Hormonal and biochemical anomalies in dairy cows affected by retained fetal membranes

Mohamed A. Hashem and Hussein A. Amer1
Departments of Clinical Pathology and Theriogenology1, Faculty of Veterinary Medicine, Zagazig University, Egypt.


The aim of this study was to compare hormonal and biochemical blood indices, as well as reproductive parameters, in cows healthy and affected with retained fetal membranes (RFM).  Twelve Holstein cows that shed the placenta soon after calving (control) and 32 that retained placenta more than 24 hours were enrolled. The cows with RFM were allocated into 4 treatment groups. Group 1 (n = 8) received 4 intrauterine antibiotic tablets (Synulox, Pfizer, Egypt) for 3 consecutive days. Group 2 (n = 8) was treated with GnRH (100 µg). Group 3 (n = 8) was treated with PGF2α (25 mg). Group 4 (n = 8) received both GnRH (100 µg) and PGF2α (25 mg).  

All cows with a rectal temperature ≥39.5oC received antibiotic marbofloxacin (Marbocyl, Vetoquinol) for 3 days. 

The hormonal profile showed a significant (P<0.05) higher levels of progesterone (P4) and cortisol, and significantly (P<0.01) lower level of oestradiol 17-β in cows with RFM. Concentration of liver enzymes was significantly (P<0.05) higher, while the serum glucose, total lipids, cholesterol, triglycerides, electrolytes and vitamins (A and β-carotene) levels were significantly lower in cows with RFM compared with the control group.

The serum protein electrophoresis revealed hyper-globulinemia in cows with RFM. Treatment of RFM cows with Synulox antibiotic, GnRH, PGF2α or GnRH+ PGF2α was effective in hastening the return of the mentioned biochemical parameters close to the control group levels. This was more evident with PGF2α or GnRH treated groups compared with others.

PGF2α or GnRH treatments alone reduced significantly (P<0.05) the period of time from calving to the 1st insemination and the number of consultations required per conception (P<0.05), followed by a significantly (P<0.05) higher conception rate.  

 In conclusion, low serum electrolytes levels and hormonal imbalance might predispose to placental retention in dairy cows. Treatment with PGF2α or GnRH alone helps the normalization of some biochemical parameters better than the antibiotic treatment.



            Retained fetal membrane (RFM) is one of the most common disorders affecting reproduction of dairy cattle (Stephen, 2008). The fetal membranes retention is defined as the failure to expel the fetal membranes within 12 to 24 hours after calving (Drillich et al., 2006). Various causes of RFM have been identified i.e. uterus paresis, abortion, stress, late or premature birth, dystocia, twinning, infections, seasonal and hormonal disorders. Additionally, some vitamin and mineral deficiencies induce or predispose animals to RFM (Akar and Yeldiz, 2005; Lotthammer, 2005). Nonetheless, the primary cause remains often equivocal. Michal et al. (2006) conclude that RFM is due to complex metabolic disturbances during the pre-partum period. Retained placenta causes reduction in the fertility which leads to a reduction in pregnancy rate, increases number of consultations per conception and consequently longer calving interval (Bella , ,  and Roberts, 2007). The higher occurrence of metritis after RFM has been identified as the main reason for reduced fertility of cows with RFM (Gröhn and Rajala-Schultz, 2000). Low serum calcium, zinc, magnesium and potassium in cows before parturition might cause or increase the risk of RFM (Stancioiu and Constantinescu, 1983; Zhang et al., 1992). Inadequate supplementation of a ration with vitamins A and E, β-carotene, iodine, selenium, copper and zinc may also induce abortion in cows with increase incidence of RFM (Markiewicz et al., 2001; Farzaneh et al., 2002). Uterine involution is delayed in old females whit postpartum disorders (retained placenta, metritis, etc.). Higher cholesterol level during postpartum induces lengthening of uterine involution (Quintela et al., 2003). Average incidence of RFM ranges from 4 to 11% of calving (Eiler, 1997). Many cows with RFM have elevated body temperature indicative of metritis. For dairy cows with RFM, the incidence of fever (≥39.5oC) varies between 36 and 95% (Drillich et al., 2003). It causes considerable economic losses in the herd due to decreased milk production, illness and treatment cost, beside a decrease market value (Ahmed et al., 1999; Semacan and Sevinc, 2005).

The aim of the present work was to understand the relationship between hormonal and biochemical parameters, as well as the reproductive outcomes, in cows affected with RFM. 




            The study was conducted in a commercial dairy herd in Sharkia Province from June 2006 to February 2008. A total of 342 Holstein dairy cows were housed in free stall barns with slotted floors and cubicles. At least 1 week before expected calving, cows were fed a total mixed ration and milked 3 times daily. Herd average milk yield was between 7,600 and 8,850 kg per lactation. The cows that delivered the placenta within 12 hours after calving were assigned to the untreated control cows (n=12), while those with undelivered placenta within the first 24 hours were assigned to RFM group (n=32 cows). The cows with RFM showed either only RFM (n=12) or RFM plus other complications and a body temperature ≥39.5oC i.e. fever (n=20).

Clinical examination and Treatment design

            During the first 10 days post-partum (PP), rectal body temperature was measured daily in all cows enrolled in the study. Cows of all groups with a rectal temperature ≥39.5oC received Marbocyl antibiotic treatment (Vetoquinol Co., France) in a dose of 1 ml/50kg body weight IM for 3 consecutive days.

Untreated control group (n=12), included cows with non retained fetal membranes, and without fever, did not receive any antibiotic treatment and no attempt was made to remove the fetal membranes.

Group 1 (n = 8) received 4 intrauterine tablets (Synulox TM, Pfizer, Egypt, each tablet contains 100mg clavulanic acid and 400mg amoxicillin), 24 hours PP for 3 consecutive days.  No attempt was made to remove the fetal membranes manually.

Group 2 (n = 8) was treated with 100 µg of GnRH (Receptal, Intervet, Germany) IM on day 12 PP Group 3 (n = 8) was treated with 25 mg of PGF2α (Lutalyse, Pharmacia and Upjohn Co., USA) IM between day 18 and 26 PP and again between day 32 and 38 PP. 

Group 4 (n = 8) received a combination of 100 ug GnRH on day 12 PP and 25 mg of PGF2α IM between day 18 and 26 PP.

Collection of blood samples

            Blood samples were collected 12 hours after calving from jugular veins of control cows (untreated control, n = 12) or at 24 hours from those with RFM (n = 8) after calving. In addition, 8 blood samples were collected from RFM cows treated with Synulox antibiotic, GnRH, PGF2α or a combination (GnRH + PGF2α), 24 hours after medications. Serum was kept frozen at -20oC until hormonal and biochemical analysis was performed.

Hormonal analysis

            Serum concentrations of progesterone, estradiol 17β and cortisol levels were determined by immuno-enzymatic methods (Biodata Diagnostic tests). The sensitivities of these tests were 0.15ng/ml, 8.0pg/ml and 0.36 ng/ml with intra-assays C. vs. of 3.9, 5.30 and 3.70 % respectively.

Biochemical analysis

            The serum alkaline phosphatase (ALP), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), glucose, total lipid, cholesterol, triglyceride, total protein, albumin, globulins (calculated), calcium, inorganic phosphorus, magnesium were colorimetrically determined using commercial test kits from Diamond/Egypt. Serum zinc (Zn) and selenium (Se) concentrations were analyzed with an atomic absorption spectrophotometer (Perkin Elmer 370 Model), while serum potassium (K) with a flame photometer (Petracourt PFP1). Serum vitamin A and β-carotene levels were measured using spectrophotometric method (Schimadzu UV-1208, UVVIS spectrophotometer). 

Postpartum reproductive parameters

The waiting period varied among the herd from 60 to 90 days PP. Cows were bred on observed estrus. No timed breeding protocols (e.g., targeted breeding or Ovsynch) were used. Cows not pregnant after breeding and cows not observed in estrus within 30 days after the end of the voluntary waiting period were examined by rectal palpation by a veterinarian. Cows with a palpable corpus luteum received PGF2α to induce estrus. All cows included in the study were followed up for pregnancy determination. The reproductive performance variables monitored and documented were: days from calving to 1st insemination, days from calving to conception, services required per conception, first-service conception rate and total conception rate (number of cows pregnant by total number of inseminations). Pregnancy was determined by rectal palpation on day 35-40 after the service.

Statistical analysis

The differences between groups were determined with ANOVA (SAS, 2002), significance of differences with Duncan’s test. The results are expressed as mean ±SE. A level of P < 0.05 was considered statistically significant. Comparison between groups was done by Chi-square (x2).



Thirty-two out of 342 cows (9.35%) were suffering from RFM and the remaining cows delivered placenta normally within 12 hours. The most common clinical signs are the malodorous or foul smelling discharge and the unsightly mass of tissue hanging from the genital tract.

Hormonal analysis in cows with RFM showed a significant (P<0.05) higher serum levels of progesterone (1.72 ± 0.23 ng/ml) and cortisol (8.46 ± 0.22 ng/ml) than those of control (0.98 ± 0.02 and 5.99 ± 0.13 ng/ml, respectively). The progesterone level was similar to control level in all treated cows except those treated with Synulox, which had higher levels (1.22 ± 0.17 ng/ml) than controls, but lower than RFM cows. The cortisol concentration did not show statistical differences between treated RFM and control animals. Compared to control cows (43.55 ± 5.2 pg/ml), the serum estradiol 17β level was significantly (P<0.01) decreased in the RFM group (19.66 ± 1.73 pg/ml) and also (P<0.05) in the animals treated with Synulox (38.45 ± 0.3 pg/ml), while other treated animals showed non significant change (Fig. 1).

Cows with RFM showed a significant serum decrease of glucose, total lipid, cholesterol and triglyceride (Table 1), whereas ALP, AST and GGT activities were increased. The RFM cows treated with Synulox antibiotic, GnRH, PGF2α and a combination of GnRH + PGF2α showed a higher (P<0.05) levels of glucose, lipids, with lower liver enzyme concentrations than those of RFM cows, close to the control group levels. Total serum proteins and albumins were not different in the two groups, treated RFM and untreated control cows. Only globulins were significantly higher in RFM cows than those of control cows. Protein profile returned close to control level in treated groups.


Electrolytes analysis in the animals with retained placenta revealed a significant (P<0.01) decrease in serum calcium, phosphorus, magnesium, zinc, selenium, vitamins A and β-carotene, in comparison with control cows (Table 2). There were no statistical differences in these electrolytes between treated and untreated control, however serum magnesium and selenium were still decreased in antibiotic treated cows.

Table 1. Serum biochemical parameters (Mean ± standard error) in cows with or without retained fetal membranes and those subjected to different therapeutic treatments.


Untreated Control


RFM before



RFM after treatment

Synulox antibiotic




PGF2α n=8






± 2.60 c


± 5.11 a


± 4.80bc


± 5.01 c


± 6.22 c


± 3.50 bc

AST (U/l)


± 9.00 c


± 19.00 a


± 15.50 b


± 16.00 c


±19.00 c


± 17.04 c

GGT (U/l)


± 2.10 c


±7.80 a


± 5.22 b


± 8.80 c


±6.55 c


± 6.14 c

Glucose (mg/100ml)


± 4.51 a


±  2.11 c


±  5.01 ab


±  8.00 a


± 4.66 a


±  4.12 a

Total lipids (mg/100ml)


± 18.77 a


± 11.55 d


± 15.47 c


±16.10 ab


±19.85 a


± 5.47 a

Cholesterol (mg/100ml)


± 15.45 a


± 7.80 c


± 14.70 a


± 11.00 a


± 17.2 a


± 12.00 b

Triglyceride (mg/100ml)


± 7.52 a


± 5.66 b


± 9.25 a


± 8.05 a


± 9.22 a


± 6.55 a

Total proteins (gm/100ml)


± 1.58 a


±0.44 a


±0.44 a


± 0.44 a


±0.35 a


± 0.44 a

Albumin (gm/100ml)


± 0.22 a


±0.25 ab


±0.25 a


± 0.25 a


±0.11 a


± 0.25 a

Globulins (gm/100ml)


±0.32 b


±0.05 a


± 0.32 b


± 0.32 b


±0.09 b


± 0.32 ab

Means within the same row having different alphabetical superscript letters are significantly different (P < 0.05).



Table 2. Serum electrolytes and vitamins (Mean ± standard error) in cows with or without retained fetal membranes and those subjected to different therapeutic treatments.


Untreated Control


RFM before



RFM after treatment

Synulox antibiotic




PGF2α n=8

GnRH + PGF2α


Calcium (mg/100ml)


± 1.22 a


± 0.21 b


± 0.17 a


±1.20 a


±1.40 a


±0.89 a

Phosphorus (mg/100ml)


± 0.12 a


±0.16 b


±0.32 ab


± 0.21 a


±0.30 a


± 0.22 ab

Magnesium (mg/100ml)


± 0.06 a


±0.11 b


± 0.22 b


± 0.15 a


±0.15 a


± 0.22 a

Zinc (mg/100ml)


± 0.15 a


± 0.20 b


± 0.51 a


± 0.12 a


±0.19 a


± 0.42 a

Selenium (mg/l)


± 0.03 a


± 0.01 c


± 0.05 b


± 0.04 a


± 0.03 a


± 0.04 a

Potassium (mEq/l)


±  0.23 a


±0.28 ab


± 5.21 a


± 5.21 a


± 5.21 a


± 5.21 a

Vitamin A (µg/100ml)


± 2.04 a


± 1.12 b


± 2.01 ab


±1.91 a


± 1.04 a


± 2.00 a

β-carotene (µg/100ml)


± 12.97 a


± 8.28 c


± 11.30 ab


±10.44 a


±12.55 a


± 11.34 a

Means within the same row having different alphabetical superscript letters are significantly different (P < 0.01).




            The effect of treatments is shown in Table 3. The cows in PGF2α or GnRH treatment significantly (P<0.05) reduced the rest period from calving to the 1st insemination and to conception than the GnRH+ PGF2α or antibiotic treated groups. Meanwhile, the mean number of consultations required per conception was the lowest (P<0.05) in GnRH+ PGF2α treated group than the other groups, which nearly reached that of the untreated controls. However, no significant difference in conception rate after 1st artificial insemination was found between the Synulox antibiotic, GnRH+ PGF2α or untreated control groups, but a significant (P<0.05) difference was obtained with PGF2α or GnRH groups in comparison to the others. The highest (P<0.05) total conception rate was obtained after treatment of the RFM cows with PGF2α, then GnRH than the other two treated groups.


 Table 3. Reproductive performance in cows with retained fetal membranes subjected to   different therapeutic treatments.



Untreated Control


Therapeutic treatments of cows with RFM

Synulox antibiotic






GnRH + PGF2α


Days from calving

to 1st  insemination

76.00 ±24.70 c


±27.90 a


± 6.8 c


±27.0 c


± 23.40 b

Days from calving

to conception

116.70   ±62.60 b


±80.90 a


±59.50 b


±58.30 c


±38.30 a

Services per



±1.00 c


±1.57 a


±1.23 b


±1.12 b


±0.69 c

1st insemination

conception rate (%)











Total conception rate

n (%)











Means within the same row having different alphabetical superscript letters are significantly different (P < 0.05).




Confirming previous reports on bovine RFM and concomitant high progesterone and cortisol (Sabry et al., 1997; Michal et al., 2006) and low estrogens (Kankofer et al., 1998) levels in the blood, animals in this study showed a homogeneous increase in the progesterone and cortisol, and a decrease in the estradiol 17β levels. Similar results were also noticed by Kornmatitsuk et al. (2000) in cows with RFM after induced calving.


Increased progesterone level in RFM may be due to failure of the placenta to produce specific steroidal enzymes that help in progesterone metabolism and its conversion to estrogen (Matton et al., 1987), which in turn may induce the accumulation of immunosuppressive proteins in the uterine lumen which make the uterus susceptible to infection and persistence of bacteria (Königsson et al., 2002).


Increased cortisol may be related to the stress in cows with RFM (Dobson and Smith 2000). Similar data were previously recorded by Sabry et al. (1997), Kornmatitsuk et al. (2000), Farzaneh et al. (2002) and Kandeil et al. (2002).


In this study, the serum estradiol 17β was significantly decreased in RFM animals when compared with the control group. El-Nemer et al. (2000) observed that during the week before parturition, the level of estradiol reaches its maximum level and this help the uterus to get rid of any remnant of fetal membranes and also to prevent endometriosis. Therefore, a decreased level of estrogen may be indicated as a factor enhancing RFM.


Increased liver enzyme (ALP, AST & GGT) activities in cow with retained placenta in this study were in accord with the recorded association to the fatty liver observed in cows with retention of placenta (Semacan and Sevinc, 2005). The fat infiltration of the liver is associated with an increase of hepatic enzymes and a serum decrease of glucose, total lipid, cholesterol, triglyceride and electrolytes. Mild to moderate fatty liver might result in a liver dysfunction without hepatocytes destruction and a subsequent increase in liver enzyme activities (Sevinc et al., 2001; Bülent et al., 2006). In cows with RFM this may be due to the accumulation of lipids in the hepatocytes. Endotoxins released in infectious diseases such as endometriosis cause destruction and necrosis of the liver with various degrees of hepatic dysfunction (Semacan and Sevinc, 2005).


Hypoglycemia found in cows with RFM is in agreement with the hypothesis that glucose in late pregnancy act as a predictive indicator of RFM risk (Patel, 1999). An alternative explanation is that low glucose levels may be due to increased nutritional requirements by the fetus and for the production of colostrum (Bell, 1995). Hypoglycemia can be related to the high level of cortisol associated with RFM and postpartum metritis (PM), either by direct or indirect way (Michal et al., 2006). The hypoglycemia during the last month of pregnancy is a predisposing factor for RFM and PM (Markiewicz et al., 2001).


The present study showed that the serum levels of total lipid, cholesterol, and triglyceride of cows with RFM were lower than those of control group. The decreased cholesterol level may be owing to the persistence utilization of cholesterol for progesterone synthesis or due to increase breakdown of cholesterol (Kandeil et al., 2002). Decreased total lipid and triglyceride may be related to disturbed lipid metabolism, increased tissue lipolytic enzymes or due to the interfering factor of ketosis in the affected animals (Michal et al., 2006).

The abnormal lipid concentrations are often associated with liver disorders (Grummer, 1993; Van DenTop et al., 1995; Semacan and Sevinc, 2005). Blood proteins are unchanged in cows with RFM, except for evident hyper-globulinemia, which may be due to bacterial infection.


             Minerals are important in the prevention of RFM (Wilde, 2006). Hypocalcemia, hypophosphatemia and hypomagnesemia were present in cows with RFM in the present study. Imbalance of calcium and phosphorus metabolism was correlated positively with RFM in previous studies (Zhang et al., 1992; El-Hanafy, 1998). Such hypocalcemia might play an important role in the incidence of RFM and associated uterine infections (Semacan and Sevinc, 2005). It seems that low calcium, phosphorus, zinc, magnesium and selenium values during the pre-calving and post-calving periods predispose to RFM in cows (Bari et al., 1996; Sabry et al., 1997; Akar and Yeldiz, 2005; Tillard et al. 2008). However, prepartum calcium supplementation prevents RFM mainly improving health condition and enhancing of the myometrial sensitivity (Sosa and Nasr, 1995). It was found that RFM animals show concurrent decreased zinc, selenium and vitamins A and beta carotene, as well. Altogether, this complex of factors might lead to RFM (Hattab and Abdel Moghney, 1994; Markiewicz et al., 2001; Farzaneh et al., 2002; Roche, 2006).



In this study, administration of Synulox antibiotic, GnRH, PGF2α or GnRH+PGF2α combinations to cows with RFM was effective in hastening the return of the mentioned biochemical parameters nearly to the normal level in cows. This was more evident in RFM cows treated with GnRH or PGF2α, compared with other groups. Treatment with GnRH or PGF2α helps the normalization of electrolytes and vitamins, as well. Early treatment of abnormal cows with GnRH improves the reproductive efficiency through normalization of serum progesterone (Foote and Riek, 1999).         Antibiotic and hormonal therapies in bovine RFM have been an issue of controversy for many years (Drillich et al., 2003). The trial described here is one of the largest control field studies performed to check the efficacy of different treatment protocols for RFM in many years (Stevens et al., 1995). Local administration of antibiotics was not effective to prevent fever in all cows. More than 70% of the cows developed fever even when they received local antibiotics. Because there was no control group of cows with untreated fever, it remains unclear if the reduced number of cows with fever after treatment with Marbocyl was a result of the antibiotic treatment or a consequence of a spontaneous cure. An alternative explanation is that fever itself indicates the reaction of the immune system against the inflammatory noxa and therefore can be regarded as a sign for an intact immune response (Laroux, 2004).


Administration of PGF2α or GnRH alone produced the best improvement in the reproductive performances of these cows. Although, the number of services required per conception was the lowest in GnRH + PGF2α treated group, a significant higher rate of conception after the 1st insemination and total conception rate were obtained with PGF2α or GnRH. Treatment by GnRH alone in early PP increased the interval from calving to conception unless PGF2α is given 9 days after GnRH treatment (Stevenson and Call, 1988). In contrast, a decreased interval from calving to conception was seen in cows with abnormal puerperium treated with GnRH from day 10 to 14 PP (Benmrad and Stevenson, 1986).

In this study, the use of GnRH followed 14 days later by PGF2α treatment did not improve any of the reproductive traits studied in cows affected with RFM, as previously observed in the study by by Richardson et al. (1983) in which PGF2α was administered 10 days after GnRH.

We expected that GnRH treatment would hasten ovulation and establish a corpus luteum that could be lysed when PGF2α, was administered. Potentially, the induction of a new estrous cycle could aid uterine involution and improve fertility (Benmrad and Stevenson 1986). This was not.


In short, biochemical, hormonal and electrolytes profiles can be used as predictive and prognostic criteria in cows with RFM. Higher progesterone and cortisol with lower estrogen concentrations seem to be the main hormonal changes in retained placental cows. The RFM has detrimental effects on reproductive efficiency in PP cows characterized by delayed interval to first insemination and poor conception rate of first PP insemination. Treatment of retained placental cows by GnRH or PGF2α was better in hastening the return of the liver function tests and improving the postpartum performance than a combination of GnRH and PGF2α or Synulox antibiotic alone.



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Copyright Priory Lodge Education Ld. 2008

First Published September 2008



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