Non-steroidal anti-inflammatory (COX-2 selective and non-selective) did not alter the luteal function and pregnancy rates of water buffalo embryo recipientsABSTRACT
The objective was to evaluate the influence of nonsteroidal anti-inflammatory drugs on the duration of the luteal phase, progesterone concentrations and pregnancy rates in buffaloes undergoing embryo transfer. Twenty-nine buffaloes were synchronized. After estrus detection, the recipients were divided into three groups: Control (uterine manipulation), Flunixum Meglumine (manipulation + administration) and Firocoxib (manipulation + administration). Ultrasound evaluations and hormonal dosages were performed on days 6, 12 and 18 to determine the plasma concentration of P4 and the functional duration of the corpus luteum. The control group presented a larger CL area on day 0 than the FM group, there was a significant increase in plasma progesterone concentration between D6 and D12 (P < 0.05), and a significant decrease between D6-D18 and D12-D18. Additionally, 8 buffalo cows underwent in vitro embryo transfer and were divided into three groups: CONT (manipulation of the reproductive tract), FM group (manipulation + administration) and FIRO group (manipulation + administration). Pregnancy diagnosis was performed at 30 and 60 days. No statistically significant differences in pregnancy rates were observed between groups. It was concluded that a single application of FM and FIRO at the time of embryo transfer did not significantly increase conception rates.
Keywords:
buffaloes; estrus; embryo recipients; folliculogenesis
RESUMO
O objetivo deste estudo foi avaliar a influência dos anti-inflamatórios não esteroides na duração da fase lútea, nas concentrações de progesterona e nas taxas de prenhez em búfalas submetidas à transferência de embriões. Foram sincronizadas 29 búfalas. Após a detecção do estro, as receptoras foram divididas em três grupos: controle (manipulação uterina), flunixum meglumine (manipulação + administração) e firocoxib (manipulação + administração). Avaliações ultrassonográficas e dosagens hormonais foram nos dias seis, 12 e 18, para determinar a concentração plasmática de P4 e a duração funcional do corpo lúteo. O grupo controle apresentou a maior área de CL no dia 0 em relação ao grupo FM; houve aumento significativo na concentração plasmática de progesterona entre D6 e D12 (P<0,05) e diminuição significativa entre D6-D18 e D12-D18. Adicionalmente, oito vacas búfalas foram submetidas à transferência de embriões produzidos in vitro e divididas em três grupos: CONT (manipulação do trato reprodutivo), FM (manipulação + administração) e FIRO (manipulação + administração). O diagnóstico de gestação foi realizado aos 30 e 60 dias. Não foram observadas diferenças estatisticamente significativas nas taxas de gestação entre os grupos. Concluiu-se que uma única aplicação de FM e FIRO no momento da transferência do embrião não aumentou significativamente as taxas de concepção.
Palavras-chave:
búfalas; estro; receptoras de embriões; foliculogênese
INTRODUCTION
The buffalo (Bubalus bubalis) has made a significant contribution to the global agricultural economy in the form of milk, meat, hides, and draft power (Young et al., 2019). The duration of the estrous cycle in buffaloes can range from 16 to 33 days, with an average duration of 21 to 24 days, varying between breeds and other factors such as climate, nutritional management, and genetics (Siqueira et al., 2009; Vale and Ribeiro, 2005). Animals with insufficient nutritional management, energy, protein, and minerals experience shorter or longer estrus, as is the case with thermal stress (Vale and Ribeiro, 2005). In addition to similarities in general anatomy and physiology with cattle, the reproductive endocrine parameters and all hormones involved in the hypothalamic-pituitary-gonadal axis during cyclical ovarian activity are similar (Drost, 2007; Terzano et al., 2012).
Understanding the estrous cycle and follicular dynamics in buffaloes allows for the development of hormonal treatments aimed at improving the indices of protocols used in embryo transfer, based on the inhibition of luteolysis with the use of progestogens, gonadotropin-releasing hormone (GnRH), and luteinizing hormone (LH), or by increasing the production of bovine interferon (bST) (Mann and Lamming, 2001a).
Luteolysis is directly related to the synthesis of prostaglandin 2 α (PGF2α), which in turn is related to the expression of the enzyme cyclooxygenase 2 (COX-2). There are attempts to increase pregnancy rates with linoleic acid, which has the property of inhibiting endometrial prostaglandin synthesis via inhibition of COX-2 activity, or the use of fatty acids that would compete with arachidonic acid for the action of enzyme classes in the synthesis of PGF2α, phospholipase A2 (PLA2), and cyclooxygenase (COX) (Cardoso et al., 2020).
Manipulation of the reproductive tract of bovine and buffalo females during embryo transfer can also cause the endometrium to stimulate the release of PGF2α (Scenna et al., 2005; Wann and Randel, 1990). Thus, when evaluating the negative effects of PGF2α release during embryo transfer, it can be assumed that strategies aimed at specifically inhibiting the enzymes involved in prostaglandin synthesis could increase the pregnancy rate of buffalo embryos (Mann and Lamming, 2001b).
Flunixin meglumine (FM) is part of the group of non-steroidal anti-inflammatory drugs (NSAIDs), which act in the peripheral circulation with anti-inflammatory, analgesic, antithrombotic, and antiendotoxic effects, and in the central nervous system with antipyretic and analgesic actions. These actions are due to the inhibitory activity generated by substances that metabolize arachidonic acid, COX, and lipoxygenase. Firocoxib is a highly selective COX-2 inhibiting NSAID with little effect on COX-1 enzymes, reducing the complications associated with the inhibition of the physiological pathway (Mann and Lamming, 2001b).
Thus, the synthesis of PGF2α would be directed towards the production of other less bioactive compounds without evidence of involvement in luteolysis. Another alternative to inhibit luteolysis would be the administration of NSAIDs, such as flunixin meglumine (FM), diclofenac, and firocoxib (FIRO) (Wasfi et al., 2015).
From the literature reviewed, no articles were found reporting the efficiency of these drugs in buffalo species during the estrous phase. Therefore, the objective of experiment 1 was to evaluate the application of flunixin meglumine and firocoxib at the time of reproductive tract manipulation in buffalo cows on the sixth day of the estrous cycle during the luteal phase, with the hypothesis of a possible reduction in the duration of the luteal phase and a possible decrease in plasma P4 concentration caused by reproductive tract manipulation. Experiment 2 aimed to evaluate the effect of FM and FIRO application as prostaglandin synthesis inhibitors at the time of embryo transfer on the pregnancy rate in buffalo embryo recipients at 30 and 60 days of gestation.
MATERIAL AND METHODS
This study was conducted in accordance with the ethical principles recommended by the National Council for the Control of Animal Experimentation (CONCEA) and approved by the Institutional Animal Use Ethics Committee of the School of Veterinary Medicine and Animal Science, Unesp - Botucatu Campus, under protocol 0109/2019.
For the experiment 1 were used twenty-nine Murrah buffalo females (Bubalus bubalis), aged 4 to 8 years (multiparous), with a body condition score equal to or greater than 3 (scale of 0 to 5) (Houghton et al., 1990), exhibiting cyclical ovarian activity and a postpartum period of more than 90 days, were used. These animals were from the herd of the Research and Development Unit of Registro, São Paulo (Regional Polo of Vale do Ribeira - Paulista Agribusiness Agency Agency), located in the State of São Paulo, Brazil, during the favorable reproductive season (autumn and winter). The animals were kept on Brachiaria sp. pasture, receiving mineral salt and water ad libitum.
The animals were synchronized on day 0 (D0) with estradiol benzoate 2mg/kg (Sincrodiol®, Ourofino-S.A., Cravinhos, São Paulo, Brazil) and an intravaginal progesterone implant (Sincrogest®, Ourofino-S.A.). On day 9 (D9), the intravaginal progesterone device was removed, and PGF2α 2mg/kg (Sincrocio®, Ourofino-S.A.) and eCG 2mg/kg (Sincro eCG®, Ourofino-S.A.) were administered. On day 10 (D10), estradiol benzoate 1mg/kg (Sincrodiol®, Ourofino-S.A.) was administered. The synchronization and estrus detection protocol is schematized in Fig 1.
Protocol applied in female buffaloes for synchronization and detection of estrus. D-10: first day of estrous synchronization; D-2: nine days after the beginning of synchronization; D-1: ten days after the beginning of estrous synchronization; D0*: ovulation day; D6: six days after ovulation, day of manipulation of the reproductive tract of females and application of treatments; D12: twelve days after ovulation; D18: eighteen days after ovulation; EB: estradiol benzoate; P4: application of the progesterone pessary; PGF2α: prostaglandin F 2α analog; eCG - equine chorionic gonadotrophin; Ov: ovulation day; [] P4: measurement of plasma levels of progesterone concentration; ØCL: estimation of the area of the CL.
Six days after estrus manifestation (D6), the 29 females underwent ultrasonographic evaluation of the reproductive tract to determine the characteristics of the corpus luteum (CL) and to assess the uterine wall for possible presence of content in the uterine lumen. Additionally, blood was collected on the same day to measure plasma P4 concentration. The 29 females were randomly divided into three groups: CONT (control + manipulation, n=9), FM (manipulation + FM, n=10), and FIRO (manipulation + FIRO, n=10) (Fig. 1).
The females in Group 1 underwent reproductive tract manipulation on D6, similar to embryo transfer, immediately after ultrasonographic evaluation and blood collection. Prior to manipulation, the animals received a caudal epidural anesthesia with 4 ml of 2% lidocaine (Anestésico L®, Pearson Saúde Animal). After anesthesia, the vulva and perineal region were cleaned with water and paper towels. A sanitized sheath-protected insemination device was then inserted into the vaginal fundus of the buffalo, and with the aid of rectal palpation, the device was guided through the cervical rings. After passing the cervix, the sanitized sheath was broken, allowing direct contact between the insemination device and the uterine wall. Finally, the insemination device was positioned in the final third of the uterine horn ipsilateral to the ovary that exhibited the CL on palpation. All manipulations were scored for difficulty from 1 to 3 (1 = insemination device easily positioned in the final third of the uterine horn; 3 = great difficulty positioning the insemination device in the final third of the uterine horn). The animals in the CONT group underwent ultrasonographic examinations and hormonal assessments between D6 and the end of the luteal phase (D12, D18) to determine plasma P4 concentration and functional duration of the CL.
The females in the FM and FIRO groups underwent reproductive tract manipulation on D6, immediately after ultrasonographic evaluation and blood collection, simulating embryo transfer identical to the technique described for the CONT group. However, at the end of the manipulation, the animals received FM 1.1 mg/kg intravenously (Flunixin Injectable®, Chemitec, Ipiranga, São Paulo, Brazil) in Group 2 (FM) and FIRO 0.1 mg/kg intravenously (Firo®, Botupharma, Botucatu, São Paulo, Brazil) in Group 3 (FIRO). The FM and FIRO groups, as well as the CONT group, underwent ultrasonographic examinations and blood collections for hormonal assays between D6 and the end of the luteal phase to determine plasma P4 concentration and functional duration of the CL.
Transrectal ultrasonographic examinations were performed with the animals restrained in a standing position, using an Aloka®8 ultrasound machine, model SSD 500, with a 5 MHz frequency transducer. The volume of luteal tissue and the luteal cavity were calculated using the mathematical formula: V= 4/3 π.a/2. (b/2)², where a = longitudinal axis and b = transverse axis. The final volume was considered as the total CL volume minus the cavity volume.
Blood samples were collected immediately after the ultrasonographic examinations (D6, D12, and D18) via jugular venipuncture using 21GX19 needles and deposited in vacuum blood collection tubes containing ethylenediaminetetraacetic acid (EDTA). The tubes with samples were kept in a refrigerated cooler until centrifugation at 2500 x g for 15 minutes. The obtained plasma was placed in labeled plastic tubes and stored in a -80°C freezer until hormonal assays were performed.
The blood sampling for plasma P4 quantification was performed immediately after the ultrasound examinations (D6, D12, and D18). For this, 5 mL blood samples were collected from the jugular vein using 21G gauge needles and stored into tubes containing ethylenediaminetetraacetic acid (EDTA). The tubes were kept in a refrigerated Styrofoam box until the moment of centrifugation. The samples were then centrifuged at 2.500 RPM for 15 min. The plasma was collected in labeled 2 mL tubes and stored at - 80° C until utilization. The plasma P4 concentrations were determined by means of commercial solid-phase kits and readings by radioimmunoassay method (model 1470 Gamma. Counter, Perkin Elmer) following the manufacturer's instructions (variation intra-assay of 8.15%; variation inter-assay of 8.66% and analytical sensitivity of 0.03ng/mL.
Estimates of CL area in mm² and plasma progesterone concentrations (ng/dL) were compared between days within each group by repeated measures analysis of variance, with contrasts by the Tukey method and p-value adjustment by the Bonferroni method. The same variables were compared between groups within each time point by one-way analysis of variance. All analyses were conducted using the R Program, with a significance level of 5% (R Development Core Team, 2020).
For experiment 2 were used eight buffalo embryo recipients, adult (multiparous), with a body condition score equal to or greater than 3 (scale of 0 to 5) (Houghton et al., 1990), exhibiting cyclical ovarian activity and a postpartum period of more than 60 days, were used. These animals were from herds in the Registro, São Paulo region. The animals were kept on Brachiaria sp. pasture with free access to mineral salt and water throughout the experimental period and during the period preceding embryo transfers.
Eight embryos produced in vivo at the São Paulo Agency for Agribusiness Technology (APTA), located in Registro, São Paulo, were used. The embryos were at the developmental stages of morula 1, morula 2, early blastocyst (BI), blastocyst (BL), and expanded blastocyst (BX), all of grade 1 quality (high quality). Due to difficulty in obtaining embryos at the blastocyst stage, embryos at the morula 1 and 2 stages were used (Fig. 2).
A- Embryos transferred into buffalo recipient females at D6 in the developmental stage of morula 1 (larger yellow arrow) and morula 2 (smaller red arrow). B - Embryo transferred into buffalo recipient females at D6 in the developmental stage of blastocyst (black arrow).
The superovulation protocol began on D0 with the insertion of an intravaginal progesterone implant (Sincrogest®, Ourofino-S.A.) and the administration of 2mg/kg estradiol benzoate (Sincrodiol®, Ourofino-S.A.) in the morning. On D4, two doses of 80 mg/kg FSH (Folltropin-V®, Bioniche, Canada) were administered, one in the morning and another in the afternoon. On D5, two doses of 60mg/kg FSH (Folltropin-V®, Bioniche, Canada) were administered, one in the morning and another in the afternoon. On D6, two doses of 40mg/kg FSH (Folltropin-V®, Bioniche, Canada) were administered, one in the morning and another in the afternoon, followed by an administration of 20mg/kg PGF2a (Sincrocio®, Ourofino-S.A). On D7, 20mg/kg FSH (Folltropin-V®, Bioniche, Canada) was administered in the morning, followed by an administration of 2mg/kg PGF2a (Sincrocio®, Ourofino-S.A) in the afternoon and the removal of the intravaginal progesterone implant (Sincrogest®, Ourofino-S.A). On D8 in the afternoon, 20µg/kg GnRH (Sincroforte®, Ourofino-S.A) was administered, followed by 0.53mg/kg PGF2a (Sincrocio®, Ourofino-S.A). On D9, artificial insemination was performed, followed by an administration of 0.53mg/kg PGF2a (Sincrocio®, Ourofino-S.A) in the morning and afternoon. On D10, 0.53 mg/kg PGF2a (Sincrocio®, Ourofino-S.A) was administered in the morning. On D14, the embryos were collected followed by an administration of 0.53mg/kg PGF2a (Sincrocio®, Ourofino-S.A).
This superovulation protocol, involving sequential administrations of FSH, PGF2a, and GnRH, is designed to enhance the production and collection of high-quality embryos for transfer.
The animals were synchronized on D0 with estradiol benzoate 20 mg/kg (Sincrodiol®, Ourofino-S.A) and an intravaginal progesterone implant (Sincrogest®, Ourofino-S.A) was inserted. On D9, the intravaginal progesterone device was removed, and PGF2α 2mg/kg (Sincrocio®, Ourofino-S.A) and eCG (Sincro eCGr®, Ourofino-S.A) were administered. On D10, estradiol benzoate 1mg/kg (Sincrodiol®, Ourofino-S.A) was administered, and on D18, fixed-time embryo transfer (FTET) was performed.
Recipients were used between the 6th and 9th day after estrus observation. On the day of embryo transfer, recipients were selected via rectal palpation and ultrasonographic examination of the reproductive tract. Recipients with a corpus luteum (CL) and no fluid content in the uterus were selected to receive the embryos. After selection, the suitable recipients were restrained in chutes for the following procedures: caudal epidural anesthesia with 4mL of 2% lidocaine (Anestésico L®, Pearson Saúde Animal); vulva and perineal region sanitation with water and paper towel; introduction of a sanitary sheath-protected embryo transfer device into the vaginal fornix, and with the aid of rectal palpation, the device was passed through the cervical rings. Once through the cervix, the sanitary sheath was broken, allowing direct contact between the embryo transfer device and the uterine wall; positioning the device in the final third of the uterine horn ipsilateral to the ovary with the CL, where the embryos were deposited.
All embryo transfers were classified according to the degree of difficulty, scored as follows: score 1 - no difficulties in passing the cervix and positioning the embryo transfer device in the final third of the uterine horn; score 2 - moderate difficulty in performing the procedure; score 3 - significant difficulty in performing the procedure. Following these procedures, the recipients were randomly divided into three groups: CONT - control + manipulation: embryo transfer, FM group - embryo transfer + FM administration, and FIRO group - embryo transfer + FIRO administration.
After uterine flushing, the storage filter was washed with phosphate-buffered saline (PBS) for embryo recovery. The remaining solution from the filter was placed in a Petri dish for subsequent embryo location and selection. The Petri dish was marked with a permanent marker, and the location process was conducted under a stereomicroscope. The dish was mapped, and when an embryo was located, it was recovered and selected according to the International Embryo Technology Society (IETS) code. Subsequently, the embryo was maintained in TE-Hepes medium (a medium that does not alter pH with the environment) and used for embryo packaging. Before packaging, the embryos underwent a series of washes, an average of 5 washes, in TE-Hepes medium. Packaging was done in a 0.25 mL straw, divided into 5 columns: 1st - TE-Hepes medium; 2nd - air; 3rd - TE-Hepes medium + embryo; 4th - air; and the last - TE-Hepes medium.
Pregnancy diagnosis was performed through transrectal ultrasonographic examinations on days 30 and 60 of gestation, using an Esaote®4 ultrasound machine, model Aquila ProVet, with a bifrequency transducer (6-8 MHz). Pregnancy at 30 days was confirmed by the visualization of the embryo, appearing as a hyperechoic structure without any visible differentiation, with cardiac activity observed (Pierson and Ginther, 1984). A similar evaluation was performed at 60 days.
To compare the pregnancy frequencies in buffaloes subjected to ET, the Chi-square test with Yates' correction was used. All analyses were conducted using the R Program, with a significance level of 5% (R Development Core Team, 2020).
RESULTS
In experiment 1 the manipulation scores in relation to the degree of procedural difficulty are represented in Fig. 3.
Pie chart demonstrating the percentage of difficulty scores in the procedure (Score 1 - no difficulties (18/29), uterine; Score 2 - moderate difficulty (4/29); Score 3 - great difficulty (7/29).
Based on the analyses performed, it was interesting to observe that while the treated groups maintained similar CL size (mm2) throughout the experiment (P>0.05, Fig. 4), the Control group presented a significant reduction (P<0.05) when comparing D6 and D12 to D18.
Means ± SEM (vertical lines applied to the bars) of the estimated areas for the corpus luteum in buffaloes submitted to reproductive tract manipulation, according to treatment and time after manipulation (days). Different letters indicate P<0.05).
When considering the serum progesterone concentration (ng/mL) it was observed that for all groups, a significant increase (P<0.05) was observed on D12, followed by an important decrease on D18 (P<0.05) (Fig. 5). However, it’s important to indicate that both FM and FIRO group at D18 were able to maintain a higher progesterone concentration when compared to Control group (P<0.05, Fig. 5).
Means ± SEM (vertical lines applied to the bars) of the serum P4 dosages in buffaloes subjected to uterine manipulation, according to treatment and time after manipulation (days). Legend: FIRO = Firocoxib, FM = flunixin-meglumine; CL = corpus luteum.
Regarding the pregnancy rate of the recipients, no differences were observed between the experimental groups according to the treatment used in the study (experiment 2: “Effect of flunixin meglumine and firocoxib on the pregnancy rate of recipients of buffalo embryos”), so that of the 8 embryos transferred, only one pregnancy was confirmed by ultrasound after 30 days.
DISCUSSION
While there are few publications on the impact of difficulty levels during embryo transfer on conception rates in buffalo females, it is known that these animals exhibit specific characteristics. These include factors such as the number of calvings, intervals between calvings, body condition scores, and the experience of the inseminator.
In this study, the highest conception rates were observed in score 1 (no difficulty), followed by scores 2 and 3, respectively. This result aligns with other studies that evaluated conception rates based on the difficulty of passing the cervix during artificial insemination (AI) in buffalo females. These studies categorized difficulty levels as: 0 (no difficulty), 1 (intermediate difficulty), and 2 (high difficulty), concluding that score 0 was predominant among the evaluated animals (Crudeli et al., 2008; Guevara, 2012).
Additional factors to consider in an AI program for buffalo include body condition score, seasonality, nutritional and sanitary management, animal age (multiparous vs. primiparous), climate, among others (Guevara, 2012). Generally, buffaloes respond well to superovulation, showing good ovulation rates and corpus luteum formation despite individual variability (Ohashi et al., 2017).
Excessive manipulation of the reproductive tract can lead to trauma and subsequent local inflammatory processes, during which PGF2α may be released from endometrial cells (Noronha et al., 1999; Scenna et al., 2005; Wann and Randel, 1990). This release of PGF2α can result in increased corpus luteum size, explaining the significant difference between the CONT group and the FM group on day 6. Other factors, such as the inseminator's lack of experience and animals with difficulty scores of 2 and 3, may also contribute to these changes (Cardoso et al., 2020).
On the 16th day of the estrous cycle, heifers treated with FM (2.5 mg/kg every 8 hours) showed a reduction in the amplitude of PGF2α pulses detected by peaks in 13,14-dihydro-15-keto-PGF2α (PGFM) plasma levels before and during luteolysis, delaying the onset of progesterone decline (Pugliesi et al., 2012). The effects of NSAIDs, particularly FM, in prolonging the estrous cycle in heifers are well-documented (Aké-López et al., 2005; Odensvik and Johansson, 1995).
On days 12 and 18 of the estrous cycle, an increase in the average volume of corpora lutea was observed in the FM and FIRO groups. This gradual increase was noted approximately on the 6th and 9th days post-ovulation (Acosta et al., 2003; Borges et al., 2003; Grygar et al., 1997; Kastelic et al., 1990; Sartori et al., 2004; Viana et al., 2000). The use of NSAIDs in this study likely slowed the regression of the corpus luteum on days 12 and 18. Similar effects were noted in goats, where NSAIDs were used to prevent early luteal regression in superovulated females (Salles et al., 1998; Traldi et al., 1996). This suggests that FM and FIRO effectively reduce premature CL regression by inhibiting prostaglandin synthesis.
Administration of prostaglandin synthesis inhibitors on the day of ovulation and embryo recovery can significantly reduce CL regression and increase the number of transferable embryos (Battye et al., 1988; Traldi et al., 1996). The dosages, frequency, and duration of FM and FIRO treatments used in this study might explain the results. The half-life of FM after a single intramuscular application in cattle is approximately four hours, extending to 26 hours after four daily applications (Odensvik and Johansson, 1995).
Studies on firocoxib revealed high bioavailability, prolonged half-life (18h ± 8h), and a large volume of distribution after a single 0.5mg/kg intravenous dose in calves (Stock et al., 2015). The oral form reached absolute plasma bioavailability within 3.9 hours, binding to plasma proteins with a long half-life of 30 hours due to its lipophilic nature, making it effective for treating soft tissue inflammation (Kvaternick et al., 2007). In bovine embryo recipients, these drugs helped inhibit prostaglandin synthesis caused by uterine manipulation, thereby improving pregnancy rates (Hockett et al., 2004; McNaughtan, 2004; Scenna et al., 2005).
Although all groups showed an increase in plasma progesterone concentration between days 6 and 12, only the CONT group showed a significant increase. This may be due to the small sample size and natural variation among animals. In this study, progesterone levels did not significantly differ between treated and untreated animals despite varying trends in progesterone decline during luteolysis (day 18). The CONT group experienced a sharp decline, while treated animals showed a gradual decrease. Similar results were found in studies evaluating FM effects on progesterone levels during the estrous cycle in hybrid heifers and cows (Pinto-Neto et al., 2008).
Other studies corroborate the findings that reproductive tract manipulation in heifers and cows treated or not with FM resulted in lower P4 levels in untreated animals compared to those treated with NSAIDs (Cardoso et al., 2020). Although PGF2α levels were not quantified in this study, animals undergoing reproductive tract manipulation had lower average plasma P4 concentrations than those receiving FM and FIRO at the time of manipulation, indicating effective PGF2α synthesis inhibition (Cardoso et al., 2020).
Studies on heifers treated with FM (2.2mg/kg orally three times daily) for nine days showed decreased progesterone concentrations during luteolysis compared to control animals from day 14 of the cycle (Odensvik and Johansson, 1995). Other studies confirmed that FM maintained normal progesterone levels while reducing 15-ketodihydro-PGF2α, a prostaglandin metabolite, and delaying luteolysis (Aiumlamai et al., 1990; Aké-López et al., 2005; Pinto-Neto et al., 2008). This study did not measure this prostaglandin metabolite.
No research specifically investigates firocoxib use in reproductive management of ruminants. However, its use has been documented in horses (Friso et al., 2019; Kvaternick et al., 2007; Okada et al., 2019; Orsini et al., 2012), pre-weaning calves (Schade et al., 2021; Stock et al., 2015), adult goats (Stuart et al., 2019), and dogs (Davila et al., 2013; Mira and Figueiredo, 2011). In mares, FM, firocoxib, and meloxicam effects on embryo mobility were studied, indicating that firocoxib minimally affected embryo mobility, making it the safest NSAID for early pregnancy, especially towards the end of gestation and during parturition (Okada et al., 2019). In this study, FIRO results were comparable to FM despite not showing statistically significant differences.
The initial hypotheses of this study were not confirmed, as flunixin meglumine and firocoxib did not increase conception rates in embryo recipient buffaloes. Research on anti-inflammatory use during bovine maternal recognition of pregnancy has yielded mixed results. While some studies indicate these drugs do not improve conception rates (Erdem and Guzeloglu, 2009; Hirsch and Philipp, 2009), others suggest NSAIDs as an alternative method to enhance herd reproductive performance (Guzeloglu et al., 2007; Merrill et al., 2007; von Krueger and Heuwieser, 2010).
This study demonstrated that 50% of embryo transfers had a difficulty score of 1 (no difficulty). However, reproductive tract manipulation during embryo transfer stimulates endometrial PGF2α release (Scenna et al., 2005), compromising pregnancy rates in embryo recipients (Hockett et al., 2004). The embryo development stage most susceptible to prostaglandin's deleterious effects is the transition from morula to blastocyst/expanded blastocyst stage (Hockett et al., 2004; Maurer and Beier, 1976; Schrick et al., 2003; Wiltbank et al., 1989). Embryonic losses occurred because the embryo may not have sufficiently inhibited PGF2α secretion (Thatcher et al., 2001). In ruminants, interferon-tau (INFτ) initiates the luteolytic mechanism (Bazer et al., 2015), and less developed embryos produce minimal or undetectable interferon levels (Mann and Lamming, 2001b).
In vivo embryo transfer (ET) from superovulation (SOV) programs is inefficient in buffaloes, making in vitro embryo production (IVP) more relevant (Liang et al., 2008). This inefficiency is linked to failures in FSH-based superovulation protocols, low recruitable follicle populations, and failure of oocyte capture by the infundibulum (Carvalho et al., 2011). Low embryo recovery rates in buffaloes may result from embryos lost in the abdominal cavity or retained in uterine folds, hindering their recovery via uterine flushing (Baruselli et al., 1997) and the high incidence of follicular cysts caused by superovulation (Zicarelli, 1994).
Other contributing factors include the lack of selection based on reproductive capacity for donors and recipients, insufficient knowledge on gonadotropin dosages and metabolism, and the absence of a defined superovulation and AI protocol (Misra et al., 1993).
Regarding the pregnancy rate of the recipients, our study showed that the use of FM and Firocoxib did not result in an increase in conception rates. This result may have been influenced by the small sample size (n = 8 embryos) due to the low recovery of embryos in superovulated animals.
There are no published studies on using flunixin meglumine and firocoxib as prostaglandin synthesis inhibitors in buffalo embryo transfer and conception rates. Further research with larger animal samples is necessary.
CONCLUSION
Flunixin meglumine and firocoxib administration did not have detrimental effects on the estrous cycle of buffaloes, effectively inhibiting abrupt declines in plasma progesterone concentrations and delaying corpus luteum regression. However, their administration during embryo transfer did not affect pregnancy rates in buffalo embryo recipients.
ACKNOWLEDGMENTS
We thank APTA (Agribusiness and Technology Agency from the state of São Paulo, Brazil), UNESP (São Paulo State University, Botucatu, São Paulo, Brazil), FAPESP (Research Support Foundation from São Paulo State), National Council for Scientific Development (CNPq), Coordination for the Improvement of Higher Education Personnel (CAPES) and Botupharma Veterinary Products (Botucatu, São Paulo, Brazil).
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FUNDING
This work was supported by the FAPESP (Research Support Foundation from São Paulo State) for the concession of a graduate-level research scholarship (2019/14353-9), financial support for study development (2018/21211-3) and Coordination for the Improvement of Higher Education Personnel (CAPES).
Legend: FIRO = Firocoxib, FM = flunixim-meglumine.
