter et al. \n1989, Kamimura et al. 1993, Sakaguchi et al. 2004, \nButler et al. 2006, Tanaka et al. 2008, Rajmon et al. \n2012). Most studies are conducted with cows that have \nno negative disorders postpartum. In the literature,  \nthe first ovulation in dairy cows is well characterized, \nbut there is no information on what proportion of cows \nhave problems with uterine diseases. There is a lack  \nof information about how the dairy cow estrous cycle \nrecovers after puerperal metritis, and how derivatives  \nin the ovaries (follicles and corpus luteum (CL)) change \nin early lactation for cows after puerperal metritis.\nThe aim of this study was to determine how puer -\nperal metritis and its treatment with antibiotic Ceftiofur \n(1 mg/kg s.c. Cevaxel RTU, Ceva, France) influences \nthe first ovulation and the rate of double ovulation  \nin healthy cows and cows after treatment for puerperal \nmetritis, the difference between cows with single and \ndouble ovulation on progesterone (P 4) concentration \nand CL volume on day 7 after the first ovulation bet-  \nween healthy cows and cows after puerperal metritis, \nand the difference in the first follicular wave after  \nthe first ovulation between healthy cows and cows  \nafter puerperal metritis treatment. \nMaterials and Methods\nThe study was carried out on lactating Holstein \ncows from a commercial dairy farm located in Lithua -\nnia, milking approximately 1100 cows, and averaging \napproximately 33 kg of milk/d. This study was per -\nformed from 2018 September to July 2019. The cows \nwere housed in freestall barns with access to fresh water \nad libitum  and were fed a total mixed ration supple -\nmented with the concentrate based on milk yield.  \nThe cows were milked with Lely Astronaut® A3 milk-\ning robots with free traffic. To motivate the cows  \nto visit the robot, 2 kg/day of concentrate was fed  \nto them by a milking robot. Data for daily milk yield \nand lactation days data were collected from the Lely \nT4C management program for analysis.\nThe study was carried out in compliance with EU \nlegislation. The procedures complied with the criteria \ngiven by the Lithuanian animal welfare regulations \n(No. B1-866, 2012; No. XI-2271, 2012) and the decree \nof the director of the State Food and Veterinary Service, \nRepublic of Lithuania (No. B6-(1.9)-855, 2017). \nFor this study, 85 cows (41 H and 44 M) were  \nenrolled, and 72 (38 H and 34 M) cows were used at the \nend of the analyses. The 13 cows not used were exclu-  \nded from the study because of the following disorders \nuntil first ovulation: mastitis (n=7), lameness (n=2) and \nleft displaced abomasum (n=4). Puerperal metritis was \ndiagnosed in 10 of the 13 cows. These cows were not \nincluded in our data analyses. All 72 cows were divided \ninto groups after their first ovulation: HSO (n=29) – \ncows without signs of puerperal metritis and with  \na single ovulation; MSO (n=21) – cows after puerperal \nmetritis treatment with a single ovulation; HDO (n=9) \n– cows without signs of puerperal metritis and with  \na double ovulation; and MDO (n=13) – cows after puer-\nperal metritis treatment with a double ovulation.\nIn the present study, the cows were selected bet-  \nween day 5 to 14 after calving (day 0 = day of calving). \nThe cows were divided into two different groups: mul -\ntiparous cows after puerperal metritis treatment (M) \nand multiparous cows without signs of puerperal metri-\ntis (H). There was no significant difference in parity \namong the groups, H group – 2.38±0.55 days and M \ngroup – 2.32±0.58 days. \nInformation about the reproductive history of the \nexperimental cows was also recorded. M group cows \ndelivered 58.8% (20/34) male calves before the study, \nwhile 36.8% (14/38) of male calves were delivered  \nby H group cows. 17.6% (6/34) of M group cows had \nthe incidence of dystocia compared to 5.3% (2/38)  \nof H group cows. Furthermore, M group cows had more \nstillbirths than H group cows (11.8% (4/34) and 5.3% \n(2/38), respectively) and there were more cows with  \n\n621\nInfluence of puerperal metritis on the first ovulation ...\nretained placenta in the M group compared to the H \ngroup (20.6% (7/34) and 0% (0/38), respectively).\nPuerperal metritis was diagnosed by rectal tempera-\nture and if the relevant uterine discharge was present \n(Sheldon et al. 2009). All the cows with signs of puer -\nperal metritis (abnormally enlarged uterus and a fetid, \nreddish brown, watery uterine discharge detectable in \nthe vagina with the presence of pyrexia (≥39.5°C) were \ntreated with Ceftiofur (1 mg/kg s.c. Cevaxel RTU, \nCeva, France) for 5 days. All the cows from the M \ngroup were checked after the treatment with Ceftiofur \nto evaluate the process of recovery. All these cows were \nchecked again on day 30. After uterus massage, cows \nwhich had purulent uterine discharge (>50% of pus) \n(20.6% (7/34) of M group cows) seen in the vagina \nwere treated with Clamoxyl metritis® intra-uterine  \nantibiotic infusion (Amoxicillin 0.84 g, Pfizer animal \nhealth, Belgium). At the end of the voluntary waiting \nperiod on this farm (50 days postpartum), all M group \ncows (also, additionally treated with Clamoxyl  \nmetritis® intra-uterine infusion) were considered  \nto be healthy if they had no vaginal discharge on day 50. \nAll cows were subdivided into two subgroups after \nthe first ovulation as follows: cows which ovulated one \ndominant follicle (SO) and cows which had double  \novulation (DO). All cows were examined from day  \n5 (day 0 = day of calving) three times per week (Mon -\nday, Wednesday, Friday) until the first ovulation and  \non the 7th day after this ovulation to evaluate cyclicity. \nThe changes in ovaries were examined using  \na digital diagnostic ultrasound scanner (Dramiński  \niScan, Dramiński S.A., Olsztyn, Poland) at a frequency \nof 7.5 MHz, using a linear rectal transducer.\nThe first dominance of the follicle postpartum  \nwas recorded when at least one of the follicles reached \n8.5 mm in diameter. To detect follicle ovulation,  \nthe cows were monitored by ultrasound machine three \ntimes a week (Monday, Wednesday, Friday). Ultraso -\nnography was started on day 5 postpartum and was con-\ntinued until the follicle ovulation was diagnosed.  \nThe follicle ovulation was diagnosed when a dominant \nfollicle (DF), which had been seen at previous examina-\ntion, was no longer visible. A CL with a diameter  \nof more than 7 mm was deemed to be the result of ovu-\nlation two days previously (day of ovulation = day 1), \nand the cycle stage was designated as day 3. When the \nDF was no longer present, and CL was not visible  \nor was smaller than 7 mm, the previous day was consi-  \ndered the day of ovulation (Struve et al 2013). The last \nsize measure of an ovulated follicle was recorded, and \nultrasonography was repeated on day 7 after ovulation \n(day of ovulation = day 0) to measure the volume of CL \nand the size of the follicles. Ultrasound measurements \nof CL were used to calculate average diameters (aver -\nage of length (L) and width (W) and volume (V).  \nThe volume of CL was calculated using the formula  \nV=4/3×π×R3 using a radius (R) calculated by the for -\nmula R=(L/2+W/2)/2 and π=3.14. For CL with a fluid- \n-filled cavity, the volume of the cavity was calculated \nand subtracted from the total CL volume (Sartori et al. \n2004). Results from mm3 were converted to cm3.\nBlood samples were collected into tubes without \nanticoagulants via a puncture from the median caudal \nblood vessels. The first blood sample was collected on \nday 7 postpartum and then every 7 days until ovulation, \nand on day 7 after ovulation (day of ovulation = day 0). \nTo take the blood samples at the correct time, the farm \nwas visited every day. Ovulation was determined  \nby ultrasonography. After collection, blood samples \nwere taken to the laboratory and centrifuged (2000 × g, \n20 minutes at 4°C), and serum was collected and stored \nat –20°C until analysis. The serum progesterone (P 4) \nconcentration was analyzed using chemiluminescent \nassay (Immulite, Siemens, Wales, UK) in an accredited \nlaboratory (Segalab, Portugal). The minimum detection \nlevel for P4 was 0.2 ng/mL.\nStatistical analysis was performed using SPSS 22 \ncomputer software. Averaged experimental results are \nreported as mean ± standard error of the mean.  \nAll the results between the groups were compared using \nthe independent t-test. The level of significance was  \nset at p<0.05.\nResults\nIn the present study, the proportion of cows that had \nSO in their first ovulation postpartum was similar bet-  \nween M and H groups, 61.8% and 76.4%, respectively \n(p>0.05). Also, the same tendency was observed in the \ncows with DO after the first ovulation, M group 38.2% \nand H group 23.6% (p>0.05).\nThe mean time to the first follicle deviation (selec -\ntion of the DF during follicular wave from the size  \nof 8.5 mm) postpartum was higher in the MSO group \ncompared with the HSO group, 8.9±1.6 and 6.8±1.8 \ndays postpartum, respectively (p<0.05). The same ten -\ndency was observed in the MDO and HDO groups, \n9.5±1.3 and 7.0±1.4 days postpartum, respectively \n(p<0.05). Overall, the mean time to the first follicle  \ndeviation inside M groups was longer (9.2±1.5 days), \ncompared to H group cows (6.9±1.6 days) (p<0.05).\nThere was no significant difference between HSO \nand MSO groups at the resumption of cyclicity on the \ndays with regard to the first DF ovulation postpartum \nand on the diameter of the ovulatory follicle at the first \nfollicular wave follicle ovulation (Table 1). Meanwhile, \nHDO group cows ovulated their follicle during the first \n\n622\nV . Juodžentis et al.\nfollicular wave faster compared to the MDO group \n(p<0.05) (Table 1). Also, HDO group cows had wider \ndiameter of the ovulatory follicle compared to the MDO \ngroup (p<0.05) (Table 1). Furthermore, we found that \nMDO cows ovulate their first dominant follicle less  \nfrequently compared with the HDO group (p<0.05)  \n(Table 1). Cows that did not ovulate their first DF post-\npartum, ovulated their DF’s from the second or later \nfollicular waves. We did not find any significant diffe-  \nrence in later ovulations between HSO and MSO,  \nnor between HDO and MDO groups at the time of ovu-\nlation and the diameter of the ovulatory follicle (p>0.05) \n(Table 1).\nComparing HSO and MSO groups and HDO and \nMDO groups, there were no significant differences bet- \nween the diameter of the ovulatory follicles, CL volume \nat day 7 and P4 concentration at day 7 (p>0.05) (Fig. 1). \nComparing total 1st follicular and 2nd and later fol-\nlicular waves ovulatory follicle diameters, in H and M \ngroups , we found that HSO and MSO group cows had \nsignificantly larger ovulatory follicles than the cows  \nin the HDO and MDO groups, respectively (p<0.05) \n(Fig. 1). We did not find any significant difference in the \nCL volume between HSO and HDO groups at day 7 \nafter ovulation. Meanwhile, there was a significant dif-\nference in the CL volume between cows from the MSO \nand MDO groups at day 7 after ovulation (8.05±2.40 \ncm3 and 10.91±3.71 cm 3, respectively, p<0.05). Com -\nparative analysis revealed no difference in the P4 con -\ncentration between SO and DO cows in both H and M \ngroups (p>0.05) (Fig. 1).\nA double-dominance of the first follicle wave after \nthe first ovulation was also observed. It was found that \nMSO and MDO group cows had a significant difference \nTable 1. First ovulation after calving in cows.\nCategory\nGroups\nH M\nHSO (n/n) HDO (n/n) MSO (n/n) MDO (n/n)\n1st follicular wave follicle ovulation\n % of cows which ovulate during their 1 st follicle wave (n/n) 41.4 (12/29) 55.6a (5/9) 47.6 (10/21) 23.1b (3/13)\nDays 15.9±3 11.4±2.7c 18.8±6.3 20±1d\nDiameter of the ovulatory follicle (mm) 22.5±3.5 15.3±1.9e 19.2±3.5 17.3±1.7f\n2nd and later follicular waves ovulation\n% of cows which ovulate during their 2 nd and later follicle waves (n/n) 58.6 (17/29) 44.4 (4/9) 52.4 (11/21) 76.9 (10/13)\nDays 37±8.9 30.5±7.8 35.9±10 32.9±6.5\nDiameter of the ovulatory follicle (mm) 19.8±3.9 15.3±1.9 20.1±4.7 16.5±2.7\nHSO – cows without signs of puerperal metritis – single ovulation, MSO – cows after puerperal metritis – single ovulation, HDO – cows \nwithout signs of puerperal metritis – double ovulation, MDO – cows after puerperal metritis – double ovulation. Significant difference \nbetween groups with the letters a, b; c, d; e, f (p<0.05).\nTable 2. Differences in the follicle size, corpus luteum volume and progesterone concentration between H and M groups of cows  \non day 7 post ovulation.\nItem\nGroups\nH M\nHSO (n/n) HDO (n/n) MSO (n/n) MDO (n/n)\nDominant follicle size at day 7 post ovulation (mm)\nOne dominant follicle 14.6±1.1 (27/29) 14.3±0.9 (7/9) 14.1±0.7a (14/21) 13.8±0.4 (4/13)\nTwo dominant follicles 13.5±0.6 (2/29) 13.5±0.6 (2/9) 13.4±0.5b (7/21) 13.6±0.9 (9/13)\nCorpus luteum volume at day 7 post ovulation (cm3)\nOne dominant follicle 9.51±4.01 (27/29) 12.14±4.83 (7/9) 8.29±2.59 (14/21) 9.44±3.72 (4/13)\nTwo dominant follicles 10.95±6.50 (2/29) 13.78±0.33 (2/9) 7.55±2.04 (7/21) 11.56±3.71 (9/13)\nProgesterone concentration at day 7 post ovulation (ng/mL)\nOne dominant follicle 2.8±1.6 (27/29) 3.1±0.9 (7/9) 2.8±1.3 (14/21) 3.1±0.6 (4/13)\nTwo dominant follicles 2.6±1.1 (2/29) 2.7±0.8 (2/9) 2.5±0.7 (7/21) 3.3±0.9 (9/13)\nHSO – cows without signs of puerperal metritis – single ovulation, MSO – cows after puerperal metritis – single ovulation, HDO – cows \nwithout signs of puerperal metritis – double ovulation, MDO – cows after puerperal metritis – double ovulation. \nH – cows without signs of puerperal metritis, M – cows after puerperal metritis. Significant difference between subgroups with the letters \na, b; (p<0.05).\n\n623\nInfluence of puerperal metritis on the first ovulation ...\nfor double follicle dominance on day 7 after the first \novulation compared to HSO and HDO groups, respec -\ntively; between MSO and HSO groups (33.3% and \n6.9%, respectively, p<0.05), and between MDO and \nHDO groups (69.2% and 22.2%, respectively, p<0.05).\nThe diameter of the follicles on day 7 after ovula -\ntion was compared in all four groups (HSO, HDO, \nMSO and MDO). There was no significant difference  \nin follicle diameter between groups with one or two \ndominant follicles in groups HSO, HDO and MDO \n(p>0.05) (Table 2). Meanwhile, dominant follicle  \ndiameter was wider for cows which had one dominant \nfollicle in the group MSO (p<0.05) (Table 2). Compa-  \nrative analysis revealed no difference at day 7 in all the \ngroups on CL volume between cows with one and two \ndominant follicles (p>0.05) (Table 2). Furthermore, \nthere was no significant difference on progesterone con-\ncentration on day 7 in all the groups between cows with \none dominant follicle and two dominant follicles \n(p>0.05) (Table 2).\nDiscussion\nIn the postpartum period, dairy cows face many \nchallenges. Fast recovery of ovarian activity at the time \nof postpartum in high-producing dairy cows is critical \nfor good reproduction. One of the criteria for a postpar-\ntum cow to have good reproductive health is the time  \nof the first ovulation. A shorter interval to the first ovu-\nlation leads to a reduced interval to conception and  \nincreased conception rates (Tanaka et al. 2008). How -\never, such disease as puerperal metritis leads to nega -\ntive effects which can compromise fertility.\nPuerperal metritis is highly prevalent in some dairy \nfarms and can vary from 7% to 20% for multiparous \ndairy cows (Benzaquen et al. 2007, Giuliodori et al. \n2013, Armengol and Fraile 2015). It is well known that \nthe impact of puerperal metritis on dairy cow repro-  \nduction can cause significant losses in production  \n(Benzaquen et al. 2007, Dubuc et al. 2010, Martinez  \net al. 2012, Giuliodori et al. 2013, Lima et al. 2014, \nArmengol and Fraile 2015, Lopez-Helguera et al. \n2016). In the present study, the incidence of puerperal \nmetritis for multiparous cows in the herd was 9.2%.  \nOur results ware those obtained by Benzaquen et al. \n(2007) and Armengol and Fraile (2015). They found \nthat the cases of puerperal metritis were 7.5% and 7.9%, \nrespectively (Benzaquen et al. 2007, Armengol and \nFraile 2015).\nNowadays, modern dairy cows produce great quan-\ntities of milk because of genetic improvement and nutri-\ntional management optimized through lactation  \n(Friggens et al. 2010). Such changes in production  \nare related to changes in reproduction physiology.  \nThe greatest incidence of double ovulation is when \ncows produce >40 kg/d. It is clear that such cows have \nincreased metabolism of hormones which leads to dou-\nble ovulation (Lopez et al. 2005). However, in relation \nto the first ovulation after calving when preovulatory \nfollicles are growing in sub-luteal P4 concentrations, \nthere is no difference between cows with single or dou-\nble ovulation on milk production (Lopez-Helguera et al. \n2016, Macmillian et al. 2018). A possible reason for \ndouble ovulations at the first ovulation after calving  \nfor cows with uterine disease could be lipopolysaccha -\nrides (LPS) in the uterus. LPS decreases LH pulse fre -\nquency and decreases estradiol, and such cows, by the \ntime of deviation, have higher levels of FSH than nor -\nmal, which leads to co-dominance of two or more folli-\nFigure 1. Comparison within the H and M groups of cows. HSO – cows without signs of puerperal metritis – single ovulation, MSO – \ncows after puerperal metritis – single ovulation, HDO – cows without signs of puerperal metritis – double ovulation, MDO – cows after \npuerperal metritis – double ovulation. Significant difference between groups with the same tags (p<0.05)\n\n624\nV . Juodžentis et al.\ncles in the follicular wave (Wiltbank et al. 2000, Lavon \net al. 2008). This explains our results, as M group cows \nhad more double ovulation at the time of first ovulation \ncompared to H group cows, but the difference was not \nsignificant. Both M and H group results are in agree -\nment with other studies where incidence of DO follow-\ning anestrus is from 29 to 58.8% (Lopez et al. 2005, \nStevenson 2016, Kusaka et al. 2017, Macmillan et al. \n2018). However, Stevenson et al. (2006) claim that DO \nincidence in the first ovulation following anestrus  \nis lower and reaches 15%.The results of the present \nstudy support the idea that infection slows down follic-\nular growth for cows with uterine disease by reducing \ncirculating estradiol (E 2) and perturbing prostaglandin \nsignaling (Sheldon et al. 2002, Herath 2007). Probably, \nfor this reason, M group cows showed the first DF  \napproximately 2 days later than the healthy cows \n(p<0.05). We also observed that cows with uterine dis -\nease emerge follicle waves at the same time as healthy \ncows as in other studies (Sheldon et al. 2002, Herath \n2007). The present study results from groups H and M \nare in agreement with previous studies, where deviation \noccured between 5 to 10 days postpartum (Savio et al. \n1990, Crowe 2008, Tanaka et al. 2008). On the other \nhand, Rajmon et al. (2012) found that the first follicle \ndeviation in multiparous cows was seen from day 15±3. \nAccording to Savio et al. (1990), Sakaguchi et al. \n(2004) and Kawashima et al. (2006), 38–73% of a herd \nhas first wave follicle ovulation. We can confirm this \nstatement, as the findings in the HSO, MSO and HDO \ngroups were similar. According to Crowe (2008),  \nthe first wave follicles could ovulate in 50–80%  \nof a herd by day 20. Tanaka et al. (2008) claim that 83% \nof multiparous dairy cows ovulated their first wave DF. \nHowever, Rajmon et al. (2012) claim that only 29%  \nof multiparous cows ovulated their first DF. Our study \nresults of group MDO support the results of Rajmon  \net al. (2012). However, in the above studies, there was \nno information about cows which had double ovulation \nat the time of the first DF ovulation or which had puer-\nperal metritis.\nThe mean time of the first DF ovulation postpartum \nwas observed in the groups by day 20. According to \nButler et al. (2006), the first DF ovulation in mature \nHolstein cows was on day 16.6±1.6 postpartum. Kami-\nmura et al. (1993), Kawashima et al. (2006), Sakaguchi \net al. (2004) and Tanaka et al. (2008) reported the first \novulation on day 17±1, 17±4, 18.1±1 and 17.3±6.3,  \nrespectively. Similar results were observed during our \nstudy in groups HSO, MSO and MDO. HDO group \ncows had their first DF ovulation on day 11.4±2.7, \nprobably due to the high feed intake and low negative \nenergy balance after calving which ensure appropriate \nLH pulse frequency and plasma IGF-1 level. This leads \nto the fast recovery of ovarian activity and first ovula -\ntion after calving (Beam and Butler 1999). The second \nand later DF ovulation usually occurs between 36±4 \nand 38±7 days postpartum as revealed in the HSO and \nMSO groups during our study (Kamimura et al. 1993, \nKawashima et al. 2006, Rajmon et al. 2012). According \nto the results, it is clear that most cows ovulate their first \nwave DF postpartum by day 20. \nWe also observed the size of the first ovulating fol-\nlicle postpartum. The follicles of the HSO group \n(22.5±3.5 mm) were larger compared to the MSO group \n(19.2±3.5 mm) at the time of ovulation. Butler et al. \n(2006) and Rajmon et al. (2012) found that multiparous \ncows ovulated their first DF at the size of 18.6±1.2 mm \nand 16.0±0.6 mm, respectively. Our results from  \nthe HDO and MDO groups are in agreement with those \nof Rajmon et al. (2012). Moreover, these two studies \nfrom Butler et al. (2006) and Rajmon et al. (2012) could \nexplain the results in our groups if we combine them \ntogether (HSO, MSO, HDO, MDO – 18.5±3.9 mm),  \nbecause these studies did not mention the cow’s health \nstatus and the double ovulation rate. The smaller follic-\nular size in the MSO group could be explained by defi-\nciency of luteinizing hormone (LH) pulses caused  \nby a more pronounced negative energy balance than  \nin healthy cows (Beam and Butler 1999). Besides, lipo-\npolysaccharides (LPS), which are produced by bacteria \nsuch as E. coli, could also contribute to the deficiency \nof LH pulses by decreasing gonadotropin realizing  \nhormone (GnRH) secretion from the hypothalamus  \n(Peter et al. 1989, Sheldon et al. 2009, Kass é et al. \n2016). For this reason, cows with postpartum uterine \ndisease have slower growth of the first postpartum DF \nand lower peripheral plasma E 2 concentrations around \nthe time of the maximal follicle diameter (Sheldon et al. \n2002, Williams et al. 2007). However, the follicle size \nin the MDO group was larger compared with that in  \nthe HDO group. This could be explained by the time  \nof the first DF ovulation. In the HDO group the first DF \novulation was earlier than in the MDO group (11.4±2.7 \nand 20±1 days), so the DF had less time to grow.\nInflammation of the uterus is associated with smal- \nler first CL postpartum (Williams et al. 2007, Strüve  \net al. 2013). The results of the present study support this \nidea; the size of the first CL on day 7 after the first ovu-\nlation in the MSO and MDO groups were smaller than \nin the HSO and HDO groups. As with the other authors, \nwe did not notice a significant difference between the \ngroups in the P4 concentration on day 7 after ovulation \n(Strüve et al. 2013). During our study, CL volume and \nP4 concentration were higher in the double ovulation \ngroup compared to the single ovulation group. Accor-  \nding to other authors, cows which had double ovulation \n7 days later had a lower P 4 concentration compared to \n\n625\nInfluence of puerperal metritis on the first ovulation ...\ncows after single ovulation (2.5±0.3 vs. 3.2±0.1 ng/mL) \ndespite a greater CL volume (8.29±0.51 vs. 6.40±0.15 cm3) \n(Lopez et al. 2005). Our results may differ, because we \nstarted to analyze cows at the beginning of lactation \n(until the first ovulation and 7 days after first ovulation); \ntherefore, it is possible that at this time the metabolism \nof hormones is not so high. \nAccording to Sartori et al. (2002), significantly larger \nfollicles were found for single ovulators. The same  \ntendency was observed between HSO and HDO, and \nbetween MSO and MDO groups in our study. Also, \nduring our study the CL volume at day 7 after ovulation \nwas larger in the MSO group compared to the MDO \ngroup, as in Sartori et al. (2002) (p<0.05). Comparative \nanalysis revealed no difference in the P4 concentration \nbetween SO and DO cows in both H and M groups as in \nSartori et al. (2002) experiment (p>0.05) (Fig. 1). \nThe double dominance of the first follicle wave  \nafter the first ovulation was also observed in MSO and \nMDO groups compared to HSO and HDO groups,  \nrespectively (p<0.05). According to the literature,  \nthe average incidence of multiple ovulations in healthy \nlactating dairy cows ranges from 10.3% to 22.4% \n(Bleach et al. 2004, Lopez et al. 2005, Lopez-Gatius  \net al. 2005, Stevenson et al. 2006, Stevenson 2016). \nHowever, cows which have uterine infections have  \na higher risk of multiple ovulations. Uterine infections \nsoon after calving delay the return to cyclicity. Such \ncows have a lower P 4 environment before and after  \ndeviation, increased FSH and LH before the deviation, \nand increased E2 after deviation (Macmillan et al. 2018).\nConclusions\nDairy cows which have had puerperal metritis need \nmore time until the first ovulation. Also, metritic cows \nhave a higher risk of double dominance in the first fol -\nlicular wave, after the first ovulation.\nAcknowledgements\nThis study was supported by the Science Founda -\ntion of the Lithuanian University of Health Sciences \n(LUHS).\nReferences\nArmengol R, Fraile L (2015) Comparison of two treatment \nstrategies for cows with metritis in high-risk lactating \ndairy cows. Theriogenology 83: 1344-1351.\nBeam SW, Butler WR (1999) Effects of energy balance on \nfollicular development and first ovulation in postpartum \ndairy cows. J Reprod Fertil Suppl 54: 411-424.\nBell MJ, Roberts DJ (2007) The impact of uterine infection on \na dairy cow’s performance. 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