BAD基因在敖汉细毛羊发情不同时期卵巢表达规律及对生殖激素的影响

本实验研究旨在探究BAD基因在敖汉细毛羊中的不同发情时期卵巢中表达量的变化规律以及对生殖激素的影响,为探讨其与绵羊发情的关系奠定基础。首先利用放射免疫方法测定敖汉细毛羊血液中促卵泡激素(FSH)、促黄体生成素(LH)、雌二醇(E_2)和孕激素(P_4)的浓度,然后利用qRT-PCR检测乏情期、发情前期、发情间期和发情期卵巢BAD基因的表达量变化。结果表明:卵巢中BAD基因在发情前期的表达量极显著高于乏情期、发情期和发情间期(P0.01),在发情期的表达量极显著高于乏情期和发情间期(P0.01),乏情期和发情间期的表达量基本相同(P0.05);E_2和LH浓度在BAD基因表达量最高的发情前期维持较高水平,P_4的浓度维持较低水平。综上可知,BAD基因的表达可能促进了E_2和LH浓度升高,抑制了P_4产生,进而影响细毛羊发情,可为绵羊发情的研究提供借鉴。     

转化生长因子βⅠ型受体基因在敖汉细毛羊不同组织及发情时期卵巢中的表达分析

试验旨在探究转化生长因子βⅠ型受体（transforming growth factor-beta receptorⅠ,TGF-βRⅠ）基因在敖汉细毛羊中的组织表达情况,以及在不同发情时期卵巢中表达量的变化规律,为探讨其与绵羊发情的关系奠定基础。以24只季节性发情的敖汉细毛羊为研究对象,分为乏情期、发情间期、发情前期和发情期4组,每组6只。首先利用实时荧光定量PCR检测其发情期甲状腺、下丘脑、垂体、卵巢、肾上腺、心脏、肝脏、脾脏、肺脏、肾脏、胰腺、肌肉12个组织中TGF-βRⅠ基因的表达情况,其次对4个不同发情时期卵巢TGF-βRⅠ基因的表达量变化进行研究。结果显示,TGF-βR Ⅰ在各组织中均有表达,在卵巢和甲状腺中表达量最高,极显著高于其他组织（P<0.01）;在下丘脑、垂体、胰腺、肾上腺、脾脏和肺脏组织表达量较高,显著高于心脏、肝脏、肾脏和肌肉组织（P<0.05）。卵巢中TGF-βRⅠ基因在发情前期表达量最高,极显著高于其他3个时期（P<0.01）,发情间期表达量最低,极显著低于其他3个时期（P<0.01）。综上所述,卵巢组织中TGF-βRⅠ基因在发情前期时可能对排卵前卵泡的成熟起促进作用。     

Immunohistochemical and qPCR determination of the expression and serum level of anti‐Müllerian hormone in pre‐pubertal,intact and ovarian remnant syndrome detected bitches

The aim of this study was to determine the serum concentrations, ovarian presence and expression of anti‐Müllerian hormone (AMH) in pre‐pubertal, bitches with signs of ovarian remnant syndrome (ORS) and intact bitches. In addition, we aimed to verify the suitability of serum AMH concentrations for diagnostic purposes in sterilized bitches and/or in suspected cases of ORS in the field of veterinary medicine. For this purpose, 36 healthy female dogs divided into six groups: proestrus, oestrus, dioestrus, anoestrus, pre‐pubertal and ORS. Serum AMH concentrations were determined by electrochemiluminescence immunoassay, and ovarian presence and distribution of AMH was confirmed by immunohistochemical and qPCR techniques. According to the results of qPCR, while the expression values of AMH were at the highest concentrations in the proestrus and oestrus, there was a statistically significant decrease in these values at the later stages of the cycle (p < 0.05). According to hormone analysis, the serum AMH values of the ORS group had decreased significantly compared with the proestrus and oestrus (p < 0.05). Although serum AMH levels of ORS group were increased compared with anestrus and pre‐pubertal groups, this increase was statistically non‐significant (p > 0.05). Immunohistochemically, AMH expression was first observed in the granulosa cells of primordial follicles in folliculogenesis. Expression values were the highest in the proestrous and oestrus groups, but values from bitches in later stages of the cycle were statistically significant decrease in comparison with these groups (p < 0.05). As a result, AMH concentration and expression were found to be higher in proestrus and oestrus than in other periods (p < 0.05). In addition, the measurable level of AMH concentration in bitches with ORS is an indication that it can be used in the diagnosis of ORS.     

Expression of oestrogen receptor,androgen receptor and progesterone nuclear receptor in sheep uterus during the oestrous cycle

Oestrogen, androgen and progesterone are involved in the regulation of uterine physiological functions, with the participation of the following proteins: oestrogen receptor (ER), androgen receptor (AR) and progesterone nuclear receptor (PGR). In this study, we used immunohistochemistry to detect the localization of ERα, ERβ, AR and PGR in sheep uterus. Additionally, we used real‐time polymerase chain reaction (RT‐qPCR) and Western blot technique to analyse their expression profiles at different stages of sheep oestrous cycle in the endometrium and myometrium. Immunohistochemical analysis showed that ERα, ERβ, AR and PGR were present in sheep uterus in oestrus, mainly in the uterine luminal epithelium, stroma, gland and myometrium. Real‐time polymerase chain reaction results showed that in the endometrium, ERα expression level was highest in oestrus. ERβ and PGR, instead, were highly expressed in pro‐oestrus. In the myometrium, ERα was highly expressed in both oestrus and pro‐oestrus, and ERβ was highly expressed in oestrus and dioestrus. Progesterone nuclear receptor expression was highest in oestrus, followed by metoestrus. In the endometrium, both receptors ERα and ERβ were abundant in pro‐oestrus, while the maximum AR protein content was found in oestrus. At this stage of the oestrous cycle, PGR protein concentration in the myometrium was significantly lower than those observed in other stages. These results suggest that these receptors are important for sheep reproductive function, as their expression at mRNA and protein levels exhibits particular time‐ and tissue‐specific profiles along the oestrous cycle.     

Endometrial cytology of the normal bitch throughout the reproductive cycle

Samples for endometrial cytology were collected both from live bitches using transcervical uterine cannulation (n=48) and postmortem (n=10). The cells identified were endometrial epithelial cells, leucocytes, erythrocytes, spermatozoa, bacteria and cervical or vaginal cells. The endometrial epithelial cells varied morphologically throughout the reproductive cycle and had signs of degeneration during late dioestrus and during early and mid-anoestrus following dioestrus and postpartum. Neutrophils were the most common leucocytes observed during pro-oestrus, oestrus, dioestrus and early pregnancy, and lymphocytes during anoestrus. Macrophages were frequently seen during anoestrus. Erythrocytes were found in variable numbers at all stages of the reproductive cycle. Spermatozoa were detected in samples collected during oestrus and early pregnancy in bitches which had their last mating one to three days previously. Bacteria were commonly observed during pro-oestrus and oestrus. Cornified cervical or vaginal cells were present during pro-oestrus and oestrus. This study demonstrated that the numbers, types, proportions and morphology of ceils in endometrial cytological samples from normal bitches varied throughout the reproductive cycle.     

Anti‐Müllerian Hormone in the Domestic Dog during the Anestrus to Oestrous Transition

The reproductive cycle of the domestic dog features a long period of relative ovarian inactivity or anestrus. The mechanism of anestrous termination/oestrous resumption is not yet fully understood, which presents a challenge to the development of oestrous induction protocols. In this study, we assess the possibility that anti‐Müllerian hormone (AMH) might play a role in this transition by characterizing its patterns of expression in the circulation during the transition from anestrus to oestrous and in all stages of ovarian follicular growth. Serum samples from five beagles (2.0–4.5 years) were collected three times per week at least 30 days prior to the onset of oestrous and assessed for AMH concentrations. Serum AMH concentration increased significantly during the transition from anestrus to proestrus and then declined back to the anestrous baseline beginning on day ?4 before the luteinizing hormone surge, which was determined by changes in serum progesterone concentrations. Cortical sections of ovaries from females undergoing routine ovariohysterectomy (aged 8 months–5 years, n = 4) were evaluated for AMH by immunohistochemistry. Pre‐antral and small antral follicles were most strongly immunoreactive for AMH. These data suggest that the increase in the number of antral follicles is associated with the rise in serum AMH as the anestrous period comes to an end. The rise in AMH might be useful in predicting the onset of oestrus and therefore assist with the optimization of oestrous induction protocols and possibly other assisted reproductive technologies.     

非繁殖季节中国美利奴羊血清E2、P4、FSH、LH激素变化规律研究

为了在非繁殖季节判断绵羊的发情状况,试验以新疆高海拔地区中国美利奴羊为研究对象,采用酶联免疫测定(ELISA)法测定绵羊血清中雌二醇(E2)、孕酮(P4)、促卵泡素(FSH)、促黄体生成素(LH)的含量,研究激素诱导发情羊、发情症状不明显羊、不发情羊体内激素分泌规律的差异。结果表明:激素诱导发情羊的E2含量比不发情羊高,差异显著(P<0.05);但是在P4、FSH、LH水平上,激素诱导发情羊要低于不发情羊,差异不显著(P>0.05)。发情羊与发情不明显羊在4种激素含量上差异不显著(P>0.05),说明公羊试情存在一定缺陷,而通过测定E2含量判定绵羊发情状态将成为未来研究的重点。     

不同繁殖季节绵羊卵巢组织中piRNAs序列特征及表达差异分析

为了分析绵羊卵巢组织中piRNAs的序列特征及不同繁殖季节绵羊卵巢组织中piRNAs的表达差异,本试验采用Solexa测序技术对休情季节（休情期）和发情季节（卵泡期和黄体期）滩羊卵巢组织进行高通量测序,采用生物信息学方法筛选绵羊卵巢中的piRNAs,对piRNAs的长度分布、首位碱基偏好、基因组来源、染色体分布、链特异性、ping-pong循环特征、piRNAs比对重复序列类型进行分析,并对发情季节与休情季节绵羊卵巢之间差异表达的piRNAs进行靶基因预测及其靶基因的GO功能和KEGG Pathway富集分析。结果显示,绵羊卵巢中不同长度的piRNAs首位碱基偏好性不一致;3个时期（休情期、黄体期和卵泡期）的piRNAs均主要比对到基因内含子、编码区及lncRNA区域,而比对到重复序列的piRNAs相对较少;绵羊卵巢piRNAs在染色体上呈不均匀分布;绵羊piRNAs簇存在很强的链特异性,但首位碱基偏好性不明显;3个时期的piRNAs均无明显的ping-pong循环特征。KEGG富集分析发现,差异piRNA靶基因主要富集在RNA运输通路（RNA transport pathway）、嘌呤代谢通路（purine metabolism pathway）、黏着斑通路（focal adhesion）、PI3K-Akt信号通路（PI3K-Akt signaling pathway）、Hippo信号通路（Hippo signaling pathway）等与绵羊繁殖相关的通路。本研究揭示了不同繁殖季节绵羊卵巢组织中的piRNAs序列特征,暗示绵羊卵巢组织中的piRNAs可能通过BAD等基因和黏着斑通路等调控绵羊季节性繁殖,为深入解析绵羊季节性发情分子机制提供一定参考。     

AANAT基因在滩羊不同繁殖时期卵巢中的转录差异及DNA甲基化程度分析

试验旨在检测5-羟色胺-N-乙酰基转移酶(AANAT)基因在绵羊休情季节和繁殖季节(卵泡期和黄体期)卵巢组织中的转录差异,并分析转录差异是否由DNA甲基化修饰程度改变所导致。试验采用自然环境条件和饲养管理一致,且体重差异在0.5 kg范围内的空怀母滩羊作为试验动物,采集其休情期、卵泡期和黄体期(每个时期3只)的卵巢组织,采用SYBR染料法进行实时荧光定量PCR检测AANAT基因在滩羊不同繁殖时期卵巢组织中的转录水平。随后针对转录水平有差异的两个时期(休情期和卵泡期)的样本,利用MethPrimer 2.0在线软件预测AANAT基因启动子区和第一外显子区的CpG岛;用重亚硫酸盐测序法(BSP法)检测AANAT基因启动子区及第一外显子区的甲基化程度。试验结果显示,滩羊休情期卵巢组织中AANAT基因转录水平显著低于卵泡期的AANAT基因转录水平(P<0.05),休情期与黄体期滩羊卵巢组织中AANAT基因的转录水平差异不显著(P>0.05)。滩羊卵巢组织中AANAT基因启动子区上存在着一个长度为173 bp的CpG岛,第一外显子区存在着一个长度为118 bp的CG岛。然而,两个甲基化岛区内的单个CpG位点甲基化程度在滩羊休情期和卵泡期之间均不存在显著差异,暗示AANAT基因的表达受甲基化修饰外的因素调控。本研究结果可为进一步探讨AANAT基因在季节性发情和卵泡成熟中的功能提供参考资料。     

AANAT基因在滩羊不同繁殖时期卵巢中的转录差异及DNA甲基化程度分析

试验旨在检测5-羟色胺-N-乙酰基转移酶（AANAT）基因在绵羊休情季节和繁殖季节（卵泡期和黄体期）卵巢组织中的转录差异,并分析转录差异是否由DNA甲基化修饰程度改变所导致。试验采用自然环境条件和饲养管理一致,且体重差异在0.5 kg范围内的空怀母滩羊作为试验动物,采集其休情期、卵泡期和黄体期（每个时期3只）的卵巢组织,采用SYBR染料法进行实时荧光定量PCR检测AANAT基因在滩羊不同繁殖时期卵巢组织中的转录水平。随后针对转录水平有差异的两个时期（休情期和卵泡期）的样本,利用MethPrimer 2.0在线软件预测AANAT基因启动子区和第一外显子区的CpG岛;用重亚硫酸盐测序法（BSP法）检测AANAT基因启动子区及第一外显子区的甲基化程度。试验结果显示,滩羊休情期卵巢组织中AANAT基因转录水平显著低于卵泡期的AANAT基因转录水平（P<0.05）,休情期与黄体期滩羊卵巢组织中AANAT基因的转录水平差异不显著（P>0.05）。滩羊卵巢组织中AANAT基因启动子区上存在着一个长度为173 bp的CpG岛,第一外显子区存在着一个长度为118 bp的CG岛。然而,两个甲基化岛区内的单个CpG位点甲基化程度在滩羊休情期和卵泡期之间均不存在显著差异,暗示AANAT基因的表达受甲基化修饰外的因素调控。本研究结果可为进一步探讨AANAT基因在季节性发情和卵泡成熟中的功能提供参考资料。     

Immunohistochemical Studies on Oestrogen Receptor Alpha (ERα) and the Proliferative Marker Ki-67 in the Sow Uterus at Different Stages of the Oestrous Cycle

In order to better understand physiological changes during the different stages of the oestrous cycle, immunohistochemistry was used in the present study to investigate the distribution of oestrogen receptor alpha (ERα) as well as the proliferative marker Ki‐67, in the sow uterus during the oestrous cycle. Uterine samples were collected from multiparous sows with normal reproductive performance at selected stages of the oestrous cycle: at late dioestrus (d 17), prooestrus (d 19), oestrous (d 1), early dioestrus (d 4) and dioestrus (d 11–12), respectively. The tissue samples were fixed in 10% formaldehyde, embedded in paraffin and subjected to immunohistochemistry using monoclonal antibodies against ERα (C‐311) and Ki‐67 (MM‐1). In general, the immunostaining of both ERα and Ki‐67 was confined to nuclei of the target cells. Variations were seen, not only at the different stages of the oestrous cycle, but also in the different tissue compartments of the uterus. In the epithelia, the strongest ERα staining and highest amount of positive Ki‐67 cells were found at early dioestrus. In the myometrium, the highest levels of staining of both ERα and Ki‐67 positive cells were found at pro‐oestrus and oestrus. For the proliferative marker, Ki‐67, no positive cells were found at dioestrus and late dioestrus in the epithelium and myometrium. In the connective tissue stroma (subepithelial layer), the highest number of ERα positive cells were found at oestrus, which was significantly different compared with other stages (p≤0.05), whereas the levels of Ki‐67 positive cells were relatively low and did not differ between the stages examined. Significant correlations between the number of ERα positive cells in the stroma and Ki‐67 positive cells in the epithelia were observed. This suggests indirect regulatory mechanisms on epithelial proliferation via ERα in the stroma. In conclusion, these findings in the sow uterus show that the presence of ERα as well as Ki‐67 protein varies not only between different stages of the oestrous cycle but also between different tissue compartments of the uterus. These findings indicate various regulatory mechanisms and stress the importance of localising ERα and proliferating cells in different uterine tissues.     

Expression of messenger RNA coding for 5-HT receptor, alpha and beta adrenoreceptor (subtypes) during oestrus and dioestrus in the bovine uterus

Serotoninergic and adrenergic receptors (5-HTR and AR) are involved in the regulation of uterine contractility. The objective of this study was to compare mRNA levels of 5-HTR(1A), 5-HTR(1B), 5-HTR(1D), 5-HTR(1F), 5-HTR(2A), 5-HTR(2B), 5-HTR(2C), 5-HTR(4) and alpha(1A), alpha(1B), alpha(1D), alpha(2AD), alpha(2B), alpha(2C), and beta(1), beta(2), beta(3)-AR in oestrus and dioestrus, and at three uterine locations (tip, middle and base) using quantitative real-time polymerase chain reaction. Uterine specimens consisting of endometrium and myometrium including vessels and serosa were collected from cows in oestrus (n = 10) and dioestrus (n = 15) respectively. Levels of 5-HTR and AR mRNA were expressed relative to the geometric mean of ribosomal RNA (18S), ubiquitin and glyceraldehyde phosphate dehydrogenase by the mean values of geNorm algorithm. 5-HTR(1A), 5-HTR(2C) and beta(3)-AR mRNA could not be detected in uterine tissues. The mRNA levels of 5-HTR(1F) and 5-HTR(2B) were lower (P < 0.05), but of 5-HTR(4) were higher (P < 0.05) in oestrus than in dioestrus. The mRNA levels of alpha(1A)-AR, alpha(2AD)-AR, alpha(2B)-AR were lower (P < 0.05), but of alpha(2C)-AR and beta(2)-AR were higher (P < 0.05) in oestrus than dioestrus. The mRNA levels of 5-HTR(1B) and 5-HTR(1D) (oestrus) and of alpha(2AD)-AR (dioestrus) differed among uterine locations (base > middle > tip; P < 0.05). The mRNA expression of 5-HTR and AR (subtypes) in bovine uterus was associated with cycle activity and varied according to uterine location. Additional studies on protein level will be carried out in order to elucidate the role of these receptor families on uterine contractility, which may then help to clarify clinical relevance.     

Vaginal and Cervical Anatomic Modifications During the Oestrus Cycle in Relation to Transcervical Catheterization in the Domestic Cat

In a previous study we observed that it is possible to reach the cervix in all queens with a 1 mm diameter probe only. So, we developed both a new technique and a catheter (1 mm diameter) to allow transcervical insemination [Zambelli and Castagnetti 2001]. The aims of this study were to investigate vaginal and cervical anatomic modifications during the various stages of the oestrus cycle and to test the previously described technique of transcervical catheterization during the various stages of the oestrus cycle. In experiment 1, silicon impression moulds were obtained from the reproductive tracts of 21 queens' cadavers and vaginal and cervical measures were taken. The results showed that there are some significant anatomic modifications during the various stages of the oestrus cycle in vaginal and cervical anatomy, principally related to the dorsal medial fold increase induced by the follicular phase. In experiment 2, transcervical catheterization was attempted in 95 queens at various stages of oestrus cycle both during reproductive and non-reproductive season. After catheterization, methylene blue solution was injected through the cervical catheter. Successful catheterization was assessed during surgery, when colour was observed in the uterine horns. It was possible to perform transcervical catheterization during non-reproductive season in 16 of 20 anoestrus queens and in 12 of 15 induced oestrus queens; during reproductive season in nine of 21 interoestrus queens, in eight of 13 dioestrus/pregnancy queens, in four of 18 oestrus queens and in seven of eight queens in first oestrus during lactation.     

Resident Macrophages and Lymphocytes in the Canine Endometrium

Resident immune cells play a major role in endometrial immunity and in tissue homoeostasis. This study aimed to analyse the distribution of macrophages, B and T lymphocytes (respectively, Mø, B‐Lym and T‐Lym) in the canine endometrium throughout the oestrous cycle and in late involution (at the proestrus stage post‐parturition). An immunohistochemistry technique was used on samples from 50 post‐pubertal healthy female dogs, of which five in late post‐partum. The distribution of resident immune cells was analysed in three endometrial layers (superficial, intermediate and basal areas). Mø, B‐Lym and T‐Lym were demonstrated to reside in the endometrium in all the stages of the canine cycle; their numbers being considerably higher during late involution. T‐Lym were scattered in the stroma or amidst the glandular epithelium, constituting the predominant immune cell population in anestrus and proestrus, but decreased in number at all other stages. Endometrial B‐Lym remained fairly constant during the canine cycle, although its numbers were higher in late involution. Mø counts were higher during anestrus compared to the other stages, the cells being displaced into the superficial endometrial layer. Mø demonstrated the highest level in late involution samples, forming small aggregates below the surface epithelium. The number of immune cells was not normally distributed, suggesting the influence of individual factors, such as age or parity, not explored herein due to limited sample availability. Still, this study provides important information for the interpretation of endometrial biopsies in dogs and for the understanding of the increased susceptibility to uterine infection during dioestrus found in the bitch.     

Effects of Oxytropis glabra DC Poisoning on the Reproductive Organs in Hetian Sheep Ewes

The study was aimed to research the effects of Oxytropis glabra DC (O.glabra DC) poisoning on reproductive organs coefficent, reproductive performance and related gene expression in Hetian sheep ewes. The sera, hypothalamus, pituitary and ovary of Hetian sheep ewes cases poisoning of O. glabra DC were collected, the organ exponent and contents of GnRH, FSH, LH, E₂ and P₄ in serum were measured, then the mRNA expression of reproductive genes were measured. The result demonstrated that the index of hypothalamus, pituitary and ovary were extremely significantly higher than normal sheep (P < 0.01),the follicle number on the surface of the ovaries in poisoning groups were significantly lower than normal sheep (P < 0.05). The result of hematoxylin-eosin staining showed that the nuclear of neuronal cells in hypothalamus were pyknosis and hyperchromatic; The nuclear of cells in pituitary deformation and hyperchromatic, cytoplasm decreased; The primary oocyte in ovary dissolved, disappeared, interstitial blood vessel expanded. The content of GnRH, FSH, LH, E₂ and P₄ in serum in poisoned Hetian sheep ewes were extremely significantly lower than normal sheep (P < 0.01). The mRNA expression levels of Kiss-1, GPR54, ERα in hypothalamus, GnRHR in pituitary and FSHR and LHR in ovary were extremely significantly lower than normal sheep (P < 0.01). The result showed that O. glabra DC poisoning could affect Hetian sheep ewes reproductive system by affecting microstructure and ultrastructure of hypothalamus-pituitary-ovarian axis.     

小花棘豆中毒对和田羊母羊繁殖器官的影响

试验旨在探讨小花棘豆（Oxytropis glabra DC）中毒对和田羊母羊繁殖器官指数、性激素水平和相关基因mRNA表达量的影响。以小花棘豆中毒和田羊母羊为研究对象,采集血清后屠宰,采集试验羊的丘脑、垂体和卵巢组织,测定其脏器系数,检测血清中促性腺激素释放激素（GnRH）、促卵泡素（FSH）、促黄体素（LH）、雌二醇（E₂）和孕酮（P₄）含量的变化,并检测各组织中相关繁殖基因的表达。结果显示,小花棘豆中毒和田羊母羊丘脑、垂体和卵巢指数均极显著升高（P < 0.01）,且卵巢表面卵泡数显著下降（P < 0.05）,HE染色表明丘脑神经元细胞固缩、浓染;垂体中细胞核变形、浓染,胞浆减少;卵巢中初级卵母细胞溶解、消失,间质血管扩张。小花棘豆中毒和田羊母羊血清中GnRH、FSH、LH、E₂和P₄含量均极显著下降（P < 0.01）,丘脑中Kiss-1、GPR54、ERα mRNA,垂体中GnRHR mRNA和卵巢中FSHR、LHR mRNA表达量均极显著下降（P < 0.01）。结果表明,小花棘豆毒性成分可通过丘脑-垂体-性腺轴影响和田羊母羊的生殖系统。     

Qualitative analysis and functional classification of the uterine proteome of mares in oestrus and dioestrus

Quantitative analysis of the uterine flush fluid proteome of mares in oestrus and dioestrus has been previously reported. The objectives of this study were to: a) evaluate qualitative differences in the uterine flush fluid proteome between mares in oestrus and mares in dioestrus and b) perform a functional classification of proteins either unique to each stage or common between the two stages. Uterine flush fluid samples were collected from 8 light breed mares in either oestrus (n = 5) or dioestrus (n = 3). Proteomic analysis of the samples was conducted using liquid chromatography–tandem mass spectrometry. Proteins exclusively detected in oestrus or dioestrus and those common to both stages were identified using the Scaffold software (version 4.4.8, Proteome Software Inc., Portland, OR). The identified proteins were classified into gene ontology (GO) categories (cellular component [CC], molecular function [MF] and biological process [BP]) using the PANTHER ( www.pantherdb.org ) classification system version 14.0. Of 172 proteins identified, 51 and 28 were exclusively detected in mares in oestrus and dioestrus, respectively, and 93 proteins were common to both stages. The most represented terms in various GO categories were similar among the three subsets of proteins. The most represented CC terms were extracellular region and cell, the most represented MF terms were catalytic activity and binding, and the most represented BP terms were metabolic process and cellular process. In conclusion, proteomic analysis of the uterine flush fluid enabled the identification of subsets of proteins unique to oestrus or dioestrus, or common to both stages. The results of this study can serve as a baseline for future research focused on finding stage-specific protein markers or evaluating differences in the uterine flush fluid proteome between normal mares and those with uterine disease.