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Effect of flunixin and administration on conception rate and estrous cycle characteristics of Egyptian Baladi cows during hot season

Ahmed Ismail Damarany1 & Nasser Ghanem2

Received: 7 August 2019 /Accepted: 1 June 2020 # Springer Nature B.V. 2020

Abstract The current investigation aims to evaluate the effects of flunixin meglumine (FM) and aspirin as non-steroid anti-inflammatory drug (NSAID) administration on estrous cycles characteristics and conception rate of Egyptian Baladi cows during hot season. In the first phase, 30 cows were divided into 3 groups, 10 cows for each treatment. The first group was treated with FM at the rate of 1.1 mg/kg body weight (BW) intramuscular, while the second group was administrated aspirin solution orally at the rate of 50 mg/kg BW. The third group was assigned as control (CG) that has no treatment. The FM group was administrated on day 14 after mating, while aspirin was given on day 14 and day 15 post-mating. All cows were mated naturally after showing estrus signs. Pregnancy diagnosis was carried 60 days after mating by rectal palpation. In the second phase, cows were monitored for estrus behavior by visual observation twice a day. The length of normal estrous cycles was 20, 23, and 22 days in cows treated with FM, aspirin, and control cows, respectively. There was no significant effect of treatment on the length of normal estrous cycles in Egyptian cows (P < 0.05). Proportions of long cycles in Egyptian cows that treated with FM or aspirin and control were 75, 67.7, and 57.1%, respectively. Short cycles were completely absent in cows that treated with FM or aspirin, but it was 29% in CG. Mounting behavior and tail rising were not detected in CG compared to 0 and 33% in FM or 25 and 33% in aspirin treated cows, respectively. Conception or pregnancy rate were 60, 40, and 30%, respectively, in FM, aspirin treated, and CG. Treatment cows whether FM or aspirin group did not influence (P < 0.05) progesterone concentration during the 14 days and 21 days from estrous cycle in pregnant and non-pregnant Egyptian Baladi cows than CG. In conclusion, the results of this study clearly indicated beneficial effect of FM and aspirin administration on intense of displayed estrous behavior and conception rate of Egyptian Baladi cows during the hot season.

Keywords Flunixin meglumine . Aspirin . Conception rate . Egyptian Baladi cows

Introduction viability of dairy farms. Early research work confirmed incidence of embryonic loss for heifers (40%) and Embryonic loss considered as one of the most important moderate-producing dairy cattle (70 to 80%) between problems in dairy cows that has an impact on the eco- days 8 and 16 post-insemination (Diskin and Sreenan nomic return of cattle breeding. Therefore, research ef- 1980; and Sreenan and Diskin 1986). Although anti- forts should be focused on minimizing embryonic losses inflammatory drugs are used widely in dairy farms, by finding possible treatment, which is essential for many researchers have concerned on the effect of non- steroid anti-inflammatory drugs on pregnancy rate in cattle (Kaveh et al. 2011;Giammarcoetal.2016; * Ahmed Ismail Damarany Barkhori-Mehnietal.2018). Flunixin meglumine (FM) [email protected] and aspirin, considered as a strong non-steroid anti-in- flammatory drugs (NSAIDs), these drugs inhibits syn- 1 Department of Animal and Poultry Production Faculty of Agriculture thesis of (COX-1 and COX- and Natural Resources, Aswan University, Aswan, Egypt 2) and transformation of to prostaglan- 2 Department of Animal Production, Faculty of Agriculture, Cairo din F2α (Purcelletal.2005;Gearyetal.2010; University, Giza, Egypt Sanchez-Rodriquez et al. 2011)). Incidence of embryo Trop Anim Health Prod loss during the time of maternal recognition of pregnan- Climatic conditions and farm location cy (MRP) is consequence of embryos to inhibit secre- tion of F2α (Thatcher et al. 1994). Scenna The study was carried out in Aswan governorate. The farm et al. (2004) reported negative effect of PGF2α on via- was located in Kom Ombou city (32°, 31′ 23″ east and 22°, bility and development of bovine embryos produced 28′ 09″ north). Ambient temperature and relative humidity in vitro pre- and post-compaction period. Von Krueger during hot season are presented in Table 1. Temperature hu- and Heuwieser (2010) showed that FM administration in midity index (THI) was calculated using the formula proposed dairy cattle led to delayed luteolysis and enhanced em- by Mader et al. (2006): bryonic survival. Positive trend observed in pregnancy THI ¼ ðÞþ0:8  Tdb ½ŠþðÞÂRH=100 ðÞTdb−14:4 46:4 rate of cows and heifers when administrated NSAIDs (Sanchez-Rodriquez et al. 2011; Heuwieser et al. 2011; Antanaitis et al. 2018). Noteworthy, viability of in vivo Temperature-humidity index (THI) = 0.8 × ambient tem- recovered embryo was reduced from 59 to 27% during perature + [(% relative humidity) 100) × (ambient tempera- the period from day 7 to 14 of pre-implantation devel- ture − 14.4)] + 46.4. opment in the hot season compared with those collected According to livestock weather safety index (LCI in cold season (52 vs. 60%). Furthermore, pregnancy 1970), THI less than 74 and more than 84 are consid- rate was lower (21%) in hot than cold season (36%). ered as normal and emergency, respectively, while THI Therefore, the aim of this study was to evaluate the upto79isalertzoneandbetween79to84isconsid- effects of FM and aspirin administration as NSAIDs ered as danger zone. on conception rate and estrous cycle characteristics of Egyptian Baladi cows during the hot season. Management of animals

Material and methods All cows were free from any disease and no manifested pathological signs were observed. The cows were natu- Experimental design ral mated by the bull after displaying signs of estrus during post-partum period (40th day of parturition). Cow’s parity ranged between the 2nd and 5th parity. Experiments were conducted in two phases using 30 local The mean BW at mating was 369.3 ± 43.8 kg. Cows Egyptian cows (Fig. 1). In the first phase, cows were divided were reared in traditional farm as semi-shaded yards. into 3 equal groups of 10 each to evaluate the effect of differ- Cows were fed alfa-alfa, hay wheat, corn fodder and ent treatments on pregnancy rate. The first group injected with concentrate ration consisted corn grain and wheat bran FM at the rate of 1.1 mg/kg BW intramuscularly on day 14 during the period from April to September. The animals after mating according to Geary et al. (2010). The second were fed corn fodder and alfa-alfa ad libitum. All cows group orally administered aspirin solution at the rate of kept under the same managerial and environmental con- 50 mg/kg BW on day 14 (twice) and day 15, 12 h apart, ditions according the farm routine work. post-mating according to Spencer et al. (2016). The final group was untreated and served as a control group. During Heat detection and pregnancy diagnosis second phase, cows were monitored for estrous behavior, such as vaginal mucus discharge, mounting behavior, bellowing, Visual observation of cows was conformed at the morn- tail rising, and standing frequency by visual observation twice ing and night at 6:00 am and 6:00 pm. Cows were aday. considered in heat based on any sexual demeanor signs described by El-Wardani (1995). Conception rate was estimated from number of cows that did not return to estrus after first mating over total number of cows mat- ed. Pregnancy was diagnosed by rectal palpation (60 days post-mating) as described by Arthur (1964). Pregnancy rate was calculated as the number of cows, which conceived from the total served post-partum using the following formula:

Pregnancy rate¼ðÞNumber of pregnant cows=Total number of mated cows Â100: Fig. 1 Egyptian Baladi cows Trop Anim Health Prod

Table 1 Mean of the ambient o temperature (°C), relative Hot months Average ambient temperature ( C) Average relative humidity (RH %) THI humidity (%), and temperature humidity index during the hot Min Max months April 19.1 35.0 18 79.2 May 23.1 41.2 16 83.6 June 25.3 41.3 16 83.7 July 26.2 41.3 18 84.3 August 26.0 41.0 20 84.6 September 24.2 39.4 22 83.8

but it was 29% in CG. There was no significant effect of Analysis of blood progesterone treatment on the length of normal estrous cycles of Egyptian local cows (P < 0.05). Number of cows with estrus signs ob- Blood from jugular vein was collected 10 ml during days 7, served in FM or aspirin and CG was 4, 6, and 7. The greater 14, and 21 after mating in heparinized tubes. Samples were number of animals expressed estrus signs in CG reflects repeat centrifuged at 3000 rpm for 20 min and separated plasma. breeding of animals in this group compared to FM or aspirin Plasma was stored at − 20 °C until estimation of progesterone groups. There was no effect of high THI as indicator of heat (P4) hormone using radioimmunoassay kit (Immunotech, stress on estrus manifestation of Egyptian cattle. Among dif- France). Sensitivity value reported to be according to manu- ferent estrus signs, standing behavior was more frequent being facturer information. The intra- and inter-assay variation co- 100 in control (CG) or treated groups (TGs). The mounting efficients were 5.3 and 11.2%, respectively. behavior and tail raising were not detected in CG compared with cows administrated FM (50% and 33.3%) and aspirin Statistical analysis (25% and 33.3%). While, vaginal mucus discharge and bellowing was more frequent in cows treated with FM (75% The statistical design was included one factor (that was effect and 75%) and aspirin (66.7% and 66.7%) compared to control of FM or aspirin on conception rate) and tested the variance by group (57.1% and 57.1%) (Table 3). following model significant variation between means was tested using Duncan’s multiple range test. Chi-square was Conception, pregnancy rate, and progesterone used to test the significance of the percentage values. concentration Statistical analysis was carried using software (SAS 2002). The following model was used: Conception rate in cows treated with FM or aspirin and CG P Yij ¼ μ þ Ti þ Rj þ eij were 60, 40, and 30%, respectively ( < 0.05) and similar was pregnancy rate (P < 0.05) (Table 4). The profile of P4 was at where minimum level during estrus (0.24 ng/ml), increased at day 7 Yij = the observation trait (2.30 ng/ml) and continued to increase until day 14 μ = overall mean (4.15 ng/ml) at diestrus. There was no significant difference Ti = effect of treatment (treatment = 1, control = 2) between CG and TGs (Table 5). The concentration of P4 Rj = effect of replicates (1,2,…eg) reached maximum level at day 21 in cows treated with FM eij = experimental error (6.72 ng/ml), aspirin (5.54 ng/ml) and control (4.84 ng/ml). However, P4 concentration declined (0.21 and 0.24 ng/ml) at day 21 in cows of CG and TGs those did not conceived, Results respectively.

Estrous cycle length and behavior Discussion The length of normal estrous cycles observed was 20, 23, and 22 days in cows treated with FM, aspirin, and control cows, Majority of embryonic wastage occurs meantime the peri- respectively (Table 2). Compared to FM-treated cows, lower implantation phase of pregnancy, possibly due to reduced oo- percentage of long cycles was recorded in cows treated by cyte quality and reduced embryo viability. The viability of aspirin but both were higher than CG. Short cycles were embryos is compromised due to uterine dysfunction, failure completely absent in cows that treated with FM or aspirin, to communicate with maternal tissue (oviduct and uterus) Trop Anim Health Prod

Table 2 Effect of treatment with flunixin meglumine and aspirin on estrous cycles length (days) of Baladi cows during the experimental period

Baladi Treatment cows Flunixin meglumine Aspirin Control

Types of estrous cycles length

Normal Long Short Normal Long Short Normal Long Short 18–24 days >24days <18days 18–24 days >24days <18days 18–24 days >24days <18days

No. of 13– 24– 142 cycles X ± SE 20.0 ± 0.0a 26.7 ± 2.3b – 23.0 ± 1.4a 28.0 ± 2.6b – 22.0 ± 0.0a 28.3 ± 1.7b 15.0 ± 0.7 % 25 75 33.3 66.7 14.3 57.1 28.6

Values within the same row having different letters are significant at P <0.05 appropriately and/or, lack of pregnancy recognition signal to pregnancy approximately 14 days post-mating that block uter- undergo implantation and placentation (Wiltbank et al. 2016). ine secretion of F2α. It was demonstrated that embryonic loss is nearly 40% for Aspirin is widely used as anti-inflammatory and heifers and moderate-producing dairy cattle with maximum (James et al. 2007; Schistermann et al. 2009). Two research (70 to 80%) losses happening between days 8 and 16 post- groups (Bertoni et al. 2004; Sanchez-Rodriguez et al. 2011) insemination (Diskin and Sreenan 1980; Dunne et al. 2000; reported that treatment of cows with aspirin lead to improved Sreenan and Diskin 1986). Our results indicated that pregnan- pregnancy rate from 27 to 50% after first insemination and cy rate was distinctive in FM-treated cows, but aspirin-treated reduced percentage of culling cows for infertility reason. The cows were marginally comparable to CG. Similar trend was beneficial effect of aspirin is dependent on its ability to inhibit reported by Guzeloglu et al. (2007) who found that injection synthesis of COX-2 responsible for of FM in heifers increased pregnancy rate on days 29 and 65 PGF2α biosynthesis (Pugh et al. 2004). post-mating in treated (77 and 69%, respectively) than untreat- Progesterone plays important roles in enhancing uterine ed heifers (50 and 46%, respectively). In recent years, many secretion required for embryo growth and development, in- other workers also reported encouraging result in pregnancy ducing inhibition of contractility of myometrium to avert rate after treating dairy cows with FM (Pfeifer et al. 2016; abortion and preservation of embryo versus maternal immune Giammarco et al. 2016; Barkhori-Mehni et al. 2018). The system during pregnancy (Bazer 1975; Lewis 2003; Soloff improvement of pregnancy rate in the treated cows with FM et al. 2011). The profile of P4 peak concentration in this study is probably because of suppression of the prostaglandins F2α is lower (5.2–6.7 ng/ml) than that suggested by many re- secretion from the uterus (Guzeloglu et al. 2007) that was searchers (El-Gaafarawy et al. 2000; El- Gaafarawy and linked with greater developmental rate of conceptus (Kim Abdel-Kaber, 2001) for Egyptian Baladi cattle. Results of et al. 2014; Merrill et al. 2007). In support to this idea, the current study indicated that treatment cows whether with Thatcher et al. (2001) suggested that embryonic wastage is FM or aspirin group recorded insignificant higher (P <0.05) likely due to conceptus failure to produce adequate amount progesterone concentration during days 14 and 21 from es- of interferon tau (IFN-τ)toinitiatematernalrecognitionof trous cycle in pregnant and non-pregnant cows than control cows. Spencer et al. (2016) observed similar trend of in- creased progesterone concentration and luteolysis duration in Table 3 Effect of treatment with flunixin meglumine and aspirin on estrus signs of Baladi cows during the experimental period aspirin treated compared to CG. It was suggested that in- creased pregnancy rate of lactating dairy cows treated with Estrus signs Treatment FM is due to increased progesterone concentration (Barkhori-Mehni et al. 2018; Clemente et al. 2009; Green Flunixin meglumine Aspirin Control et al. 2005; Mann and Lamming 1999; Masoumi et al. 2012; No. of cases 4 6 7 Masoumi et al. 2017; McNeill et al. 2006; Santos et al. 2004; Vaginal mucus discharge 75 66.7 57.1 Leroy et al. 2008; Motavalli et al. 2017). Increased progester- Mounting behavior 50 33.3 ND one profile was linked with an advancement of conceptus Bellowing 75 66.7 57.1 elongation and increased interferon tau (IFN-τ) production Tail raising 25 33.3 ND (Inskeep 2004; Stronge et al. 2005; McNeill et al. 2006). In Standing behavior 100 100 100 addition, FM prolonged the life span of the corpus luteum through suppression of PGF2α release (Elli et al. 2001; Trop Anim Health Prod

Table 4 Conception and pregnancy rates (%) of Baladi Parameters Treatment Overall mean cows after treatment with flunixin meglumine and aspirin compared Flunixin meglumine Aspirin Control with control Number of served cows 10 10 10 30 Number of conceived cows 6 4 3 13 Conception rate (%) 60a 40ab 30abc 43.3 Pregnancy rate (%) 60a 40ab 30abc 43.3

Values within the same row having different letters are significant at P <0.05

Geary et al. 2010). Kaveh et al. (2011) reported that treatment This result is in agreement with the finding of Rekwot et al. of heat stressed dairy cows with FM after-insemination be- (2000) who showed that nearly 51% of ovarian cycles of Zebu tween days 2–5 and 10–13 led to maintaining of corpus cows and the reported in Friesian cows (Savio et al. 1990). It luteum. This data are in accordance with our results that was suggested that persistency of the corpus luteum contrib- showed increased THI during hot months in Egypt, which uted to prolonging estrous cycle length over 21 days (Fonseca negatively reduced conception rate of Egyptian local cattle et al. 1983; Mukasa-Mugerwa et al. 1991; Stagnaro et al. that did not administrated with FM or aspirin at the time of 1994). Wilson et al. (1998) reported that dairy heifers exposed pregnancy recognition. On the other hand, Diskin and Morris to heat stress led to delay in corpus luteum regression. (2008) reported that lower progesterone concentration However, our results are not agreement with this finding as throughout the early luteal phase, or late in the normal rise the Egyptian cows were exposed to severe heat stress as indi- has been associated with decline of embryo survival. The pos- cated by high THI value, but estrous cycle length did not itive effect of FM and aspirin on conception rate recorded in change which may reflect reasonable adaptability of this this study could be due to maintenance of corpus luteum with- breed. In addition, administration of FM or supplementing out changing its secretory activity of progesterone. feeding with FM granules increased estrous cycle length in There was no significant effect of treatment on the length of heifers(Aiumlamaietal.1990; Odensvik et al. (1998). The normal estrous cycles in Egyptian local cows. The present same effect was observed after oral administration aspirin result is similar to that reported by El-Banna et al. (2005) (Spencer et al. 2016) due to the increased conceptus secretion and Damarany (2017) in Egyptian Baladi cows and in agree- of IFN-τ required for maternal pregnancy recognition ment with normal ovulation of this breed (Barkawi et al. (Thatcher et al. 1995; Bazer et al. 1997). 2006). Percentage of long cycles in cows that treated with Standing behavior was more frequent in Egyptian local FM were (75%), however, lower percentage of long cycles cows compared with other symptoms of estrus sings were recorded in cows that treated by aspirin 66.7%. Short (100%). Tail raising and mounting behavior is listed the cycles were completely absent in cows that treated with FM lowest frequency in addition to the vaginal mucous or aspirin in the present study. FM or aspirin treatment may discharge, bellowing and standing behavior. Foot (1974) affect the lifespan of corpus luteum, which lead to extent of and Baker and Seidel (1985) reported that one of the more cycle’s length. Wide-variation in percentage of long estrous important heat sign is the standing behavior for bull and cycles (9 to 41%) was observed in Egyptian local cows 70 to 90% of mounted cows were being in estrus (Hurnik (Damarany 2000; El-Gaafarawy and Abdel-Khaber 2001). et al. 1975; Chicoteau et al. 1989; Phillips 1993). It was

Table 5 Effect of treatment with flunixin meglumine and aspirin on progesterone concentration (ng/ml) at estrus, 7, 14, and 21 days post-estrus Egyptian Baladi cows during the experimental period

Days of estrous cycle Treatment

Flunixin meglumine Aspirin Control

Pregnant Non-pregnant Pregnant Non-pregnant Pregnant Non-pregnant

At estrus X ± SE 0.34 ± 0.03 0.20 ± 0.02 0.25 ± 0.029 0.21 ± 0.02 0.27 ± 0.03 0.17 ± 0.02 At day 7 X ± SE 2.62 ± 0.18 2.10 ± 0.10 2.56 ± 0.179 2.04 ± 0.31 2.65 ± 0.16 1.85 ± 0.21 At day 14 X ± SE 4.79 ± 0.75 4.03 ± 0.48 4.74 ± 0.679 3.68 ± 0.59 4.26 ± 0.19 3.41 ± 0.14 At day 21 X ± SE 6.72 ± 0.64a 0.24 ± 0.05 b 5.54 ± 0.56 a 0.24 ± 0.04b 4.84 ± 0.43a 0.21 ± 0.01b

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