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Hemostatic profile during late pregnancy and early postpartum period in mares
Theriogenology 81 (2014) 639–643
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Theriogenology journal homepage: www.theriojournal.com
Hemostatic profile during late pregnancy and early postpartum period in mares M. Bazzano, C. Giannetto, F. Fazio, S. Marafioti, E. Giudice, G. Piccione* Department of Veterinary Science, University of Messina, Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2013 Received in revised form 28 November 2013 Accepted 6 December 2013
Hemostasis is a physiological process that prevents excessive blood loss and represents a protective mechanism at the time of delivery. Peripartum hemorrhage is a recurring hazardous condition to mare’s health; therefore, we aimed to study mares’ hemostatic profile to investigate whether physiological adjustments occur during late pregnancy and early postpartum. Fifteen pregnant mares have been monitored from the 34th week of pregnancy until the third week after foaling. Fifteen nonpregnant mares were used as control group. Jugular blood samples were analyzed for platelet count (Plt), prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fb). Platelet count showed significant changes at foaling (P < 0.05) and a negative correlation (r ¼ 0.968; P ¼ 0.032) with postpartum. Prothrombin time changed (P < 0.05) showing a significant correlation (r ¼ 0.675; P ¼ 0.016) with late pregnancy. Fibrinogen concentrations changed throughout the experimental period (P < 0.0001). The linear regression model revealed a positive correlation (r ¼ 0.9210; P < 0.0001) between Fb and late pregnancy and a negative correlation (r ¼ 0.9583; P ¼ 0.042) between Fb and early postpartum. The shortening in PT recorded in the imminence of parturition along with the increase in Plt and Fb at foaling might reflect a physiological hypercoagulable state that constrains excessive bleeding, enhancing mares’ odds of surviving. Our research improves the knowledge about blood coagulation in periparturient mares providing specific information on routine coagulation tests that may support in monitoring mare’s hemostatic profile during late pregnancy and early postpartum. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Mare Pregnancy Postpartum Hemostasis
1. Introduction Pregnancy is a physiological condition that significantly influences animal metabolism [1]. Most adjustments occur during late gestation and postpartum after hormonal changes [2,3]. Although several studies dealt with the assessment of blood coagulation in pregnant women [4–6], few researches concerned with hemostatic profile in domestic species such as cows [7,8], sows [9], mares [10], and dogs [11–13] during pregnancy. Hemostasis is a physiological process that prevents excessive
* Corresponding author. Tel.: þ39 0903503584; fax: þ39 0903503975. E-mail address: [email protected] (G. Piccione). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.12.003
blood loss from damaged vessels and represents a protective mechanism at the time of delivery [14]. In women, normal pregnancy is often associated with major changes in blood coagulation and fibrinolysis causing hypercoagulability. This event is probably because of oestradiolinduced triglyceride changes and protects women from fatal hemorrhage during delivery [5]. Also in equine species, the peripartum hemorrhage is one of the most common problems. The rupture of the middle uterine, iliac, utero–ovarian, pudendal or vaginal artery usually occurs during or after parturition. However, few mares may hemorrhage during midgestation to late gestation or up to several days after delivery [15]. The vascular damage can lead the mare to a rapid and profound blood loss resulting in hypovolemic shock and death [15–18]. A few
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M. Bazzano et al. / Theriogenology 81 (2014) 639–643
research dealt with physiological modifications in pregnant mares and it is well known that significant changes occur within this period [16,19,20]. Specific attention was paid to hormonal changes around parturition, and mare’s hormonal profile is widely recognized [2,3,21]. Hematological and biochemical changes have also been studied during pregnancy [19], whereas little is known about mare’s coagulation profile during the peripartum period [10]. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and platelet count (Plt) are the minimum-recommended laboratory tests for hemostasis. Prothrombin time reflects the function of the extrinsic pathway and aPTT monitors the changes in the intrinsic system and common pathway [22]. We assessed fibrinogen (Fb) to obtain further information about blood coagulation as well. Peripartum hemorrhage represents a recurring hazardous condition to mare’s health; therefore, we aimed to study mares’ hemostatic profiles to investigate whether physiological adjustments occur during late pregnancy and early postpartum.
2. Materials and methods 2.1. Animals Thirty healthy mares of different breed and age (Table 1) were enrolled in the study with the informed owner consent. The study was realized from November 2012 to June 2013 in Sicily (latitude 37.46 N; longitude 14.93 E). Animals were housed in individual straw-bedded boxes (4.0 3.5 m) at the same breeding center and were kept under natural environmental conditions. Fifteen pregnant mares (group A) were monitored from the 34th week of pregnancy until the third week after foaling. Fifteen nonpregnant mares (group B) were used as control group. All deliveries occurred within March and midMay. Mares from group A were subjected to daily clinical examination over the first 3 days after foaling. During postpartum, transrectal ultrasound exams were performed weekly to
ensure the normal involution of the uterus using the M-Turbo ultrasound system (FUJIFILM SonoSite, London, UK). Animals were fed twice a day (7 AM and 5 PM) and water was available ad libitum. Diet consisted of 6 1 kg/ day hay and 5 0.5 kg/day concentrates (crude protein 16%, crude fat 6%, crude fiber 7.35%, ash 10.09%, sodium 0.46%, lysine 0.85%, methionine 0.35%, and omega-3 0.65%). Animals were allowed to go to pasture during the day (10 AM–4 PM) as well. All treatments, housing and animal care were carried out in accordance with the standards recommended by the EU Directive 2010/63/EU for animal experiments. 2.2. Data collection Sampling was performed weekly on the same day in the morning (8 AM) until the time of parturition. All mares delivered within a week (5 2 days) from the last prepartum sampling. Therefore, we expressed each time point before foaling (BF) as weeks BF. Additional samples were taken from each mare within 24 12 hours from foaling (F) and then 7, 14, and 21 days after foaling (AF). Blood samples were collected by jugular venipuncture into 3.6-mL vacutainer tubes containing 3.8% of sodium citrate (Terumo Corporation, Tokyo, Japan) and 3-mL vacutainer tubes containing EDTA (Terumo Corporation). Whole blood with sodium citrate was centrifuged at 1500 g for 15 minutes within 30 minutes from the collection. Citrated plasma and EDTA whole blood samples were placed on ice, delivered to the laboratory, and processed within 2 hours. Platelet count was performed on EDTA whole blood samples using the HeCoVet automatic analyzer (SEAC, Florence, Italy). Sodium citrate plasma was analyzed for PT, aPTT, and Fb using standard kits for Clot 2 coagulometer (SEAC) as described by Casella, et al. [23]. 2.3. Statistical analysis All data are expressed as means standard error of the mean. Two-way repeated measures analysis of variance
Table 1 Breed and age (years) of experimental (group A) and control (group B) mares. Gestation length (days) and parity (þ, multiparous; , primiparous) have been indicated for group A. No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Breed
Gestation length (days)
Parity
Group A
Group B
Group A
Age (y) Group B
Group A
Group A
Selle Français Standardbred Thoroughbred Italian Saddle Paint Italian Saddle Thoroughbred Thoroughbred Italian Saddle Italian Saddle Italian Saddle KWPN Rheinlander KWPN Italian Saddle
Holsteiner Italian Saddle Standardbred KWPN Italian Saddle Thoroughbred Thoroughbred Standardbred Quarter Italian Saddle KWPN Italian Saddle Standardbred Holsteiner Italian Saddle
17 4 12 16 8 6 5 5 9 10 3 17 14 10 12
12 4 6 10 7 6 4 8 7 18 12 4 6 15 18
337 360 346 341 350 356 335 323 333 344 345 328 338 331 339
þ þ þ þ þ þ þ þ þ þ þ
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Table 2 Mean values standard error of the mean with statistical significances of Plt, PT, aPTT, and Fb recorded for group A throughout the experimental period. Time (wk)
Plt (103/mL)
12 11 10 9 8 7 6 5 4 3 2 1 Foaling þ1 þ2 þ3
141.0 141.0 142.1 145.2 148.5 149.4 149.8 147.1 145.7 146.4 141.0 148.4 170.8 159.9 143.7 140.8
8.666a 8.464a 6.906a 6.506a 8.147a 8.374a 7.673a 8.231a 9.620a 6.475a 7.376a 8.742a 6.229 6.128a 4.948a 6.884a
PT (s) 11.43 11.50 11.73 11.67 11.68 11.71 11.71 11.51 11.49 11.23 11.10 10.81 11.16 11.38 11.15 10.94
aPTT (s)
0.201 0.250 0.135e 0.296e 0.222e 0.251e 0.259e 0.175 0.173 0.294 0.172 0.171 0.194 0.216 0.118 0.082
42.32 41.35 41.15 41.32 42.44 42.24 43.42 42.63 41.55 42.30 42.33 41.37 42.16 41.92 41.77 40.44
Fb (mg/dL) 0.526 0.673 1.274 0.192 1.686 0.522 0.915 1.107 1.103 0.903 0.944 0.990 1.788 1.180 1.183 0.540
139.2 149.2 148.3 152.4 150.0 153.8 166.7 159.1 162.2 185.8 190.0 190.6 204.0 185.3 180.2 170.6
5.283abcdef 4.598abcdef 8.637abcdefg 2.796abcefg 6.903abcdef 3.022abcefg 5.311aefgi 3.690abcefgi 4.184abefgi 6.561hi 5.464d 4.290d 4.607 3.262a 3.743a 4.353a
Significance: P < 0.05. a versus Foaling; b versus þ1; c versus þ2; d versus þ3; e versus 1; f versus 2; g versus 3; h versus 11; i versus 12. Abbreviations: aPTT, activated partial thromboplastin time; Fb, fibrinogen; Plt, platelet count; PT, prothrombin time.
(ANOVA) and Bonferronis post hoc comparison were applied to evaluate statistically significant effects of the experimental period on Plt, PT, aPTT, and Fb levels. A linear regression model (y ¼ a þ bx) was applied to determine the degree of correlation between each clotting parameter and the time before and after foaling, respectively. P-values less than 0.05 were considered statistically significant. Statistical analysis was performed using the PRISM 4 statistical package (GraphPad Software Inc., San Diego, CA, USA). 3. Results Animals included in group A showed no clinical signs of disease throughout their pregnancy. All subjects delivered healthy, viable foals at full-term gestation, without human assistance. All mares had shed the placenta spontaneously within 2 hours and achieved the complete involution of the uterus within 2 weeks after foaling. Table 2 showed means standard error of the mean of Plt, PT, aPTT, and Fb obtained from pregnant mares throughout the experimental period.
3.1. Platelet count Platelets reached the highest concentration at F in group A (Fig. 1A). This increase was statistically significant (P < 0.05) compared with the other time points recorded during the experimental period. The linear regression model showed a negative correlation (r ¼ 0.968; P ¼ 0.032) between Plt and AF (Fig. 2). No correlation was found between Plt and BF.
3.2. Prothrombin and activated partial thromboplastin times Two-way ANOVA showed a significant influence of late pregnancy on PT that reached the lowest values at one BF (Fig. 1B). The linear regression model showed a significant
correlation (r ¼ 0.675; P ¼ 0.016) between PT values and BF (Fig. 3). No statistical significant difference was found in aPTT values throughout the study (Fig. 1C).
3.3. Fibrinogen Two-way ANOVA revealed a significant influence of the experimental period on Fb concentration (P < 0.0001) that reached its peak (204.0 4.607 mg/dL) at F (Fig. 1D). The linear regression model revealed a positive correlation (r ¼ 0.9210; P < 0.0001) between Fb levels and BF (Fig. 4A) and a negative correlation (r ¼ 0.9583; P ¼ 0.042) between Fb and AF (Fig. 4B). 4. Discussion In the present study, significant changes occurred in Plt, PT, and Fb levels in periparturient mares. Platelet count showed no change during pregnancy, whereas a significant increase occurred at foaling. However, the negative correlation between Plt and the postpartum period displayed a return to Plt basal levels within the third week after foaling. This variance might represent an adaptive response to blood loss that usually occurs at the time of delivery. Actually, a common misconception is that blood loss causes thrombocytopenia. In contrast, Plt may even increase during the first few hours after blood loss as a result of the splenic contraction that is most prominent in horses [24]. Prothrombin time and aPTT values showed no pathological alterations and stayed within the reference range [25]. However, we found a significant shortening in PT levels around parturition with the lowest value recorded at the last time point before foaling. The same PT trend was found in healthy pregnant women [6] suggesting that a decrease in PT time might be a common condition during late pregnancy. Another study showed a decrease in PT values around parturition in mares [10] although it was not considered statistically significant. In addition, PT and
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M. Bazzano et al. / Theriogenology 81 (2014) 639–643
Fig. 1. Trend standard error of the mean of Plt (A), PT (B), aPTT (C), and Fb (D) recorded for experimental (group A) and control (group B) mares during the study. Time was expressed as weeks before foaling (BF), foaling time (F) and weeks after foaling (AF). aPTT, activated partial thromboplastin time; Fb, fibrinogen; Plt, platelet count; PT, prothrombin time.
aPTT mean values recorded by Gentry, et al. [10] were higher when compared with our results. This gap might be because of the differences in samples’ storage and assay methods [23,26]. However, sampling procedures and assay methods we used in the present study complied with Shalm’s laboratory testing of coagulation disorders [27]. Among hemostatic parameters, Fb exhibited the most significant changes throughout the study, showing a positive correlation with pregnancy and a negative one with postpartum period. In our study, the highest Fb concentration was recorded at the time of delivery as observed previously in women [6], cows [7,8], sows [9], bitches [11], and mares [10]. In women, it has been postulated that this increase in Fb levels, observed at parturition, is because of
an increased synthesis related to the local use of Fb in uteroplacental circulation [28]. Also in mares this might be a contributing factor [10]. However, besides providing a substrate for fibrin formation, Fb is also a positive acute phase protein [29]. It may react at parturition [30] because this time represents a critical stage that requires mare to carry out a great physical effort [19]. In addition, several research investigated the relationship between stress and coagulation. Their results strongly suggest that healthy organisms tend to develop a physiological hypercoagulable state in response to acute stress [14]. On the basis of this knowledge, the shortening in PT recorded in the imminence of parturition along with the increase in Plt count and Fb plasma concentrations at foaling might
Fig. 2. Linear regression between Plt count and postpartum period (weeks AF) in group A. AF, after foaling; Plt, platelet.
Fig. 3. Linear regression between PT and late pregnancy (weeks BF) in group A. BF, before foaling; PT, prothrombin time.
M. Bazzano et al. / Theriogenology 81 (2014) 639–643
Fig. 4. Linear regression between Fb and late pregnancy (A), and between Fb and postpartum period (B) in group A. AF, after foaling; BF, before foaling; Fb, fibrinogen.
reflect a physiological hypercoagulable state that constrains excessive bleeding enhancing mares’ odds of surviving. 4.1. Conclusions Our research improves the knowledge about blood coagulation in periparturient mares, showing how hemostatic parameters change at this stage. Actually, both pregnancy and early postpartum represent paraphysiological conditions and reference ranges for hemostatic assays based on healthy male or nonpregnant female might be inappropriate. In effect, they may hinder the accurate diagnosis and effective treatment of hemostatic disorders that may occur during pregnancy. Therefore, our study provides specific information on routine coagulation tests that may support in monitoring mare’s hemostatic profile during the peripartum period. References [1] Filipovi c N, Stojevi c Z, Prvanovi c N, Tucek Z. The influence of late pregnancy and lactation on bone metabolism in mares. Res Vet Sci 2010;88:405–10. [2] Berg EL, McNamara DL, Keisler DH. Endocrine profiles of periparturient mares and their foals. J Anim Sci 2007;85:1660–8. [3] Vivrette S. The endocrinology of parturition in the mare. Vet Clin North Am Equine Pract 1994;10:1–17. [4] Paniccia R, Prisco D, Bandinelli B, Fedia S, Giustia B, Pepe G, et al. Plasma and serum levels of D-dimer and their correlations with other hemostatic parameters in pregnancy. Thromb Res 2002;105: 257–62. [5] Bremme A. Haemostatic changes in pregnancy. Best Pract Res Clin Haematol 2003;16:153–68.
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[6] Liu J, Yuan E, Lee L. Gestational age-specific reference intervals for routine haemostatic assays during normal pregnancy. Clin Chim Acta 2012;413:258–61. [7] Gentry PA, Liptrap RM, Black WD. Changes in blood coagulation profiles of dairy cows during pregnancy and in heifer calves after hormone treatment. Can J Anim Sci 1979;59:503–10. [8] Heuwieser W, Kautni J, Biesel M, Grunert E. Coagulation profile of dairy cattle in the periparturient period. J Vet Med A 1990;37: 8–15. [9] Liptrap RM, Gentry PA. Interrelationships between blood coagulation factors and plasma hormone levels in the periparturient sow. Can J Anim Sci 1984;64:655–65. [10] Gentry PA, Feldman BF, O’Neill SL, Madigan JE, Zinkl JG. Evaluation of the haemostatic profile in the pre- and post parturient mare, with particular focus on the perinatal period. Equine Vet J 1992;24:33–6. [11] Gentry PA, Liptrap RM. Influence of progesterone and pregnancy on canine fibrinogen values. J Small Anim Pract 1981;22:185–94. [12] Concannon PW, Gimpel T, Newton L, Castracane VD. Postimplantation increase in plasma fibrinogen concentration with increase in relaxin concentration in pregnant dogs. Am J Vet Res 1996; 57:1382–5. [13] Gunzel-Apel AR, Hayer M, Mischke R, Wirth W, Hoppen HO. Dynamics of haemostasis during the oestrous cycle and pregnancy in bitches. J Reprod Fertil Suppl 1997;51:185–93. [14] von Känel R. Hemostasis and stress. In: Fink G, editor. Encyclopedia of stress. 2nd ed. Oxford: Elsevier Academic Press; 2007. p. 300–5. [15] LeBlanc MM. Peripartum problems in the mare. J Equine Vet Sci 2008;28:709–15. [16] Le Blanc MM. Disease of the reproductive system: the mare. In: Colahan PT, Mayhew IG, Merritt AM, Moore J, editors. Equine medicine and surgery. Goleta: American Veterinary Publications Inc.; 1991. p. 949–1081. [17] Evans JW, Borton A, Hintz H, Va Vleck LD. Anatomy and physiology of reproduction in the mare. In: Evans JW, Borton A, Hintz H, Va Vleck LD, editors. The horse. 2th ed. New York: Freeman WH and Company; 1997. p. 315–53. [18] Zent WW. Postpartum complications. In: Robinson E, editor. Current therapy in equine medicine. 2nd ed. Philadelphia: WB Saunders Company; 1987. p. 554–7. [19] Aoki T, Ishii M. Hematological and biochemical profiles in peripartum mares and neonatal foals (heavy draft horse). J Equine Vet Sci 2012;32:170–6. [20] Aoki T, Honda H, Ishii M. Immunologic profiles of peripheral blood leukocytes and serum immunoglobulin G concentrations in perinatal mares and neonatal foals (heavy draft horse). J Equine Vet Sci 2013;33:989–95. [21] Ousey JC. Peripartal endocrinology in the mare and foetus. Reprod Dom Anim 2004;39:222–31. [22] Criswell KA. Preclinical evaluation of compound-related alteration in hemostasis. In: Weiss DJ, Wardrop KJ, editors. Shalm’s veterinary hematology. 6th ed. Ames: Wiley-Blackwell; 2010. p. 92–7. [23] Casella S, Giannetto C, Fazio F, Giudice E, Piccione G. Assessment of prothrombin time, activated partial thromboplastin time, ad fibrinogen concentration on equine plasma samples following different storage conditions. J Vet Diagn Inverst 2009;21:674–8. [24] Tvedten H. Laboratory and clinical diagnosis of anemia. In: Weiss DJ, Wardrop KJ, editors. Shalm’s veterinary hematology. 6th ed. Ames: Wiley-Blackwell; 2010. p. 152–61. [25] Thrall MA, Baker DC, Campbell TW, DeNicola D, Fettman MJ, Lassen ED, et al. Diagnosis of disorders of hemostasis. In: Thrall MA, Baker DC, Campbell TW, DeNicola D, Fettman MJ, Lassen ED, et al., editors. Veterinary hematology and clinical chemistry. Philadelphia: Lippincott Williams & Wilkins; 2004. p. 179–96. [26] Alesci S, Borggrefe M, Dempfle CE. Effect of freezing method and storage at 20 C and 70 C on prothrombin time, aPTT, and plasma fibrinogen levels. Thromb Res 2009;124:121–6. [27] Lubas G, Caldin M, Wiinberg B, Kristensen AMT. Laboratory testing of coagulation disorders. In: Weiss DJ, Wardrop KJ, editors. Shalm’s veterinary hematology. 6th ed. Ames: Wiley-Blackwell; 2010. p. 1082–100. [28] Hathaway WE, Bonnar J. Perinatal coagulation. New York: Grune & Stratton; 1978. [29] Cray C, Zaias J, Altman NH. Acute phase response in animals: a review. Comp Med 2009;59:517–26. [30] Gruys E, Toussaint MJM, Niewold TA, Koopmans SJ. Acute phase reaction and acute phase proteins. J Zhejiang Univ SCI 2005;6B: 1045–56.
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Theriogenology journal homepage: www.theriojournal.com
Hemostatic profile during late pregnancy and early postpartum period in mares M. Bazzano, C. Giannetto, F. Fazio, S. Marafioti, E. Giudice, G. Piccione* Department of Veterinary Science, University of Messina, Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 September 2013 Received in revised form 28 November 2013 Accepted 6 December 2013
Hemostasis is a physiological process that prevents excessive blood loss and represents a protective mechanism at the time of delivery. Peripartum hemorrhage is a recurring hazardous condition to mare’s health; therefore, we aimed to study mares’ hemostatic profile to investigate whether physiological adjustments occur during late pregnancy and early postpartum. Fifteen pregnant mares have been monitored from the 34th week of pregnancy until the third week after foaling. Fifteen nonpregnant mares were used as control group. Jugular blood samples were analyzed for platelet count (Plt), prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen (Fb). Platelet count showed significant changes at foaling (P < 0.05) and a negative correlation (r ¼ 0.968; P ¼ 0.032) with postpartum. Prothrombin time changed (P < 0.05) showing a significant correlation (r ¼ 0.675; P ¼ 0.016) with late pregnancy. Fibrinogen concentrations changed throughout the experimental period (P < 0.0001). The linear regression model revealed a positive correlation (r ¼ 0.9210; P < 0.0001) between Fb and late pregnancy and a negative correlation (r ¼ 0.9583; P ¼ 0.042) between Fb and early postpartum. The shortening in PT recorded in the imminence of parturition along with the increase in Plt and Fb at foaling might reflect a physiological hypercoagulable state that constrains excessive bleeding, enhancing mares’ odds of surviving. Our research improves the knowledge about blood coagulation in periparturient mares providing specific information on routine coagulation tests that may support in monitoring mare’s hemostatic profile during late pregnancy and early postpartum. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Mare Pregnancy Postpartum Hemostasis
1. Introduction Pregnancy is a physiological condition that significantly influences animal metabolism [1]. Most adjustments occur during late gestation and postpartum after hormonal changes [2,3]. Although several studies dealt with the assessment of blood coagulation in pregnant women [4–6], few researches concerned with hemostatic profile in domestic species such as cows [7,8], sows [9], mares [10], and dogs [11–13] during pregnancy. Hemostasis is a physiological process that prevents excessive
* Corresponding author. Tel.: þ39 0903503584; fax: þ39 0903503975. E-mail address: [email protected] (G. Piccione). 0093-691X/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.theriogenology.2013.12.003
blood loss from damaged vessels and represents a protective mechanism at the time of delivery [14]. In women, normal pregnancy is often associated with major changes in blood coagulation and fibrinolysis causing hypercoagulability. This event is probably because of oestradiolinduced triglyceride changes and protects women from fatal hemorrhage during delivery [5]. Also in equine species, the peripartum hemorrhage is one of the most common problems. The rupture of the middle uterine, iliac, utero–ovarian, pudendal or vaginal artery usually occurs during or after parturition. However, few mares may hemorrhage during midgestation to late gestation or up to several days after delivery [15]. The vascular damage can lead the mare to a rapid and profound blood loss resulting in hypovolemic shock and death [15–18]. A few
640
M. Bazzano et al. / Theriogenology 81 (2014) 639–643
research dealt with physiological modifications in pregnant mares and it is well known that significant changes occur within this period [16,19,20]. Specific attention was paid to hormonal changes around parturition, and mare’s hormonal profile is widely recognized [2,3,21]. Hematological and biochemical changes have also been studied during pregnancy [19], whereas little is known about mare’s coagulation profile during the peripartum period [10]. Prothrombin time (PT), activated partial thromboplastin time (aPTT), and platelet count (Plt) are the minimum-recommended laboratory tests for hemostasis. Prothrombin time reflects the function of the extrinsic pathway and aPTT monitors the changes in the intrinsic system and common pathway [22]. We assessed fibrinogen (Fb) to obtain further information about blood coagulation as well. Peripartum hemorrhage represents a recurring hazardous condition to mare’s health; therefore, we aimed to study mares’ hemostatic profiles to investigate whether physiological adjustments occur during late pregnancy and early postpartum.
2. Materials and methods 2.1. Animals Thirty healthy mares of different breed and age (Table 1) were enrolled in the study with the informed owner consent. The study was realized from November 2012 to June 2013 in Sicily (latitude 37.46 N; longitude 14.93 E). Animals were housed in individual straw-bedded boxes (4.0 3.5 m) at the same breeding center and were kept under natural environmental conditions. Fifteen pregnant mares (group A) were monitored from the 34th week of pregnancy until the third week after foaling. Fifteen nonpregnant mares (group B) were used as control group. All deliveries occurred within March and midMay. Mares from group A were subjected to daily clinical examination over the first 3 days after foaling. During postpartum, transrectal ultrasound exams were performed weekly to
ensure the normal involution of the uterus using the M-Turbo ultrasound system (FUJIFILM SonoSite, London, UK). Animals were fed twice a day (7 AM and 5 PM) and water was available ad libitum. Diet consisted of 6 1 kg/ day hay and 5 0.5 kg/day concentrates (crude protein 16%, crude fat 6%, crude fiber 7.35%, ash 10.09%, sodium 0.46%, lysine 0.85%, methionine 0.35%, and omega-3 0.65%). Animals were allowed to go to pasture during the day (10 AM–4 PM) as well. All treatments, housing and animal care were carried out in accordance with the standards recommended by the EU Directive 2010/63/EU for animal experiments. 2.2. Data collection Sampling was performed weekly on the same day in the morning (8 AM) until the time of parturition. All mares delivered within a week (5 2 days) from the last prepartum sampling. Therefore, we expressed each time point before foaling (BF) as weeks BF. Additional samples were taken from each mare within 24 12 hours from foaling (F) and then 7, 14, and 21 days after foaling (AF). Blood samples were collected by jugular venipuncture into 3.6-mL vacutainer tubes containing 3.8% of sodium citrate (Terumo Corporation, Tokyo, Japan) and 3-mL vacutainer tubes containing EDTA (Terumo Corporation). Whole blood with sodium citrate was centrifuged at 1500 g for 15 minutes within 30 minutes from the collection. Citrated plasma and EDTA whole blood samples were placed on ice, delivered to the laboratory, and processed within 2 hours. Platelet count was performed on EDTA whole blood samples using the HeCoVet automatic analyzer (SEAC, Florence, Italy). Sodium citrate plasma was analyzed for PT, aPTT, and Fb using standard kits for Clot 2 coagulometer (SEAC) as described by Casella, et al. [23]. 2.3. Statistical analysis All data are expressed as means standard error of the mean. Two-way repeated measures analysis of variance
Table 1 Breed and age (years) of experimental (group A) and control (group B) mares. Gestation length (days) and parity (þ, multiparous; , primiparous) have been indicated for group A. No.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Breed
Gestation length (days)
Parity
Group A
Group B
Group A
Age (y) Group B
Group A
Group A
Selle Français Standardbred Thoroughbred Italian Saddle Paint Italian Saddle Thoroughbred Thoroughbred Italian Saddle Italian Saddle Italian Saddle KWPN Rheinlander KWPN Italian Saddle
Holsteiner Italian Saddle Standardbred KWPN Italian Saddle Thoroughbred Thoroughbred Standardbred Quarter Italian Saddle KWPN Italian Saddle Standardbred Holsteiner Italian Saddle
17 4 12 16 8 6 5 5 9 10 3 17 14 10 12
12 4 6 10 7 6 4 8 7 18 12 4 6 15 18
337 360 346 341 350 356 335 323 333 344 345 328 338 331 339
þ þ þ þ þ þ þ þ þ þ þ
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Table 2 Mean values standard error of the mean with statistical significances of Plt, PT, aPTT, and Fb recorded for group A throughout the experimental period. Time (wk)
Plt (103/mL)
12 11 10 9 8 7 6 5 4 3 2 1 Foaling þ1 þ2 þ3
141.0 141.0 142.1 145.2 148.5 149.4 149.8 147.1 145.7 146.4 141.0 148.4 170.8 159.9 143.7 140.8
8.666a 8.464a 6.906a 6.506a 8.147a 8.374a 7.673a 8.231a 9.620a 6.475a 7.376a 8.742a 6.229 6.128a 4.948a 6.884a
PT (s) 11.43 11.50 11.73 11.67 11.68 11.71 11.71 11.51 11.49 11.23 11.10 10.81 11.16 11.38 11.15 10.94
aPTT (s)
0.201 0.250 0.135e 0.296e 0.222e 0.251e 0.259e 0.175 0.173 0.294 0.172 0.171 0.194 0.216 0.118 0.082
42.32 41.35 41.15 41.32 42.44 42.24 43.42 42.63 41.55 42.30 42.33 41.37 42.16 41.92 41.77 40.44
Fb (mg/dL) 0.526 0.673 1.274 0.192 1.686 0.522 0.915 1.107 1.103 0.903 0.944 0.990 1.788 1.180 1.183 0.540
139.2 149.2 148.3 152.4 150.0 153.8 166.7 159.1 162.2 185.8 190.0 190.6 204.0 185.3 180.2 170.6
5.283abcdef 4.598abcdef 8.637abcdefg 2.796abcefg 6.903abcdef 3.022abcefg 5.311aefgi 3.690abcefgi 4.184abefgi 6.561hi 5.464d 4.290d 4.607 3.262a 3.743a 4.353a
Significance: P < 0.05. a versus Foaling; b versus þ1; c versus þ2; d versus þ3; e versus 1; f versus 2; g versus 3; h versus 11; i versus 12. Abbreviations: aPTT, activated partial thromboplastin time; Fb, fibrinogen; Plt, platelet count; PT, prothrombin time.
(ANOVA) and Bonferronis post hoc comparison were applied to evaluate statistically significant effects of the experimental period on Plt, PT, aPTT, and Fb levels. A linear regression model (y ¼ a þ bx) was applied to determine the degree of correlation between each clotting parameter and the time before and after foaling, respectively. P-values less than 0.05 were considered statistically significant. Statistical analysis was performed using the PRISM 4 statistical package (GraphPad Software Inc., San Diego, CA, USA). 3. Results Animals included in group A showed no clinical signs of disease throughout their pregnancy. All subjects delivered healthy, viable foals at full-term gestation, without human assistance. All mares had shed the placenta spontaneously within 2 hours and achieved the complete involution of the uterus within 2 weeks after foaling. Table 2 showed means standard error of the mean of Plt, PT, aPTT, and Fb obtained from pregnant mares throughout the experimental period.
3.1. Platelet count Platelets reached the highest concentration at F in group A (Fig. 1A). This increase was statistically significant (P < 0.05) compared with the other time points recorded during the experimental period. The linear regression model showed a negative correlation (r ¼ 0.968; P ¼ 0.032) between Plt and AF (Fig. 2). No correlation was found between Plt and BF.
3.2. Prothrombin and activated partial thromboplastin times Two-way ANOVA showed a significant influence of late pregnancy on PT that reached the lowest values at one BF (Fig. 1B). The linear regression model showed a significant
correlation (r ¼ 0.675; P ¼ 0.016) between PT values and BF (Fig. 3). No statistical significant difference was found in aPTT values throughout the study (Fig. 1C).
3.3. Fibrinogen Two-way ANOVA revealed a significant influence of the experimental period on Fb concentration (P < 0.0001) that reached its peak (204.0 4.607 mg/dL) at F (Fig. 1D). The linear regression model revealed a positive correlation (r ¼ 0.9210; P < 0.0001) between Fb levels and BF (Fig. 4A) and a negative correlation (r ¼ 0.9583; P ¼ 0.042) between Fb and AF (Fig. 4B). 4. Discussion In the present study, significant changes occurred in Plt, PT, and Fb levels in periparturient mares. Platelet count showed no change during pregnancy, whereas a significant increase occurred at foaling. However, the negative correlation between Plt and the postpartum period displayed a return to Plt basal levels within the third week after foaling. This variance might represent an adaptive response to blood loss that usually occurs at the time of delivery. Actually, a common misconception is that blood loss causes thrombocytopenia. In contrast, Plt may even increase during the first few hours after blood loss as a result of the splenic contraction that is most prominent in horses [24]. Prothrombin time and aPTT values showed no pathological alterations and stayed within the reference range [25]. However, we found a significant shortening in PT levels around parturition with the lowest value recorded at the last time point before foaling. The same PT trend was found in healthy pregnant women [6] suggesting that a decrease in PT time might be a common condition during late pregnancy. Another study showed a decrease in PT values around parturition in mares [10] although it was not considered statistically significant. In addition, PT and
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Fig. 1. Trend standard error of the mean of Plt (A), PT (B), aPTT (C), and Fb (D) recorded for experimental (group A) and control (group B) mares during the study. Time was expressed as weeks before foaling (BF), foaling time (F) and weeks after foaling (AF). aPTT, activated partial thromboplastin time; Fb, fibrinogen; Plt, platelet count; PT, prothrombin time.
aPTT mean values recorded by Gentry, et al. [10] were higher when compared with our results. This gap might be because of the differences in samples’ storage and assay methods [23,26]. However, sampling procedures and assay methods we used in the present study complied with Shalm’s laboratory testing of coagulation disorders [27]. Among hemostatic parameters, Fb exhibited the most significant changes throughout the study, showing a positive correlation with pregnancy and a negative one with postpartum period. In our study, the highest Fb concentration was recorded at the time of delivery as observed previously in women [6], cows [7,8], sows [9], bitches [11], and mares [10]. In women, it has been postulated that this increase in Fb levels, observed at parturition, is because of
an increased synthesis related to the local use of Fb in uteroplacental circulation [28]. Also in mares this might be a contributing factor [10]. However, besides providing a substrate for fibrin formation, Fb is also a positive acute phase protein [29]. It may react at parturition [30] because this time represents a critical stage that requires mare to carry out a great physical effort [19]. In addition, several research investigated the relationship between stress and coagulation. Their results strongly suggest that healthy organisms tend to develop a physiological hypercoagulable state in response to acute stress [14]. On the basis of this knowledge, the shortening in PT recorded in the imminence of parturition along with the increase in Plt count and Fb plasma concentrations at foaling might
Fig. 2. Linear regression between Plt count and postpartum period (weeks AF) in group A. AF, after foaling; Plt, platelet.
Fig. 3. Linear regression between PT and late pregnancy (weeks BF) in group A. BF, before foaling; PT, prothrombin time.
M. Bazzano et al. / Theriogenology 81 (2014) 639–643
Fig. 4. Linear regression between Fb and late pregnancy (A), and between Fb and postpartum period (B) in group A. AF, after foaling; BF, before foaling; Fb, fibrinogen.
reflect a physiological hypercoagulable state that constrains excessive bleeding enhancing mares’ odds of surviving. 4.1. Conclusions Our research improves the knowledge about blood coagulation in periparturient mares, showing how hemostatic parameters change at this stage. Actually, both pregnancy and early postpartum represent paraphysiological conditions and reference ranges for hemostatic assays based on healthy male or nonpregnant female might be inappropriate. In effect, they may hinder the accurate diagnosis and effective treatment of hemostatic disorders that may occur during pregnancy. Therefore, our study provides specific information on routine coagulation tests that may support in monitoring mare’s hemostatic profile during the peripartum period. References [1] Filipovi c N, Stojevi c Z, Prvanovi c N, Tucek Z. The influence of late pregnancy and lactation on bone metabolism in mares. Res Vet Sci 2010;88:405–10. [2] Berg EL, McNamara DL, Keisler DH. Endocrine profiles of periparturient mares and their foals. J Anim Sci 2007;85:1660–8. [3] Vivrette S. The endocrinology of parturition in the mare. Vet Clin North Am Equine Pract 1994;10:1–17. [4] Paniccia R, Prisco D, Bandinelli B, Fedia S, Giustia B, Pepe G, et al. Plasma and serum levels of D-dimer and their correlations with other hemostatic parameters in pregnancy. Thromb Res 2002;105: 257–62. [5] Bremme A. Haemostatic changes in pregnancy. Best Pract Res Clin Haematol 2003;16:153–68.
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