Early pregnancy detection in ruminants: challenges and innovations

Abstract

Precise and early pregnancy detection is crucial for better breeding management and enhancing the overall production of ruminant livestock. Throughout the years, numerous methods have evolved for pregnancy detection in ruminants, each possessing specific advantages and limitations. This review thoroughly discusses both traditional and emergent diagnostic methods, emphasizing their principles, implementation, merits and challenges. Behavioral observation, rectal palpation and ultrasonography are the traditional approaches widely used because of their accessibility and direct detection of pregnancy conditions. Progesterone measurement, pregnancy-associated glycoprotein detection, and estrone sulfate examination are the hormonal assays that provide biochemical proof at specific phases of gestation. Recently, the analysis of interferon-stimulated gene expression and circulating microRNAs has shown promising roles in early pregnancy detection at the genetic and transcriptomic levels. The investigation of volatile organic compounds is a novel approach in pregnancy diagnosis, though it is non-invasive, and further confirmation is required for regular application. This review highlighted the importance of incorporating multiple examination strategies to enhance the accuracy and reliability of pregnancy detection in ruminants. Future research should center on the refinement and field application of advanced technologies to ensure their proper implementation in diverse ruminant production systems.

Introduction

Efficient reproductive management is essential for successful ruminant (cattle, buffalo, goats, and sheep) production. Early and precise pregnancy detection plays an important role in maximizing reproductive efficiency, reducing calving or kidding interval, improving productivity and minimizing economic losses related to reproductive failure [1,2,3,4]. On the other hand, identification of non-pregnant animals permits timely re-breeding and facilitates proper nutritional and health management to support fertility and maternal health [5]. Over the years, a number of diagnostic methods have evolved for pregnancy detection in ruminants (Table 1). Behavioral observation, rectal palpation and ultrasonography are the traditional approaches commonly used due to their feasibility and direct examination of reproductive status [6]. However, these methods require a certain skill level, precise timing and cause stress to the animals. To overcome these constraints, numerous molecular and biochemical methods have been established, including hormonal and gene expression changes.

Table 1. Comparison of different pregnancy diagnosis methods.

Methods	Species	Applicable time point (Post-estrus)	Necessity of expertise	Time required for detection	Degree of accuracy	References
Palpation	Large ruminants	35–50 days	Yes	Immediately	Moderate	[20]
Ultrasound	All	26–30 days	Yes	Immediately	High	[32, 33, 44]
Radiography	Small ruminants	70 days	Yes	Immediately	High	[61]
Seed Germination Test	All	20 days	No	72–96 h	Low	[69]
Blood Test (PAG)	All	28–30 days	No	1–4 days	High	[75, 76, 79, 83, 84]
Milk Progesterone Test	All	18–24 days	No	1–2 days	Moderate	[13, 84, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96]
Estrone Sulfate	All	Mid to late stage of gestation	Yes	Few hours	Moderate	[106, 107, 108, 113, 114]
Rosette Inhibition Test	All	Within a few weeks	Yes	1–2 days	Moderate	[119]
RT-qPCR (RNA-based test)	All	18–25 days	Yes	1–3 days (lab-based)	High	[122, 123, 127, 129]
miRNAs	All	16–18 days	Yes	1–3 days (lab-based)	High	[10, 134, 135]
Volatile Organic Compounds	Large ruminants	18 days	No	Immediately	High	[139, 140]

Progesterone-based methods, estrone sulfate quantification, and pregnancy-associated glycoprotein (PAG) detection represent biochemical strategies for pregnancy detection. Each of these methods offers different levels of specificity depending on the species and stage of pregnancy [7]. Recent molecular approaches, such as expression of interferon-stimulated gene (ISG), have been developed based on conceptus-derived production of interferon tau (IFNT) at the time of maternal recognition of pregnancy [8, 9]. The differentially upregulation of ISG15, MX1, MX2, and OAS1 genes has shown promise as an early pregnancy biomarker, especially in cattle and buffaloes. The disclosure of circulating microRNAs (miRNAs) in maternal blood has opened new avenues for early diagnosis of pregnancy. miRNAs modulate gene expression and play critical roles in embryo implantation, maternal immune modulation and placental development [10]. The differential expression of miRNAs during early pregnancy has been described in cattle, buffaloes, goats and sheep, which indicate their potential as pregnancy markers. The analysis of volatile organic compounds (VOCs) delivered through breath, feces or urine during pregnancy is another emerging field for pregnancy diagnosis.

Despite this progress, the implementation of early pregnancy detection methods among different ruminant species still meets challenges related to accessibility, cost, sampling and accuracy. This review aims to provide a comprehensive overview of the available methods for early pregnancy diagnosis in ruminants with highlighting their limitations and future perspectives for incorporating these technologies into reproductive management programs.

History of Pregnancy Detection in Ruminants

Several methods of pregnancy detection have been applied in ruminants for the past few centuries (Fig. 1). Pregnancy detection in ruminants has progressed over the past century. The early methods rely on physical changes and behavioral observations, while modern methods engage ultrasonographic, biochemical and molecular approaches. Historically, the earliest methods for pregnancy diagnosis in ruminants are based on behavioral signs such as non-return to estrus, changes in temperament, or increased feed intake. These features were often untrustworthy because of their variability between species and animals.

Fig. 1.

Methods of pregnancy detection in animals.

Burgess [11] revealed that the first use of rectal palpation in the early 20^th century brought a breakthrough to physically assess changes in the reproductive tract and detect pregnancy (Fig. 2). This technique became a foundation of field-based pregnancy detection in cattle and is still universally used today because of its low cost and instant results [6]. Researchers developed hormone-based methods for measuring progesterone levels in blood and milk samples to detect pregnancy with the development of radioimmunoassay (RIA) technology in the 1960s [12]. A high progesterone concentration after 21–24 days of post-insemination indicates the maintenance of the corpus luteum and pregnancy. However, these tests may give false positives because of the prolonged luteal function in non-pregnant animals [13]. The identification and characterization of pregnancy-specific proteins and PAGs opened a new window to detect early pregnancy in ruminants [14]. These proteins, produced by the trophoblast cells of the growing embryo and detectable in maternal blood from days 24 to 28 post-estrus that offer a more direct and pregnancy-specific molecular marker [15].

Fig. 2.

The evolution of pregnancy diagnosis techniques in animals.

The introduction of real-time B-mode ultrasonography in the 1980s transformed pregnancy diagnosis. Ultrasonography enables visualization of the ovaries, uterus, embryo and placentomes and thereby allowing confirmation of pregnancy as early as day 26–30 post-estrus [16]. The worldwide availability of portable ultrasound machines has made this method progressively accessible to animal farms. The last two decades have seen the emergence of interferon-stimulated genes (ISG15 and MX1) during early pregnancy in animals. These genes are differentially upregulated in response to IFNT that is secreted by the conceptus during maternal recognition of pregnancy in ruminants [17]. Recently, lab-on-chip systems that integrated with laboratory functions on a microfluidic chip to allow a rapid, on-farm biochemical or molecular analyses and biosensor-based technologies have been investigated for on-farm setup pregnancy detection [18]. These include optical and electrochemical biosensors, which can detect progesterone or PAGs in milk or blood samples in less than an hour. These innovations aim to bridge the gap between laboratory precision and field applicability of sensor based methods of pregnancy detection.

Pregnancy Diagnosis in Ruminant Animals by Various Techniques

1. Direct methods

Visual method

Visual observation is the most traditional and convenient system for pregnancy diagnosis in ruminants, especially in field ambience and smallholder livestock farming systems. Detection of external signs such as end of estrus behavior, increase in body size, abdominal enlargement, udder enlargement and changes in vulvar ejection or behavioral temperament are the main characteristics of pregnancy by this method. Historically, farmers and ranchers relied mainly on such signs due to a deficiency of access to pregnancy detection aids or technologies. The absence of estrus cycles for more than 21 days after insemination is believed to be a tentative sign of pregnancy in cattle and buffaloes. The behavioral signs are often indistinct, with lessening mounting activity and modest abdominal changes being noticed after 40–60 days of gestation in goats and sheep [6].

The visual diagnosis has some major limitations, including that it is highly subjective, affected by the level of experience of the technician, breed variation and environmental circumstances. Silent heat, pseudopregnancy and anestrus are the common reasons to give a false pregnancy result. Additionally, numerous physical signs, such as abdominal expansion, are only apparent in mid to late gestation in different species and breeds, making it inappropriate for early pregnancy detection. While the method is inexpensive and easy to implement, its low accuracy can lead to missed pregnancies and misclassifications, which may ultimately cause significant economic losses due to prolonged calving intervals.

Clinical method

The most typical clinical methods of pregnancy diagnosis in ruminants are rectal palpation, radiography, ultrasonography and laparoscopy.

1) Rectal palpation

Rectal palpation is one of the ancient and extensively used methods for pregnancy diagnosis in large ruminants, especially cattle and buffaloes. The rectal palpation was first reported in cattle in the early 20^th century. In this technique, the uterus is manually examined across the rectal wall to detect the signs of pregnancy, such as amniotic fluid accumulation, uterine enlargement and the existence of fetal membranes or the fetus itself [19]. The experienced practitioners can faithfully detect pregnancy from 30 to 35 days post-estrus by identifying amniotic vesicles and implementing the fetal membrane slip technique in cattle [20]. The deeper pelvic anatomy in buffaloes makes it difficult to detect pregnancy as early as in cattle and typically confirms pregnancy around 45 days of gestation. The rectal palpation is not commonly feasible in goats and sheep due to their smaller body size and limited rectal capacity, making this method unsuitable for pregnancy diagnosis in these species [21]. To overcome these constraints, several alternative methods have been developed. The placing of a lubricated glass rod into the rectum of the ewe helps elevate the uterus and detect pregnancy [22]. Additionally, Kutty [23] described a bimanual palpation method, where one hand is pushed into the rectum while the other hand is used for palpating the abdomen externally. This technique improves pregnancy detection accuracy by simultaneously examining the uterus from both the abdominal and rectal sides. Despite these improvements, the safety and accuracy of rectal palpation techniques in small ruminants remain unsettled.

This method is cost-effective, ensures immediate results, and provides the benefit of examining additional reproductive status, such as ovarian structures and uterine tone. It requires the least equipment and can be done in field conditions, which makes it popular for many livestock producers [21]. However, the technique requires high skill and experience to prevent misdiagnosis and the risk of causing embryonic damage or fetal loss. Several studies have demonstrated that rectal palpation escalates the risk of pregnancy loss if not accomplished correctly [24, 25]. Additionally, the physical requirements of the procedure may lead to operator tiredness, particularly in large herds, and there is a possibility of injuries among practitioners [26]. In addition, the rectal-abdominal palpation system has been interlinked with risks such as abortions, rectal and uterine injuries, especially when performed by inexperienced personnel [27, 28]. However, the rectal palpation is still considered a valuable technique in reproductive management when executed by skilled and experienced professionals within the defined gestational age.

2) Ultrasonography

Ultrasonography has become a crucial tool in early pregnancy detection in various ruminant species. It is a rapid, non-invasive, efficient and accurate method for reproductive assessment of livestock. Initially, this method was developed for human purposes [29]. The application of ultrasonography in the livestock industry began in the late 20^th century, later with significant advancements of this method, enhancing its utility in livestock. The detection of pregnancy using transrectal ultrasonography can be done as early as 28 days post-estrus in cattle [30]. The significant advantages of this method are that it permits visualization of the embryo proper, embryonic vesicle and fetal heartbeat, therefore providing information on fetal age and viability [31]. Thus, ultrasonography provides an accurate result of embryonic or fetal loss in inseminated females. In addition, it enables the examination of ovarian and uterine structures and recognition of multiple pregnancies, to improve reproductive efficiency [6]. It has been demonstrated that transrectal ultrasonography is efficient for detecting pregnancies from day 25 post-estrus in buffaloes, with the added benefit of observing ovarian activity and reproductive disorder diagnostics [32].

Transabdominal ultrasonography is the preferred method for pregnancy diagnosis in small ruminants like sheep and goats due to their anatomical considerations. Pregnancy can be detected from day 25 to 30 of gestation, with the determination of fetal number, viability and age [33]. This kind of information is important for managing nutrition during pregnancy and avoiding pregnancy and nutrition-related problems such as toxemia [34]. The transvaginal ultrasonography is also being practiced now-a-days for small ruminants since it was first reported in 1988 by Pieterse et al. [35]. This method is more hygienic and safer than transrectal ultrasonography. The transvaginal ultrasound approach can detect the number of fetuses more accurately than other methods of ultrasonography [36]. This method has a number of advantages, for example, dispensability of a full bladder for diagnosis, it can be used in overweight patients, and this method can overcome the obstacles such as bones, gas-filled intestines, and extensive adhesions of tissues in the pelvic area. The demerit of this technique is limitations in the handling of the probe [37]. However, ultrasonography requires unique equipment and skilled personnel, which limits its application in resource-constrained livestock settings. In addition, the perfection of the diagnosis technique is variable depending on the operator’s experience and the equipment used.

A-mode ultrasonography: A-mode ultrasonography (Amplitude-mode) is the foremost ultrasound technique applied in livestock reproductive diagnostics. It displays a one-dimensional graph by transmitting a single ultrasound beam into the body and evidence the amplitude of echoes returned from tissue interfaces. This technique was initially adopted for pregnancy detection in sheep in the 1970s, where it identified fluid-filled uterine anatomy, which is an indicator of gestation [38]. In practice, these devices emit sound waves that differ when encountering fluid-filled spaces, such as the amniotic sac, than from those of solid tissues and provide information on pregnancy status.

Its one-dimensional result lacks detailed anatomical visualization, making it less effective in differentiating between pregnancy and other circumstances that may provide similar echo patterns, such as fluid accumulations or uterine infections. In addition, it can detect pregnancy after 60 days of gestation in small ruminants, which restricts its application largely [39]. The depth and anatomical structure of the reproductive tract in larger ruminants, such as cattle and buffalo, make A-mode less practical. Although A-mode ultrasonography played an underlying role in livestock pregnancy diagnostics, its use has declined with the more advanced technologies that give higher accuracy and diagnostic detail.

Real time B-mode ultrasonography: Real-time B-mode ultrasonography (brightness-mode ultrasonography) is the most commonly used imaging system for pregnancy diagnosis in livestock. This system has been applied from human medicine to animal applications since the 1980s and has become a basis for reproductive management in ruminants. B-mode ultrasonography produces a two-dimensional, real-time grayscale depiction by changing the amplitude of returned echoes into different intensities of brightness on the projected screen. This allows for direct judgement of reproductive organs (ovaries and uterus), early embryonic conditions, and even later-stage fetuses [40, 41]. This technology has significantly advanced in identifying and describing the follicular development in cattle [42, 43].

The transrectal B-mode ultrasonography is routinely used for early pregnancy diagnosis in cattle from 28 to 35 days post-estrus [44]. Within this time, the embryonic vesicle emerges as a fluid-filled anechoic area in the horn of the uterus. Later, with the progression of gestation, the embryo and its heartbeat can also be visualized, which verifies fetal viability [30, 44, 45]. This technology also permits the identification of multiple pregnancies and assists in identifying uterine pathological conditions, such as late embryonic loss or pyometra [46]. In buffaloes, due to anatomical differences (such as thicker rectal walls and deeper pelvic positioning), the practical application of B-mode ultrasonography often begins slightly later, from day 30 to 40 after estrus [47, 48]. However, this technology has proven highly precise in early pregnancy diagnosis and examining ovarian activity in buffaloes, which is important in managing buffalo reproductive inefficiencies [32].

In small ruminants, transabdominal B-mode ultrasonography is more preferable because of anatomical constraints for rectal scanning. Pregnancy can be detected reliably earlier than in large ruminants at day 25–30 after estrus [49]. The fluid-filled uterine sacs are the earliest index of pregnancy, followed by visualization of the embryo and its heartbeat at 28–30 days post-estrus [50]. This method is mainly useful for counting fetal number and gestational age, which is necessary for nutritional management during pregnancy and prevention of pregnancy toxemia.

Real-time B-mode ultrasonography is a non-invasive, rapid and safe technique that allows direct visualization of the ovarian structure, embryonic vesicle, number of embryos, fetal heartbeat, placental structures and early embryonic or fetal death. However, it requires sophisticated equipment and skilled personnel, which restricts its use in low-resource or field settings. The diagnosis is also compromised by operator inexperience, equipment, animal obesity, or intestinal gas. However, the overall benefits of ultrasonography make it a preferred technique in modern ruminant reproductive management.

D-mode ultrasonography: D-Mode Ultrasonography (Doppler-mode ultrasonography), particularly known as color Doppler ultrasonography (CDUS), assesses blood flow kinetics in reproductive structures, offering insight into the vascularization of the uterus and corpus luteum [51]. CDUS has been effectively utilized in cattle to examine luteal blood perfusion as a sign of pregnancy status [52]. Studies have found that pregnant cows show higher corpus luteum blood flow compared to non-pregnant cows. This technique detects pregnancy as early as 20 days post-breeding [53]. Similarly, Holton et al. [52] reported that CDUS precisely distinguishes non-pregnant cows at day 20–22 after estrus, without false negatives, indicating its potential role for early detection. Similarly, Fontes [54] highlighted the role of CDUS in examining luteal function and predicting pregnancy in bovines. The application of Doppler ultrasonography for diagnosing pregnancy status and reproductive disorders has been explored in buffaloes [55, 56]. El-Sayed et al. [57] utilized a Doppler ultrasonographic tool to examine uterine blood flow in Egyptian buffalo cows, providing information on potential pregnancy and reproductive health. For small ruminants, the application of Doppler ultrasonography is less widespread because of their anatomical constraints.

D-mode ultrasonography requires specialized and expensive equipment and also skilled personnel trained in interpreting vascular flow patterns. Operator individuality and variability in assessing blood flow can lead to incorrect results. In addition, external factors like handling, stress, and ambient temperature may impact luteal and uterine blood flow, thereby affecting diagnostic accuracy.

3) Laparoscopy

Laparoscopy is a minimally invasive surgical method that has been adapted for various reproductive applications, including pregnancy diagnosis. In this method, a laparoscope is inserted through a small incision in the abdomen that allows direct inspection of the reproductive organs. In larger ruminants, laparoscopy is less commonly used for pregnancy detection but is used for the diagnosis of ovarian pathology [58]. In small ruminants, laparoscopy has been applied to assess reproductive health, although the invasiveness and need for specialized equipment and expertise make it less effective for pregnancy diagnosis [59].

The advantages of the laparoscopy method include direct observation of the reproductive organs, which provides precise diagnosis of reproductive status and conditions. However, the technique is invasive, requires anesthesia or sedation, trained personnel and specialized equipment. These factors limit their application for routine pregnancy diagnosis in field conditions. In addition, the methods have risks of injury to internal organs and infection.

4) Laparotomy

Laparotomy is a surgical method that involves an incision into the abdominal cavity of animals. It has been used for pregnancy diagnosis historically in ruminants, especially for research contexts. This method permits the direct visualization and palpation of the uterus and its contents, facilitating assessment of pregnancy status.

Laparotomy is performed using a paramedian or flank approach with anesthesia or sedation in small ruminants [60]. The uterus is exposed, allowing direct palpation or observation of the fetus or fetal membranes to confirm pregnancy [61]. This method is highly accurate in detecting pregnancy in early gestation stages when other non-invasive techniques are less reliable. However, this method is invasive, requires surgical facilities and may cause complications. Laparotomy is less common for pregnancy diagnosis in larger ruminants due to the size and depth of the abdominal cavity, which makes laparotomy challenging and complicated. In addition, due to its requirements for anesthesia, sterile conditions, and post-operative care, it is unsuitable for large-scale or field-based applications [21]. The risk of hemorrhage, postoperative adhesions and infection gives rise to a concern for animal welfare and productivity.

5) Radiography

Radiography, also known as X-ray imaging, has been utilized in small ruminants for pregnancy diagnosis [61]. This technique works based on the recognition of mineralized fetal skeletons by radiography as gestation progresses. Typically, fetal skeletal mineralization starts around day 58 of gestation in small ruminants; thus, this method of pregnancy diagnosis confirms the stage onward [62]. Radiography is also useful for counting the fetal numbers in late gestation to manage parturition and neonatal care. It is a non-invasive, highly accurate fetal count. However, its effectiveness is constrained until fetal mineralization has occurred, making it unsuitable for early pregnancy diagnosis. Additionally, ionizing radiation requires safety protocols for both animals and personnel. The necessity for trained personnel and specialized equipment further restrained its application in field conditions. Moreover, in large ruminants, there are challenges in getting clear radiographic images due to their depth and size of the abdominal cavity.

2. Indirect methods

The direct methods provide physical or visual justification for pregnancy diagnosis, whereas indirect methods depend on the hormonal, physiological, or biochemical changes associated with pregnancy. The indirect methods include quantification of hormone (e.g., progesterone, PAGs), metabolite profiles, and protein markers, which offer practical alternatives for early pregnancy detection both in laboratory and field conditions [14, 63]. However, the diagnostic accuracy depends on species, breed, sampling time, parity and assay conditions. Sometimes, the false-positive results are exhibited due to persistent corpus luteum, embryonic loss and false negatives from biomarkers. So, improvements and standardizations in assay protocols, sampling strategies and biomarker validation will enhance the reliability of indirect methods.

Seed germination test

The seed germination test (Punyakoti test) is a prehistoric method for pregnancy diagnosis in ruminants. This non-invasive method involves examining the repressing effect of females’ urine on the germination and growth of seeds, such as mung beans (Vigna radiata), wheat (Triticum aestivum), or barley (Hordeum vulgare). In this method, seeds are soaked in diluted urine for a duration of 72–96 h, followed by examination of germination rates and root growth [64]. The seed germination is easily accessible for farmers who can perform it at their homes with affordable materials and without any skills.

During pregnancy, hormonal changes take place in the animal body that alter the urine composition and affect seed germination, which is the basic principle of the test [65]. Skálová et al. [66] observed that mung bean seeds soaked in pregnant heifers’ urine exhibited reduced seed germination rates and shorter shoot lengths. The urine of pregnant cows contains abscisic acid, which is responsible for suppressing the germination and growth of seeds [67, 68]. The seed germination test’s accuracy is 68% on day 28 and 100% from days 35 to 45 of pregnancy in cattle [69]. In small ruminants, this test has also been found to have potential as a pregnancy detection tool [39, 61]. Previously, we applied the technique to diagnose pregnancy in indigenous sheep and Black Bengal goats, finding that the test could effectively distinguish between pregnant and non-pregnant animals based on wheat seed germination and shoot length patterns [70]. Although the test is inexpensive and simple, its relatively low and variable diagnostic accuracy can result in misclassification, leading to inappropriate management decisions and economic losses [66]. In addition, seed quality, environmental conditions, nutrition, and disease affect the result of this method.

Pregnancy associated glycoprotein (PAG)

The identification of pregnancy-specific proteins in maternal circulation was first reported in 1982. At that time, Butler et al. [71] used immunoelectrophoresis to isolate pregnancy-specific protein-A (PSP-A) and pregnancy-specific protein-B (PSP-B), two distinct proteins from the bovine placenta. These proteins, later acknowledged as PAGs, make the pathway for beginning a new technique for pregnancy diagnosis in animals [14, 15, 71,72,73,74]. The work of Zoli et al. [72] led to the evolution of RIA for measuring PAGs in maternal blood. These initial studies mainly focused on cattle and demonstrated the potential of PAGs as markers for pregnancy detection and trophoblastic activity. The application of enzyme-linked immunosorbent assays (ELISA) enhances their sensitivity and specificity, and helps in widespread application [75]. Later, rapid test kits were developed to detect pregnancy in animals based on the PAGs. The mechanism of the PAG pregnancy diagnosis rapid test is shown in Fig. 3.

Fig. 3.

The mechanism of pregnancy diagnosis by rapid PAG detection test.

Till now, more than 20 different PAG isoforms have been characterized in bovine and classified based on phylogenetic and expression profile into ancient and modern groups [76]. Among these groups, PAG-1 is detectable in pregnant animal blood as early as day 28 of gestation, while PAG-2 appears at a slightly later stage of gestation and persists longer [75, 76]. The effectiveness of PAGs for pregnancy detection depends on their isoform and animal species; for example, PAG-1 assays are highly effective in cattle but less sensitive in small ruminants [77]. Studies have documented that the level of PAGs correlates with embryonic viability, litter size and pregnancy losses [78, 79]. The PAG levels work as an index of embryonic mortality between 25 and 40 days of gestation in buffaloes [80, 81].

In goats and sheep, ELISA tests confirm pregnancy detection at 28–30 days post-estrus using caprine PAG-56 and ovine PAG-55, respectively [82]. In goats, the presence of PAGs in maternal blood has been confirmed at 28 days post-estrus [83]. Whereas in sheep, the PAGs are detected 30 days post-estrus [78, 84]. This method is non-invasive, highly sensitive, and detects pregnancy at an early stage. Additionally, PAG assays can be tested in milk or blood samples, which provides flexibility in sample collection. However, the residual PAGs (persist up to 60 days postpartum) may lead to false-positive results [77, 85]. Moreover, the necessity for laboratory facilities and experienced personnel limits the widespread application of PAG detection in field setting conditions.

Hormonal assay

1) Progesterone hormone

Progesterone-based hormone assays are the most comprehensively applied biochemical methods for diagnosing pregnancy in ruminants. These systems measure the increased progesterone levels either in milk or blood, which work as an indicator for the presence of a functional corpus luteum (CL) and confirm potential pregnancy [86]. Progesterone secreted from the CL after ovulation is required for the maintenance of pregnancy. The progesterone levels remain higher than the luteal phase in pregnant animals due to the anti-luteolytic activity of IFNT. IFNT inhibits prostaglandin F_2α release, hence preventing CL regression.

The pregnancy status can be determined around 18–24 days post-estrus by this method [13, 87]. In cattle, elevated milk or blood progesterone concentrations at 21 days post-estrus suggest pregnancy [88,89,90]. Studies have demonstrated that milk progesterone concentrations of more than 10 ng/ml are an indicator of pregnancy [14]. In buffaloes, progesterone measurement has become a reliable diagnostic tool for pregnancy detection [91,92,93]. The milk progesterone level concentrations more than 10 ng/ml between days 22 and 26 post-breeding are also associated with pregnancy in goats and sheep [84, 94,95,96].

Various commercial kits have been developed for progesterone-based pregnancy detection [87, 97, 98]. RIA is a highly sensitive and laboratory-based method for progesterone detection that requires sophisticated equipment and radioactive materials [99]. ELISA is widely used for progesterone-based pregnancy detection because of its sensitivity and specificity. In addition, some human ELISA kits have even been authenticated for use in animals that offer cost-effective alternatives [98, 100,101,102,103,104]. The latex agglutination tests are field-friendly, although they have lower accuracy compared to the ELISA-based protocol [98, 105]. Progesterone-based assays are non-invasive, enabling early pregnancy detection and on-farm application. The accuracy rates for this assay are 80–95%, depending on species, sample collection, and the assay method used [97]. However, persistent CL or ovarian luteal cysts may give false-positive results. Conversely, declining progesterone levels due to early embryonic loss may give false negative results.

2) Estrone sulfate (E1S)

E1S is a conjugated estrogen synthesized by the fetoplacental tissues in ruminants. The presence of E1S in maternal fluids plays a role in placental function and fetal viability. It can be used as a biomarker for pregnancy diagnosis, especially from mid to late gestation [106,107,108]. E1S can be detectable in maternal milk, serum and urine from 50 days post-estrus with increasing concentration along the parturition process [109,110,111]. The level of E1S is significantly higher in twin pregnancies compared to single pregnancies, reflecting the enhanced estrogen production correlated with multiple fetuses [106]. In buffaloes, E1S concentrations markedly increased after the second month of gestation, thus confirming pregnancy after 100 days of breeding [112]. E1S assays detect pregnancy at day 50–70 post-estrus in goat and sheep, respectively [113, 114].

The measurement of E1S is non-invasive, easy for sampling, correlates with fetal number and facilitates on-farm testing. However, the late onset of distinguishable E1S levels limits its applicability for early pregnancy diagnosis. In addition, E1S concentrations are influenced by breed, parity, environmental conditions and maternal weight [115].

3) The Rosette inhibition test (RIT)

RIT was developed to detect pregnancy by immunological assay. In this assay, transient immunosuppressive protein known as early pregnancy factor (EPF) is measured, that is produced in maternal serum shortly after fertilization in mammals. EPF adjusts the maternal immune response to aid embryo survival and can be distinguished in maternal serum within a few hours after fertilization, which makes it the earliest method for pregnancy detection [116,117,118]. RIT effectively differentiates inseminated cows within the first week of their pregnancy [117]. In sheep, this method can detect pregnancy as early as 24 h after mating [119]. This early detection of pregnancy is pivotal for reproductive management and decision-making in livestock production. However, this test needs laboratory equipment and expertise that make it less applicable for on-farm use. Furthermore, the transient character of EPF indicates that its detection time is shorter.

Interferon-stimulated gene (ISG) expression

The concept of using ISG expression for pregnancy diagnosis started after the first discovery of IFNT as the maternal recognition signal in ruminants in the late 1980s [120]. Roberts et al. [121] explained the role of IFNT in preventing luteolysis through suppression of prostaglandin F_2α secretion. Green et al. [9] and Han et al. [122] demonstrated that IFNT regulates the expression of ISGs in maternal blood leukocytes. These discoveries set the foundation for developing ISG-based methods for pregnancy detection in animals. This method is based on the mechanism of maternal recognition of pregnancy, where the embryo secretes IFNT at 15–17 days after fertilization [123]. IFNT prevents luteolysis and induces the expression of ISGs. It has been shown that ISG15, MX1, MX2, and OAS1 are upregulated in response to IFNT during early pregnancy [124]. The quantification of mRNA levels for these genes through quantitative real-time PCR provides a means of pregnancy examination after 18–20 days of post-insemination.

Green et al. [9] reported that ISG15 expression levels in pregnant cows were significantly higher compared to non-pregnant cows on day 18 post-insemination. Han et al. [122] confirmed that the 85–90% sensitivity of ISG15 expression in cows is between days 18 and 22 post-insemination. In addition, primiparous cows showed higher ISG15 expression than multiparous cows [125]. Breukelman et al. [126] highlighted the benefit of this method in cows undergoing embryo transfer. In buffaloes, ISG15, MX2, and OAS1 are differentially expressed during early pregnancy with a sensitivity of 80–90% [127]. Mishra and Sarkar [128] reported that ISG15 and MX2 expressions markedly increase in pregnant buffaloes between days 18 to 24 post-estrus, with a peak at day 21.

ISG15 mRNA levels in pregnant goats are significantly increased than those in non-pregnant goats by day 20 post-estrus [129]. In another study, the upregulation of ISG15 in goats has been shown as early as day 18 post-estrus [130]. However, the regulation of many ISGs is not noted in the sheep during early pregnancy [131,132,133]. The advantages of ISG expression include that it is non-invasive and detects pregnancy at an early stage. However, it requires laboratory equipment and expertise that limit its widespread adoption in field settings.

MicroRNA (miRNA)

miRNAs are small and non-coding RNAs that control gene expression at the post-transcriptional level and are used as biomarkers for pregnancy detection in ruminants. The investigation of miRNAs for pregnancy detection was initiated from studies acknowledging their role in ovarian function, implantation, embryo development and placental formation. Their inherent stability in blood and the ability to reflect physiological changes make them suitable for pregnancy diagnosis. In cattle, various studies have illustrated that circulating miRNAs (miR-26a, miR-486, and miR-1249) are differentially expressed during early pregnancy as early as 16 days after post-insemination [10, 134]. In buffaloes, miR-181a and miR-486 have been detected by day 18 post-insemination in maternal plasma as early pregnancy markers [135]. These miRNAs correlate with conceptus signaling and trophoblast activity, which supports their usefulness in buffalo pregnancy monitoring. miR-143 has been differentially expressed during early gestation in goats [136]. Numerous circulating miRNAs, including oar-miR-218a, miR-34c, oar-miR-1185-3p, miR-183, miR135a, and miR-379 hold their potential for early pregnancy determination in sheep [137, 138]. These findings indicate that multiple miRNAs are associated with pregnancy among different species. mRNA assay methods have several advantages, including early pregnancy detection, being non-invasive and being easy for sample collection under various conditions. However, the requirement for quantitative PCR, microarray, or next-generation sequencing escalates the cost of assays, thus limiting their routine use at the field level.

Volatile organic compounds (VOCs)

The detection of VOCs is a novel method for pregnancy diagnosis in ruminants. VOCs include ketones, aldehydes, hydrocarbons and alcohols, which are the metabolic byproducts that can be identified in feces, urine, breath and saliva. The metabolic and hormonal changes during pregnancy influence the production and release of these VOCs. Gas chromatography-mass spectrometry (GC-MS) analyzes VOC profiles between pregnant and non-pregnant animals. The increased levels of ketones and aldehydes during early pregnancy have been demonstrated in cattle. Pluta et al. [139] reported the invention of a sensor-based device for VOCs to detect estrus and ovulation in cattle. Barman et al. [140] reported estrus-specific VOCs in feces, urine, and saliva of buffaloes. Studies on goat and sheep regarding VOC-based pregnancy detection are currently sparse.

The use of VOCs for pregnancy detection is a non-invasive, early detection method and is suitable for on-site analysis using smart device. However, VOCs profiles are influenced by breed, diet, environment, and animal health, leading to variation in diagnostic accuracy. Moreover, GC-MS requires laboratory infrastructure that makes it less practical for field use. Standardization of protocols, characterization of reliable pregnancy-specific VOCs biomarkers, and validation among species remain essential for advancing the practical application of this method.

Conclusion

Over the decades, a number of pregnancy diagnosis techniques have been developed and refined. No single diagnostic technique can be appraised as ideal for all ruminant species and management contexts. Therefore, integrating multiple approaches ensures the accuracy of pregnancy diagnosis. Future research should focus to develop precise, non-invasive and rapid techniques of pregnancy diagnosis to be applied in ruminants. Such technology will not only boost up reproductive outcomes but also contribute to animal production, welfare, and economic profit in ruminant farming systems.

Conflict of interests

The authors declare that no conflict of interest could be perceived as prejudicing the impartiality of the review reported.