Importance of Monitoring the Peripartal Period to Increase Reproductive Performance in Dairy Cattle

Abstract

Parallel with the successful genetic selection for higher milk production in Holstein-Friesian cows, a dramatic decline in fertility rates has been observed around the world. Therefore, to achieve an optimum herd reproductive performance, we must focus on the first 100 days postpartum. During and after calving, a cow overcomes a series of physiological hurdles before becoming pregnant. By selecting accurate diagnostic devices and/or methods, such as predicting the onset of calving, monitoring activity and rumination time to determine cows for early treatment of clinical metritis and/or metabolic diseases, long-term measurement of reticuloruminal pH to monitor subclinical acidosis, perform metabolic profile tests to diagnose subclinical metabolic diseases at the herd level, estrous detectors and/or detection aids, on-farm/in-line P4 test to monitor specific events in the postpartum periods, diagnosis of early pregnancy and pregnancy loss using ultrasonography to correctly identify problems and their potential causes to enable these issues are to be rectified. Despite higher milk production, acceptable fertility results can be achieved, even on large-scale dairy farms, if the impacts of the above factors that contribute to reduced fertility can be moderated. The advantages and disadvantages of the different diagnostic methods are discussed to help the dairy select the most accurate method.

1. Introduction

Since 1960 due to successful genetic selection of Holstein-Friesian cows for higher milk production, the average milk production in the United States has exceeded 11,000 kg/year. Parallel with this, the reproductive performance of dairy cows declined; however, only the conception rate was considered for the comparison to milk production [1, 2, 3]. Others could not confirm this antagonistic relationship between milk production and fertility [4, 5, 6]. LeBlanc [5] emphasizes that individual (age at first calving, parity, body condition, and diseases) and herd-level (herd size, nutrition, season, environment, herd/reproductive management, and skilled farm personnel) factors may substantially influence the production of a dairy farm. At the same time, any shortage in individual or herd-level factors in a dairy farm may increase the average number of days open (calving to conception), the number of services per conception, and the number of cows culled for infertility [2]. It is important to emphasize that reproductive performance in heifers was not affected [7]. It is essential to improve our reproductive management practices to decrease the longer lactations and the number of cows culled for reproductive reasons [7].

Concentrated management activities, especially during the first 100 days following calving, are needed to achieve an optimal calving interval (less than 400 days) with higher milk production per lactation and the birth of more calves [8]. Correct reproductive management can significantly contribute to reducing production costs.

The following diagnostic activities should be pursued during the early postpartum period to achieve or approach the optimal calving interval: prediction of the onset of calving, early diagnosis of postparturient uterine and metabolic diseases, accurate detection of estrus, determining the optimal time for artificial insemination (AI), and accurate diagnosis of early pregnancy and pregnancy losses.

2. Prediction of the onset of calving in dairy cows

One of the essential reproductive management goals is to reduce the number of calving assistances, which may negatively affect the acid-base balance of newborn calves and thus increase the number of stillbirths [9, 10, 11] and may subsequently affect the reproductive performance of the dam [12, 13]. Therefore, we must decrease the prevalence of neonatal asphyxia at calving, since instruments suitable for the reliable clearing of respiratory passages and artificial respiration of newborn calves are not yet widely available under farm conditions [14, 15].

In the case of dystocia, we must select the mode and time of calving assistance according to the profitability factors. Before applying traction, we must evaluate the soft birth canal, and it must be expanded nonsurgically or surgically (episiotomy lateralis). With using obstetric lubricants, we must avoid traction of longer than 2–3 min [16] and rib or vertebral fractures due to excessive traction [17, 18]. If prolonged traction is expected, we should perform a Cesarean section to save the calf and prevent maternal birth canal injuries. Previous studies have shown that before selecting the mode of calving assistance (traction or Cesarean section), it would also be essential to measure the acid-base balance of the fetus to be born [19, 20, 21]. The routine treatment of newborn calves with severe asphyxia may reduce postnatal calf losses [14, 15]. However, particular attention to the ingestion of sufficient qualities and quantities of colostrum must also be paid [22, 23], since an increased susceptibility accompanies poor colostrum uptake to Escherichia coli infections [24, 25].

While it is not possible to eliminate dystocia, adequate management of heifers during the development (adequate feeding, selection of a sire with a negative expected progeny difference for birth weight, or using sexed semen for AI) and close observation of calving heifers and cows are crucial for reducing the prevalence of stillbirth [14]. Since the behavioral signs of calving in some cases are not expressed, it is not easy to recognize the onset of calving, especially on large dairy farms. Inserting a vaginal thermometer into the vagina (e.g., Vel’Phone^®) may help decrease the prevalence of stillbirth by sending an alarm about the imminent start of the second stage of labor with the rupture of the allantochorionic sac [26].

In our recent experiment, 241 single calvings were monitored using a vaginal thermometer (Vel’Phone^®), which was inserted into the vagina by a vaginal applicator about 5 days before expecting to calve. The stillbirth rate was 1.7% for heifer and 0.5% for cow calvings, respectively [26]. Similar results were reported by others [27]. Imminent calving can be predicted without false alarms (Figure 1) [28], and in this way, it can minimize the time spent on standby by the workers [29].

Figure 1.

Accuracy of prediction of calving by an SMS message generated by using an intravaginal thermometer adapted from [28].

At the same time, it is essential to mention that in contrast to direct indicators (vaginal thermometers), calving predictors such as Ruminact^® HR-tag or Moocall^® calving sensor cannot inform about the exact time of calving; however, they can help optimize worker efficiency [29]. It is also essential to avoid birth injuries and infection of the reproductive tract, which may more likely develop in cows with inappropriately timed obstetrical assistance (less than 50 min after amniotic sac appearance) and dystocia [30]. Namely, premature obstetrical assistance may lead to a high prevalence of dystocia, impairs postpartum health of the dam, and poses a potential risk to calf survival [30]. At the same time, Villettaz Robichaud et al. [31] reported that systematic early obstetrical assistance at calving (15 min after the first sight of the calf’s two front hooves) that does not present signs of calving difficulties did not adversely affect calves’ likelihood of being stillborn, vigor at birth, or transfer of passive immunity. It is essential to mention that obstetrical lubricant was applied liberally to the cow’s vagina before performing the exam and providing assistance. Pumping copious amounts of sterile obstetrical lubricant around the fetus before each assisted delivery seems that the target prevalence of stillbirth (1–3%; [32]) can also be approached in the field [33].

3. Early diagnosis of postparturient uterine diseases in dairy cow

The aim of monitoring postparturient uterine diseases such as clinical metritis, clinical endometritis (pyometra), and subclinical endometritis (SCE) in a dairy farm is to diagnose [34, 35, 36, 37] and treat them as soon as possible after calving to decrease their negative effects on pregnancy rates, open days, and culling rates, which may increase economic losses in dairy farms [38, 39].

Besides minimizing stress and careful sanitation during calving, cows having dystocia, stillbirth, retained fetal membranes (RFMs), metabolic disorders (hypocalcemia, ketosis), or twins are more likely to contract uterine infections than cows calving normally. Although retained fetal membrane (RFM) is not a disease per se [39], its early treatment is greatly recommended to decrease the risk for the development of different uterine diseases.

Clinical metritis and endometritis should be diagnosed and treated as early and as intensively as possible to shorten the conception interval. Recently, new cow programs have been developed based on monitoring cow temperatures each morning for the first 10 (first 13 [40] or 14 [41]) days after calving, thus allowing for early treatment [42]. Monitoring milk production (calving milk deviation of more than 12%) or failure to increase milk yield by at least 4% (primiparous) or 7% (multiparous) per day in the first 20 days after calving [39], rumination time [43, 44, 45], cow activity [46, 47] and/or body temperature measured by ear tag, neck collar, vaginal-mounted type biosensors [48], or ventral tail base surface temperature sensors [49] may contribute to the early diagnosis of clinical metritis in the field.

Clinical endometritis can be diagnosed by transrectal palpation, transrectal ultrasonography, manual vaginal examination, vaginoscopy, and/or Metricheck^® from Day 21 after calving. In the absence of a gold standard, it seems that vaginoscopy or Metricheck^® is preferred as a cow-side diagnostic tool for diagnosing clinical endometritis in the field. Subclinical endometritis (SCE) is defined as an inflammation of the uterine endometrium that can be detected by histology (biopsy) or cytology (samples collected by uterine lavage, cytobrush, or cytotape techniques) in the absence of purulent material in the vagina [37, 50, 51, 52].

Routine treatment of clinical metritis with intrauterine antimicrobial agents (oxytetracycline; ampicillin; and cloxacillin), antiseptic chemicals (iodine solutions: 2% Lugol’s iodine immediately after calving and again 6 h later as a preventive measure), systemic antibiotics (penicillin or one of its synthetic analogs; ceftiofur/third-generation cephalosporin/for 3–5 days; a single dose of ceftiofur s.c. in the base of the ear within 24 h after abnormal calving), intrauterine ozone treatment [53], supportive therapy (nonsteroidal anti-inflammatory drugs (NSAIDs)) such as flunixin meglumine, fluid therapy in case of dehydration, therapy with calcium and energy supplements in case of depressed appetite, and/or hormone therapy (oxytocin, PGF_2α, or its synthetic analogs) are very variable [52]. According to our present knowledge, since intrauterine antibiotics and antiseptics may irritate the endometrium, it is not recommended. Routine use of prostaglandins is also controversial and requires further confirmation. Presently, systemic antibiotics (ceftiofur) and supportive therapy can be recommended for dairy farms [54, 55].

Cows with clinical endometritis having a palpable CL, treated by intrauterine infusion of cephapirin or PGF_2α, had no significant difference in time to pregnancy [56]. However, higher pregnancy rates were detected in the treated groups than in untreated cows. Several reports have suggested that PGF2α treatment for clinical endometritis is at least as effective as any other alternative therapies with Lugol, polyvinylpyrrolidone-iodine, meta-cresol sulfuric acid, Lotagen, dextrose [57], or N-acetylcysteine combined with amoxicillin and clavulanic [58] and presents a minimal risk of harm to the uterus or presence of residues in milk or meat [52]. The treatment efficacy of clinical endometritis without an active corpus luteum, solely with prostaglandin, is limited; however, according to Lewis [59], such a treatment may be advantageous by stimulating the self-defense mechanism. The presence of bacteria in the uterus can be accurately diagnosed using an on-farm bacteriological culture system (Tri-plate). This way, we can contribute to our antibiotic use to be as rational as possible on our dairy farm [60].

Treatment of subclinical endometritis with antibiotics and/or PGF_2a or nonsteroidal anti-inflammatory drugs (NSAIDs) has been tried. However, controversial results were achieved; further examinations are required [51, 61]. Intrauterine lavage with 500–600 ml of sterile physiological saline (35–40°C) on Day 30 after calving or intrauterine infusion with 50 ml of boiling sterile water (~100°C; “Samia-treat; SAT”) of repeat breeding cows may improve pregnancy rate; however, they require further large-scale confirmations [62, 63].

4. Early diagnosis of postparturient metabolic disease in dairy cow

Ruminal fluid is one of the essential sources of energy metabolism in dairy cattle because they can ferment volatile fatty acids (acetate, propionate, and butyrate), which may complete some 60–70% of the energy requirements. Dairy cattle are usually in a negative energy balance (NEBAL) in the initial weeks of lactation. The energy intake during this period is less than half the energy requirements for milk production. Therefore, dairy cattle, through increased nonesterified fatty acid (NEFA) production, try to meet the gap between energy input and output during early lactation. The suppressed feed intake results in a lack of gluconeogenesis, which causes a lack of glucose for the complete oxidation of NEFA. The incomplete oxidation of fatty acids contributes to the increased production of ketone bodies (β-hydroxybutyrate/BHB/, acetone, and acetoacetate), which may cause ketosis and fatty liver [64, 65]. It is essential to mention that primiparous cows are more susceptible to metabolic stress during the transition period (3 weeks before to 3 weeks after calving) than multiparous cows [66]. According to Iwersen et al. [67], the electronic handheld BHBA measuring system using whole blood is a more valuable and practical tool for diagnosing subclinical ketosis than the commonly used chemical dipsticks, for example, Ketostix or Ketorolac. Szelényi et al. [68] reported that BHB concentrations measured at the farm by a portable handheld device (Precision Xtra, Abbot Laboratories) showed a significant correlation (r > 0.92, P < 0.001) with the results of samples evaluated in the laboratory before and after freezing.

More recently, metabolic health disorders (e.g., subclinical ketosis [69], subclinical hypocalcemia [70]) can be predicted with high accuracy during the transition period by using different wearable wireless biosensors such as ear-tag, halter (noseband), neck collar, or leg-tag-type sensors by measuring eating, ruminating, lying, and/or standing time reviewed recently by Cocco et al. [44] and Lee and Seo [48]. Paudyal et al. [71] suggested using “two indices that could identify different health disorders satisfactorily using animal level and pen level comparisons. The cow level index compared daily rumination with the 7-day rolling average of the same cow, and the pen level index compared daily rumination time with the average of the cows in the same herd. This approach utilized deviations in rumination, which accounts for variations in rumination time between the cows and daily variation within the same cow.” Cows can be treated before developing clinical diseases, and in this way, costs associated with prolonged treatment and reduced milk yield can be decreased. The importance of early treatment of different metabolic disorders can be emphasized by the fact that dairy cows, after calving, may sacrifice their immune function to maintain lactation [69]. Therefore, they are also more sensitive to different infectious diseases such as metritis and/or mastitis [44, 45, 72].

Acute or subacute ruminal acidosis may develop due to decreased salivation during calving due to reduced period and intensity of chewing, especially when the ratio of concentrate is not limited to the days surrounding calving. Rumen acidosis may also negatively affect rumen motility and appetite. It can be diagnosed by measuring the pH value of the rumen fluid collected by a stomach tube or by rumenocentesis in the field. However, the accuracy of diagnosing subacute ruminal acidosis is limited. Long-term measurement of reticuloruminal pH value using an indwelling and wireless data transmitting unit enables the evaluation of dietary composition. It allows for adjustments in feeding management in the field [73]. However, the currently available commercial bolus sensor systems with a pH sensor have an operational lifetime of no more than a few months; therefore, its general use in daily practice is presently limited [48].

As mentioned previously, a negative energy balance (NEBAL) can develop several days before calving and usually reaches its most negative level (nadir) about 2–3 weeks later and is used to be extended 10–12 weeks until the beginning of the usual breeding period [74]. A spontaneously NEBAL in dairy cows can represent a physiological state of undernutrition. The severity and duration of NEBAL are primarily related to differences in dry matter intake and its rate of increase during early lactation.

In the absence of precious livestock biosensors, it is essential to evaluate the body condition score (Table 1) using the 5-point condition scoring system (scale 0–5, in 0.25-point increments) to control nutritional management on the farm [75, 76]. Calving in moderate condition (3–3.5) and maintaining feed intake during the periparturient transition period are critical factors in reducing NEBAL and avoiding metabolic and reproductive disorders that are harmful to performance. Different levels of body condition score changes (ΔBCS) on fertility, milk yield, and survival of Holstein-Frieasian cows diagnosed with reproductive disorders (dystocia, twins, retained fetal membranes, metritis, and clinical endometritis), and other health disorders (subclinical ketosis, left displaced abomasum, lameness, clinical mastitis, and respiratory disease) between Days 5 ± 3 and Day 40 ± 3 after calving were examined in an extensive dataset involving almost 12 thousands dairy cows. It turned out that there were no significant interactions between body condition score changes and different health-related events. At the same time, excessive loss of BCS and reproductive diseases decreased reproductive performance and survival compared with other ΔBCS categories and health groups. It is essential to mention that excessive loss of BCS during early postpartum was characterized as having a higher milk yield [77].

Body region	BCS
	2.00	2.25	2.50	2.75	3.00	3.25	3.50	3.75	4.00	4.25	4.50	4.75	5.00
Thurl	"V” in appearance					"U” in appearance					flat		rounded
Ileal tuberosity	Angular				Rounded							Just visible	Not visible
Ischial tuberosity	Angular		Fat pad palpable	Rounded							Not visible
Transverse processes of lumbar vertebrae	>0.5 visible	0.25 to 0.50 visible				0.10 to 0.25 visible		Only tips visible		Tips not visible
Coccygeal ligament	Visible						Just visible	Not visible
Sacral ligament	Visible							Just visible	Not visible

Table 1.

The decision chart for body condition score (BCS) suggested by Ferguson et al. [75].

Since body condition score is a strong predictor of subcutaneous fat reserves but, to a lesser degree, of skeletal muscle reserves, in periparturient dairy cows, a more precise evaluation of those reserves can be reached by separate ultrasonic examinations [78]. According to Schröder and Staufenbiel [79], backfat measurements can be done by placing a linear array transducer “lightly on the sacral area, vertically on an imaginary line connecting the pin (tuber ischii) and the hook (tuber coxa), at the point corresponding to the cranial end of the first coccygeal vertebra. The backfat measurements always include the skin thickness, and the profound fascia can be used as a landmark to distinguish backfat from the gluteal muscle.” According to van der Drift et al. [80], “longissimus dorsi muscle thickness measurements can be done by placing linear-array transducer perpendicularly to the vertebral column on the transverse process of the fourth lumbar vertebra, at the site of the larger diameter of the muscle between the fasciae corresponding to the lateral edge of the multifidus dorsi muscle.” The examination sites must be brushed to remove debris but not clip, and ultrasound gel must be applied to couple the probe surface with the skin [78]. Quantifying dairy cow body morphological traits by automatically processing images taken in a 3-D single-camera vision system makes it possible to predict body weight in dairy cows automatically. However, this model is unsuitable for monitoring short-term body weight variation or detecting anomalies in a cow’s health status [81]. According to a recent review, while current research shows promising results in dairy cattle, there are still many avenues to be explored for better automation of the whole-body weight estimation process [82].

Following parturition, regardless of NEBAL, a wave of follicular development begins 5–7 days after calving due to elevated plasma follicle-stimulating hormone (FSH). Three types of follicular development (Table 2) have been described and can be diagnosed in the field using ultrasonography [83]. The re-establishing pulsatile LH secretion can induce ovulation of a dominant follicle during early lactation [84]. Conversely, the developing NEBAL in early postpartum may suppress pulsatile luteinizing hormone (LH) secretions and reduce ovarian responsiveness to LH stimulation, thereby deterring ovulation. Non-ovulatory dominant or cystic follicles may prolong the interval for the first ovulation to 40–50 days after calving [84, 85]. It is important to mention that prolonged anovulatory anestrus may occur in 11–38% of dairy herds and can be associated with reduced fertility caused by NEBAL [86]. NEBAL can influence the timing of first postpartum ovulation, which negatively affects fertility [84, 87]. If a cow remains anovulatory for >50 days of lactation, it will be less likely to become pregnant during lactation and will be culled [88].

First dominant follicle (FDF)	FDF will ovulate 16–20 days after calving
	A turnover followes non-ovulation, and a new follicular wave will start
	FDF fails to ovulate and becomes cystic

Table 2.

Three types of follicular developments can be found immediately after calving [83].

Plasma progesterone (P4) concentrations generally rise during the first two or three postpartum ovulatory cycles [89, 90]. NEBAL may reduce or moderate the rate of increase in P4 [89, 90]. Meanwhile, the metabolic clearance of P4 in high-yielding dairy cows can be increased by high energy and protein intake. As P4 plays an essential role in conceptus development and growth, a slower increase in P4 after ovulation may decrease embryo growth by Day 16 and may cause early embryonic mortality [91, 92].

Early postpartum NEBAL may adversely impact the quality of oocytes during the first 80–100 days after calving, which exerts another carryover effect on fertility [93, 94]. However, it is not easy to reconcile the impact of NEBAL on follicles and oocytes with the impact of high dietary energy on oocyte quality and the development of blastocysts in dairy cows [95, 96]. Extremes in BCS may negatively influence fertility [84].

Metabolic, endocrine, and postpartum health statutes may influence together fertility in dairy cows. Energy imbalance seems to be one of the most important factors, though we should consider the complex interactions of the factors mentioned earlier to improve fertility in our dairy farm [84]. Similarly, BCS, glucose, NEFA, or Insulin-like growth factor 1 (IGF-I) concentration from calving to AI cannot explain the low fertility rate [97, 98]. In contrast, Saby-Chaban et al. [99] have found a significant correlation between the prevalence of biochemical ketosis (BHB >0.15 mmol/l) measured by in-line in milk and fertility.

To prevent metabolic disorders in the periparturient period, such as milk fever, ketosis, fat cow syndrome, or rumen acidosis is essential to provide challenge fed during the dry-off period and early lactation. These diseases can increase the prevalence of reproductive disorders and reduce reproductive performance. Therefore, prevention is preferable to treatment and requires close attention to nutrition and management. Treatment of different metabolic diseases (hypocalcemia and ketosis) has been reviewed recently by Oetzel [100] and Mann et al. [101]. In addition, maintaining good body condition at calving and providing a high-density energy diet that does not produce a fatty liver in early lactation are also essential in minimizing the detrimental effects of NEBAL on the return of the estrous cycle after calving.

5. Accurate detection of estrus in dairy cow

Estrous detection rate may contribute to low fertility results because of the low detection rate [102]. Van Vliet and Van Eerdenburg [103] reported that cow factors might also be contributing to low detection rates. Due to the relatively small size of the average dairy herds in several European countries (<50 cows) and the year-round calving pattern, the chances of having more than one cow in estrus simultaneously are somewhat limited. In this way, they cannot stimulate the intensity and length of each other estruses [103]. Another point of concern is the short duration of estrus. A previous study [103] showed that a substantial number of animals (40%) showed estrous signs for less than 12 h. The mean duration of estrus was 13.7 h in their study, in which they observed the cows every 2 h for 30 min. Others found that the high-yielding dairy cows (46.4 ± 0.4 kg milk/day) had a shorter duration of estrus (6.2 ± 0.5 h vs. 10.9 ± 0.7 h), fewer standing events (6.3 ± 0.4 vs. 8.8 ± 0.6), and shorter standing time (21.7 ± 1.9 s vs. 28.2 ± 1.9 s) than lower-producing dairy cows (33.5 ± 0.3 kg milk/day) measured at the same conditions [104].

The short duration of estrus on modern dairy farms emphasizes the importance of correctly determining the optimum time for artificial insemination [105]. Simple observation of the herd in the morning before and after milking, at midday, and late in the evening for 30 min is greatly recommended to determine estrus accurately under usual management circumstances. The use of traditional aids such as tailhead markings with chalk, paint, or crayon (the pin bones and the tailhead are painted), pressure-sensitive mount detectors using a colored fluid that fills a container when pressure is applied (these devices are fixed with adhesive to the hair over the midline just in front of the tailhead), camera-based recognition system for pressure-sensitive devices [106], estrous detection strip (applied to the sacrum) with a reflective strip which can be detected with an overhead camera [107], and/or detector animals (vasectomized or surgically altered bulls or androgenized nonlactating cull cows, heifers, or freemartin heifers with chin-ball marking harness) [108] may contribute to detect estrus accurately in the field. The recent development of a pressure-sensitive device is that when a certain threshold on mounts is reached, a light is activated on the device. Different flashing light patterns can determine whether a cow is in suspect heat, standing heat, or when it is ideal for AI [108]. Recently developed activity meters (activity behavior is classified as lying, standing, walking, active, or inactive/resting/) such as leg bracelets, neck collars, or ear tags [48], and/or electronic pressure-sensitive mount detectors [109] may improve the accuracy of estrous detection. The combined use of monitoring of estrous behavior and one or more estrous detection aids may enhance its efficiency. Similarly, combined use of biosensor data of animal activity with in-line monitoring of milk yield, milk flow rate, milk temperature, and electrical conductivity of milk [110, 111], in-line progesterone measurement [112, 113], and/or ruminating time and eating time (eating bouts) may increase the accuracy of estrous detection in the farm [48].

It is essential to emphasize that when standing heat is used as a predictor for ovulation (26.4 ± 5.2 h), only a limited number of cows display standing heat (58%), especially when few animals are in estrus at the same time. The onset of mounting behavior shown in 90% of estruses is the best predictor for ovulation (30.0 ± 5.1 h); however, its limitation is that it cannot yet be assessed by estrous detection aids [114].

There are several other methods to detect estrus, for example, direct electronic sensing of the odors of estrous pheromones [115, 116], continuous measurement of vaginal temperature and conductivity [117], ventral tail base skin surface temperature [118], rumen reticular temperature [119], or external auditory canal temperature [120]; however, they need further developments before introducing them into the daily practice.

6. Determining the optimal time for artificial insemination

The mean duration of an estrous cycle in dairy cows is 21 days (between 18 and 26 days), and ovulation occurs 25–32 h after the onset of standing heat [108]. According to Roelofs et al. [114], the duration between the onset of estrus and ovulation is 29.3 ± 3.9 h, while the onset of the first standing estrus and the time of ovulation is 27.6 ± 5.4 h [121]. Since the chances for pregnancy after artificial insemination (AI) are much higher when ovulation occurs within the survival time of sperm [105], it is essential to inseminate the cow within 12 h after the onset of estrus, namely during the last half of standing heat; therefore, the a.m./p.m. rule was developed as a guide for AI. This guideline recommends that cows observed in estrus in the morning should be inseminated in the afternoon, and cows observed in estrus during the afternoon should be inseminated the following morning [105]. In contrast, if we inseminate the cows at a similar time each day or employ an inseminator service, there is no need to follow the a.m./p.m. rule if heat detection is accurate, the insemination technique is good, and semen fertility is high on the farm [122, 123, 124].

The optimal time for artificial insemination (AI) after the onset of increased activity measured by pedometers is between 5 and 17 h [114], while according to Maatje et al. [125] and Yoshioka et al. [126], it is between 6–17 h and 10–18 h, respectively. When neck-mounted collars were used to detect estrus, the highest pregnancy rates were reached for primiparous and multiparous cows when they were inseminated between 13–16 h and 9–12 h after the onset of estrus, respectively [127]. At the same time, this difference between primiparous and multiparous cows could not be confirmed by Roelofs et al. [114]. When pressure sensing devices were used to detect estrus, the optimal time for AI felt between 4–12 h [109] and 12–18 h [128] after the onset of estrus, respectively. In comparison, artificial insemination had proven to be the most effective when cows were inseminated at 12 h after the onset of estrus [129].

According to Van Eerdenburg et al. [130], cows (n = 100) were detected with a scoring system in estrus. Of these animals, 50% showed standing heat (58% reported by Roelofs et al. [131]), and only 64 of the 100 cows achieving a score of >50 were presented for insemination; 98% did indeed ovulate. The other 36 were <45-day postpartum and were not inseminated [132]. The milk yield, parity, follicular size, and ovulation time were not correlated with the estrous behavior score. The animals that ovulated 0–24 h after the first ultrasonographic examination scored more than twice the number of points (188 versus 65 points) as those that ovulated 24–48 h after the first scan (P = 0.045). If ovulation occurred >48 h after AI, only 15% of the cows became pregnant (Figure 2). Cows that did not show overt signs of estrus and thus scored <100 points in the scoring system had a high chance of ovulating after 24 h and should therefore be inseminated again or given GnRH (or agonist) at the time of insemination [132].

Figure 2.

Pregnancy rates at Day 28 concerning ovulation time after AI. Ovulation time <0 indicates that the cow had ovulated before the initial ultrasonographic examination adapted from [132].

Ultrasonography can also detect ovulation, since it is characterized by the abrupt disappearance of the large ovulatory follicle [132, 133]. The duration between the onset of the increased number of steps and ovulation can be 29.3 ± 3.9 h. In contrast, the period between the end of the increased number of steps and ovulation is 19.4 ± 4.4 h, measured by a pedometer. Pedometers may detect estrus accurately (83%) and appear to be a promising tool for predicting ovulation in dairy cows [131], while monitoring P4 alone is not sufficient to predict ovulation [134].

A progesterone (P4) assay of plasma or milk as an indication of true estrus clearly demonstrated that 7–22% of cows showing estrus had abnormal levels of P4 at the time of AI [135]. Bulman and Lamming [136] found that 15% of cows were inseminated during inappropriate stages of the follicular phase. However, a further 15% were inseminated during the luteal phase, while according to O’Connor [137] up to 15% of the cattle presented for insemination are really not in heat. When such cows are inseminated, they do not conceive, or it leads to abortion if they have been pregnant [135]. The number of artificial inseminations performed at the wrong time in the practice can be reduced by performing ultrasonographic examination [132, 133] or by using different diagnostic kits such as on-farm milk progesterone tests [138], in-line progesterone measurements [113, 139], or on-farm heat detection kits for detecting lactoferrin in cervical mucus [140] or by measuring the electrical resistance of vaginal fluid [137].

To eliminate the requirement for estrous detection and to optimize the timing of insemination relative to ovulation, different fixed timed artificial insemination (TAI) protocols were introduced into daily practice (Table 3). The TAI protocols may provide similar pregnancy rates per AI when compared with those of classical reproductive management systems, based on estrous detection and hormonal therapy when necessary. However, before selecting any protocol, it is always very important to compare the results with the traditional methods used on the dairy farm. When estrous detection on the farm is good, PGF_2α treatment and AI at the observed estrus are recommended [155], while when estrous detection is poor, TAI protocols may be recommended. Recently published reviews [156, 157, 158, 159, 160, 161] and meta-analyses [162, 163, 164] may contribute to selecting from an economic and management point of view the most suitable and most effective TAI protocol(s) for our dairy farm.

Protocol names	Treatments	TAI	References
OvSynch	Day 0: GnRH, Day 7: PGF, Day 9: GnRH	Day 10	Pursley et al. [141]
Modified OvSynch-1	Day 0 a.m.: GnRH, Day 7 a.m.: PGF, Day 9 p.m. (30–36 h after PGF): GnRH	Day 10 a.m. (16–20 h after GnRH)	Pursley et al. [142]
Modified Ovsynch-2	Day 0 a.m.: GnRH, Day 7 a.m.: PGF, Day 8 a.m.: PGF, Day 9 p.m. (30–36 h after PGF): GnRH	Day 10 a.m. (13–16 h after GnRH)	Rheinberger et al. [143]
Shortened Ovsynch	Day 0: PGF, Day 2: GnRH	Day 3; (16–20 h after GnRH)	Stevenson et al. [144]
Double-Ovsynch	Day 0 a.m.: GnRH, Day 7 a.m.: PGF, Day 10 a.m.: GnRH, Day 17 a.m.: GnRH, Day 24 a.m.: PGF, Day 26 p.m.: GnRH	Day 27 a.m.	Ribeiro et al. [145]
Cosynch	Day 0 a.m.: GnRH, Day 7 a.m.: PGF	Day 9 p.m.; +GnRH	Geary et al. [146]
Presynch-14 − Ovsynch 10	Day 0: PGF, Day 14: PGF, Day 24: GnRH, Day 31: PGF, Day 33: GnRH	Day 34	Stevenson et al. [147]
Presynch-14 − Ovsynch-12	Day 0 p.m.: PGF, Day 14 p.m.: PGF, Day 26 a.m.: GnRH, Day 33 a.m.: PGF, Day 35 p.m.: GnRH	Day 36 a.m.	Martínez et al. [148]
Presynch-14 − Ovsynch 14	Day 0 p.m.: PGF, Day 14: PGF, Day 28: GnRH, Day 35 a.m.: PGF, Day 37 p.m.: GnRH (56 h after PGF)	Day 38 a.m.; (16–20 h after GnRH)	Giordano et al. [149]
G-4-G − Ovsynch	Day 0: PGF, Day 2: GnRH, Day 6: GnRH, Day 13: PGF, Day 15: GnRH	Day 16; (16 h after GnRH)	Bello et al. [150]
G-5-G − Ovsynch	Day 0: PGF, Day 2: GnRH, Day 7: GnRH, Day 14: PGF, Day 16: GnRH	Day 17; (16 h after GnRH)
G-6-G − Ovsynch	Day 0: PGF, Day 2: GnRH, Day 8: GnRH, Day 15: PGF, Day 17: GnRH	Day 18; (16 h after GnRH)
Modified G-6-G – Ovsynch	Day 0: PGF, Day 2: GnRH, Day 8: GnRH, Day 15: PGF, Day 17 p.m. (56 h after PGF): GnRH	Day 18 a.m.; (16 h after GnRH)	Pursley and Martins [151]
PG-3-G − Ovsynch	Day 0: PGF, Day 3: GnRH, Day 10: GnRH, Day 17: PGF, Day 19: GnRH	Day 20	Stevenson et al. [147]
Ovsynch + PD: progesterone device (CIDR/PRID)	Day 0: GnRH + PD, Day 7: PGF − PD, Day 9: GnRH	Day 10; (16–20 h after GnRH)	El-Zarkouny et al. [152]
Cosynch-5 + PD	Day 0: GnRH + PD, Day 5: PGF − PD, +6–8 h: PGF	Day 8; +GnRH	Santos et al. [153]
Modified Cosynch-5 + PD	Day 0: PD, Day 5: PGF − PD	Day 8; +GnRH	Colazo and Amdrose [154]

Table 3.

Fixed-timed artificial insemination (TAI) protocols.

7. Accurate diagnosis of pregnancy and pregnancy loss in dairy cow

Pregnancy diagnosis plays an essential role in decreasing days open on dairy farms. Therefore, it is essential to select an accurate method for diagnosing early pregnancy and pregnancy loss (late embryonic and early fetal mortality) because the cost of each day open 100 days after calving may reach $4.00 [165] or €2.5–6.5 [166], respectively. Besides traditional pregnancy diagnosis (rectal palpation of the uterus or progesterone tests) [167, 168, 169], there are several new possibilities to diagnose early pregnancy on dairy farms. However, before introducing any new diagnostic test on our dairy farm, we must evaluate the accuracy of that particular test. Their results must be confirmed by the old diagnostic method to decrease the adverse effects of false-negative diagnoses. This can be caused by prostaglandin treatment to reduce the interval to the next AI service [167] or by using new resynchronization protocols in our dairy farm [170, 171, 172].

One of the most recent techniques for diagnosing early pregnancy on the dairy farm is B-mode ultrasonography. Under field conditions, ultrasonography may achieve accurate results from Days 25 to 30 after AI [173, 174, 175]. However, the accuracy of the transrectal ultrasonographic diagnoses greatly depends on the frequency of the transducer used, the surgeon’s skill, the criterion used for a positive pregnancy diagnosis, and the position of the uterus in the pelvic inlet [176]. For example, if during ulrasonographic examinations performed between Days 24 and 38 after AI, we can find a uterus far cranial to the pelvic inlet compared with those cases when the uterus is located within or close to the pelvic inlet, we can make more incorrect nonpregnancy diagnoses [177].

Nonpregnant animals can be selected accurately by evaluating blood flow in the corpus luteum around Day 20 after AI, meaning we can substantially improve the reproductive efficiency of our herd [169].

Pregnancy protein RIA assays such as pregnancy-specific protein B/PSPB/, pregnancy-associated glycoprotein/PAG/, and PSP60, commercial ELISA, or rapid visual ELISA tests may provide an alternative method to ultrasonography for determining early pregnancy and pregnancy loss in dairy cows. However, the relatively long half-life after calving and pregnancy loss may limit the effectiveness of these laboratory methods for early pregnancy diagnosis in the field, especially when compared with a direct method such as transrectal ultrasonography [176]. Linear array/sector B-mode [178] and Doppler ultrasonography [169] may exceed the other diagnostic methods in the amount of information collected from each animal during scanning. However, their accuracies greatly depend on the operator’s proficiency and availability [178].

A new technology (in-line milk analysis system) has already made the automatic collection of milk samples at milking robots or in the milking parlor to analyze progesterone to detect early pregnancy and pregnancy loss, respectively [113, 179, 180]. Bruinjé and Ambrose [113] reported high sensitivity (>95%) from Day 27 after AI, while the specificity was somewhat lower before Day 40 after AI. Any new biomarkers discovered for early pregnancy diagnosis may make it possible to diagnose pregnancy loss much earlier, which may significantly 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 nonpregnant animals on time 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 [181].

Although fertilization in the cow can be detected by measuring the early pregnancy factor with the rosette inhibition test, it is not a practical method; therefore, it needs further development. Recently found biomarkers such as interferon-tau-stimulated genes or microRNAs may help us diagnose early pregnancy in dairy cows; however, these tests need further development before their general use in the dairy practice [176].

8. Conclusion

Besides providing high-quality food for milk production and preventing metabolic disorders, it is essential to pay special attention to the first 100 days after calving. This is because the aim of our activity during the transition period is to prevent calving complications and uterine diseases and, if it is not possible, to treat them accordingly as soon as possible. Furthermore, it is essential to emphasize that if we would like to select a new diagnostic method or treatment protocol, it is always necessary to compare the results with the previously used test or treatment protocol.

Conflict of interest

The authors declare no conflict of interest.