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Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares
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Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares U. Tuppits a,∗ , T. Orro a , S. Einarsson b , K. Kask a , A. Kavak a a Department of Therapy, Institute of Veterinary Medicine and Animal Science, Estonian University of Life Sciences, Kreutzwaldi 62, 51014 Tartu, Estonia b Division of Reproduction, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 7054, 75007 Uppsala, Sweden
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Article history: Received 19 February 2013 Received in revised form 30 January 2014 Accepted 7 February 2014 Available online xxx
Keywords: Serum amyloid A Haptoglobin Fibrinogen Endometritis Mare
a b s t r a c t The aim of this study was to investigate the clinical relevance of measuring blood concentrations of serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fib) in horse reproductive management, and changes in response to artificial insemination (AI) with frozen–thawed semen. Standardbred mares (n = 18) with different reproductive status (eight healthy mares in first postpartum oestrus, five healthy barren mares and five mares with endometritis) were inseminated with frozen–thawed semen. Endometritis was evaluated during oestrus by bacteriological culture, cytology and presence of ultrasonically visible intrauterine fluid during oestrus. Concentrations of SAA, Hp and Fib were analysed in the blood in every 48 h during oestrus and until 5, 6 or 7 days after AI. The day of sampling and number of blood samples varied between mares because of length of the oestrus and time of AI. Changes in concentrations of SAA, Hp and Fib were evaluated based on the day of sampling regard to AI and classification of the mares. There were no differences in SAA, Hp and Fib concentrations over time before or after AI or between the groups of mares. The insemination of mares with frozen–thawed semen did not increase the plasma concentrations of SAA, Hp and Fib above clinical threshold concentration and there were no differences between susceptible or healthy mares. © 2014 Published by Elsevier B.V.
1. Introduction Artificial insemination or natural breeding causes local inflammation in the uterus of mares (Kotilainen et al., 1994). Inflammation and tissue injury are followed by a systemic response of the organism called acute phase responses (APR) (Baumann and Gauldie, 1994). One of the features of APR is hepatic production of different plasma
∗ Corresponding author. Tel.: +372 5167817; fax: +372 7313706. E-mail address: [email protected] (U. Tuppits).
proteins named acute phase proteins (APP) and initiation and course of APR can be evaluated by measuring plasma concentrations of APP. The main signalling molecule for APP synthesis in hepatocytes is the pro-inflammatory interleukin (IL)-6 originating mainly from inflamed tissues. Glucocorticosteroids and cytokines, such as tumour necrosis factor alpha (TNF-␣), IL-1, interferon gamma (IFN-␥), and transforming growth factor beta (TGF-) influence the process started by IL-6 (MacKay, 2000). Different studies have demonstrated a significant induction of gene expression as evidenced by amounts of mRNA for IL-6 in the mares’ endometrium post-breeding (Fumuso et al., 2003;
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Please cite this article in press as: Tuppits, U., et al., Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.007
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Christoffersen et al., 2012) as well as after intrauterine infusion of different extenders (Palm et al., 2008). Neutrophils (PMN) enter the uterine lumen within 30 min after AI of mares (Katila, 1995), which is a characteristic of local inflammation response and indicates pro-inflammatory cytokines activation. Based on studies about cytokines and PMN in equine endometrium, different concentrations of APP could be expected in mares before or after AI. The purpose of the present study was to investigate the possible influence of endometrial inflammatory reaction on the APR in mares after AI with frozen–thawed semen during normally managed oestrous cycle, evaluated by three APP: fibrinogen (Fib), haptoglobin (Hp) and serum amyloid A (SAA). The mares in first postpartum oestrus were compared to barren mares, because the condition of the uterus and breeding management differ (Blanchard and Macpherson, 2011). 2. Materials and methods 2.1. Animals and management A total of 18 Standardbred mares with known reproductive history and belonging to a private stud farm in Middle-Estonia were examined during the breeding season (April–May). The barren mares were kept on the pasture about 12 h during the day and stabled in individual boxes during the night; foaled mares were stabled in individual boxes with the foal and kept in smaller paddocks in groups of three to four mares for 5 h (9.00–14.00). Barren mares were fed a standard portion of grain twice and foaled mares were fed three times a day. Both groups were fed hay ad libitum in the stable and outside. The age of the mares varied from 5 to 19 years. Median day for the AI of foaled mares was Day 12 and not later than Day 17 post-partum. Of 18 oestrous cycles, nine were the first oestrous cycle after foaling and nine were the second cycle of the breeding season in barren mares. Three barren mares had more than 10 PMN per microscopic field, one also had a positive bacterial culture; one barren mare had less than 10 PMN per microscopic field and a positive bacterial finding and one mare in first postpartum oestrus had more than 10 PMN per microscopic field. These five mares were classified as “endometritis”-mares. 2.2. Experimental design Prior to study all mares were individually examined for the clinical health, to exclude mares with any sign of injury, swelling or other form of health problem. The genital tract was trans-rectally examined using a real-time ultrasonic scanner and a linear-array 5-MHz transducer (Tringa Linear, Esaote Pie Medical, Italy) every 12 h from Day 7 after foaling or when increasing oedema was detected in barren mares. Starting when either endometrial oedema decreased or the pre-ovulatory follicle matured or both, the mares were examined every 6 h until ovulation. The ovarian follicle was considered as mature, when it stopped growing, and shape and turgidity changed. Size of the dominant follicle, intensity of endometrial oedema and presence of free fluid were monitored and recorded. The
amount of intra-uterine fluid was measured at the deepest point of uterus and was considered as relevant if the amount exceeded 2 cm in width. Trans-rectal guided insemination with 2 mL of frozen–thawed semen was performed into the tip of the horn ipsilateral to the ovary from which ovulation occurred with a 70 cm sterile insemination plastic catheter (Equivet insemination catheter, Kruuse, Marslev, Denmark) after the occurrence of ovulation was detected. The frozen semen from two stallions was commercially processed in an EU-certified breeding centre. No uterine or anti-inflammatory systemic treatment was performed during the sampling period. Neither the beginning of oestrus nor time of ovulations was synchronized. In barren mares, the uterine sampling started when endometrial oedema was increasing and growth of the dominant follicle was detected. Sampling of mares with foals started from Day 9 post-partum. Endometrial samples for cytology were obtained with a guarded culture swab (Guarded Endometrial Culture Swab, Minitüb, Landshut, Germany) at 48 h intervals until ovulation and once every 24 h after insemination. Blood samples were collected from beginning of oestrus in barren mares and from Day 9 post-partum in foaled mares from the jugular vein into EDTA containing vacuum tubes (Vacuette Greiner Bio-one, Kremsmünster, Austria) for APP analysis. Sampling was repeated in every 48 h, in the mornings. Because of different insemination times, the sampling time and number of samples were different for each mare before and after AI.
2.3. Laboratory analyses Cytological samples were rotated on the slide, air-dried, stained by Diff-Quick stain (Dade Behring AG, CH 3186 Switzerland) and evaluated microscopically. The number of PMN per 10 fields at a magnification 400× was counted. Additionally by first sampling, the swab was retracted back into the inner tube, sealed with sterile plastic caps at both ends and sent for bacteriological examination within 1 h. Each sample was cultured on blood–esculin agar and incubated for 48 h at 37 ◦ C. The plates were examined after 24 and 48 h of incubation. A minimum of five colonies of the same type of bacterium was recorded as bacteriologically positive, and growth of more than two types of bacterial colonies was categorized as mixed growth. No bacterial growth was recorded when fewer than five colony-forming units were detected during 48 h of incubation. The first sample collected (EDTA) was sent to the laboratory within 4 h for automated haematological examination. Blood samples for SAA and Hp were allowed to coagulate for 1 h and then centrifuged at (1200 × g for 10 min). The plasma was decanted and stored at −20 ◦ C until laboratory analysis. Samples for fibrinogen analysis were sent to laboratory. A total of 85 samples were collected from 18 mares. The average number of samples per mare was 4.4 with a minimum of one sample and with a maximum of ten samples, covering the period 12 days before and 7 days after ovulation. Fibrinogen was measured in 75 samples, SAA and Hp in 85 samples. The concentrations on three APP are presented in Table 1.
Please cite this article in press as: Tuppits, U., et al., Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.007
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Table 1 Median (minimum and maximum) plasma concentrations of serum amyloid A (SAA), fibrinogen (Fib) and haptoglobin (Hp) of 18 mares before and after artificial insemination (AI) (−12 to 7 days). Mares are divided according to their reproductive status; mares in first postpartum oestrus (n = 8), barren mares (n = 5) and “endometritis”-mares (n = 5). Reproductive status of mares (n of mares)
SAA (mg/L) median (min; max) (n of samples)
Fib (g/L) median (min; max) (n of samples)
Hp (g/L); median (min; max) (n of samples)
Mares in first postpartum oestrus (n = 8) Barren mares (n = 5) “Endometritis”-mares (n = 5)
0.14 (0.02; 11.41) (n = 38) 0.23 (0.05; 10.16) (n = 15) 0.30 (0.02; 1.81) (n = 32)
3.4 (2.3; 5.2) (n = 31) 3.2 (1.9; 4.2) (n = 15) 3.3 (1.5; 5.7) (n = 26)
1.54 (0.15; 2.21) (n = 38) 1.67 (1.32; 2.26) (n = 15) 1.60 (1.06; 2.31) (n = 32)
Fibrinogen concentration in whole blood was measured using the heat precipitation method (Millar et al., 1971) within 12 h after sample collection. Plasma Hp was determined using commercial haemoglobin binding assay (Tridelta Development Ltd. Kildare, Ireland) according to the manufacturer’s instructions. Intra-assay coefficients of variation were <7%. This assay has been used for measuring horses Hp blood concentrations in several studies (Cywinska et al., 2010a; Casella et al., 2012; Pihl et al., 2013). Plasma SAA concentrations were measured with a multi-species ELISA kit (Phase SAA kit, Tridelta Development Ltd. Kildare, Ireland) according to the manufacturer’s instructions for horses. The detection limit of the assay for equine samples was 0.02 mg/L. Intra- and inter-assay coefficients of variation were <13 and <12%, respectively. This assay has been used for measuring horses SAA blood concentrations in several studies (Cywinska et al., 2010b; Lavoie-Lamoureux et al., 2012; Suagee et al., 2013). 2.4. Statistical analysis Polynomial linear random-intercept models were used to explore time-trend changes around insemination time within mare groups and the differences in concentrations of the acute phase proteins between 3 groups: mares in first postpartum oestrus (n = 8), barren mares (n = 5) and “endometritis”-mares (n = 5). Mares were included as random intercepts and polynomials of time (linear and square term) in days and their interactions with groups were added as fixed effects. Overall time-trend differences between groups were tested with the F test. As the time between sampling was not the same in all mares, an isotropic spatial exponential correlation structure was used for modelling serial correlations of repeated measurements at the within-mare level. Assumptions of all models were verified by scatter and normality plots of standardized residuals, and logarithmical transformation for SAA were used. The NLME package (Pinheiro et al., 2006) with statistical software R 2.13.0 was used for fitting these polynomial linear random-intercept models. 3. Results None of the mares had signs of systemic illness during the study period and no mare had elevated clinical whole blood parameters at the beginning of oestrus. Fluid that was visible with ultrasonic assessments was found in three mares before insemination (two mares at first postpartum oestrus and one barren mare with endometritis) and in three mares at 24 h after insemination (one from each group). The amount of intrauterine free fluid did not
exceed 2 cm in width in any mare before or after insemination. At 24 h after AI, three mares had more than 10 PMN (in the group of foaled healthy mares), and one barren healthy mare had between 1 and 10 neutrophils per microscopic field. There were no significant time changes within the groups or significant overall time changes between the groups for any APP plasma concentration (Fig. 1). 4. Discussion The results of the present study indicate that measuring systemic concentrations of APP before or after artificial insemination in mares does not provide relevant clinical information about the status of the uterus and insemination with frozen semen at the time of ovulation does not cause a systemic APR. The role of systemic concentrations of SAA has been investigated previously in horse reproduction studies. SAA has several pro- and anti-inflammatory properties, including protective roles in pathogen defence (Uhlar and Whitehead, 1999). Christoffersen et al. (2010) have reported increases of SAA in peripheral circulation after induction of endometritis with Eecherichia coli culture during dioestrus. Nash et al. (2009) inseminated mares with frozen semen at the time of ovulation and did not report an increase of systemic SAA. The response to endometritis after mating continued to be localized in the uterus in the previous study without a spreading of the response to infection beyond this site. Urieli-Shoval et al. (2000) hypothesized that locally produced APP can be present in uterus without evoking a systemic response under inflammatory conditions. It can be hypothesized that the ability of local inflammation in the uterus to cause a systemic acute phase response depending on the time of the inflammation. Christoffersen et al. (2010) demonstrated that systemic APR occurred when inflammation was induced during the period when the reproductive organs were under the influence of progesterone. Based on numerous studies over 50 years, progesterone is the agent which clearly changes the uterus from an infectionresistant organ to an infection-susceptible organ (Lewis, 2004). Causey (2006) presented, in a previous review, clear evidence of progesterone’ negative influence on clearing an infection from the uterus. The action of ovarian steroid hormones on endometrium cells can be the key-factor in determining if local endometritis turns into a systemic inflammatory response. In normal healthy individuals the concentration of APP is less (Eckersall and Bell, 2010), but increases or decreases at least 25% after the onset of APR in response to local inflammation (Gabay and Kushner, 1999). The concentration of SAA has been measured in previous studies by the
Please cite this article in press as: Tuppits, U., et al., Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.007
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Serum amyloid A (mg/l)
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12 7 2 1.5 1.0 0.5 0.0
-12 -10 -8
-6
-4
-2
0
2
4
6
2
4
6
2
4
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Time (days)
Fibrinogen (g/l)
6.0 4.5 3.0 1.5 0.0
-12 -10 -8
-6
-4
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0
Time (days)
Haptoglobin (g/l)
2.5 2.0 1.5 1.0 0.5 0.0
-12 -10 -8
-6
-4
-2
0
Time (days) Fig. 1. Serum concentrations of acute phase proteins (APP): serum amyloid A (SAA), fibrinogen (Fib) and haptoglobin (Hp) from 18 mares during time period before and after artificial insemination (AI) (Day 0). Mares are divided into three groups: mares in first postpartum oestrus (♦, n = 8), barren mares (, n = 5) and “endometritis”-mares (×, n = 5). Lines represent overall time changes in APP concentrations within the mares in first postpartum oestrus (. . .), non-foaling mares (—) and problem-mares (-----) evaluated from linear random-intercept models. There were no statistical differences in overall time changes of all APP between groups.
same method as in the present study and median concentrations of 0.15 mg/L (Pollock et al., 2005) and 0.41 mg/L (Cywinska et al., 2010b) in healthy adult animals have been reported. The median SAA concentrations in the present study were similar to those in previous studies. Production of Fib and SAA in the liver is stimulated by the same agents (Baumann and Gauldie, 1994). Concentration of Fib increases in horses during inflammation, remaining under 4–7 g/L in healthy horses (Hulten, 1999). In the present
study, the mean concentration of Fib remained less than that in the previous study at all time-points where concentrations were ascertained. The concentration of Hp varies widely in healthy horses with mean values of 0–9 mg/mL reported (Pollock et al., 2005). The concentration of Hp did not differ between the groups in the present study. Changes in APP concentrations during oestrus and until 5, 6 or 7 days after AI were investigated in the present study. The three APP differ in time of increase in concentrations after the initiation of inflammation. Pollock et al. (2005) studied the effects of surgeries on APP concentrations in horses and found the concentration of SAA reached a maximal amounts between 24 and 48 h and began to decrease after 48 h. The concentrations of Hp and Fib also were maximal between 24 and 48 h, but did not start to decrease during the 72 h period after surgeries (Pollock et al., 2005). The concentration of SAA did not increase until 24 h after insemination with frozen thawed semen (Nash et al., 2009). In previous reports, it has been speculated that the time to increase in APP response can be delayed under some circumstances after insemination. However, in the present study there was no increase of APP during several days after insemination. The concentration of Hp has been investigated in postpartum cows and the researchers concluded that uterine involution and bacterial contamination can be associated with elevation of Hp concentrations (Sheldon et al., 2001). Hp analysis related to endometrial conditions has not been reported in mares. The post-partum size and fluid content of the mare uterus decreases to normal values during 7 days after uncomplicated foaling, but histological involution does not take place until 14 days post-partum (Stanton, 2011) and a positive uterine culture accompanied by inflammatory cytological findings at the time of the first postpartum oestrus is not unusual (Gygax et al., 1979). Taira et al. (1992) demonstrated elevation in Hp concentrations in pregnant mares and during the period of parturition. In the present study, changes in HP concentrations of mares in first postpartum oestrus were investigated, but no differences were found.
5. Conclusions In the present study, no systemic inflammatory response after insemination was detected in mares supporting the hypothesis that the breeding associated inflammation in the uterus is a local process without evoking a systemic response. Therefore, the measuring the concentrations of SAA, Fib and Hp is not relevant in horse reproduction management and does not help to detect mares susceptible to infection between groups.
Acknowledgements We are grateful to professor Terttu Katila from Helsinki University for revising the manuscript and to the mare owners at Stud-farm Tooma for providing the mares to collect the samples. Research support was provided for authors by the Estonian Science Foundation, grant no 7539.
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Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares U. Tuppits a,∗ , T. Orro a , S. Einarsson b , K. Kask a , A. Kavak a a Department of Therapy, Institute of Veterinary Medicine and Animal Science, Estonian University of Life Sciences, Kreutzwaldi 62, 51014 Tartu, Estonia b Division of Reproduction, Department of Clinical Sciences, Faculty of Veterinary Medicine and Animal Science, Swedish University of Agricultural Sciences, P.O. Box 7054, 75007 Uppsala, Sweden
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Article history: Received 19 February 2013 Received in revised form 30 January 2014 Accepted 7 February 2014 Available online xxx
Keywords: Serum amyloid A Haptoglobin Fibrinogen Endometritis Mare
a b s t r a c t The aim of this study was to investigate the clinical relevance of measuring blood concentrations of serum amyloid A (SAA), haptoglobin (Hp) and fibrinogen (Fib) in horse reproductive management, and changes in response to artificial insemination (AI) with frozen–thawed semen. Standardbred mares (n = 18) with different reproductive status (eight healthy mares in first postpartum oestrus, five healthy barren mares and five mares with endometritis) were inseminated with frozen–thawed semen. Endometritis was evaluated during oestrus by bacteriological culture, cytology and presence of ultrasonically visible intrauterine fluid during oestrus. Concentrations of SAA, Hp and Fib were analysed in the blood in every 48 h during oestrus and until 5, 6 or 7 days after AI. The day of sampling and number of blood samples varied between mares because of length of the oestrus and time of AI. Changes in concentrations of SAA, Hp and Fib were evaluated based on the day of sampling regard to AI and classification of the mares. There were no differences in SAA, Hp and Fib concentrations over time before or after AI or between the groups of mares. The insemination of mares with frozen–thawed semen did not increase the plasma concentrations of SAA, Hp and Fib above clinical threshold concentration and there were no differences between susceptible or healthy mares. © 2014 Published by Elsevier B.V.
1. Introduction Artificial insemination or natural breeding causes local inflammation in the uterus of mares (Kotilainen et al., 1994). Inflammation and tissue injury are followed by a systemic response of the organism called acute phase responses (APR) (Baumann and Gauldie, 1994). One of the features of APR is hepatic production of different plasma
∗ Corresponding author. Tel.: +372 5167817; fax: +372 7313706. E-mail address: [email protected] (U. Tuppits).
proteins named acute phase proteins (APP) and initiation and course of APR can be evaluated by measuring plasma concentrations of APP. The main signalling molecule for APP synthesis in hepatocytes is the pro-inflammatory interleukin (IL)-6 originating mainly from inflamed tissues. Glucocorticosteroids and cytokines, such as tumour necrosis factor alpha (TNF-␣), IL-1, interferon gamma (IFN-␥), and transforming growth factor beta (TGF-) influence the process started by IL-6 (MacKay, 2000). Different studies have demonstrated a significant induction of gene expression as evidenced by amounts of mRNA for IL-6 in the mares’ endometrium post-breeding (Fumuso et al., 2003;
http://dx.doi.org/10.1016/j.anireprosci.2014.02.007 0378-4320/© 2014 Published by Elsevier B.V.
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Christoffersen et al., 2012) as well as after intrauterine infusion of different extenders (Palm et al., 2008). Neutrophils (PMN) enter the uterine lumen within 30 min after AI of mares (Katila, 1995), which is a characteristic of local inflammation response and indicates pro-inflammatory cytokines activation. Based on studies about cytokines and PMN in equine endometrium, different concentrations of APP could be expected in mares before or after AI. The purpose of the present study was to investigate the possible influence of endometrial inflammatory reaction on the APR in mares after AI with frozen–thawed semen during normally managed oestrous cycle, evaluated by three APP: fibrinogen (Fib), haptoglobin (Hp) and serum amyloid A (SAA). The mares in first postpartum oestrus were compared to barren mares, because the condition of the uterus and breeding management differ (Blanchard and Macpherson, 2011). 2. Materials and methods 2.1. Animals and management A total of 18 Standardbred mares with known reproductive history and belonging to a private stud farm in Middle-Estonia were examined during the breeding season (April–May). The barren mares were kept on the pasture about 12 h during the day and stabled in individual boxes during the night; foaled mares were stabled in individual boxes with the foal and kept in smaller paddocks in groups of three to four mares for 5 h (9.00–14.00). Barren mares were fed a standard portion of grain twice and foaled mares were fed three times a day. Both groups were fed hay ad libitum in the stable and outside. The age of the mares varied from 5 to 19 years. Median day for the AI of foaled mares was Day 12 and not later than Day 17 post-partum. Of 18 oestrous cycles, nine were the first oestrous cycle after foaling and nine were the second cycle of the breeding season in barren mares. Three barren mares had more than 10 PMN per microscopic field, one also had a positive bacterial culture; one barren mare had less than 10 PMN per microscopic field and a positive bacterial finding and one mare in first postpartum oestrus had more than 10 PMN per microscopic field. These five mares were classified as “endometritis”-mares. 2.2. Experimental design Prior to study all mares were individually examined for the clinical health, to exclude mares with any sign of injury, swelling or other form of health problem. The genital tract was trans-rectally examined using a real-time ultrasonic scanner and a linear-array 5-MHz transducer (Tringa Linear, Esaote Pie Medical, Italy) every 12 h from Day 7 after foaling or when increasing oedema was detected in barren mares. Starting when either endometrial oedema decreased or the pre-ovulatory follicle matured or both, the mares were examined every 6 h until ovulation. The ovarian follicle was considered as mature, when it stopped growing, and shape and turgidity changed. Size of the dominant follicle, intensity of endometrial oedema and presence of free fluid were monitored and recorded. The
amount of intra-uterine fluid was measured at the deepest point of uterus and was considered as relevant if the amount exceeded 2 cm in width. Trans-rectal guided insemination with 2 mL of frozen–thawed semen was performed into the tip of the horn ipsilateral to the ovary from which ovulation occurred with a 70 cm sterile insemination plastic catheter (Equivet insemination catheter, Kruuse, Marslev, Denmark) after the occurrence of ovulation was detected. The frozen semen from two stallions was commercially processed in an EU-certified breeding centre. No uterine or anti-inflammatory systemic treatment was performed during the sampling period. Neither the beginning of oestrus nor time of ovulations was synchronized. In barren mares, the uterine sampling started when endometrial oedema was increasing and growth of the dominant follicle was detected. Sampling of mares with foals started from Day 9 post-partum. Endometrial samples for cytology were obtained with a guarded culture swab (Guarded Endometrial Culture Swab, Minitüb, Landshut, Germany) at 48 h intervals until ovulation and once every 24 h after insemination. Blood samples were collected from beginning of oestrus in barren mares and from Day 9 post-partum in foaled mares from the jugular vein into EDTA containing vacuum tubes (Vacuette Greiner Bio-one, Kremsmünster, Austria) for APP analysis. Sampling was repeated in every 48 h, in the mornings. Because of different insemination times, the sampling time and number of samples were different for each mare before and after AI.
2.3. Laboratory analyses Cytological samples were rotated on the slide, air-dried, stained by Diff-Quick stain (Dade Behring AG, CH 3186 Switzerland) and evaluated microscopically. The number of PMN per 10 fields at a magnification 400× was counted. Additionally by first sampling, the swab was retracted back into the inner tube, sealed with sterile plastic caps at both ends and sent for bacteriological examination within 1 h. Each sample was cultured on blood–esculin agar and incubated for 48 h at 37 ◦ C. The plates were examined after 24 and 48 h of incubation. A minimum of five colonies of the same type of bacterium was recorded as bacteriologically positive, and growth of more than two types of bacterial colonies was categorized as mixed growth. No bacterial growth was recorded when fewer than five colony-forming units were detected during 48 h of incubation. The first sample collected (EDTA) was sent to the laboratory within 4 h for automated haematological examination. Blood samples for SAA and Hp were allowed to coagulate for 1 h and then centrifuged at (1200 × g for 10 min). The plasma was decanted and stored at −20 ◦ C until laboratory analysis. Samples for fibrinogen analysis were sent to laboratory. A total of 85 samples were collected from 18 mares. The average number of samples per mare was 4.4 with a minimum of one sample and with a maximum of ten samples, covering the period 12 days before and 7 days after ovulation. Fibrinogen was measured in 75 samples, SAA and Hp in 85 samples. The concentrations on three APP are presented in Table 1.
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Table 1 Median (minimum and maximum) plasma concentrations of serum amyloid A (SAA), fibrinogen (Fib) and haptoglobin (Hp) of 18 mares before and after artificial insemination (AI) (−12 to 7 days). Mares are divided according to their reproductive status; mares in first postpartum oestrus (n = 8), barren mares (n = 5) and “endometritis”-mares (n = 5). Reproductive status of mares (n of mares)
SAA (mg/L) median (min; max) (n of samples)
Fib (g/L) median (min; max) (n of samples)
Hp (g/L); median (min; max) (n of samples)
Mares in first postpartum oestrus (n = 8) Barren mares (n = 5) “Endometritis”-mares (n = 5)
0.14 (0.02; 11.41) (n = 38) 0.23 (0.05; 10.16) (n = 15) 0.30 (0.02; 1.81) (n = 32)
3.4 (2.3; 5.2) (n = 31) 3.2 (1.9; 4.2) (n = 15) 3.3 (1.5; 5.7) (n = 26)
1.54 (0.15; 2.21) (n = 38) 1.67 (1.32; 2.26) (n = 15) 1.60 (1.06; 2.31) (n = 32)
Fibrinogen concentration in whole blood was measured using the heat precipitation method (Millar et al., 1971) within 12 h after sample collection. Plasma Hp was determined using commercial haemoglobin binding assay (Tridelta Development Ltd. Kildare, Ireland) according to the manufacturer’s instructions. Intra-assay coefficients of variation were <7%. This assay has been used for measuring horses Hp blood concentrations in several studies (Cywinska et al., 2010a; Casella et al., 2012; Pihl et al., 2013). Plasma SAA concentrations were measured with a multi-species ELISA kit (Phase SAA kit, Tridelta Development Ltd. Kildare, Ireland) according to the manufacturer’s instructions for horses. The detection limit of the assay for equine samples was 0.02 mg/L. Intra- and inter-assay coefficients of variation were <13 and <12%, respectively. This assay has been used for measuring horses SAA blood concentrations in several studies (Cywinska et al., 2010b; Lavoie-Lamoureux et al., 2012; Suagee et al., 2013). 2.4. Statistical analysis Polynomial linear random-intercept models were used to explore time-trend changes around insemination time within mare groups and the differences in concentrations of the acute phase proteins between 3 groups: mares in first postpartum oestrus (n = 8), barren mares (n = 5) and “endometritis”-mares (n = 5). Mares were included as random intercepts and polynomials of time (linear and square term) in days and their interactions with groups were added as fixed effects. Overall time-trend differences between groups were tested with the F test. As the time between sampling was not the same in all mares, an isotropic spatial exponential correlation structure was used for modelling serial correlations of repeated measurements at the within-mare level. Assumptions of all models were verified by scatter and normality plots of standardized residuals, and logarithmical transformation for SAA were used. The NLME package (Pinheiro et al., 2006) with statistical software R 2.13.0 was used for fitting these polynomial linear random-intercept models. 3. Results None of the mares had signs of systemic illness during the study period and no mare had elevated clinical whole blood parameters at the beginning of oestrus. Fluid that was visible with ultrasonic assessments was found in three mares before insemination (two mares at first postpartum oestrus and one barren mare with endometritis) and in three mares at 24 h after insemination (one from each group). The amount of intrauterine free fluid did not
exceed 2 cm in width in any mare before or after insemination. At 24 h after AI, three mares had more than 10 PMN (in the group of foaled healthy mares), and one barren healthy mare had between 1 and 10 neutrophils per microscopic field. There were no significant time changes within the groups or significant overall time changes between the groups for any APP plasma concentration (Fig. 1). 4. Discussion The results of the present study indicate that measuring systemic concentrations of APP before or after artificial insemination in mares does not provide relevant clinical information about the status of the uterus and insemination with frozen semen at the time of ovulation does not cause a systemic APR. The role of systemic concentrations of SAA has been investigated previously in horse reproduction studies. SAA has several pro- and anti-inflammatory properties, including protective roles in pathogen defence (Uhlar and Whitehead, 1999). Christoffersen et al. (2010) have reported increases of SAA in peripheral circulation after induction of endometritis with Eecherichia coli culture during dioestrus. Nash et al. (2009) inseminated mares with frozen semen at the time of ovulation and did not report an increase of systemic SAA. The response to endometritis after mating continued to be localized in the uterus in the previous study without a spreading of the response to infection beyond this site. Urieli-Shoval et al. (2000) hypothesized that locally produced APP can be present in uterus without evoking a systemic response under inflammatory conditions. It can be hypothesized that the ability of local inflammation in the uterus to cause a systemic acute phase response depending on the time of the inflammation. Christoffersen et al. (2010) demonstrated that systemic APR occurred when inflammation was induced during the period when the reproductive organs were under the influence of progesterone. Based on numerous studies over 50 years, progesterone is the agent which clearly changes the uterus from an infectionresistant organ to an infection-susceptible organ (Lewis, 2004). Causey (2006) presented, in a previous review, clear evidence of progesterone’ negative influence on clearing an infection from the uterus. The action of ovarian steroid hormones on endometrium cells can be the key-factor in determining if local endometritis turns into a systemic inflammatory response. In normal healthy individuals the concentration of APP is less (Eckersall and Bell, 2010), but increases or decreases at least 25% after the onset of APR in response to local inflammation (Gabay and Kushner, 1999). The concentration of SAA has been measured in previous studies by the
Please cite this article in press as: Tuppits, U., et al., Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.007
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Serum amyloid A (mg/l)
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12 7 2 1.5 1.0 0.5 0.0
-12 -10 -8
-6
-4
-2
0
2
4
6
2
4
6
2
4
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Time (days)
Fibrinogen (g/l)
6.0 4.5 3.0 1.5 0.0
-12 -10 -8
-6
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Time (days)
Haptoglobin (g/l)
2.5 2.0 1.5 1.0 0.5 0.0
-12 -10 -8
-6
-4
-2
0
Time (days) Fig. 1. Serum concentrations of acute phase proteins (APP): serum amyloid A (SAA), fibrinogen (Fib) and haptoglobin (Hp) from 18 mares during time period before and after artificial insemination (AI) (Day 0). Mares are divided into three groups: mares in first postpartum oestrus (♦, n = 8), barren mares (, n = 5) and “endometritis”-mares (×, n = 5). Lines represent overall time changes in APP concentrations within the mares in first postpartum oestrus (. . .), non-foaling mares (—) and problem-mares (-----) evaluated from linear random-intercept models. There were no statistical differences in overall time changes of all APP between groups.
same method as in the present study and median concentrations of 0.15 mg/L (Pollock et al., 2005) and 0.41 mg/L (Cywinska et al., 2010b) in healthy adult animals have been reported. The median SAA concentrations in the present study were similar to those in previous studies. Production of Fib and SAA in the liver is stimulated by the same agents (Baumann and Gauldie, 1994). Concentration of Fib increases in horses during inflammation, remaining under 4–7 g/L in healthy horses (Hulten, 1999). In the present
study, the mean concentration of Fib remained less than that in the previous study at all time-points where concentrations were ascertained. The concentration of Hp varies widely in healthy horses with mean values of 0–9 mg/mL reported (Pollock et al., 2005). The concentration of Hp did not differ between the groups in the present study. Changes in APP concentrations during oestrus and until 5, 6 or 7 days after AI were investigated in the present study. The three APP differ in time of increase in concentrations after the initiation of inflammation. Pollock et al. (2005) studied the effects of surgeries on APP concentrations in horses and found the concentration of SAA reached a maximal amounts between 24 and 48 h and began to decrease after 48 h. The concentrations of Hp and Fib also were maximal between 24 and 48 h, but did not start to decrease during the 72 h period after surgeries (Pollock et al., 2005). The concentration of SAA did not increase until 24 h after insemination with frozen thawed semen (Nash et al., 2009). In previous reports, it has been speculated that the time to increase in APP response can be delayed under some circumstances after insemination. However, in the present study there was no increase of APP during several days after insemination. The concentration of Hp has been investigated in postpartum cows and the researchers concluded that uterine involution and bacterial contamination can be associated with elevation of Hp concentrations (Sheldon et al., 2001). Hp analysis related to endometrial conditions has not been reported in mares. The post-partum size and fluid content of the mare uterus decreases to normal values during 7 days after uncomplicated foaling, but histological involution does not take place until 14 days post-partum (Stanton, 2011) and a positive uterine culture accompanied by inflammatory cytological findings at the time of the first postpartum oestrus is not unusual (Gygax et al., 1979). Taira et al. (1992) demonstrated elevation in Hp concentrations in pregnant mares and during the period of parturition. In the present study, changes in HP concentrations of mares in first postpartum oestrus were investigated, but no differences were found.
5. Conclusions In the present study, no systemic inflammatory response after insemination was detected in mares supporting the hypothesis that the breeding associated inflammation in the uterus is a local process without evoking a systemic response. Therefore, the measuring the concentrations of SAA, Fib and Hp is not relevant in horse reproduction management and does not help to detect mares susceptible to infection between groups.
Acknowledgements We are grateful to professor Terttu Katila from Helsinki University for revising the manuscript and to the mare owners at Stud-farm Tooma for providing the mares to collect the samples. Research support was provided for authors by the Estonian Science Foundation, grant no 7539.
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Please cite this article in press as: Tuppits, U., et al., Influence of the uterine inflammatory response after insemination with frozen–thawed semen on serum concentrations of acute phase proteins in mares. Anim. Reprod. Sci. (2014), http://dx.doi.org/10.1016/j.anireprosci.2014.02.007