Anatomy of placenta in dairy cows
The surface of the endometrium in a pregnant cow has four rows of specialized endometrial regions known as caruncles along the long axis of both horns, with a total of 60 to 80 convex and ovoid structures (Duello et al., 1986; Kohan-ghadr, 2011) .
Hydrostatic pressure of the allantoic and trophoblastic membrane against the uterine wall is believed to facilitate chorioallantoic attachment to the caruncle and stimulate the fetal chorion to vascularise and hypertrophy. As the cotyledons mature, the remodeling of the endometrium that leads to the development of the caruncles is necessary to accommodate the specialized folding of the chorioallantois. The surface of contact between maternal and fetal tissues is increased by the development of outgrowths on the surface of chorion, known as the villi. These chorionic villi consist of vascular mesenchymal cones surrounded by cuboidal trophoblastic and giant binucleate cells bring the fetal (allantoic) vessels into proximity with the maternal blood vessels. The temporal and spatial changes of the extracellular matrix (ECM) during apposition, adhesion and attachment increase the complexity of placental formation and implantation process (Kohan-ghadr, 2011; Santos et al., 2017) .
Placentomes:
Placentomes appear as smooth, flattened, and semicircular domed elevations. At first, they are detected only in the area immediately surrounding the embryo. By Day 60, they are present in the uterine horn, both proximal and distal to the embryo, although those near the embryo are larger. Placentomes are not macroscopically observable until approximately Day 37. Initially, from Days 37 to 40, a maximum of 20 placentomes are present on the chorionic surface in the embryo. From Days 40 to 50, the number of placentomes triples. Subsequently, the number increases gradually up to Day 70, averaging approximately 80 to 90 (range, 20–120), based on the number of cotyledons observed on the chorioallantoic surface (Neto et al., 2010., Adeyinka, 2012; Lemley and Camacho, 2021).
The placentomes are linked together by areas of flat apposition between trophoblast and glandular uterine epithelium (interplacentomal areas). Gland openings are covered by domes of phagocytic trophoblast, which facilitate histotrophic uptake of endometrial gland secretions by small endocytotic vesicles or much larger phagocytic vesicles. The trophectoderm is remarkably versatile, with great capacity for invasion, cell fusion, hormone production, specific nutrient absorption, selective transport, active metabolism, and finally for its ability to resist maternal immunological attack. This epithelium, together with internal membranes and blood vessels, form the chorioallantoic placental structures from Day 30 to term (Peter, 2013) .
The sizes of the placentomes vary depending on location. For example, placentomes closer to the middle of the uterine horn containing the fetus and nearest to the attachment of the middle uterine artery are the largest (Roberts, 2004., Blankenvoorde, 2011; Redifer et al., 2021).
Placentomes are larger and more numerous closer to the fetus and in the horn containing the fetus relative to those located in the uterine extremities and those found in the contralateral horn. Total placentome weight and length increase until Day 190, attaining 13 approximately 4.5 kg and 10 to 12 cm (Kohan-ghadr, 2011; Schlafer et al., 2000) with most acquiring a mushroom-like shape with an occasional flat configuration (Miles et al., 2004).
The total number of placentomes does not correlate with the increased fetal nutritional demands of late pregnancy suggesting that there is an alteration in the pattern of vasculature to increase feto/maternal exchange without an increase in placentome number (Laven and Peters, 2001; Leiser et al., 1997) .
Fetal membrane
The placenta is literally a "sac" covering and allowing the fetus to be nourished and developed until the process of parturition. The placenta is considered an essential organ for the prenatal transfer of nutrients and oxygen from the dam to the fetus. The other functions of the placenta are to provide a reservoir of blood for the fetus (Elmetwally and Bollwein, 2017; Gohar et al., 2018). Nutritional delivery to the fetus is determined by several essential parameters, including placental growth and development, utero-placental blood flow, nutrient availability, and placental metabolism and transport capacity (Dunlap et al., 2015). Previous research from Dr. Fuller W. Bazer's lab and others has highlighted the importance of amino acids and their metabolites in normal fetal growth and development, as well as the importance of amino acid transporters in nutrient delivery to the fetus (Elmetwally et al., 2022) .
During the first stages of pregnancy, the placenta secretes amino acids. It is well known that amino acids are essential for the conceptus's (the embryo/fetus and related placental membranes) growth (Elmetwally et al., 2018; Lenis et al., 2018). Amino acids are essential for the production of tissue proteins and also act as cell signalling molecules, hormone secretion regulators, antioxidants, and hormone secretion regulators (Wu et al., 2013). Biologically significant non-protein molecules such as nitric oxide, polyamines, neurotransmitters, amino sugars, purine and pyrimidine nucleotides, creatine, carnitine, porphyrins, melatonin, melanin, and sphingolipids are all synthesised using amino acids as key building blocks (Wu, 2009).
The main hormones produced by the placenta are human chorionic gonadotropin, equine chorionic gonadotropin, lactogenic hormone, progesterone, and estrogen hormone. When the fetus is born, the placenta normally attaches within a short time and is expelled. That is why it is referred to as "afterbirth”. (Ball and Peters, 2004; Napso et al., 2018).
The drop of fetal membranes postpartum is a physiological process that involves loss of feto-maternal adherence, combined with contraction of the uterine musculature, and is usually accomplished within 6 hours of calving. (El-Malky et al., 2010 and Gaafar et al., 2010).
During pregnancy, nitric oxide (NO), a byproduct of arginine catabolism and secreted by the trophectoderm cells, is essential for controlling placental angiogenesis and fetal-placental blood flow (Chen and Zheng, 2014; L P Reynolds et al., 2005). polyamines (polycationic compounds made from ornithine) was ptoved to control cell division, differentiation, and function as well as controlling DNA and protein synthesis, ion channel activity, signal transmission, and gene expression, (Wu et al., 2005). It should be highlighted that a thorough collection of studies looking at the function of specific nutrients and their transporters during the early peri-implantation period in the ruminant conceptus' development have been reported (Berkane et al., 2017; Raguema et al., 2020; Reynolds et al., 2005).
Retained placenta in Holstein dairy cows
Retained placenta is a pathological condition defined as failure to expel fetal membranes within 24 hr after parturition (Dervishi et al., 2016). Retained placenta is one of the most common puerperal disorders affecting the reproductive performance of dairy cows (LeBlanc, 2008; Mahnani et al., 2021b). The incidence varies from 4.0-16.1% but can be much higher in problem herds (Mahnani et al., 2021a) Abortion, stillbirth, and twin calving resulted in increased incidence rates to 25.6, 16.4, and 43.8%, respectively (Tucho and Ahmed, 2017).
Fetal membrane retention and metritis are linked to substantial immunological and metabolic alterations that predispose cows to periparturient illnesses. Kimura et al. (1999) revealed that parturition-related colostrogenesis/lactogenesis impacted CD62L expression prepartum, leukocyte count postpartum and neutrophil killing activity postpartum (Kimura et al., 1999). Cows with a negative energy balance during early lactation exhibit lower blood polymorphonuclear leukocyte (PMNL) phagocytosis, chemotaxis, and diapedesis than cows with a positive energy balance by mid-lactation (Stevens et al., 2011). As a result, it is not unexpected that roughly 60% of viral illnesses affecting dairy cows during lactation are observed in the first 30 days of lactation (Pinedo et al., 2020).
Retained placentae typically lead the cow to miss the next pregnancy by 2–6 months, have a later calving date the following year, and may result in an open cow the following year. The prevalence of retained placenta in dairy cows is related to breed, age, parity, and body condition score (Newby et al., 2014).
Risk factors affecting the retained placentae in dairy cows:
Metritis
Metritis is considered a symptom of a poorly functioning immune system during the transition period (Goff, 2008), and it predisposes to an increase in the incidence of RFM. Shortly after parturition, foetal membranes become a "foreign body," and their ejection is dependent on the maternal immune system detecting and destroying them (Gunnink, 1984). Cotyledons from RFM cows were less leukocyte chemoattractant than cotyledons from cows that ejected the placenta regularly after calving (Gunnink, 1984). Furthermore, in vitro tests revealed that leukocytes from cows with RFM were less able to detect cotyledon tissue in a chemotaxis experiment before, during, and after calving than leukocytes from cows without RFM (Gunnink, 1984).
From the prepartum period until 1–2 weeks after calving, polymorphonuclear leukocytes PMNL from cows with RFM have diminished reactivity to chemoattractants and lower phagocytic capability (Cai et al., 1994; Kimura et al., 2002). Delayed foetal membrane evacuation exposes the uterus to environmental infections and pollution as well as increasing the risk of metritis. Furthermore, the majority of cows have postpartum uterine contamination, (LeBlanc, 2010), and macrophages and PMNL serve as the first line of defense against this contamination, moving from the bloodstream to the uterus. The immune system is unable to quickly clear bacterial contamination in immunocompromised cows, and bacterial proliferation in the uterus causes metritis (LeBlanc, 2010) .
Metritis is distinguished by a foul-smelling, red-brown watery discharge from the uterus within the first 21 days following calving, which may or may not be followed by systemic symptoms (i.e. pyrexia, anorexia, depression). Hammon et al., (2006) found that PMNL function was decreased during the periparturient phase in cows with puerperal metritis and subclinical endometritis compared to healthy cows.
During the last few decades, many hypotheses have been proposed and a great number of studies have been conducted to understand the etiological factors and pathobiology of RP. In addition, several blood biomarkers have been proposed as indicators of the risk of RP in dairy cows (Dervishi et al., 2016) .Retention of the placenta (ROP) denotes failure of the fetal villi to separate from the maternal crypts i.e. the lack of placental dehiscence. (Tucho and Ahmed, 2017).
The effects of infectious diseases, hormonal imbalance, and inflammatory status on the incidence of retained placenta in cows:
Infectious disease plays an important role as a causative agent for retained placenta in dairy cows. In order to preserve the placenta, four primary ideas have been offered throughout the years: uterine atony, chorionic villi edema, inflammatory conditions, and neutrophil inactivation (Elmetwally, 2018; Lai et al., 2022). It was proposed that the decrease in neutrophil functions prior to parturition supported the latter idea (Kimura et al., 2002; Mordak and Stewart, 2015). Later, LeBlanc (2008) and Wagner et al. (2017) proposed that RP, metritis, and endometritis are illnesses of immune function that begin at least 2 weeks before birth (LeBlanc, 2008; Wagener et al., 2017). Additionally, Ametaj et al., (2010) postulated that endotoxin may be involved in all four states by decreasing uterine atony, generating chorionic villi edema, commencing the inflammatory state, and promoting neutrophilia due to the neutrophil's inability to migrate to inflammatory tissues (Ametaj et al., 2010).
Pathogenic bacteria that induce localized tissue damage and inflammation are present in the uterus after calving, which is a key characteristic of uterine disorders. The bacteria Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella melaninogenica are frequently cultured from cows with uterine illness (Sheldon et al., 2020).
Infectious causes are associated with Brucellosis, Salmonellosis, Leptospirosis, and Listeriosis. Such retention creates a number of problems by allowing microorganisms to grow inside the uterus, causing inflammation, fever, weight loss, decreased milk yield, longer calving intervals, and possibly an open cow during the next year; if the infection is so bad, the animal may actually die (Han and Kim, 2005).
Steroids are important in pregnancy maintenance and parturition because they regulate prostaglandin production. Progesterone promotes the production of prostaglandin E2 (PGE2), and estrogens raise the levels of prostaglandin F2 (PGF2) in the uterus (Wango et al., 1992; Wooding et al., 1996). Although progesterone levels are higher in animals with placental retention (Rasmussen et al., 1996), estrogen levels are lower (Beagley et al., 2010). Of note, cows with retained fetal membranes had higher cortisol and lower estrogen levels in late pregnancy, which affect immune function by exerting local and systemic immunosuppressive effects. Uncomplicated fetal membrane retention is unattractive and difficult for animal workers and milkers, but it is often not damaging to the cow (Hartmann et al., 2013; Kindahl et al., 2002).
The effect of management, nutrition, and genetic traits on the incidence of retained placenta in cows:
Managemental causes of retained placentas include stress, genetic traits, inbreeding, and obesity (Joosten et al., 1991). Lack of exercise and hypocalcemia are the most frequent causes of decreased myometrial contractility. Stress (Transportation, rough handling, poor feed conditions, isolation from group, lameness,) results in elevated corticosteroids and increased risk of placental retention. Dairy producers have suggested that poor health management in herds can predispose animals to the retention of the placenta (Fricke, 2001).
In addition to this deficiency of antioxidants, vitamin E and selenium may decrease chemotaxis and leukocyte numbers at the fetomaternal junction, thus contributing to the retention of fetal membranes (Amin and Hussein, 2022; Bourne et al., 2007). Over-condition and under the condition as well as managemental defects and environmental factors can result in the retention of the placenta (Hayirli et al., 2002).
Retained placenta incidence would be reduced through dietary management of older cows for optimum body condition and minimal incidences of milk fever. The most crucial management factors for preventing retained placenta in heifers are proper growth rates that cause heifers to calve at 600 kg and the selection of calving ease sires (Barański et al., 2021; Mahnani et al., 2021a).
Nutritional causes of RP are primarily due to the deficiency of feed during the last 6 to 8 weeks before calving, especially when there is a deficiency in the content of minerals and vitamins in the diet. (Alšić et al., 2008; Spears and Weiss, 2008)
Heavy grain feeding may be associated with both higher milk production and an increased risk of reproductive disorders such as dystocia, retained placenta, cystic ovaries, and metritis. (Jorritsma et al., 2003). Vitamin and mineral deficiency conditions such as selenium, vitamin E and vitamin A, β-carotene, and a disturbed Ca/P ratio can impair general immunity, alter the competence of the cellular self-defense mechanism, and increase the risk for placental retention and metritis (Ahmed et al., 2009). High milking cows with a greater degree of negative energy balance prepartum and higher NEFA concentrations were more likely to suffer from RP (LeBlanc, 2010).
On the other hand, over-conditioned cows were shown to be more sensitive to a retained placenta and subsequent infertility than cows with normal body condition scores. (Madushanka and Ranasingha, 2016; Markusfeld et al., 1997).
No significant differences were found in the incidence of the retained placenta during green (winter) and dry (summer) feeding (26.20% vs. 22.90%), respectively. Also, Deyab, (2000.) and Gabr, et al., (2005) found non-significant differences between feeding systems in the percentage of retained placenta.
In regards to the genetic traits, it was proven in the past, a concentration on production traits resulted in declining trends in health, reproduction, and longevity traits, which can lead to a decrease in herd profitability (Fleming et al., 2019, Erasmus and van Marle-Köster, 2021; Luo et al., 2021). To minimize the harmful impact of years of choosing primarily productivity and conformation traits, several countries began to integrate health traits into their breeding objectives (Egger-Danner et al., 2015).In Egypt, the dairy herd health monitoring system has tracked fertility-related diseases and reprodutive abnormalities, and RP was first incorporated with other health features (Hamed and Kamel, 2021).
Cow’s Body Weight, Calves’ Birth Weight:
The percentage of retained placenta increases significantly with increasing live body weight in cows due to the increment in fat adipose tissues (Madushanka and Ranasingha, 2016; Markusfeld et al., 1997), which may result in trapping the steroid sex hormones. With increasing fetal birth weight, a major rise in retained placental issues occurs. (Gaafar et al., 2010). The cause may be due to fetal pressure on the placenta and fetal membrane. This increases the incidence of placental retention to reinforce the bond between the cotyledons and the fetal membrane.
Estrone sulphate content did not significantly correlate with gestational length, calf birth weight, placenta weight, or newborn calves' survivability. These findings suggest that variations in plasma estrone sulphate content between Japanese beef cattle and Holstein dairy cattle are very similar. Estrone sulfate's plasma concentration is related to the breed of the pregnant cow, and depending on the breed of bull, it is also influenced by calf birth weight. In Japanese beef cattle, it appears to be possible to predict the incidence of retained placenta but not the calf birth weight or survivability of newborn calves (Isobe et al., 2003).
Twining and Sex of calves:
It suggested that with twinning birth, the retained placenta incidence (37.9% vs. 24.20 in Friesian cows was higher than with singleton %) (Gaafar et al., 2010). Increased management of twin-producing dams and their calves during the calving season is required in order to achieve increased productivity with twinning in cattle due to the shorter gestation period and the increased incidence of retained placenta and/or dystocia. The periparturient identification of twin pregnancies can enable obstetrical help to allow delivery of twin calves and to increase newborn survival (Echternkamp and Gregory, 1999).
In Friesian cows, the percentage of retained placenta was insignificantly higher with born male rather than female calves (26.50 vs. 23.20) respectively; the slight increase in the percentage of retained placenta observed with born male calves may mean that the fetal androgenic hormone from the fetal testes may have partially affected the placenta retention process. (Gaafar et al., 2010).
Parity, body condition scores and seasons:
Parity had a major impact on the occurrence of RFM milk for cow, 5th parity were reported as the highest occurrences of retained placenta (Islam et al., 2012). Furthermore, it was proven that calf birth weight has a positive correlation with placental weight, (Echternkamp and Gregory, 1999).
On the other hand, dystocia and stillbirth in primiparous cows raised the odds ratios (OR) of RP by 4.30 and 3.33 times, respectively. Dystocia, twinning, and stillbirth in multiparous cows raised the OR of RP by 4.36, 3.94, and 1.29 times, respectively (Mahnani et al., 2021a).
The state of the body had a major influence on the incidence of RFM following the birth of milk cows. In fair, good, and bad conditions, the incidence of retained placenta in cows is 7.1%, 3.2%, and 3.1%, respectively. However Sarder et al., (2010) reported that in good health, the prevalence of retained placenta was higher and lower in fair and other cows in bad condition, respectively. These variations may be due to several risk factors, such as uterine contractility, last type of calving, pregnancy body condition, postpartum estrus period of onset, stillbirth, twin birth, calving month and season, hereditary, gestation duration, dietary deficiency, difficult delivery, age and parity of the cow, and other uterine infections that predispose the cows to retention after birth, carotene, vitamin A, progesterone, and estrogen imbalance at parturition. (Sarder et al., 2010)
Researchers have found that in fall partiurtions, the incidenec of retained incidence is less than 40% relative to spring (Binabaj et al., 2014; Echternkamp and Gregory, 1999). The occurrence of retained placenta was lower in the cold months of the year than in the warm months. (DuBois and Williams, 1980; Mahnani et al., 2021a). In contrast, Ghavi Hossein-Zadeh and Ardalan, (2011) and Wetherill, (1965) reported that the incidence of retained placenta in partiurtions was lower in the summer in comparison to the winter and spring.
Birth comparisons were made between the weights of calves born during cold and warm times. Calves born in cold periods have a higher birth weight relative to calves born in warm periods (1.12 kg) (Binabaj et al., 2014). Moreover, heavier birth weights of calves born during the cold period suggest that they have a larger, more physiologically distinguished, and mature placenta, resulting in decreased placenta retention. (Echternkamp and Gregory, 1999).
The effects of gestational length on the incidence of retained placenta in dairy cows:
The increased risk of placenta physiological immaturity at a delivery time is due to the shortening of the gestation period because the relations of cotyledon and caruncle do not fully separate and part of the placenta remains in the genital period.
It was reported that the gestation period in cows with retained placenta was 3.3 to 5.25 days shorter than in cows without retained placenta (Muller and Owens, 1974., DuBois and Williams, 1980). A gestation period shorter than 274 days caused a doubling of the incidence of retention rates (Mahnani et al., 2021a; Vieira-Neto et al., 2021). However it is often noticed that the length of the gestation period does not affect the incidence of retained placenta (Kok et al., 2021)
Induced parturition had a higher incidence of placenta retention than spontaneous parturition (21.0 vs 0.0%), but there was no difference between cows treated with prostaglandin F2α or fenprostalene (19.2 vs 22.6%). By administering prostaglandin F2α or fenprostalene together with dexamethasone, an acceptable level of parturition synchrony was achieved (Königsson and Gustafsson, 2002; Wischral et al., 2001).
Different Concepts Of Placental Separation In Dairy Cows:
Fetal membranes are normally expelled within two to eight hours of parturition. Pathological retention of fetal membranes beyond 12 hours can be considered (Hooshmandabbasi et al., 2018; Kindahl et al., 2004). Usually, the uterus contracts approximately 14 times/hour immediately after parturition, but at 42 hours, the frequency steadily decreases to one per hour. Delayed involution of the uterus is normally connected with membrane retention (Hartmann et al., 2013; Wiebe et al., 2021).
Maternal immunological recognition by trophoblast cells of fetal MHC class I proteins triggers an immune/inflammatory response that leads to placental separation during parturition. According to Tucho and Ahmed, (2017), normal separation and placenta delivery processes are multifactorial and begin before parturition. (Fig. 2).
The maturation of the placenta plays an important role in the detachment loosing and/or detachment process of the placenta in dairy cows .The decreased number of binucleate trophoblast giant cells in the fetal chorion is an important feature that facilitates the separation of the placenta (Fig. 2, Seo et al., 2019).
Significant differences between RFM cows and cows that shed their fetal membranes on time have been identified in terms of serum/plasma cholesterol, urea nitrogen, glucose, total protein, non-esterified fatty acids, and -hydroxybutyric acid (Zhang et al., 2002).
Following a normal parturition, foetal membranes should be evacuated in less than 8 hours; hence, their retention for more than 8 to 12 hours is inappropriate (Amin and Hussein, 2022; Gohar et al., 2018). RFM frequently develops from abortions that happen during the second part of pregnancy, whether they are random or contagious. When compared to normal parturitions, hydrops, uterine torsion, twinning, and dystocia generally increase the incidence of RFM (Beagley et al., 2010; Szelényi et al., 2022). It is also made worse by heat stress and peripartum hypocalcemia. RFM should be expected in cows that have been induced to calve pharmacologically, such as through the administration of exogenous corticosteroids. Nutritional factors like carotene and selenium deficiency as well as over-conditioning of dry cows have been implicated (Garcia-Ispierto et al., 2022; Klisch and Schraner, 2021).
RFM, metritis, and miscarriage are all linked to low levels of vitamin A, which occur in hyperkeratosis and polybrominated biphenyl poisoning. Low selenium cattle may be more susceptible to RFM, metritis, and cystic ovaries in selenium-deficient locations. Vitamin E, which has been demonstrated to improve neutrophil function, may potentially be at play. Selenium levels in cattle fed stored diets from selenium-deficient areas should be regularly checked, and supplements should be given as needed. Vitamin E and selenium may be linked to RFM either through altered neutrophil activity or a simple lack (Chebel, 2021a; Moghimi-Kandelousi et al., 2020). After giving birth, cows with RFM may be more susceptible to developing the condition the following year. What's more, epidemiological research indicates that cows with RFM are more likely to have metabolic disorders, mastitis, metritis, and subsequent abortions (Amin and Hussein, 2022; Chebel, 2021b; Melendez et al., 2021). Therefore, related disorders pose a risk even if many cows with RFM remain asymptomatic with regard to their immediate uterine health. RFM-related neutrophil dysfunction in periparturient cows has been linked to the condition, which helps to explain why animals with the condition have decreased resistance to uterine and other infections (Mordak and Stewart, 2015; Moretti et al., 2016).
As demonstrated by the degenerative left shift in the leukogram seen in some septic metritis patients, cattle with acute metritis associated with RFM may also have a depletion of neutrophils in the peripheral blood as a result of acute recruitment of neutrophils to the infected uterus (McNaughton and Murray, 2009). Although most calves with RFM do not develop septic metritis or chronic endometritis, the need to treat RFM stems mostly from the inability to foresee which cows may experience clinically severe consequences (Moretti et al., 2015; Pathak et al., 2015).
The idea that RFM is mediated by reduced neutrophil function starting in the late dry period is supported by recent findings (Moretti et al., 2016). In cows that later develop RFM, decreased neutrophil migration into tissue extracts of placentomes can be seen as early as two weeks before calving. These cows are also less able to perform other neutrophil tasks, such as oxidative burst, which is a part of the neutrophils' bacterial killing action (McNaughton and Murray, 2009; Mordak and Stewart, 2015). In hypocalcemic cows, impaired neutrophil function has also been seen. In fact, a large number of the etiological factors linked to RFM, such as vitamin and mineral deficiencies, heat stress, or the use of exogenous corticosteroids, have also been linked to impaired neutrophil activity (Daros et al., 2020; Ehsanollah et al., 2021).
Molecular Changes Associated With Placental Dysfunction In Dairy Cows
It was hypothesized that the separation of the placenta depends mainly on the uterine and umbilical blood perfusion during the peri-parturient period (Elmetwally and Bollwein, 2017; Hartmann et al., 2013).
Intrauterine fetal growth depends mainly on placental function and blood perfusion. Vascular remodeling of the placenta plays an important role either during the gestational period and/or directly after parturition. Even while placental weight in ruminants reaches its peak by about midgestation, placental vascular beds continue to grow throughout pregnancy (Reynolds et al., 2005 a; Reynolds et al., 1985). Rates of uterine and umbilical blood flows, as well as fetal oxygen and nutrition uptakes, rise in tandem with the formation of placental vascular beds throughout gestation (Reynolds et al., 2005b). Despite the fact that angiogenesis is crucial to placental health, little is understood about how the placental blood supply develops. However, there is some evidence to suggest that the placenta's own production of vasoactive substances, such as prostaglandins and glycosaminoglycans, may be the source of the stimulation for the development of placental vascular beds (Reynolds et al., 2005b).
Angiogenesis in particular plays a crucial part in the correct progression of healing since the newly formed blood vessels provide nutrients and oxygen to the growing tissue (Raguema et al., 2020; Song et al., 2020). The secretion of angiogenic proteins from the placenta depends mainly on the NO, INFT (interferon tau), and polyamines secreted by the placenta (trophectoderm cells) (Wu, 2013). The angiogenic response has been attributed in part to several angiogenic regulators, such as growth factors and chemokines, which can also be released by mesenchymal stromal cells (MSCs), normal T cell expressed and presumably secreted (RANTES), and VEGF in fibroblasts and macrophages (Porwal et al., 2021). RANTES, a chemokine for monocytes and activated T cells, is actively synthesized by stromal cells derived from normal endometrium and endometriosis implants. (Khorram et al., 1993).
The increase in RANTES gene expression observed in normal endometrium during the ovulatory cycle was less than that noted for endometrial vascular endothelial growth factor mRNA. Nevertheless, RANTES likely participates in the normal physiology of the endometrial immunological system. (Saed et al., 2022).
One of the most important angiogenic factors in the placenta is the VEGF, which is essential for embryonic vasculogenesis (Porwal et al., 2021) and its expression increases as gestation proceeds. The hypoxia-inducible factor 1 (HIF1A) controls the expression and transcription of VEGF, therefore, the clock network regulates angiogenesis and embryonic development (Johnson and Mowa, 2021). Under low oxygen conditions, HIF1A activates multiple transcription factors, including VEGF which promotes angiogenesis and vascularization in the placenta (Fong, 2008, Agaoglu et al., 2015).
VEGF was characterized by its ability to induce vascularity, and permeability, and promote vascular endothelial cell proliferation. Three families of VEGF proteins and their corresponding receptors have been characterized, and the main receptors involved in the first steps of signal transduction cascades comprise different tyrosine kinase receptors, such as VEGFR-1, VEGFR-2, and VEGFR-3. It was proven that miRNA-185 plays an important role in the regulation of the VEGF signaling pathway in cows suffering from a retained placenta (Zheng et al., 2018).
Across species, some VEGF family members and receptors are found in placentomes, uterus tissues, and oviducts, and in different species including humans, mice, rats, cattle, sheep, pigs, and rabbits (Cheung et al., 2017; Gabler et al., 1999; Kaczynski et al., 2020; Kalkunte et al., 2009; Kazi and Koos, 2007; Llobat et al., 2012; Pfarrer et al., 2006; Złotkowska et al., 2019).
In the fifteen days before parturition, a substantial decrease in IL-1β gene expression was observed and continued to the fifteenth day after delivery, which indicates a disrupted immune response with subsequent RP. Also, Shimizu et al., (2018) found that at 4 weeks postpartum, IL-1β gene expression in peripheral blood mononuclear cells was significantly lower in RP dairy cows than in control cows.
In the normal physiological processes of parturition, IL-6 plays a role as it is expressed in the female reproductive tract and gestational tissues and also regulates placenta growth, and helps in embryo implantation. (Gomez-Lopez et al., 2016).
It was reported that IL-6 affects low P4 levels and activates the uterus genes responsible for normal delivery (Robertson et al., 2010). Moreover, IL-6 stimulates the adaptive immune response, which may take place throughout parturition (Jaworska and Janowski, 2019).
A process called placental maturation involves a decrease in CE and a decrease in TGC numbers and is necessary for the release of bovine fetal membranes. Impaired regulation of the process can lead to the retention of fetal membranes, one of the major reproductive disorders in cattle (RFM). The condition in which the fetal membranes are not expelled from the uterus within 12–48 h postpartum is described as this disturbance. (Dilly et al., 2011).
The loosening of the adhesion of the fetal membranes in the maternal compartment could involve local factors. Both the maternal and fetal compartments are exposed during gestation to rapid growth, angiogenesis, and tissue remodeling. Proteolytic enzymes and the subsequent degradation of extracellular matrix (ECM) components are required for these processes, as well as for the proper release of fetal membranes. Matrix metalloproteinases (MMPs) play a pivotal role in tissue remodeling and ECM breakdown processes during placentation and implantation in several species. (Beceriklisoy et al., 2007).
Biochemical changes during retained placenta
For dairy cows to behave very productively and reproductively in the later postpartum phase, lactation must transition smoothly from pregnancy to lactation. However, due to hampered reproduction and productivity, a bad transition frequently causes dairy farmers to suffer significant financial losses (Roche et al., 2017). Understanding the reasons behind and effects of metabolic alterations during this time is crucial for postpartum health management (Wankhade et al., 2017).Variations in serum metabolic marker concentrations for systemic inflammation, liver function, mineral, and energy status, and blood neutrophil counts are associated with healthy postpartum dairy cow neutrophil function. (Bogado et al., 2021).
The most important biomarkers are those related to liver and kidney function, lipid profile, minerals, and oxidative stress.
Kidney and liver functions
As a result of tissue destruction and inflammation during the RFM period in attempts to expel the placenta, the production of protein in the liver is partially altered towards the increased synthesis of positive acute phase proteins and reduction of albumin, which is a negative acute phase protein, and globulin elevation, thereby reducing A/G (Hassan et al., 2019). With altered energy metabolism and systemic inflammation, there is a state of reduced liver function in postpartum dairy cows (Osorio et al., 2014). Concentrations of hepatic proteins such as albumin and globulin can alter physiological and pathological conditions. Albumin is a negative acute phase protein, and albumin synthesis in the liver is expected to decrease in the case of infection to facilitate the production of globulin (Trevisi and Bertoni, 2008; Trevisi et al., 2011).
To evaluate the source of the tissue insult, AST activity should be viewed in combination with that of a liver-specific enzyme, such as GGT, or a muscle-specific enzyme, such as CPK (creatine phosoho kinase). For clinical and subclinical diagnosis, serum activity of AST is a very sensitive predictor of liver disorders. There were higher levels of GGT and AST in cows with fatty livers. The increase in AST and GGT activity in cows with retained placenta relative to control cows may be due to lipid accumulation in hepatocytes. (Hashem and Amer, 2008; Semacan and Sevinç, 2005b).
Albumin or globulin concentrations are correlated with neutrophil activity, but total protein concentrations at 5 d postpartum were positively linked to the release of reactive oxygen species. (Bogado et al., 2021).In healthy and RP cows, albumin and urea concentrations were similar, but total protein concentrations were higher at 30 ± 4 days after calving in healthy cows. (Yazlık et al., 2019).
Serum minerals
Decreased levels of Ca can cause uterine atony, which leads to RP. Furthermore, subclinical hypocalcemia in milk cows is an issue for the first few days after parturition due to excessive demand for calcium for colostrum synthesis and milk production and an insufficient bone response to restore Ca concentration. (Reinhardt et al., 2011).
Serum total calcium concentrations at 5, 10, 14, or 21 d postpartum were > 2.2 mmol/L. So, he did not observe associations of total calcium with neutrophil function, but samples were collected after postpartum hypocalcemia would have passed in almost all cows. RFM and reduced circulating levels of calcium are believed to be associated with one another ( Vallejo-Timaran et al., 2021).
Normal ranges for blood calcium (1.97–2.5 mmol/L), blood phosphorus (4.6 to 9 mg/dL), blood magnesium (1.4 to 2.3 mg/dL), milk urea nitrogen (9 to 18 mg/dL), milk BHB (< 0.1 mM), and milk fat/protein ratio (1 to 1.2) were defined a priori (Lu et al., 2020; Vallejo-Timaran et al., 2021).
Serum Lipid Profile
Severe RP hyperlipidemia leads to lipid accumulation inside the hepatocytes and subsequently to an increase in liver enzyme activity and liver injury. (Joksimovic-Todorovic and Davidovic, 2013). Liver disorders are often associated with abnormal lipid and lipoprotein concentrations. Several authors reported that the concentrations of triglycerides, cholesterol, and HDL-cholesterol decreased in fatty liver cows. For cows with retained placenta, serum levels of cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol are lower than those of control cows. Several mechanisms, including a decrease in the conversion of VLDL to LDL, could result in a decrease in LDL-cholesterol serum concentration in cows with retained placenta. An increase in LDL catabolism could be another explanation for the decrease in LDL. The decrease in serum HDL-cholesterol levels in cows with the retained placenta may be related to lower serum cholesterol levels because HDL-cholesterol consists of about 33% cholesterol. (Civelek et al., 2011; Semacan and Sevinç, 2005a).
Different Protocols Used For The Treatment Of Retained Placenta In Dairy Cows
Retained placenta remains a therapeutic challenge in cattle. The negative effects of retained placenta include reduced milk yield, increased incidence of metritis, and impaired subsequent fertility. Therefore, effective treatments for retained placenta are crucial for improving puerperal health care in cows (Cui et al., 2014).
High levels of prostaglandin F2α or PGE2 are released from the uterus during the early post-calving time in cows and may play an important role in both placental separation and uterine involution (Slama et al., 1994).
For the prevention or treatment of retention of the placenta, injections of ecbolic drugs such as oxytocin, (PGF2α), or methylergometrine have been administered within 24 hours of parturition (Nosier et al., 2012; Solanki et al., 2019). Time elapsed from parturition to complete fetal membrane drop was shorter in cows receiving oxytocin or methylergometrine maleate/ intramuscular injection than in untreated cows (Azad, 2010; Madhwal et al., 2007).
Also, the usage of methylergometrine or PGF2α immediately postpartum reduced the incidence of retained fetal membranes and improved reproductive performance in cows (Solanki et al., 2019). Additionally, the administration of 500 µg PGF2α or 50 I.U. oxytocin via an intramuscular injection immediately after the expulsion of the fetus induces early expulsion of the placenta and improves the reproductive and productive efficiency of cows experiencing retention of fetal membranes (Abou-Aiana et al., 2019; El-Hawary et al., 2020).
Manual Removal And Antibiotic Therapy:
Many broad-spectrum antibiotics and hormonal therapies have been used to treat retained placenta in dairy cattle. However, the efficacy of these approaches is controversial, and some treatments might negatively affect subsequent reproductive performance (Drillich et al., 2006).Furthermore, the administration of antibiotics in livestock should be minimized to reduce the prevalence of resistant bacteria (Peter et al., 2018).
The manual removal of the RFM, although commonly practiced by owner of the animals (Drillich et al., 2006) has been critically discussed for many years. Among the downsides of manual removal as a routine procedure for RFM treatment includes traumatic injury to uterine mucosa, intrauterine bacterial contamination, disturbance of intrauterine cellular defenses, and impairment of subsequent fertility (Peter et al., 2018).
Although intrauterine therapy such as tetracycline/sulfonamide boluses is used in most RFM cases (Hehenberger et al., 2015), its application does not reduce the incidence of metritis or improve fertility, and it inhibits the metalloproteinase matrix and possibly perpetuates bacterial resistance (Peter, 2013).
Systemic antibiotics are believed to be beneficial in RFM cases where fever is also present, although it is not clear whether the resolution of fever is due to antibiotics or the cow’s own immune defense mechanisms (Amin et al., 2013; Drillich et al., 2006). Only antibiotic treatment is beneficial in cases of acute postpartum metritis (Chenault et al., 2004). Prostaglandins and oxytocin are the most commonly used hormones in treating RFM, which play a role in uterine contraction and are thus effective in treating RFM following uterine atony (Han and Kim, 2005; Patel and Parmar, 2016). However, it has been reported that uterine atony accounts for a very small percentage of retained placenta cases (Peters and Laven, 1996). Therefore, these hormones are not supported for their use in the treatment of RFM (Drillich et al., 2006).
Effects Of Retained Fetal Membranes On The Fetrility Traits In Dairy Cows
The retained fetal membranes are associated with a great variation in the postpartum reproductive performances in dairy cows (Gohar et al., 2018). RFM abrogates endometritis, puerperal metritis, and mastitis, and these diseases ultimately lead to a decrease in cattle fertility and milk production (Vallejo-Timaran et al., 2021).
Post-calving time (puerperium) is of paramount value in the reproductive and productive performance of cows (El-Hawary et al., 2020; Waheeb et al., 2014). Aberrations of the postpartum period, including retained placenta, cystic ovaries, metritis/ endometritis, uterine prolapse, and pyometra, are major causes of infertility in the postpartum period (Alharoon, 2018; El-Hawary et al., 2020; Waheeb et al., 2014)).
First estrus
Placenta retention increased the time between parturition and the first postpartum estrus of Friesian cows by approximately 5 days. In a previous study, it was proven that approximately 72% of the normally calved cows showed first estrus after parturition by 25 days. Meanwhile, of the delivered cows suffering from retained placenta, the corresponding percentage was 63%. There were long periods between calving and first estrus for cows with retained placenta. (Shiferaw et al., 2005).
Days open
Following RFM, the uterus becomes infected with bacteria, which has a detrimental effect on cattle's ability to reproduce, including delaying uterine involution, lengthening the time until the first service, increasing the number of services required for each conception, and lengthening the time the uterus is open (Mahnani et al., 2021b). RFM has also been linked to a higher incidence of mastitis, metritis, endometritis, and ketosis (Mahnani et al., 2022). These conditions may also result in reduced fertility and possible milk production losses. In the retained placenta, open days were longer by about 17 days relative to normal cows. This is because about 52% of normally calved animals had days open within the first three months postpartum, while only 45% of cows exhibiting retained placenta conceived during the same period. This is in addition to 28.20% of the retained cows that had been open for more than 120 days. (Shiferaw et al., 2005).
The number of services per conception
The time from parturition to the first service was longer than 42 days, while during the same period, 37.1% of the cows exhibiting retained placenta were served. (Shiferaw et al., 2005) . In Holstein cows with retained placenta, the interval from calving to the first service was extended, meaning that cows with retained placenta had longer intervals from calving to the first service (Han and Kim, 2005). The length of service was longer (P < 61 days). The corresponding percentage of retained placenta in cows was around 57%. (Gaafar et al., 2010). For cows exhibiting retained placenta, the number of services per conception was higher. These may be attributed to the longer time of service as well as the lower rate of cow conception with retained placenta, which required more services. (Gaafar et al., 2010; Komba and Kashoma, 2020).
Calving interval and conception rate
The calving period was longer than 375 days, whereas only 44% of cows with exhibition retained placentas had open days of the same duration reported (Elmetwally et al., 2016). Retained placenta causes the dairy industry to suffer significant financial losses by lengthening days open, calving to first heat interval, services per conception, and days from calving to first service (Kimura et al., 2002). With an estimated cost of $285 per case and an average incidence rate of 7.8%, RP is regarded as an economically significant problem for the dairy sector since it increases the likelihood of developing metritis, infertility, mastitis, and poorer milk supply (Dervishi et al., 2016). Retention of the placenta resulted in a reduction in conception rate of about 7% compared to a healthy one (74.10 vs. 66.70%, respectively). The conception rates of normally calved animals and those exhibiting retained placentas were 15.20 and 12.90% before 60 days, 23.60 and 16.80% for 61–90 days, 20.40 and 18.10% for 91–120 days, and 14.90 and 18.80% for more than 120 days postpartum, respectively. Moreover, the highest percentage (31.80%) of normal cows was conceived 61 to 90 days after parturition, while the highest percentage (28.20%) of retained placenta cows was conceived more than 120 days after parturition. (Gaafar et al., 2010).