Pregnancy Diagnosis in Dairy Cows: Comparison
Please note this is a comparison between Version 4 by Peter Tang and Version 5 by Peter Tang.

The PL represents a considerable biological and economic waste for the farmer, therefore, the objective of the present review is to discuss recent methods suitable for diagnosing early pregnancy and pregnancy losses in dairy cattle.

  • dairy cow
  • ultrasonography
  • pregnancy proteins
  • embryonic mortality
  • fetal mortality

1. Introduction

The extent of pregnancy loss (PL) in dairy cows can be estimated from the difference between the fertilization rate (FR) and the subsequent calving rate (CR). Therefore, 41% to 57% of pregnancies can be lost during gestation. Most of the losses (early embryonic mortality: EEM) may occur before the maternal recognition of gestation [1]. After the maternal recognition of pregnancy, the life of the corpus luteum will be extended, and embryonic mortality (further 5% to 10%) occurring between Days 16 and 42 after AI is called late embryonic mortality (LEM), while occurring between Days 42 to 90 after AI is named early fetal mortality (EFM).

Recently, Pohler et al. [2] have suggested using the terms ‘early embryonic development’ until Day 30 and ‘late embryonic development’ between Days 31 and 45 of gestation. Due to the fact that different technical terms are used for this period, perhaps it would be easier to divide the former late embryonic period between Days 16 to 42 into two stages, namely, stage 1 between Days 16 and 30 and stage 2 between Days 31 and 42/45 of gestation.

The PL represents a considerable biological and economic waste for the farmer, therefore, the objective of the present review is to discuss recent methods suitable for diagnosing early pregnancy and pregnancy losses in dairy cattle.

2. Early Pregnancy Diagnosis (PD)

During ultrasonographic examination (UE), a cow was initially considered to be pregnant when an irregularly shaped, non-echogenic black spot (or spots) were recognized within the uterine lumen, representing the chorioallantoic fluid [3]. Demonstration of an embryo and/or embryonic membranes provided additional confirmation of pregnancy [4][5]. The confirmation of ultrasonographic diagnoses was usually based on palpation per rectum of the uterus at 2 to 3 months post-AI, or upon spontaneous return to estrus after AI. A cow was also considered pregnant if an embryo proper with a beating heart was recognized at a final UE on Days 50 to 60 post-AI.

Under experimental conditions, embryonic vesicles in dairy cows can be detected as early as at 9 [6], 10 [7], or 12 days [8] of gestation; however, an accurate pregnancy diagnosis (100%) can be reached only on Days 20 and 22 of gestation [9], when the embryo (20.3 days) and its heartbeat (20.9 days) can be detected [10]. It is important to mention that fluid accumulation in the chorioallantois during early pregnancy can be easily confused with the uterine fluid within the uterus during pro-estrus and estrus [9].

Under on-farm conditions, between Days 22 to 49 after AI, the sensitivity, specificity, positive predictive value, and negative predictive value of ultrasonographic diagnoses made with a 5 MHz linear-array or sector transducer varied between 95% and 100%, 67% and 100%, 85.7% and 100%, and 84% and 100%, respectively (Table 1).

Table 1. Accuracy of ultrasonographic examinations for diagnosing early pregnancy in dairy cattle by using a 5.0 MHz linear-array or sector transducer.

Days

after AI

N

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive value

(%)

References

22–40

435

96.2

71.1

89.6

87.8

Filteau and DesCoteaux, [11]

25–29

101

98

91.3

93

97.6

Szenci et al. [12] a

[12] a

30–39

143

100

100

100

100

26 b

48

100

96.7

94.4

100

Romano et al. [5]

27 b

53

100

92.8

92.6

100

26–33

85

97.7

87.8

89.6

97.2

Pieterse et al. [3]

29 c

151

100

96.3

91.3

100

Romano et al. [5]

30 c

151

100

97.4

91.9

100

30–39

722

95

67

89

84

Hanzen and Laurent [13]

40–49

620

98

77

92

94

31–35

323

98

80

85.7

97

Munoz del Real et al. [14]

a Sector transducer, b Heifers, c Cows, NG: not given. Sensitivity = correct positive/correct positive + false negative × 100. Specificity = correct negative/correct negative + false positive × 100. Positive predictive value = correct positive/correct positive + false positive × 100. Negative predictive value = correct negative/correct negative + false negative × 100.

In contrast, Badtram et al. [15] reported that the sensitivity and specificity of the ultrasound test between Days 23 and 31 post-AI were only 68.8% and 71.7%, respectively. In a recent study, maximum sensitivity and negative predictive value were reached at Day 26 in dairy heifers and at Day 29 in dairy cows, while the specificity and positive predictive values were 96.7% and 94.4% for dairy heifers and 96.3% and 91.3% for dairy cows, respectively [5]. In contrast, according to Nation et al. [4], due to pregnancy loss, Days 28 to 35 after AI are too early for reliable detection of pregnancy in dairy cows.

Between Days 27 to 34 after AI, the sensitivity, specificity, positive predictive value, and negative predictive value of ultrasonographic diagnoses made by the use of 5–10 MHz transducer varied between 90% and 96.6%, 91.5% and 100%, 88.4% and 100%, and 92.3% and 97.8%, respectively (Table 2). When the recognition of an embryo proper with a beating heart was used as the criterion for a positive ultrasound PD, significantly (p < 0.001) more false negative and less false positive ultrasound diagnoses were made, in comparison with the recognition of chorioallantoic fluid [16]. In contrast, Silva et al. [17] reported that the presence of chorioallantoic fluid in the uterine lumen and a corpus luteum alone might lead to more false positive results than when an embryo was visualized by using a 5–10 MHz linear-array transducer on Day 27 after timed AI.

Table 2. Accuracy of ultrasonographic examinations for diagnosing early pregnancy in dairy cattle by using a high frequency linear-array transducer.

Days

after AI

N

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive Value

(%)

References

27

1673

96.5

93.4

89.7

97.8

Silva et al. [17]

(5–10 MHz transducer)

28

100

92.7

91.5

88.4

94.7

Karen et al. [18]

(6–10 MHz transducer)

28–35

497

96 a

83

91

92

Nation et al. [4]

(7.5 MHz transducer)

97 b

86

92

93

29–30

138

90.4 c

96.0

95.0

92.3

Szenci et al. [16]

(7.5 MHz transducer)

33–34

135

96.6 c

98.6

98.3

97.3

33–34

135

90.0 d

100

100

92.5

30

47

92.3

97.1

92.9

97.1

Abdullach et al. [19]

(6.5 MHz transducer)

a Observation of ≥15 mm fluid in the uterine lumen and embryonic membranes; b Observation of an embryo with beating heart; c Recognition of allantoic fluid was used as the criterion for a positive pregnancy diagnosis; d Recognition of an embryo proper with a beating heart was used as the criterion for a positive pregnancy diagnosis.

The reliability of the test greatly depends on the frequency of the transducer used, the skill of the operator [15][20], the criterion used for a positive pregnancy diagnosis (uterine fluid or embryo [3][12][18]; uterine fluid, embryonic membranes, or embryo [4]; amniotic fluid and/or embryo surrounded by an amniotic sac [14]; normal amount of chorioallantoic fluid, embryo, ipsilateral CL [17]; embryo with a heartbeat and corpus luteum [21]; embryo with a heartbeat, fluid-filled uterine horn, ipsilateral corpus luteum [22][23]; amniotic fluid, embryo, or embryonic heartbeat [24]; accumulation of intrauterine gestural fluid in non-pregnant cows [9], and the position of the uterus in the pelvic inlet [25]. More incorrect non-pregnancy diagnoses were made between Days 24 to 38 in cows in which the uterus was located far cranial to the pelvic inlet, in comparison with cows in which the uterus was located within or close to the pelvic inlet [25]. It is important to mention if our ultrasonographic pregnancy diagnoses are based on detection of uterine fluid in the uterus on Day 29 after timed AI, at that time, these cows can be classified 3.8 times more likely as not pregnant 74 days after timed AI than those cows diagnosed pregnant based on visualization of an embryo with a heartbeat [22].

Special attention must be paid to the diagnosis of twin pregnancy by clearly locating the two embryos because twin pregnancy loss and spontaneous twin reduction have been reported to occur up to Day 90 of gestation [26][27]. Possibilities for diagnosing twin pregnancy in the field have been reviewed recently by Szelényi et al. [28].

On the other hand, there is a good correlation between decreasing blood flow during CL regression and progesterone concentration [29][30]. One of the main advantages of CFDU is its high sensitivity (99%) and negative predictive value (98.5%) of diagnosing non-pregnant dairy cattle on Day 20 after AI, which results in few false negative diagnoses [31][32], by monitoring the blood flow in the CL on Day 21 after AI in contrast with measuring the progesterone concentration, non-pregnancy in dairy cows on Day 32 after AI can also be predicted with high accuracy (sensitivity: 99.8%, negative predictive value: 99.3%) [33]. have found that the reduction of blood flow in the CL takes place some days before any detectable changes in CL morphology and echotexture, and therefore, determination of the blood flow and the adjusted blood flow calculated from the ratio of luteal tissue area and blood flow were the best early predictors of non-pregnancy.

Previously three pregnancy proteins (PSPB [34], PAG [35] and PSP60 [36]) have been described, however it turned out lately that the isolated preparations of pregnancy proteins may differ in carbohydrate and sialic acid content, which may explain their minor differences in profile and disappearance from the maternal circulation after calving or EM [18][37][38]. Because the concentrations of these proteins gradually increase during pregnancy, they are good indicators of the presence of a live embryo [39]. Due to the delayed appearance of these proteins in the blood in some cows, their use for PD provides more accurate results from Days 28 to 30 onwards [18][40][41].

A further limitation after late embryonic mortality (LEM) is that protein levels may remain above the threshold level, although the concentration of both proteins decreases steadily [42][43]. This is probably related to the relatively long half-life (7–8 days for bPSPB and 3–4 days for bPAG-1) in the maternal circulation after EM [42][44].

Serrano et al. [45] reported that the herd, fetal sex, milk production, lactation number, and plasma progesterone concentrations did not significantly influence the plasma PAG-1 concentration, while twin pregnancy, the use of Limousin semen and conception during the cool period significantly increased its concentrations throughout gestation. Clone pregnancies comparing with control cows may also significantly increase pregnancy protein concentrations during the whole gestation, regardless of pregnancy outcome [46].

Originally PSPB, PAG-1, and PSP60 radioimmunoassay tests were used to detect pregnancy proteins; however, in the meantime, it turned out that there are 21 PAG family members, and PAG-1 used for pregnancy diagnosis was not the earliest pregnancy protein produced by the trophoblast [47]. After recognizing this, the first in-house sandwich ELISA protocol was developed for measuring the circulating concentration of PAGs by Green et al. [47]. Between Days 26 and 58 after AI, the sensitivity, specificity, positive predictive value, and negative predictive value of the in-house PAGs ELISA tests varied between 94% and 100%, 77% and 94.2%, 90.7% and 97.8%, and 91.2% and 97.1%, respectively (Table 3).

Table 3. Accuracy of in-house early-pregnancy associated glycoproteins (PAGs) ELISA tests for diagnosing early pregnancy in dairy cattle.

Days

after AI

N

Type of Sample

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive Value

(%)

References

26–58

169

Serum

97.8

91.2

97.8

91.2

Piechotta et al. [48] a

27–35

2129

Plasma

95.1

89.0

90.1

94.5

Sinedino et al. [49] b

31–35

209

98.7

88.1

83.7

99.1

26–30

106

Serum *

94

77

NG

NG

Friedrich and Holtz [50]

31–35

88

95

93

NG

NG

36–40

57

95

93

NG

NG

>40

128

100

92

NG

NG

27

1673

Plasma

95.4

94.2

90.7

97.1

Silva et al. [17] b

28

100

Plasma

95.3

88.3

NG

NG

Thompson et al. [51] b

30

100

Plasma

100

90.9

NG

NG

* using a threshold of 2 ng/mL; a Used an in-house ELISA assay described by Friedrich and Holtz [50]; b Used an in-house ELISA assay described by Green et al. [52] (2005); NG: not given.

Mercadante et al.[53] found higher PAGs concentrations (in-house ELISA) in primiparous cows compared with multiparous pregnant cows (in agreement with Ricci et al. [54] found similar differences between heifers and lactating cows), during the second and later breeding compared with the first breeding postpartum, in cows experiencing clinical metritis, metabolic problems and left displaced abomasum after calving compared with cows not experiencing those clinical diseases and in cases of greater milk yield, while the body condition score did not influence it. [24]. The accuracy of the PAG test (commercial milk ELISA) was not influenced by parity, the number of days, since the last breeding, and the level of milk production, while Ricci et al. [23] found a negative correlation between plasma and milk PAG concentration (commercial ELISA) and milk production.

There are currently several ELISA tests are available on the market, as discussed below.

BioPRYN ELISA test for the detection of pregnancy-specific protein B in the serum as early as 28 days after breeding in cattle with no interference from a previous pregnancy as early as 73 days after calving.

Between Days 26 to 58 after AI, the sensitivity, specificity, positive predictive value, and negative predictive value of the BioPRYN ELISA test varied between 93.9% and 100%, 87% and 97.1%, 92% and 99.3%, and 91.7% and 97.8%, respectively (Table 4).

Table 4. Accuracy of commercially available ByoPRYN pregnancy ELISA tests for diagnosing early pregnancy in dairy cattle.

Days

after AI

N

Type of Sample

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive Value

(%)

References

23

544

Serum

92.8

97.0

NG

NG

Martins et al. [55]

23 a

98.0

97.0

28

100

91.6

24

206

Serum

100 b

93.6 b

93.3 b

100 b

Middleton and Pursley [56]

26–58

185

Serum

98.0

97.1

99.3

91.7

Piechotta et al. [48]

28

246

Plasma

93.9

95.5

94.7

94.7

Romano and Larson [57]

30

229

96.0

93.9

92.2

96.8

35

246

97.2

93.6

92.0

97.8

28–41

507

Serum

97.0

95.9

96.2

96.7

Breed et al. [58]

35–60

976

96.6

92.2

95.0

94.7

30–36

1742

Serum

95.1

68.6

NG

NG

Gábor et al. [59]

30–36

1336

Serum

100

87.8

NG

NG

Howard et al. [60]

a A cow was considered pregnant when there was an increase in serum PSPB from basal (Day 17) to Day 23 after AI of more than 28%; b Only non-pregnant animals were evaluated.

Martins et al. [55] compared the basal serum PSPB concentrations determined at Day 17 after AI with Day 23 results. They found that if the difference was more than 28% at that time the sensitivity, and the specificity of the ELISA test were higher (98% and 97%), compared to 92.8% and 97% obtained when only Day 23 values were evaluated. It is also important to mention that the concentrations of PSPB on Days 23 and 28 were related to pregnancy losses between Days 28 and 35 after AI, but not to those occurring in later periods (Days 35 to 56 and Days >56 to calving).

Middleton and Pursley [56] suggested comparing the results of serum PSPB samples withdrawn on Days 17 and 24 after AI to diagnose non-pregnant cows with 100% accuracy at Day 24 after AI and to predict early pregnancy loss.

DG29® Bovine Blood Pregnancy ELISA Test for the detection of specific pregnancy-related protein in serum as early as 29 days after breeding in cattle with no interference from a previous pregnancy as early as 90 days after calving. Between Days 28 and 40, after AI the sensitivity, specificity, positive predictive value, and negative predictive value of the D29 test varied between 90.2% and 100%, 66.7% and 98.3%, 91% and 97.4%, and 93.7% and 100%, respectively (Table 5).

Table 5. Accuracy of commercially available D29® bovine blood pregnancy ELISA tests for diagnosing early pregnancy in dairy cattle.

Days

after AI

N

Type of Sample

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive Value

(%)

References

28

100

serum

90.2

98.3

97.4

93.7

Karen et al. [16]

29–36

202

blood

99.4

66.7 *

92.6

96.3

Paré et al. [61]

30–40

212

plasma

100

81.3

91

100

Moussafir et al. [62]

* 100% in non-inseminated cows.

The IDEXX Bovine Pregnancy ELISA Test for the detection of early pregnancy-associated glycoproteins in the serum or plasma of cattle as early as 28 days after breeding in cows with no interference from a previous pregnancy as early as 60 days after calving. Between Days 25 and ~41, after AI the sensitivity, specificity, positive predictive value, and negative predictive value of the test varied between 92% and 100%, 87% and 100%, 84% and 100%, and 94.2% and 100%, respectively (Table 6).

Table 6. Accuracy of commercially available IDEXX bovine pregnancy ELISA tests for diagnosing early pregnancy in dairy cattle.

Days

after AI

N

Type of Sample

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive Value

(%)

References

25

61

plasma

92.0

91.6

88.4

94.2

Kaya et al. [54]

28

84

97.1

100

100

98.0

32

86

97.4

91.4

90.4

97.7

25–32

231

95.9

94.7

93.1

96.9

28

210

serum

100

94.9

94.4

100

Akkose et al. [63]

28

320

plasma

97 *

94 **

95 **

97 *

Mayo et al. [64]

30 ± 1

116

plasma

100

88.9

93.8

100

Commun et al. [65]

30 ± 1

116

serum

100

88.6

93.8

100

32

141

plasma

100

87

84

100

Ricci et al. [23]

41 ± 2

116

plasma

100

100

100

100

Commun et al. [65]

41 ± 2

116

serum

98.4

96.8

98.4

96.8

* ±1, ** ±2.

The IDEXX Milk Pregnancy ELISA Test for the detection of pregnancy-associated glycoproteins in bovine milk from 28 days after breeding in cows with no interference from a previous pregnancy as early as 60 days after calving. Between Days 28 to ~53, after AI the sensitivity, specificity, positive predictive value, and negative predictive value of the test varied between 96% and 100%, 83% and 97.9%, 79% and 98.5%, and 96% and 100%, respectively (table 7). While on Day ≥60, the sensitivity and the specificity of the test varied between 98.5–99.2% and 95.5–96.7%, respectively, the positive predictive value and the negative predictive value were 99.8% and 80.8%, respectively (Table 7). It is important to mention that the plasma PAG levels turned out to be approximately twice higher than the milk PAG levels [22].

Table 7. Accuracy of commercially available milk pregnancy ELISA tests for diagnosing early pregnancy in dairy cattle.

Days

after AI

N

Sensitivity

(%)

Specificity

(%)

Positive Predictive Value

(%)

Negative Predictive Value

(%)

References

28

320

96 *

94 *

94 *

96 *

Mayo et al. [64]

28–45

497

99

95

NG

NG

Dufour et al. [24]

28–35

1006

99.2

93.4

NG

NG

Fosgate et al. [20]

30 ± 1

116

98.1

90.3

94.5

96.6

Commun et al. [64]

32

135

98

83

79

99

Ricci et al. [23]

33–52

119

100

97.9

98.5

100

Lawson et al. [66]

41 ± 2

116

100

92.3

96.4

100

Commun et al. [65]

53 ± 2

116

98.1

96.2

98.1

96.2

≥60

683

99.2

95.5

99.8

80.8

LeBlanc [67]

≥60

602

98.5

96.7

NG

NG

Byrem et al.

[68]

NG: not given, * ±2.

The Rapid Visual Pregnancy ELISA test has been recently developed to detect early pregnancy-associated glycoproteins in bovine whole blood, plasma, or serum as early as 28 days after breeding with no interference from a previous pregnancy as early as 60 days after calving.

Between Days 25 and 45 after AI, the sensitivity, specificity, positive predictive value, and negative predictive value of the visual blood test for dairy cattle were 99.8%, 91.7%, 92.7%, and 99.7%, respectively [69]. When the IDEXX visual ELISA test was used, the sensitivity, specificity, positive predictive value, and negative predictive value of the whole blood test were 98 ± 1%, 85 ± 3%, 87 ± 3%, and 98 ± 1%, respectively [64]. The high sensitivity and negative predictive values mean that very few truly pregnant cows were misdiagnosed as not pregnant. When a microtiter plate reader was used to measure the optical density for individual wells in the ELISA plate, the overall accuracy of the test became 94 ± 1% [64].

The high overall accuracy of the test (98.9%) was reported when the BioPRYN Rapid Visual Pregnancy Test® was used in 92 dairy cattle on Day 28 in heifers and on Day 30 in cows. However, it is important to mention that there were three samples evaluated as ‘to be rechecked’; however, later, it was not possible to evaluate them, and therefore, these three samples were removed from the dataset [70]. A somewhat lower overall accuracy (90%) was reported when the Ubio quickVET rapid visual test was used for plasma samples between Days 30 and 40 after AI [62], and a much lower accuracy (70%) was obtained when the Fassisi® BoviPreg visual test kit was used for serum samples on Day 30 after AI [71].

The earliest specific indicator of fertilization and the continuing presence of a viable conceptus is a serum constituent that had originally been detected in mice [72]. This substance is known as the early pregnancy factor (EPF) and has also been described in women [73], sheep [74], cattle [75], and pigs [76].

The reported and extraordinary properties of EPF include:

Early appearance (within hours) after mating or insemination

Rapid disappearance following induced death or removal of the embryos [77][78].

These factors suggest that EPF may be the most useful tool for investigating early embryonic survival or failure [78][79][80]. According to Laleh et al. [81], the rosette inhibition test (RIT) has the potential to distinguish pregnant from non-pregnant dairy cows in the first week of pregnancy. After identifying immunosuppressive EPF with a molecular weight of approximately 200,000 and raising polyclonal antibodies against it [82], a new diagnostic test, the early conceptus factor (ECF) test, was developed in the USA for field use; however, it cannot accurately identify conception within days or at any time before Day 21 of gestation [83][84][85][86][87].

After EPF, a 10.84 kDa protein, chaperonin 10 [88] having immunosuppressive and growth factor properties [89], was identified. Chaperonin 10 belongs to the family of heat shock proteins but, unlike other members of this family, EPF is detected extracellularly [89]. Further experimental work is needed for the development of an accurate on-farm diagnostic test.

With its very low levels in extrauterine tissues and in the peripheral circulation, IFNT is regarded as a signal for the maternal recognition of bovine pregnancy. INFT contributes to the maintenance of the CL by blocking prostaglandin F2α secretion of the endometrium. Currently, there is no accurate assay for diagnosing early pregnancy based on measuring IFNT concentrations. According to a recent review [2], the relative abundance of ISG in total leukocytes, peripheral blood mononuclear cells and polymorphonuclear cells in pregnant cows from Days 18 to 20 after AI may be significantly higher than in non-pregnant cows.

Another biomarker for diagnosing early pregnancy is the measurement of circulating microRNAs; however, their use is currently limited to research investigations because standardized laboratory techniques are needed to isolate and measure them [2].

Proteomics analysis of the milk identified three possible biomarkers (lactoferrin, lactotransferrin, and alpha1G) for diagnosing early pregnancy [90], while proteomics analysis of the blood identified another three genes (Myxovirus resistance: MX1 and MX2 and oligoadenylate synthetase-1: OAS1), which can be used for early pregnancy diagnosis after validation on a large number of dairy cows [91]. Glycans may also play some critical roles in both the normal function of cells and in disease; therefore, bovine pregnancy can be predicted from a glycan biomarker present in a cow’s milk some 2–4 weeks earlier than by the standard method of pregnancy detection using ultrasonography [92]. Circulating nucleic acids (CNAs) or preimplantation factor (PIF) can be another biomarker for diagnosing early pregnancy in dairy cows [93][94], while Barbato et al. [95] suggested measuring PAG-2 mRNA in maternal blood cells, which can be detected earlier than the PAG-1 placental proteins in water buffalo and in other ruminants, as well.

These new branches of diagnostic sciences may contribute to finding molecules that may be exclusively related to maternal metabolic alterations during early embryonic development and to signaling for maternal recognition of pregnancy and continued survival [96], and may contribute to the development of an accurate early pregnancy diagnostic test in dairy cows.

A new technology (in-line milk analysis system, Herd Navigator) has already made possible the automatic collection of milk samples at milking robots or in the milking parlor to analyze progesterone, lactate dehydrogenase, and beta-hydroxybutyrate to detect estrus, tissue damage, and metabolic disorders, respectively [97]. According to Bruinjé and Ambrose [98], by using this new technology for early pregnancy diagnosis, a high sensitivity (>95%) could be reached from Day 27 after AI, while the specificity was somewhat lower before Day 40 after AI. After finding an accurate biomarker for early pregnancy diagnosis, its continuous measurements during milking will make it possible to diagnose pregnancy loss much earlier, meaning we can greatly contribute to increasing reproductive efficiency in our dairy herds. The importance of this technology would also be emphasized by its ability to identify pregnant and non-pregnant animals in a timely manner with no animal handling, because even a simple transrectal examination of dairy cows can lead to increased plasma and salivary cortisol concentrations and changes in heart rate, heart rate variability, and behavior that are indicative of pain [99].

3. Diagnosis of Pregnancy Losses (PL)

One of the advantages of UEs is that PL can be recognized by the absence of a heartbeat, the detachment of the fetal membranes, the appearance of particles in the fetal fluids, or the lack of the embryo proper [9][100]. UEs have revealed that LEM may occur in up to 23% of pregnancies [13][101]. PL (8%) diagnosed by ultrasonography in cows between Days 26 and 58 post-AI occurred at approximately Day 29 (n = 1), Day 33 (n = 3), Day 37 (n = 3), Day 40 (n = 2), Day 44 (n = 1), and Day 56 (n = 1) after AI. The exact day of occurrence of LEM/EFM could not be determined because UE was performed at intervals of 3–4 days [43].

According to Kelly et al. [102], decreased crown-rump length and progesterone concentration measured on Day 34 of gestation tended to be associated with an increased odds ratio for pregnancy loss, whereas CL perfusion and reduced blood flow of the uterine arteries evaluated by Doppler ultrasonography could not be used for predicting pregnancy loss in lactating dairy cattle.

After diagnosing spontaneous cases of LEM by ultrasonography, both plasma bPSPB and bPAG-1 levels began to decline in most cases, while the CL continued to produce progesterone [103][43][44]. This confirms the previous observations [42][104], and demonstrates that lower progesterone concentrations are not the cause of conceptus death.

Although the concentrations of both proteins decrease steadily [42][43] after spontaneous or induced LEM/EFM, they reach the threshold level only after a relatively long half-life, namely, about 7 to 8 days for the bPSPB Thus, they can contribute to the elevation of false positive pregnancy diagnoses on the farm. Although the threshold levels for these tests were not determined, they reached the basal levels in both tests approximately 5 to 7 days after inducing LEM. Based on commercial blood and milk ELISA tests, the threshold levels are reached approximately 7 to 14 days after pregnancy loss [23].

[53] found, that reduced PAG concentrations (in-house ELISA) at Day 32 after AI may predict pregnancy loss between Days 46 and 74 of gestation. Based on positive and negative predictive value analysis, a circulating concentration of PAG (in-house ELISA) below 1.4 ng/mL in lactating dairy cattle following timed AI and 1.85 ng/mL following timed embryo transfer on Day 7 was 95% accurate in predicting LEM/EFM (between Days 31 and 59) at Day 31 of gestation [105]. It has been recently reported that cows being pregnant at Day 31 of gestation and maintaining the pregnancy until Day 59 had significantly higher circulating concentrations of PAG [23]. In contrast, although there was a significant difference in the PAG concentrations measured on Day 24 after AI between pregnant and non-pregnant multiparous cows while in heifers only a tendency was detected at Day 31 after AI, the circulating concentrations of PAG at Day 24 of gestation in animals that maintained pregnancy until Day 60 compared to animals that lost pregnancy between Days 31 and 60 of gestation, were not significantly different [106].

[107] reported that low or very high plasma pregnancy-associated glycoprotein-1 (PAG-RIA) levels on Day 35 of gestation in cows were related to a subsequent pregnancy loss. [108] also found pregnancy losses in cows with high PSPB (commercial ELISA) concentrations ( >1.1ng/mL) and in cows with low concentrations of both PSPB and progesterone (0.6–1.1 and <2ng/mL, respectively), however the prevalence of pregnancy loss was significantly lower in cows with high PSPB concentrations (15%) between Days 29 and 35 of gestation than in those with low concentrations (76.3%).

It has been recently reported that different PAG ELISA assays may accurately detect pregnancy; however, the ability to predict embryo survival vs. mortality during early gestation appears to be antibody-/assay-dependent [109].

In order to be able to decrease the effect of false positive diagnoses, due to pregnancy loss on the farm, it is necessary to repeat the pregnancy tests [110]. By using commercial plasma or milk ELISA tests, the optimal time for the first pregnancy diagnosis is around Day 32 after AI, when plasma and milk PAG levels are at an early peak. After this period, all pregnant cows should be retested on Day 74 after AI or later, when plasma and milk PAG levels rebound from their nadirs [23]. In contrast to the gradual increase in PAG-1 concentration (RIA) throughout gestation [39], plasma and milk PAG levels (commercial ELISA) reached a peak at Day 32 of gestation and then started to decrease to a nadir from Days 53 to 60 for the plasma PAG level and from Days 46 to 67 for the milk PAG level, followed by a gradual increase in PAG levels from Days 74 to 102 after AI [23].

The potential clinical significance of diagnosing pregnancy loss using ultrasonography or pregnancy protein tests and treating the cows with prostaglandin as soon as possible is that these measures may reduce the number of days before re-insemination [111].

4. Future Perspectives

Pregnancy diagnosis plays an essential role in decreasing days open in dairy farms; therefore, it is very important to select an accurate method for diagnosing early pregnancy, because the cost of each day open past 100 DIM may reach $4.00 [112] or €2.5 to 6.5 [113], respectively. Besides traditional pregnancy diagnoses made by rectal palpation of the uterus from 40 to 60 days after AI and measuring the serum or milk progesterone concentration between 18 to 24 days after AI [2][114][115], there are several new possibilities to diagnose early pregnancy in dairy farms; however, it is very important to emphasize that before introducing any new diagnostic test we need to make sure the accuracy of that particular test to be able to decrease the rate of iatrogenic pregnancy losses caused by prostaglandin treatment to reduce the interval to the next AI service [114] or resynchronization of the cows [116][117]. Furthermore, the new pregnancy diagnostic results must be confirmed by the old diagnostic method to decrease the negative effects of false negative diagnoses [118]. Linear-array/sector B-mode [119] and Doppler ultrasonography [2] may exceed the other diagnostic methods in the amount of information collecting from each animal during scanning, however, their uses greatly depend on the operator proficiency and availability [119].

References

  1. Long, S. Abnormal development of the conceptus and its consequences. In Veterinary Reproduction and Obstetrics, 9th ed.; Noakes, D.E., Parkinson, T.J., England, G.C.W., Eds.; Saunders Elsevier: Edinburgh, UK, 2009; pp. 123–145.
  2. Pohler, K.G.; Reese, S.T.; Franco, G.A.; Oliveira Filho, R.V.; Paiva, R.; Fernandez, L.; Melo, G.; Vasconcelos, J.L.M.; Cooke, R.; Poole, R.K. New approaches to diagnose and target reproductive failure in cattle. Anim. Reprod. 2020, 17, e20200057.
  3. Pieterse, M.C.; Szenci, O.; Willemse, A.H.; Bajcsy, A.C.; Dieleman, S.J.; Taverne, M.A.M. Early pregnancy diagnosis in cattle by means of linear-array real-time ultrasound scanning of the uterus and a quantitative and qualitative milk progesterone test. Theriogenology 1990, 33, 697–707.
  4. Nation, D.P.; Malmo, J.; Davis, G.M.; Macmillan, K.L. Accuracy of bovine pregnancy detection using transrectal ultrasonography at 28 to 35 days after insemination. Aust. Vet. J. 2003, 81, 63–65.
  5. Romano, J.E.; Thompson, J.A.; Forrest, D.W.; Westhusin, M.E.; Tomaszweski, M.A.; Kraemer, D.C. Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle. Theriogenology 2006, 66, 1034–1041.
  6. Boyd, J.S.; Omran, S.N.; Ayliffe, T.R. Use of a high frequency transducer with real time B-mode ultrasound scanning to identify early pregnancy in cows. Vet. Rec. 1988, 123, 8–11.
  7. Curran, S.; Pierson, R.A.; Ginther, O.J. Ultrasonographic appearance of the bovine conceptus from days 10 through 20. J. Am. Vet. Med. Assoc. 1986, 189, 1289–1294.
  8. Pierson, R.; Ginther, O. Ultrasonography for detection of pregnancy and study of embryonic development in heifers. Theriogenology 1984, 22, 225–233.
  9. Kastelic, J.P.; Bergfelt, D.R.; Ginther, O.J. Ultrasonic detection of the conceptus and characterisation of intrauterine fluid on days 10 to 22 in heifers. Theriogenology 1991, 35, 569–581.
  10. Curran, S.; Pierson, R.A.; Ginther, O.J. Ultrasonographic appearance of the bovine conceptus from days 20 through 60. J. Am. Vet. Med. Assoc. 1986, 189, 1295–1302.
  11. Filteau, V.; DesCoteaux, L. Valeur predictive de l’utilisation de l’appareil echographique pour le diagnostic precoce de la gestation chez la vache laitiere. Médecin Vétérinaire Québec 1998, 28, 81–85.
  12. Szenci, O.; Piros, A.; Kovács, L. Early bovine pregnancy diagnosis by a battery operated portable ultrasonic scanner the Ultra-Scan. In Proceedings of the 16th World Buiatrics Congress, Salvador, Brazil, 13–17 August 1990; pp. 219–223.
  13. Hanzen, C.; Laurent, Y. Application de l’echographie bidimensionnelle au diagnostic de gestation et l’evaluation de l’incidence de la mortalit‚ embryonnaire dans l’espece bovine. Ann. Med. Vet. 1991, 134, 481–487.
  14. Munoz del Real, L.M.; Valencia, G.L.; Corral, A.; Renteria, T. Evaluation of ultrasound for pregnancy diagnosis between 20 and 40 days post insemination in dairy cows. J. Anim. Vet. Adv. 2006, 5, 949–957.
  15. Badtram, G.A.; Gaines, J.D.; Thomas, C.B.; Bosu, W.T.K. Factors influencing the accuracy of early pregnancy detection in cattle by real-time ultrasound scanning of the uterus. Theriogenology 1991, 35, 1153–1167.
  16. Szenci, O.; Beckers, J.F.; Humblot, P.; Sulon, J.; Sasser, G.; Taverne, M.A.M.; Varga, J.; Baltusen, R.; Schekk, G. Comparison of ultrasonography bovine pregnancy-specific protein B, and bovine pregnancy-associated glycoprotein 1 tests for pregnancy detection in dairy cows. Theriogenology 1998, 50, 77–88.
  17. Silva, E.; Sterry, R.A.; Kolb, D.; Mathialagan, N.; McGrath, M.F.; Ballam, J.M.; Fricke, P.M. Accuracy of a pregnancy-associated glycoprotein (PAG) ELISA to determine pregnancy status of lactating dairy cows twenty-seven days after timed AI. J. Dairy Sci. 2007, 90, 4612–4622.
  18. Karen, A.; De Sousa, N.M.; Beckers, J.F.; Bajcsy, Á.C.; Tibold, J.; Mádl, I.; Szenci, O. Comparison of a commercial bovine pregnancy-associated glycoprotein ELISA test and a pregnancy-associated glycoprotein radiomimmunoassay test for early pregnancy diagnosis in dairy cattle. Anim. Reprod. Sci. 2015, 159, 31–37.
  19. Abdullach, M.; Mohanty, T.K.; Kumaresan, A.; Mohanty, A.K.; Madkar, A.R.; Baithalu, R.K.; Bhakat, M. Early pregnancy diagnosis in dairy cattle: Economic importance and accuracy of ultrasonography. Adv. Anim. Vet. Sci. 2014, 2, 464–467.
  20. Fosgate, G.T.; Motimele, B.; Ganswindt, A.; Irons, P.C. Bayesian latent class model to estimate the accuracy of pregnancy diagnosis by transrectal ultrasonography and laboratory detection of pregnancy-associated glycoproteins in dairy cows. Prev. Vet. Med. 2017, 145, 100–109.
  21. Szenci, O.; Varga, J.; Bajcsy, A.C. Role of early pregnancy diagnosis be means of ultrasonography in improving reproductive efficiency in a dairy herd: A retrospective study. Bov. Pract. 1999, 33, 67–69.
  22. Giordano, J.O.; Fricke, P.M. Accuracy of pregnancy diagnosis outcomes using transrectal ultrasonography 29 days after artificial insemination in lactating dairy cows. J. Dairy Sci. 2012, 95 (Suppl. 2), 75.
  23. Ricci, A.; Carvalho, P.D.; Amundson, M.C.; Fourdraine, R.H.; Vincenti, L.; Fricke, P.M. Factors associated with pregnancy-associated glycoprotein (PAG) levels in plasma and milk of Holstein cows during early pregnancy and their effect on the accuracy of pregnancy diagnosis. J. Dairy Sci. 2015, 98, 2502–2514.
  24. Dufour, S.; Durocher, J.; Dubuc, J.; Dendukuri, N.; Hassan, S.; Buczinski, S. Bayesian estimation of sensitivity and specificity of a milk pregnancy-associated glycoprotein-based ELISA and of transrectal ultrasonographic exam for diagnosis of pregnancy at 28-45 days following breeding in dairy cows. Prev. Vet. Med. 2017, 140, 122–133.
  25. Szenci, O.; Gyulai, G.; Nagy, P.; Kovács, L.; Varga, J.; Taverne, M.A.M. Effect of uterus position relative to the pelvic inlet on the accuracy of early bovine pregnancy diagnosis by means of ultrasonography. Vet. Q. 1995, 17, 37–39.
  26. López-Gatius, F.; Hunter, R.H.F. Spontaneous reduction of advanced twin embryos: Its occurrence and clinical relevance in dairy cattle. Theriogenology 2005, 63, 118–125.
  27. Szelényi, Z.; Szenci, O.; Répási, A.; de Sousa, M.N.; Beckers, J.F. Accuracy of diagnosing double corpora lutea and twin pregnancy by measuring serum progesterone and bovine pregnancy-associated glycoprotein 1 in the first trimester of gestation in dairy cows. Theriogenology 2015, 84, 76–81.
  28. Szelényi, Z.; Szenci, O.; Kovács, L.; García-Ispierto, I. Practical aspects of twin pregnancy diagnosis in cattle. Animals 2021, 11, 1061.
  29. Herzog, K.; Brockhan-Ludemann, M.; Kasle, M.; Beindorff, N.; Paul, V.; Niemann, H.; Bollwein, H. Luteal blood flow is a more appropriate indicator for luteal function during the bovine estrous cycle than luteal size. Theriogenology 2010, 73, 691–697.
  30. Herzog, K.; Voss, C.; Kastelic, J.P.; Beindorff, N.; Paul, V.; Niemann, H.; Bollwein, H. Luteal blood flow increases during the first three weeks of pregnancy in lactating dairy cows. Theriogenology 2011, 75, 549–554.
  31. Siqueira, L.G.; Areas, V.S.; Ghetti, A.M.; Fonseca, J.F.; Palhao, M.P.; Fernandes, C.A.; Viana, J.H. Color Doppler flow imaging for the early detection of nonpregnant cattle at 20 days after timed artificial insemination. J. Dairy Sci. 2013, 96, 6461–6472.
  32. Dubuc, J.; Houle, J.; Rousseau, M.; Roy, J.-P.; Buczinski, S. Short communication: Accuracy of corpus luteum color flow Doppler ultrasonography to diagnose nonpregnancy in dairy cows on day 21 after insemination. J. Dairy Sci. 2020, 103, 2019–2023.
  33. Siqueira, L.G.; Arashiro, E.K.; Ghetti, A.M.; Souza, E.D.; Feres, L.F.; Pfeifer, L.F.; Fonseca, J.F.; Viana, J.H. Vascular and morphological features of the corpus luteum 12 to 20 days after timed artificial insemination in dairy cattle. J. Dairy Sci. 2019, 102, 5612–5622.
  34. Butler, J.E.; Hamilton, W.C.; Sasser, R.G.; Ruder, C.A.; Hass, G.M.; Williams, R.J. Detection and partial characterization of two bovine pregnancy-specific proteins. Biol. Reprod. 1982, 26, 925–933.
  35. Zoli, A.P.; Beckers, J.F.; Wouters-Ballman, P.; Closset, J.; Falmagne, P.; Ectors, F. Purification and characterization of a bovine pregnancy associated glycoprotein. Biol. Reprod. 1991, 45, 1–10.
  36. Mialon, M.M.; Camous, S.; Renand, G.; Martal, J.; Ménissier, F. Peripheral concentrations of a 60-kDa pregnancy serum protein during gestation and after calving and in relationship to embryonic mortality in cattle. Reprod. Nutr. Dev. 1993, 33, 269–282.
  37. Beckers, J.F.; Zarrouk, A.; Batalha, E.S.; Garbayo, J.M.; Mester, L.; Szenci, O. Endocrinology of pregnancy: Chorionic somatomammotropins and pregnancy-associated glycoproteins: Review. Acta Vet. Hung. 1998, 46, 175–189.
  38. Beckers, J.F.; Drion, P.V.; Garbayo, J.M.; Perényi, Z.; Zarrouk, A.; Sulon, J.; Remy, B.; Szenci, O. Pregnancy associated glycoproteins in ruminants: Inactive members of the aspartic proteinase family. Acta Vet. Hung. 1999, 47, 461–469.
  39. Zoli, A.P.; Guilbault, L.A.; Delahaut, P.; Ortiz, W.B.; Beckers, J.F. Radioimmunoassay of a bovine pregnancy-associated glycoprotein in serum: Its application for pregnancy diagnosis. Biol. Reprod. 1992, 46, 83–92.
  40. Vasquez, M.I.; Horta, A.E.M.; Marques, C.C.; Sasser, R.G.; Humblot, P. Levels of bPSPB throughout single and twin pregnancies after AI or transfer of IVM/IVF cattle embryos. Anim. Reprod. Sci. 1995, 38, 279–289.
  41. Humblot, P.; Jeanguyot, N.; Ruder, C.A.; Leriche, I.; Thibier, M.; Sasser, R.G. Accuracy of pregnancy diagnosis by PSPB RIA in the plasma of dairy cows 28 days after AI. In Proceedings of the 11th International Congress on Animal Reproduction and Artificial Insemination, Dublin, Ireland, 26–30 June 1988; Volume 2, p. 3.
  42. Semambo, D.K.N.; Eckersall, P.D.; Sasser, R.G.; Ayliffe, T.R. Pregnancy-specific protein B and progesterone in monitoring viability of the embryo in early pregnancy in the cow after experimental infection with Actinomyces pyogenes. Theriogenology 1992, 37, 741–748.
  43. Szenci, O.; Humblot, P.; Beckers, J.F.; Sasser, G.; Sulon, J.; Baltusen, R.; Varga, J.; Bajcsy, Á.C.; Taverne, M.A.M. Plasma profiles of progesterone and conceptus proteins in cows with spontaneous embryonic/foetal mortality as diagnosed by ultrasonography. Vet. J. 2000, 159, 287–290.
  44. Szenci, O.; Beckers, J.F.; Sulon, J.; Bevers, M.M.; Börzsönyi, L.; Fodor, L.; Kovács, F.; Taverne, M.A.M. Effect of induction of late embryonic mortality on plasma profiles of pregnancy associated glycoproteins in heifers. Vet. J. 2003, 165, 307–313.
  45. Serrano, B.; López-Gatius, F.; Santolaria, P.; Almería, S.; García-Ispierto, I.; Bech-Sabat, G.; Sulon, J.; de Sousa, N.M.; Beckers, J.F.; Yániz, J.L. Factors affecting plasma pregnancy-associated glycoprotein 1 concentrations throughout gestation in high-producing dairy cows. Reprod. Domest. Anim. 2009, 44, 600–605.
  46. Chavatte-Palmer, P.; de Sous, N.; Laigre, P.; Camous, S.; Ponter, A.A.; Beckers, J.F.; Heyman, Y. Ultrasound fetal measurements and pregnancy associated glycoprotein secretion in early pregnancy in cattle recipients carrying somatic clones. Theriogenology 2006, 66, 829–840.
  47. Green, J.A.; Xie, S.; Quan, X.; Bao, B.; Gan, X.; Mathialagan, N.; Beckers, J.F.; Roberts, R.M. Pregnancy-associated bovine and ovine glycoproteins exhibit spatially and temporally distinct expression patterns during pregnancy. Biol. Reprod. 2000, 62, 1624–1631.
  48. Piechotta, M.; Bollwein, J.; Friedrich, M.; Heilkenbrinker, T.; Passavant, C.; Branen, J.; Sasser, G.; Hoedemaker, M.; Bollwein, H. Comparison of commercial ELISA blood tests for early pregnancy detection in dairy cows. J. Reprod. Dev. 2011, 57, 72–75.
  49. Sinedino, L.D.P.; Lima, F.S.; Bisinotto, R.S.; Cerri, R.L.A.; Santos, J.E.P. Effect of early or late resynchronization based on different methods of pregnancy diagnosis on reproductive performance of dairy cows. J. Dairy. Sci. 2014, 97, 4932–4941.
  50. Friedrich, M.; Holtz, W. Establishment of an ELISA for measuring bovine pregnancy-associated glycoprotein in serum or milk and its application for early pregnancy detection. Reprod. Domest. Anim. 2010, 45, 142–146.
  51. Thompson, I.; Cerri, R.; Kim, I.; Green, J.; Santos, J.; Thatcher, W. Effects of resynchronization programs on pregnancy per artificial insemination, progesterone, and pregnancy-associated glycoproteins in plasma of lactating dairy cows. J. Dairy Sci. 2010, 93, 4006–4018.
  52. Green, J.A.; Parks, T.E.; Avalle, M.P.; Telugu, B.P.; McLain, A.L.; Peterson, A.J.; McMillan, W.; Mathialagan, N.; Hook, R.R.; Xie, S.; et al. The establishment of an ELISA for the detection of pregnancy-associated glycoproteins (PAGs) in the serum of pregnant cows and heifers. Theriogenology 2005, 63, 1481–1503.
  53. Mercadante, P.M.; Ribeiro, E.S.; Risco, C.; Ealy, A.D. Associations between pregnancy-associated glycoproteins and pregnancy outcomes, milk yield, parity, and clinical diseases in high-producing dairy cows. J. Dairy Sci. 2016, 99, 3031–3040.
  54. Kaya, M.S.; Köse, M.; Bozkaya, F.; Mutlu, H.; Uçar, E.H.; Atli, M.O. Early pregnancy diagnosis using a commercial ELISA test based on pregnancy-associated glycoproteins in Holstein-Friesian heifers and lactating cows. Turk. J. Vet. Anim. Sci. 2016, 40, 694–699.
  55. Martins, J.; Wang, D.; Mu, N.; Rossi, G.; Martini, A.; Martins, V.; Pursley, J. Level of circulating concentrations of progesterone during ovulatory follicle development affects timing of pregnancy loss in lactating dairy cows. J. Dairy Sci. 2018, 101, 10505–10525.
  56. Middleton, E.; Pursley, J. Blood samples before and after embryonic attachment accurately determine non-pregnant lactating dairy cows at 24 d post-artificial insemination using a commercially available assay for pregnancy-specific protein B. J. Dairy Sci. 2019, 102, 7570–7575.
  57. Romano, J.E.; Larson, J.E. Accuracy of pregnancy specific protein-B test for early pregnancy diagnosis in dairy cattle. Theriogenology 2010, 74, 932–939.
  58. Breed, M.W.; Guard, C.L.; White, M.E.; Smith, M.C.; Warnick, L.D. Comparison of pregnancy diagnosis in dairy cattle by use of a commercial ELISA and palpation per rectum. J. Am. Vet. Med. Assoc. 2009, 235, 292–298.
  59. Gábor, G.; Tóth, F.; Sasser, G.; Szász, F.; Bárány, I.; Wölfling, A.; Völgyi-Csík, J. Ways of decrease the period between calvings in dairy cows. Early pregnancy detection by Biopryn ELISA-test. Magy. Állatorv. Lapja 2004, 126, 459–464.
  60. Howard, J.; Gábor, G.; Gray, T.; Passavant, C.; Ahmadzadeh, A.; Sasser, N.; Pals, D.; Sasser, G. BioPRYN, a blood-based pregnancy test for managing breeding and pregnancy in cattle. J. Anim. Sci. 2007, 58, 295–298.
  61. Paré, J.; Audet-Grenier, M.H.; Rouillier, P.; Sirard, M.A. Evaluation of the DG29 test for early detection of pregnancy in cattle. Can. Vet. J. 2008, 49, 1119–1121.
  62. Moussafir, Z.; Allaia, L.; El Khalila, K.; Essamadib, A.; El Amiri, B. Could a bovine pregnancy rapid test be an alternative to a commercial pregnancy-associated glycoprotein ELISA test in dairy cattle? Anim. Reprod. Sci. 2018, 192, 78–83.
  63. Akkose, M.; Cebi-Sen, C.; Kirmit, A.; Korkmaz, O.; Yazlik, M.O.; Gocen, M.; Kuru, M.; Akçay, E. Pregnancy-associated glycoproteins (PAGs) and progesterone concentrations in Holstein heifers following two methods of estrus syncronization. Veterinaria México OA 2019, 2019, 646.
  64. Mayo, L.M.; Moore, S.G.; Poock, S.E.; Silvia, W.J.; Lucy, M.C. Technical note: Validation of a chemical pregnancy test in dairy cows that uses whole blood, shortened incubation times, and visual readout. J. Dairy Sci. 2016, 99, 7634–7641.
  65. Commun, L.; Velek, K.; Barbry, J.-B.; Pun, S.; Rice, A.; Mestek, A.; Egli, C.; Leterme, S. Detection of pregnancy-associated glycoproteins in milk and blood as a test for early pregnancy in dairy cows. J. Vet. Diagn. Investig. 2016, 28, 207–213.
  66. Lawson, B.C.; Shahzad, A.H.; Dolecheck, K.A.; Martel, E.L.; Velek, K.A.; Ray, D.L.; Lawrence, J.C.; Silvia, W.J. A pregnancy detection assay using milk samples: Evaluation and considerations. J. Dairy Sci. 2014, 97, 6316–6325.
  67. LeBlanc, S.J. Short communication: Field evaluation of a pregnancy confirmation test using milk samples in dairy cows. J. Dairy Sci. 2013, 96, 2345–2348.
  68. Byrem, T.M.; Velek, K.; Pearse, H.L. The Detection of Pregnancy Associated Glycoproteins (PAG) in Routine Milk Recording Samples as an Indicator of Pregnancy in Dairy Cattle. 2012. Available online: https://www.icar.org/wp-content/uploads/2015/09/Byrem.pdf (accessed on 15 September 2015).
  69. Green, J.C.; Volkmann, D.H.; Poock, S.E.; McGrath, M.F.; Ehrhardt, M.; Moseley, A.E.; Lucy, M.C. Technical note: A rapid enzyme-linked immunosorbent assay blood test for pregnancy in dairy and beef cattle. J. Dairy Sci. 2009, 92, 3819–3824.
  70. Hameed, O.A.; Mustafa, M.; Madi, N.; Tate, W. Evaluation of a rapid visual pregnancy (Bio-RPD) test for detection of pregnancy specific protein B (PSPB) in cow serum. J. Anim. Sci. Livest. Prod. 2020, 4, No.3:3.
  71. Karakus, O.; Kadar, C.; Kuru, M.; Kaya, S.; Demir, M.C.; Arı, U.C.; Zonturlu, A.K.; Öztürkler, Y. Efficacy of a commercial test kit to determine early pregnancy in cows using whole blood and blood serum. Trop. Anim. Health Prod. 2021, 53, 28.
  72. Morton, H.; Hegh, V.; Clunie, G.J.A. Immunosuppression detected in pregnant mice by rosette inhibition. Nature 1974, 249, 459–460.
  73. Morton, H.; Rolfe, B.E.; Anderson, M.; Morrison, J. An early pregnancy factor detected in human serum by rosette inhibition test. Lancet 1977, 1, 394–397.
  74. Morton, H.; Clunie, G.J.A.; Shaw, F.D. A test for early pregnancy in sheep. Res. Vet. Sci. 1979, 26, 261–262.
  75. Nancarrow, C.D.; Wallace, A.L.L.; Grewal, A.S. The early pregnancy factor of sheep and cattle. J. Reprod. Fertil. 1981, 30, 191–197.
  76. Paisley, L.G.; Davis, W.C.; Anderson, P.B.; Mickelsen, W.D. Detection of early pregnancy factor in swine: A need for dialogue. Theriogenology 1982, 18, 393–401.
  77. Rolfe, B.; Cavanagh, A.; Forde, C.; Bastin, F.; Chen, C.; Morton, H. Modified rosette inhibition test with mouse lymphocytes for detection of early pregnancy factor in human pregnancy serum. J. Immunol. Methods 1984, 70, 1–11.
  78. Yamazaki, S.; Kawahata, K.; Goto, T.; Takahashi, J.; Yasuda, Y. EPF as a marker for early embryonic losses in repeat breeders. J. Reprod. Dev. 1995, 41, 129–132.
  79. Rolfe, B. Detection of foetal westage. Fertil. Steril. 1982, 37, 655–660.
  80. Koch, E. Early pregnancy factor: Its significance as an indicator of fertilization and embryonic mortality. In Embryonic Mortality in Farm Animals; Screenan, J.M., Diskin, M.G., Eds.; Martinus Nijhoff Publishers: Dordrecht, The Netherlands, 1986; pp. 74–92.
  81. Laleh, V.G.; Laleh, R.G.; Pirany, N.; Ahrabi, M.M. Measurement of EPF for detection of cow pregnancy using rosette inhibition test. Theriogenology 2008, 70, 105–107.
  82. Threlfall, W.R. Immunosuppressive early pregnancy factor (ISEPF) determination for pregnancy diagnosis in dairy cows. Theriogenology 1994, 41, 317.
  83. Adams, C.S.; Jardon, P.W. Evaluation of the early conception factor tests in cows 3–7 days post-breeding. Proc. Am. Assoc. Bov. Pract. 1999, 32, 240–241.
  84. DesCoteaux, L.; Carrière, P.D.; Bigras-Poulin, M. Evaluation of the Early Conception Factor (ECF) dipstick test in dairy cows between days 11 and 15 post-breeding. Bov. Pract. 2000, 34, 87–91.
  85. Cordoba, M.C.; Sartori, R.; Fricke, P.M. Assessment of a commercially available early conception factor (ECF) test for determining pregnancy status of dairy cattle. J. Dairy Sci. 2001, 84, 1884–1889.
  86. Gandy, B.; Tucker, W.; Ryan, P.; Williams, A.; Tucker, A.; Moore, A.; Godfrey, R.; Willard, S. Evaluation of the early conception factor (ECF™) test for the detection of nonpregnancy in dairy cattle. Theriogenology 2001, 56, 637–647.
  87. Whisnant, C.S.; Pagels, L.A.; Daves, M.G. Case study: Effectiveness of a commercial early conception factor test for use in cattle. Prof. Anim. Sci. 2001, 17, 51–53.
  88. Cavanagh, A.C. Identification of early pregnancy factor as chaperonin 10: Implications for understanding its role. Rev. Reprod. 1996, 1, 28–32.
  89. Morton, H. Early pregnancy factor: An extracellular chaperonin 10 homologue. Immunol. Cell Biol. 1998, 76, 483–496.
  90. Han, R.X.; Kim, H.R.; Diao, Y.F.; Lee, M.G.; il Jin, D. Detection of early pregnancy-specific proteins in Holstein milk. J. Proteom. 2012, 75, 3221–3229.
  91. Panda, B.S.K.; Mohapatrab, S.K.; Chaudharya, D.; Alhussienc, M.N.; Kapilab, R.; Danga, A.K. Proteomics and transcriptomics study reveals the utility of ISGs as novel molecules for early pregnancy diagnosis in dairy cows. J. Reprod. Immunol. 2020, 140, 103148.
  92. Taron, C.H.; Rudd, P.M. Glycomics: A Rapidly Evolving Field with a Sweet Future. Available online: (accessed on 15 January 2016).
  93. Mayer, J.; Soller, J.T.; Beck, J.; Purwins, V.; Wemheuer, W.; Schutz, E.; Brenig, B. Early pregnancy diagnosis in dairy cows using circulating nucleic acids. Theriogenology 2013, 79, 173–179.
  94. Ramu, S.; Stamatkin, C.; Timms, L.; Ruble, M.; Roussev, R.G.; Barnea, E.R. PreImplantation factor (PIF) detection in maternal circulation in early pregnancy correlates with live birth (bovine model). Reprod. Biol. Endocrin. 2013, 11, 105.
  95. Barbato, O.; Guelfi, G.; Barile, V.L.; Menchetti, L.; Tortiello, C.; Canali, C.; Brecchia, G.; Traina, G.; Beckers, J.F.; Sousa, N.M. Using real-time PCR to identify pregnancy-associated glycoprotein 2 (PAG-2) in water buffalo (Bubalus bubalis) blood in early pregnany. Theriogenology 2017, 89, 106–113.
  96. Balhara, A.K.; Gupta, M.; Singh, S.; Ashok, K.; Mohanty, A.K.; Singh, I. Early pregnancy diagnosis in bovines: Current status and future directions. Sci. World J. 2013.
  97. Saint-Dizier, M.; Chastant-Maillard, S. Potential of connected devices to optimize cattle reproduction. Theriogenology 2018, 112, 53–56.
  98. Bruinjé, T.C.; Ambrose, D.J. Technical note: Validation of an automated in-line milk progesterone analysis system to diagnose pregnancy in dairy cattle. J. Dairy Sci. 2019, 102, 3615–3621.
  99. Kovács, L.; Kézér, F.L.; Kulcsár-Huszenicza, M.; Ruff, F.; Szenci, O.; Jurkovich, V. Hypothalamic-pituitary-adrenal and cardiac autonomic responses to transrectal examination differ with behavioral reactivity in dairy cows. J. Dairy Sci. 2016, 99, 7444–7457.
  100. Semambo, D.K.N.; Boyd, J.S.; Taylor, D.J.; Ayliffe, T.R.; Omran, S.N. Ultrasonographic study of early embryonic loss induced by Actinomyces pyogenes in cattle. Vet. Rec. 1992, 131, 7–12.
  101. Chaffaux, S.; Reddy, G.N.S.; Valon, F.; Thibier, M. Transrectal real-time ultrasound scanning for diagnosing pregnancy and for monitoring embryonic mortality in dairy cattle. Anim. Reprod. Sci. 1986, 10, 193–200.
  102. Kelly, D.E.; Galvao, K.N.; Mortensen, C.J.; Risco, C.A.; Ealy, A.D. Using Doppler ultrasonography on day 34 of pregnancy to predict pregnancy loss in lactating dairy cattle. J. Dairy Sci. 2017, 100, 3266–3271.
  103. Giordano, J.O.; Guenther, J.N.; Lopes, G.; Fricke, P.M. Changes in serum pregnancy-associated glycoprotein, pregnancy-specific protein B, and progesterone concentrations before and after induction of pregnancy loss in lactating dairy cows. J. Dairy Sci. 2012, 95, 683–697.
  104. Kastelic, J.P.; Northey, D.L.; Ginther, O.J. Spontaneous embryonic death on days 20 to 40 in heifers. Theriogenology 1991, 35, 361–363.
  105. Pohler, K.G.; Pereira, M.H.C.; Lopes, F.R.; Lawrence, J.C.; Keisler, D.H.; Smith, M.F.; Vasconcelos, J.L.M.; Green, J.A. Circulating concentrations of bovine pregnancy-associated glycoproteins and late embryonic mortality in lactating dairy herds. J. Dairy Sci. 2016, 99, 1584–1594.
  106. Reese, S.T.; Pereira, M.H.C.; Edwards, J.L.; Vasconcelos, J.L.M.; Pohler, K.G. Pregnancy diagnosis in cattle using pregnancy associated glycoprotein concentration in circulation at day 24 of gestation. Theriogenology 2018, 106, 178–185.
  107. López-Gatius, F.; Hunter, R.H.; Garbayo, J.M.; Santolaria, P.; Yaniz, J.; Serrano, B.; Ayad, A.; De Sousa, N.M.; Beckers, J.F. Plasma concentrations of pregnancy-associated glycoprotein-1 (PAG-1) in high producing dairy cows suffering early fetal loss during the warm season. Theriogenology 2007, 67, 1324–1330.
  108. Gábor, G.; Kastelic, J.P.; Abonyi-Tóth, Z.; Gábor, P.; Endrődi, T.; Balogh, O.G. Pregnancy loss in dairy cattle: Relationship of ultrasound, blood pregnancy-specific protein B, progesterone and production variables. Reprod. Domest. Anim. 2016, 51, 467–473.
  109. Gatea, A.O.; Smith, M.F.; Pohler, K.G.; Egen, T.; Pereira, M.H.C.; Vasconselos, J.L.M.; Lawrence, J.C.; Green, J.A. The ability to predict pregnancy loss in cattle with ELISAs that detect pregnancy associated glycoproteins is antibody dependent. Theriogenology 2018, 108, 269–276.
  110. López-Gatius, F.; García-Ispierto, I. Ultrasound and endocrine findings that help to assess the risk of late embryo/early foetal loss by non-infectious cause in dairy cattle. Reprod. Domest. Anim. 2010, 45 (Suppl. 3), 15–24.
  111. Baxter, S.J.; Ward, W.R. Incidence of fetal loss in dairy cattle after pregnancy diagnosis using an ultrasound scanner. Vet. Rec. 1997, 140, 287–288.
  112. Des Coteaux, L.; Fetrow, J. Does it pay to use an ultrasound machine for early pregnancy diagnosis in dairy cows? In Proceedings of the 31st AABP Annual Meeting, Spokane, WA, USA, 24–27 September 1998; pp. 172–174.
  113. Fodor, I.; Ózsvári, L. Early pregnancy diagnosis on large dairy farms and its role in improving profitability: Literature review. AWETH 2018, 14, 22–36.
  114. Fricke, P.M.; Ricci, A.; Giordano, J.O.; Carvalho, P.D. Methods for and implementation of pregnancy diagnosis in dairy cows. Vet. Clin. Food. Anim. 2016, 32, 165–180.
  115. Reese, S.T.; Pereira, M.C.; Vasconcelos, J.L.M.; Smith, M.F.; Green, J.A.; Geary, T.W.; Peres, R.F.G.; Perry, G.A.; Pohler, K.G. Markers of pregnancy: How early can we detect pregnancies in cattle using pregnancy-associated glycoproteins (PAGs) and microRNAs? Anim. Reprod. 2016, 13, 200–208.
  116. Silva, E.; Sterry, R.A.; Kolb, D.; Mathialagan, N.; McGrath, M.F.; Ballam, J.M.; Fricke, P.M. Effect of interval to resynchronization of ovulation on fertility of lactating Holstein cows when using transrectal ultrasonography or a pregnancy-associated glycoptorein (PAG) ELISA to diagnose pregnancy status. J. Dairy Sci. 2009, 92, 3643–3650.
  117. Green, J.C.; Newsom, E.M.; Lucy, M.C. Incorporation of a rapid pregnancy-associated glycoprotein ELISA into a CIDR-Ovsynch resynchronization program for a 28 day re-insemination interval. Theriogenology 2011, 75, 320–328.
  118. Szenci, O.; Taverne, M.A.M.; Beckers, J.F.; Sulon, J.; Varga, J.; Börzsönyi, L.; Hanzen, C.; Schekk, G. Evaluation of false ultrasonographic diagnoses in cows measuring plasma levels of bovine pregnancy associated glycoprotein (bPAG). Vet. Rec. 1998, 142, 304–306.
  119. Fricke, P.M. Scanning the future—Ultrasonography as a reproductive management tool for dairy cattle. J. Dairy Sci. 2002, 85, 1918–1926.
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