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Home » Issue 2 (Summer 2016) » Project Articles » A study examining the interrelationships between blood calcium levels, metritis and days to first service in third parturition Holstein dairy cows

A study examining the interrelationships between blood calcium levels, metritis and days to first service in third parturition Holstein dairy cows

Author Names: Jessica Hagain (BSc (Hons) Bioveterinary Science) and Brian Evans

 

Abstract

A key priority within the dairy industry should be to understand and act upon any undesirable reproductive health issues within the dairy herd, with the intention of maximizing on-farm welfare and economic profits. These undesirable reproductive health issues include hypocalcaemia and metritis. Hypocalcaemia commonly affects third parturition and above dairy cows and leads to hyper-activity of the nervous system and muscle contraction reduction, which in severe cases can result in paresis. Metritis is defined as the inflammation of the uterus resulting in uterine discharge. This can progress into a systemic infection where antibiotic therapy is essential. Previous research regarding whether hypocalcaemia is associated with metritis has resulted in contradictory findings. In addition to this, even though it is well documented that metritis can contribute to prolonging the onset of the oestrus cycle, there is a lack of research into whether hypocalcaemia affects the onset of the oestrus cycle. The main aims of this study were to identify whether a relationship exists between hypocalcaemia and metritis, and to investigate if the onset of oestrus, via the number of days to first service, related to either the blood calcium categories and/or the metritis score observed. To achieve these aims, thirty multiparous cows from a UK dairy farm were recruited at calving. A blood calcium level was determined, along with a metritis score and the number of days to first service. No significant relationship was found between blood calcium levels and metritis. No significant difference was also found between blood calcium categories and days to first service or metritis and days to first service. However, due to the multifactorial aetiology of metritis and reproductive health, this may have led to the insignificant results. This may have been further aggravated by the relatively small sample size recruited due to time constraints. In future research, a study of all the third parturition cows through a 365-day study period would allow for a larger data set and consideration of more key indicators of reproductive health, as well as reducing any seasonal bias in oestrus presentation.

 

1.0 Introduction

Evaluating and understanding health effects in dairy cattle should be a key priority for the dairy farmer (Krogh and Enevoldsen, 2014) in order to improve on-farm welfare and increase economic profits (Brozos, Mavrogianni and Fthenakis, 2011). Special attention should be given to reproductive health, as successful pregnancies initiate lactation and drive milk yields (Damron, 2013; Siqueira et al., 2009). As reproductive health and thus fertility success is multi-factorial (Walsh, Williams and Evans, 2011; Ball and Peters, 2004) any undesirable factors should be minimised where possible (Damron, 2013; Rushen et al., 2008) such as hypocalcaemia and metritis.

Hypocalcaemia, more commonly known as milk fever, occurs most frequently around the time of parturition (Damron, 2013). The sudden loss of calcium; up to 20-30g daily (Allen, 2015), from producing colostrum and initiating the new lactation can result in hyper-excitability of the nervous system and reduced muscle contractions (Aspinall and Cappello, 2009), in severe cases leading to paresis (Allen, 2015). This reduction in muscle contractions can result in uterine inertia leading to dystocia and retained foetal membranes (Norkus, 2012; Tilley and Smith, 2011; Andrieu and Warren, 2009; Noakes, Parkinson and England, 2009; Mee, 2008). Hypocalcaemia can present as clinical or subclinical, with the former having more practical emphasis when symptoms present and treatment is essential. Typical incidence rates for clinically acute hypocalcaemia in cattle vary between 3.5% and 7% as established by De Garis and Lean (2008). The authors also found a 9% increased risk of milk fever as the number of lactations increased, leading to a more at risk population. The Farm Animal Welfare Council (2009) stated the cost of one case of clinical milk fever at £210 per cow, due to direct milk yield loss and treatment costs. Although this costing did not take into account any indirect costings such as fatalities including disposing of carcasses and subclinical cases resulting in sub-optimal milk production. The average UK dairy herd size was estimated at 133 milking cows in 2014 (AHBD, 2015). If the incidence risk was similar to that calculated by De Garis and Lean (2008) of up to 7%, clinical milk fever may be costing the UK dairy farmer over £2000 per annum. This cost is again discounting any subclinical hypocalcaemia cases as costs remain relatively undefined, which may lead to many farmers overlooking the disease state (Mulligan et al., 2006). During the periparturient period, it has been suggested that the unfavourable conditions generated through metabolic disorders, including hypocalcaemia (Goff and Horst, 1997), can cause the decreased activity of the immune system (Cai et al., 1994; Kehrli, Nonnecke and Roth, 1989). This may increase the likelihood of postpartum diseases, such as metritis (Kim and Kang, 2003), when common environmental pathogens (Dohmen et al., 2000) challenge the dairy cow’s immune system.

Metritis commonly affects between 20-40% of dairy cattle (Mozzi, Raya and Vignolo, 2016; Reppert, 2015; Farmers Weekly, 2014) throughout the UK (Haskell, 2008). Metritis can be defined as inflammation of the uterus causing uterine discharge with absence of fever within 10 days postpartum (Bassert and Thomas, 2013; Risco and Retamal, 2011; Rushen et al., 2008). Acute metritis is characterised by a fetid brown-red discharge and pyrexia of >39.5⁰C (Reppert, 2015; Rushen et al., 2008; Drillich et al., 2001), therefore indicating that the infection has become systemic and antibiotic treatment is necessary (Divers and Peek, 2008). Previous research has been directed primarily towards treatment methods (Giuliodori et al., 2013; Krueger et al., 2013; McLaughlin et al., 2012; Goshen and Shpigel, 2006) due to this frequent antibiotic therapy. Mozzi, Raya and Vignolo (2016) believed this to be an aspect contributing to the presence of antibiotic resistance. Cockcroft (2015) estimated the combined direct and indirect costs per case of metritis to be £213, much higher than the findings of a 2015 study conducted by Mahnani, Sadeghi-Sefidmazqi and Cabrera in Holstein dairy herds in Iran. From four dairy herds the mean cost per case was £111, however the investigation did not state all factors which contributed to determining the costs. This, along with a difference in economic values such as commercial milk price and mortality costs may begin to explain the difference in estimated values. The study also relied on on-site farm record systems, a common approach taken in many studies. A DEFRA review by Escobar (2015) found that farmers tended to rely on ‘experience and sensitivity’ when trying to improve animal husbandry and welfare, rather than referring back to farm records. Therefore, some data may not have been recorded in studies with a similar approach, not just Mahnani, Sadeghi-Sefidmazgi and Cabrera (2015).

Identified risk factors for metritis include dystocia, twins and retained foetal membranes (RFM) (Dubuc et al., 2010; Potter et al., 2010; Han and Kim, 2005), however there is some evidence that hypocalcaemia and parity may also play a part (Rajala and Gröhn, 1998; Kaneene and Miller, 1995; Markusfeld, 1987). Bruun, Ersbøll and Alban (2002) conducted a retrospective longitudinal study on metritis in 2144 cattle herds in Denmark. The incidence risk of metritis was 0.7% (733 cases), with both lactation and hypocalcaemia assessed as risk factors along with others. The authors found a significant relationship between metritis and cows in the first lactation, mainly comprising of heifers,  whereas higher parities showed no statistical significance. Hypocalcaemia was also found to have no significant relationship with metritis, however these findings are at odds with those of previous studies (Grohn et al., 1990, Markusfeld, 1987). It was concluded that metritis had several multifactorial aetiologies, but this did not include hypocalcaemia. The study relied on diagnosis and treatment records which in Denmark are recorded into a database by Veterinarians who as qualified professionals may facilitate accurate records being made. However, the farmer must first of all recognise the disease or disorder and deem it severe enough to warrant calling the Veterinarian; a decision which can be influenced by cost. Denmark may have a similar system to the UK where the farm is provided with a stock of antibiotics to allow pain relief and anti-inflammatory treatment if needed. This may lead to inaccurate records due to such treatments preventing input from the Veterinarian and subsequently no records would be made in such a system present in Denmark.

Evidence from Potter et al. (2010) backs up the findings of Bruun, Ersbøll and Alban (2002) that hypocalcaemia and metritis are not significantly related. The study was conducted in four Holstein-Friesian herds across the UK, with the aim of identifying risk factors for clinical endometritis. Endometritis is considered a disease progression of metritis, when the inflammation progresses further down into the endometrial lining of the uterus usually occurring between 14-21 days postpartum (Blowey and Weaver, 2011; Noakes, Parkinson and England, 2009). Therefore, the results may still be applicable to metritis risk factors, as endometritis is currently believed to be caused by persisting metritis cases (Hopper, 2015; Kaufman et al., 2010). The farms were selected based on convenience and good record keeping with regular veterinary input via a weekly visit. Due to these selection factors, the results cannot be deemed completely representative across the UK dairy population as a result of differing management and prevention systems in place. The methodology comprised of three parts; a vaginal examination at 21 days postpartum, extraction of information from the on-site records and a survey distributed to fifty-seven practicing Veterinarians nationwide to establish the current opinion on the twenty-one most important risk factors for clinical endometritis. The vaginal examination was undertaken by means of a universally recognised method, previously proposed by Sheldon and Dobson (2004), although by assessing at 21 days the focus of the study was to diagnose endometritis. The results showed a 27% mean prevalence across the four farms with no significant difference between individual farms. Of the risk factors assessed, clinical hypocalcaemia was found not to be significant. Subclinical hypocalcaemia was not considered as a risk factor for metritis in keeping with previous studies (Kim and Kang, 2003; Bruun, Ersbøll and Alban, 2002). The survey found conflicting results listing hypocalcaemia to be fifth out of the twenty-one most important risk factors identified by the Veterinarians. This is consistent with previous studies stating a significant relationship between hypocalcaemia and metritis (Kim and Kang, 2003; Markusfield, 1984). Nonetheless, both the study and the survey agreed that dystocia, RFM and twins were the most significant risks for the development of metritis, which remains consistent with previous research. The results of the three-part study also reinforce the conclusion that endometritis, and therefore metritis, has a multifactorial aetiology (Bruun, Ersbøll and Alban, 2002).

Conversely to Potter et al., (2010) and Bruun, Ersbøll and Alban (2002), Kim and Kang (2003) found metabolic disorders including hypocalcaemia were important risk factors for metritis. The case-control study utilised 320 cows from eight dairy herds with similar housing systems, to limit variability in management effects. Even though the results showed a significant relationship between metabolic disorders and metritis, it is not possible to state that this is a direct relationship between hypocalcaemia and metritis. This is due to metabolic disorders being considered as a whole, including ketosis and displaced abomasums, as well as hypocalcaemia. It was not specified as to which personnel undertook the data collection, which with direct visual observations may make the results unreliable. Ideally, one trained individual completing the observations would improve this factor, however this may have been impractical as distances between herds were not specified. In order for the study to have been completely objective, blood samples could have been taken to accurately determine individual metabolic disorders. The study utilised a control group to compare with the endometritis group, but there was no mention of the two groups being matched in every possible way, such as parity and incidence of RFM, which would make the results more representative of the population and therefore more reliable (Schenker, Castañeda and Rodriguez-Lainz, 2014; Weiss and Koepsell, 2014; Hulley et al., 2007).

It has been well documented that metritis can lead to negative consequences to fertility postpartum (Piccardi et al., 2016; Pinedo et al., 2015; Toni et al., 2015; Wittrock et al., 2011; Lincke, Drillich and Heuwieser, 2007; Melendez et al., 2004). Wittrock et al. (2011) concluded that multiparous cows with metritis were 50% more likely to be culled due to inability to breed again than healthy individuals; emphasising the importance of managing postpartum diseases. Mahnani, Sadeghi-Sefidmazgi and Cabrera (2015) identified the prevalence of metritis as ranging from 9.0-15.8% across four Holstein herds including over 43000 calvings over five years. This range is much lower than other estimations of 20-40% in the UK (Mozzi, Raya and Vignolo, 2016; Reppert, 2015), however this may be due to geographical differences as the study was based in Iran. Within the study, 43,000 calvings were considered, along with splitting primiparous and multiparous cows into separate cohorts. The authors concluded that reproductive effects were more prevalent in multiparous cows. The number of days to first conception was increased in the metritis cases compared with the unaffected multiparous cows. The number of artificial insemination services were also increased in metritis cows. There was no reference though as to whether metritis affected and unaffected cows were matched with herd mates, or matched to cows on other farms to allow a more accurate statistical analysis within the case-control study. Furthermore, metritis was assessed for up to twenty-one days postpartum, which may mean that some of the metritis cases were actually endometritis, therefore the results may not be completely representative of metritis effects on the onset of oestrus. Additionally, Mahnani Sadeghi-Sefidmazgi and Cabrera (2015) only considered an individual to have metritis, if one veterinary treatment had been recorded, even if visual symptoms were observed. This was similar to a case-control study by Pinedo et al. (2015) which only considered clinical metritis. By only including clinical metritis, there were no results regarding lesser grades of metritis. These lower gradings could therefore be considered as ‘subclinical’, as there may still be an effect on the production and reproductive performance if the cow’s immune system cannot overcome the inflammation. However, the authors concluded that individuals with clinical metritis required an average of 0.67 more artificial insemination services to conceive than the control group. Despite not specifying the metritis severity, both Pinedo et al. (2015) and Mahnani, Sadeghi-Sefidmazgi and Cabrera (2015) concluded multiparous cows to be more at risk of negative reproductive effects due to clinical metritis.

Previous evidence suggests that both hypocalcaemia and metritis not only affect the fertility of the individual cow, but also the indirect financial loss to the farmer due to treatment and reduced lactation (Mulligan and Doherty, 2008). However, even with significant improvements in the understanding of postparturient production diseases, including hypocalcaemia, Roche (2003) reported incidence rates of subclinical hypocalcaemia as high as 33% in dairy herds. This data was reinforced more recently by Goff (2008) who estimated that approximately 50% of multiparous cows in higher lactations have subclinical hypocalcaemia post-partum. Previous research has proven to be extremely contradictory regarding whether there is a relationship between hypocalcaemia and metritis (Houe et al., 2001) and subsequently fertility, with many studies only considering clinical hypocalcaemia as a minor risk factor for metritis. However, the majority of the studies agree that multiparous cows are the most at risk of hypocalcaemia, as well as metritis, instigating more detrimental effects on their reproductive performance. Further understanding of this association, especially subclinical hypocalcaemia would be beneficial, as would identifying a direct relationship between hypocalcaemia and metritis which would enable prophylactic intervention.

 

1.1 Aims and Objectives

The first aim of the study was to determine whether a relationship exists between the blood calcium level within 24 hours post-calving and the subsequent metritis score obtained in the post-natal check 10-14 days postpartum. The second aim was to investigate if the onset of oestrus via the number of days to first service (DTFS) related to either the blood calcium category; normal, subclinical or clinical hypocalcaemia, and/or the metritis score observed.

In order to achieve these aims, the objectives of the study included measuring the blood calcium serum from 12-24 hours post calving, along with post-natal metritis scoring. In addition, the days to first service (DTFS) were extracted from the on-site farm records system to assess the effect on subsequent fertility.

 

1.2 Hypotheses

The null hypotheses are:

  • There is no relationship between blood calcium level and metritis scoring
  • The calcium category; normal, subclinical and clinical hypocalcaemia, and the metritis score have no effect on the number of days to first service (DTFS)

 

The alternate hypotheses are:

  • There is a relationship between blood calcium level and metritis scoring
  • The calcium category and metritis scores have an effect on the number of days to first service (DTFS)

 

2.0 Methodology

The study took place at Home Farm, Hartpury College (Gloucester, GL19 3BE) and focused on third parturition or above dairy cows. This was due to previous research identifying this as the most at risk group for hypocalcaemia (Blowey and Weaver, 2011; Kelton, Lissemore and Martin, 1998). The study population involved 30 multiparous cows from a 260 cow milking herd with an average milk yield of 9133 litres per cow per year. Over the 16-week study period there was over seventy calvings from 12/05/15 until 11/12/15. However, only thirty of these were included in the study due to the elimination of heifers and second parturition cows. The recruited cows were housed in a transition cow yard with other pre-partum dry cows and heifers. The yard had both heavily straw bedded and concrete areas, with ad lib water troughs available. The cows were fed the same ration as the rest of the cows in the yard which was deemed suitable to their stage of gestation.

 

2.1 Experiment Protocol

Due to time constraints, a cross sectional study was conducted. This allowed numerical data to be collated for statistical analysis and representation (Goddard and Melville, 2004). In order to achieve the aims and objectives of the study several recordings were taken from each individual including calcium blood serum measurement, metritis score, lactation number, rectal body temperature and body condition score at metritis scoring and the number of days to first service (DTFS).

 

2.1.1 Calcium Blood Serum Measurement

At 12-24 hours post calving, a blood sample from the coccygeal vein was taken with veterinary supervision. Examination gloves were worn and the cow safely restrained in a crush (Hubrecht and Kirkwood, 2010). The procedure was followed as documented by Bassert and Thomas (2014) and D’Andrea and Sjorgen (2014).

Blood samples were taken using appropriate sample tubes without preservatives (Estridge and Reynolds, 2012) which were widely available at Home Farm. Their primary use is blood chemistry; including calcium serum analysis (Holmes, 2014; Estridge and Reynolds, 2012). Haemolysis issues can occur in poorly handled samples (Holmes, 2014; Haskell, 2008), although the laboratory did not indicate any inadequate samples. The blood samples were refrigerated (Prendergast, 2015; Sirois, 2015) to preserve the blood and prevent biochemical breakdown (Hazelwood and Burgess, 2009). Samples were stored for a maximum time of 2 weeks until collection by The George Veterinary Group.

Calcium serum analysis was done using a widely available IDEXX analysis machine which analyses the blood serum calcium following centrifugation (IDEXX Laboratories, 2016). Normal serum calcium levels range from 2.2-2.6 mmol/l with clinical signs usually occurring when levels fall below 1.5mmol/l. Therefore, indicating the subclinical level range falls between 1.51-2.19mmol/l (NADIS Ltd, 2015; DeGaris and Lean, 2008).

 

2.1.2 Metritis Assessment

In order to assess for metritis, a manual vaginal examination of each cow was undertaken 10-14 days postpartum, as after 14-days metritis can progress to endometritis (Sheldon et al., 2009). The vaginal discharge characteristics were evaluated as described in previous studies (Goshen and Shpigel, 2006; Murray, Alison and Gard, 1990) using the metritis scoring system proposed by Sheldon and Dobson (2004) as a basis of evaluation (Table 1) which has been universally utilised to score both metritis and endometritis. Due to the visual nature of metritis scoring, results can vary upon individual interpretation, as Sannmann and Heuwieser (2015) found in a study with fifteen metritis assessors, concluding the results were subjective. With the aim of maintaining consistency within this study, all the scorings were carried out by one individual.

Table 1. Clinical metritis scoring system based on mucus characteristics (adapted from Sheldon and Dobson, 2004).

Hagain Figure 1

Sheldon and Dobson (2004) measured out the vaginal discharge into 50ml pots to maintain consistency of analysis, however, this may be interpreted as being impractical to both farmers and Veterinarians when assessing several cows postpartum. Like many more recent studies (Dervishi et al., 2016; Armengol and Fraile, 2015; Cui et al., 2015; McLaughlin et al., 2013) the metritis assessment was conducted on the discharge obtained through manual palpation of the vagina and the acquired discharge visually assessed. As this alteration to the procedure results in the overall examination being less time consuming and laborious, it would make it more likely for farmers to implement the procedure into their postpartum routines in the future.

A printed recording sheet, to note the previously discussed variables, was kept at Home Farm. This aimed to ensure that both the Farm and Herd Manager were fully aware of any procedures required, including systemic antibiotics for a grade 3 metritis score.

 

2.2 Statistical Analysis

Quantitative data was obtained which allowed statistical testing and descriptive statistics using IBM SPSS Statistics and Microsoft Excel. The data was examined for normality (Field, 2013) which identified if the data was parametric or non-parametric. This determined the subsequent statistical tests employed. If the data was found to be parametric, a Pearson’s Correlation was proposed to be employed between calcium serum levels and metritis scores. However, if the data was non-parametric a Spearman’s correlation was proposed to be used (Field, 2013). In order to test for differences between DTFS and calcium categories, and DTFS and metritis the subsequent statistical test used was proposed to be either the one-way ANOVA test or Kruskal-Wallis test depending on whether the data was parametric or non-parametric. Due to the relatively small sample size of 30 cows, which then decreased to 22, the Shapiro-Wilk test was chosen over the Kolmogorov-Smirnov test to assess whether the data was parametric  or non-parametric (Table 2).

 

Table 2. Results from the Shapiro-Wilk test for normality

Study Variable Normality Test Result
Calcium Serum Measurements P=0.995
Calcium Categories P=0.000
Metritis Scores P=0.002
DTFS P=0.134

 

Due to the combination of parametric and non-parametric data, further statistical analysis required the use of non-parametric specific tests, due to not all the data meeting the requirements of a more robust parametric test (Field, 2013; Petrie and Watson, 2013; Pagano, 2009). The tests used therefore were the Spearman’s Correlation Test and the Kruskal-Wallis test.

 

2.3 Ethical Considerations

Manual vaginal examinations of cows postpartum can be considered stressful (Pilz et al., 2012) therefore any discomfort was minimised where possible. The herd would undergo a vaginal examination postpartum in absence of the study taking place, therefore welfare standards were not compromised. If any infection was identified, the individual would also benefit from the subsequent treatment provided.

With regard to any scientific research, the 3 Rs (Refinement, Replacement, Reduction), described and outlined by Fenwick, Griffin and Gauthier (2009) are paramount. The importance of only using the minimum number of subjects to gain sufficient data was recognised and the use of additional subjects was avoided through only one blood sample being taken per study individual under supervision. Refinement was adhered to by only performing one vaginal examination postpartum in order to minimise any distress due to being separated from the herd and restrained in a crush. Due to the aims of the research, human or other animal participants were not suitable.

 

3.0 Results

Thirty multiparous cows had blood samples taken postpartum. Subsequent to this, two cows were removed from the study (Table 3) leaving a cohort of twenty-eight. Of the twenty-eight cows reaching the metritis scoring 10-14 days postpartum, another six cows were removed from the study for several reasons (Table 3). Therefore, only twenty-two cows were considered for assessing the second aim of the study.

 

Table 3. Reasons for removing study individuals, along with their relating calcium level

Stage of Study Removed Before Number of Cows Reason Calcium Category the individuals fell into
Metritis Score 1 Suspected Fatty Liver Clinical
Metritis Score 1 Culled to Severe Lameness Subclinical
First AI Service 2 Unable to breed again Clinical

Normal

First AI Service 2 Injury and culled for welfare reasons Subclinical

Subclinical

First AI Service 1 Displaced Abomasum Subclinical
First AI Service 1 ‘Downer’ cow, due to milk fever Clinical

3.1 Calcium and Metritis Analysis

The blood calcium serum results ranged from 0.7-2.38mmol/l with an average of 1.53mmol/l, with Figure 1 demonstrating the hypocalcaemia categories found within the study population. Even though 50% of the cows had clinical serum levels (<1.5mmol/l); only 4 cows displayed clinical hypocalcaemia symptoms requiring treatment.

 

Hagain Figure 1

Figure 1. Prevalence of hypocalcaemia in the study population

Metritis scores ranged from 0-3 with the average score being 1. Only three cows presented with clinical metritis, requiring treatment.

 

3.2 Relationship between Calcium Serum and Metritis

The Spearman’s Correlation found no relationship (r=0.151, n= 28, p= 0.443) between the two variables (Figure 2).

 

Figure 2 Hagain

Figure 2. Graphical representation of the relationship between calcium levels and the subsequent metritis score, with a line of best fit shown.

 

3.3 Calcium Categories and Metritis affecting DTFS

A Kruskal-Wallis test was performed, to test for difference between calcium categories and DTFS. No significant difference was found (P=0.444), and therefore the null hypothesis was retained, as shown graphically in Figure 3.

Figure 3 Hagain

Figure 3. A box plot showing the mean DTFS from each calcium category; 1 (normal), 2 (subclinical), 3 (clinical). The interquartile range is shown by the area of the box, with standard deviation represented as lines away from the mean. Outliers demonstrated by circles. The black line for calcium category 1 is a result of only one participant.

 

A Kruskall Wallis test was also run between DTFS and metritis scores, with no significant difference being found between the variables (P=0.181) and therefore the null hypothesis was retained. This is again demonstrated graphically in Figure 4.

Figure 4 Hagain

Figure 4. A box plot showing the mean DTFS compared to each metritis category score. With the interquartile range is shown by the area of the born, with standard deviation represented by the whiskers.

 

4.0 Discussion

The two aims of the study were to determine if there was a relationship between calcium levels postpartum and metritis and to identify if the onset of oestrus, determined by DTFS, was affected by the blood calcium category or the metritis score of the study individual. Due to the resulting data analysis showing there was no significant effects; both of the null hypotheses were retained, and the alternate hypotheses rejected.

The average calcium serum measurement was 1.5mmol/l, which is the threshold for clinical hypocalcaemia (NADIS Ltd, 2015; DeGaris and Lean, 2008). This suggested that very few of the study population had adequate calcium levels to enable them to cope with the sudden loss of calcium to produce colostrum. With 50% of the population being within the clinical range, this may be an indicator of poor on-farm mineral management within pre-partum feeding.

Metritis scores showed a much healthier range with only 10.7% of the population showing clinical metritis, compared with the national estimated average of 20-40% (Mozzi, Raya and Vignolo, 2016; Haskell, 2008) which indicates a high level of welfare. This may therefore account for the lack of a significant relationship found due to a high prevalence of clinical hypocalcaemia and a low prevalence of metritis. The low prevalence found (10.7%) may be due to using third parturition and above dairy cows, as it is well documented that primiparous cows are more susceptible to metritis compared to multiparous cows (Toni et al., 2015; Wittrock et al. 2011; Haskell, 2008; Markusfield, 1987).

The average DTFS was 57 days, which is under the target recommendation of 65 days (NADIS Ltd., 2016; DairyCo, 2010). This suggests that the study population had a decreased voluntary waiting time and increased submission rate (DairyCo, 2010). The recommended range of DTFS is 60-65 days, with the aim of achieving a 375-380 day calving interval (NADIS Ltd., 2016), which from this average, the study population were predicted to achieve.

 

4.1 The relationship between blood calcium levels and metritis scores

The main aim of the study was to identify whether a relationship exists between blood calcium levels within 24 hours post-calving and the metritis score 10-14 days later. The data analysis showed there was no relationship. Only three clinical cases of metritis were found, therefore this may have potentially led to inaccurate results. Previous hypotheses suggest that the lower calcium levels from metabolic disorders increase susceptibility to inflammatory conditions, including metritis, due to a compromised immune system (Galvão et al., 2010; Cai et al., 1994). Ducusin et al. (2013) investigated the effects of extracellular Ca²+ concentration on phagocytosis during the periparturient period, as El-Samad, Goff and Khammash (2002) had also hypothesized that the increased susceptibility to inflammatory conditions increases even more if the cow has experienced parturient paresis from hypocalcaemia. Ducusin et al. (2013) employed a similar study population to the one employed in the present study; twenty-two third parturition and above Holstein dairy cows. The cows were split into two cohort groups, one showing clinical signs of paresis and a control group which experienced no signs of hypocalcaemia or paresis. The authors concluded that when compared to clinically normal cows, hypocalcaemic cows had significantly lower levels of phagocytosis and this may partially contribute to the greater susceptibility to inflammatory infections.

There is limited consistent research regarding the presence of a relationship between hypocalcaemia and metritis; with this study’s findings coinciding with previously discussed research (Potter et al., 2010; Bruun, Ersbøll and Alban, 2002). Despite this, Martinez et al. (2012) concluded that there was a relationship through a prospective cohort study in Holstein dairy cattle. The study population was monitored daily for uterine discharge during the first twelve days in milk postpartum. This may have increased the likelihood of the cows developing metritis, as it is well documented that human intervention during calving may introduce new bacteria into the uterus (Bruun, Ersbøll and Alban, 2002). By performing vaginal examinations daily for twelve days, this interaction may have disrupted the normal microflora in the uterus and reproductive tract (Fleischer et al., 2001), predisposing the individual to developing metritis. Blood samples were also taken daily for three days. In order to identify subclinical hypocalcaemia one of the three samples had to be measured at ≤2.15mmol/l, as the authors identified this as their cut off point; just below the value considered in this study. The study found that the cows considered at low risk (normal calving) and that had subclinical hypocalcaemia had a significantly greater risk of developing metritis compared with low risk cows with normal calcium levels (40.7% vs. 14.3%). The authors concluded that overall the risk of developing metritis as a result of subclinical hypocalcaemia was 66.6%, identifying a definite relationship between hypocalcaemia and metritis. The large study population of 110 multiparous and primiparous cows may have aided the identification of a relationship due to a larger data set; unlike the present study where this was not possible. However, the study did not make any specific acknowledgment to clinical hypocalcaemia with regards as to whether clinical cases were included or excluded from the study. Although, as subclinical hypocalcaemia is often overlooked it is an important outcome that there is a relationship. In order to minimize suboptimal production, farmers should therefore consider routine blood sampling within 48 hours of calving to identify subclinical hypocalcaemic cows, as well as clinical cases.

Conversely, El-Samad, Goff and Khammash (2002) and Larsen et al. (2001) stated that nearly all dairy cows will experience some degree of hypocalcaemia post calving. Therefore, the authors suggested subclinical hypocalcaemic cows with no clinical signs of parturient paresis may be regarded as in a “normal” physiological state. This statement may begin to explain the high prevalence of subclinical hypocalcaemia found in this study of 43%, however other variables such as pre-partum diet may also play an important role (Wu et al., 2008). Also due to this statement, Ducusin et al. (2013) grouped subclinical and normal calcium levels together, resulting in no separate evidence on subclinical hypocalcaemia. Unfortunately, due to the lack of relationship found in this study, no further analysis was employed into whether clinical or subclinical hypocalcaemia had a stronger relationship with the subsequent metritis score.

 

4.1.1 Blood Calcium Category Ranges

As 50% of the study population had clinical hypocalcaemia levels, this would suggest that up to 50% presented symptoms and required treatment. However, only 13% (four cows) presented noticeable clinical symptoms; with a blood calcium serum range of 0.7-1.45mmol/l. However, the cow with 1.45mmol/l calcium showed less severe clinical symptoms than the other three, which all had calcium levels lower than 1mmol/l and displayed paresis. These results indicate that despite some cows having extremely low blood calcium levels, no clinical symptoms were displayed. This may suggest that the clinical ranges may need revising to have a further category of severely clinical hypocalcaemia of >1mmol/l where treatment is essential. If clinical symptoms do not present, the farmer would not tend to initiate treatment. This may result in a lower standard of welfare in this group of individuals due to the expectation of increasing milk yield putting more stress onto the already low calcium levels.

Another contributing factor to this would be that some clinical symptoms, such as cold extremities (especially the ears), can be difficult to diagnose in a herd situation. This may begin to explain why only four cows were considered as demonstrating clinical symptoms. The weather can also be considered a confounding factor as external temperatures affect the cow’s homeostasis, resulting in the extremities being colder in order to preserve body heat (Divers and Peek, 2008). As many of the calvings considered in the study took place in autumn/winter, the colder environmental temperature may have resulted in clinical hypocalcaemia diagnosis being more difficult.

 

4.2 Blood Calcium Categories and Metritis affecting DTFS

The second aim of the study was to identify if there was any impact of the blood calcium and metritis on the onset of oestrus, identified by the number of days to first service. Data analysis showed that there was no significant difference between metritis scores and DTFS. Piccardi et al. (2016) studied nine commercial dairy herds, with a lower yearly milk yield compared to Home Farm, across 690 calvings. Even though the authors assessed all cows available in the herd, due to splitting the population into primiparous and multiparous cows, the results can be compared to the present study. Multiparous cows considered as having a normal calving or no assistance were assessed for metritis 3-10 days postpartum, through the same protocol as the present study, undertaken by the visiting Veterinarian. The results concluded that multiparous cows with clinical metritis showed a significant difference in the DTFS of a 20.6% increase compared with multiparous cows without any signs of metritis (68 vs. 54 days). The results from Toni et al. (2015) were in agreement with Piccardi et al. (2016), who also assessed for metritis between 3-10 days postpartum on three farms. The authors concluded that the presence of metritis in multiparous cows increased the DTFS when compared to multiparous cows without metritis. Even though the present study found no significant difference, this may be due to only three cows within the study developing clinical metritis which therefore did not provide a large enough sample for statistical significance.

The analysis also showed that there was no significant difference between calcium categories and DTFS. There is a distinct lack of research in determining whether calcium categories influence DTFS. Martinez et al. (2012) concur with the findings of this study by concluding subclinical hypocalcaemia did not influence the renewal of the oestrus cycle by 38 days in milk, hence the DTFS was not affected. On the other hand, cows with subclinical hypocalcaemia had a reduced pregnancy rate compared to cows with normal calcium levels. However, Martinez et al. (2012) did not indicate whether clinical hypocalcaemia affected the renewal of the oestrus cycle, and therefore the effect of clinical hypocalcaemia on DTFS.

 

4.3 Limitations of the Study

As the study only took place over a 16-week period, only thirty multiparous cows were available to be included. Ducusin et al. (2013) utilized a smaller sample of twenty-two dairy cattle and found significant results, whereas other previously discussed studies utilised between 4 and 2144 cattle herds (Potter et al., 2010; Kim and Kang, 2003; Bruun, Ersbøll and Alban, 2002). These studies may not have had a specific time restraint which allowed large sample sizes to be obtained. In the absence of such restraint a larger study population would have been employed with the aim of improving the reliability of the results. Due to the relatively small study population size, which further decreased during the study, this may have impacted on the statistical significance as previously considered.

 

4.3.1 Weather and Seasonal Bias

Cattle are polyestrous breeders (Akers and Denbow, 2013; Houpt, 2011) therefore seasonal changes can influence oestrus expression and this therefore may have affected DTFS (Collier, Dahl and VanBaale, 2006). Along with this, the study relied on an artificial insemination (AI) technician to correctly identify when to inseminate the cows, and consequently the individual’s experience may have influenced the calculated DTFS through human error (Broom and Fraser, 2007; Gordon, 2004). However, Home Farm employ specifically trained individuals to attend the farm on a daily basis to assess for oestrus behaviour and perform inseminations where appropriate; increasing the reliability of this aspect. If the researcher had performed the fertility data themselves, it would not have improved the reliability; due to not being adequately trained in how to assess for oestrus and thus determine the optimal time for insemination. Furthermore, the researcher would not have been able to attend daily to shadow the AI technician. In addition to this, the majority of the data collection occurred during autumn months when cattle tend to be less active and therefore tend to display a smaller range of oestrus behaviours compared to summer months (Landaeta-Hernandez et al., 2002). This therefore may have affected the DTFS as oestrus may not have been picked up as early by the AI technician and subsequently served the study population later than if the study had taken place in summer months.

 

4.3.2 Methodology

With regard to the attained blood samples, they were held in the on-farm refrigerator for a maximum of two weeks. Even though it is common practice to refrigerate blood samples in order to prevent biochemical breakdown (Hazelwood and Burgess, 2009), there is a lack of research into how long blood samples can be kept before they are compromised. Due to this, it is relatively unknown whether the calculated calcium serum levels were truly representative of the individual at the time of blood collection. Although samples taken from cows that displayed severe clinical symptoms of hypocalcaemia were representative of their analyzed serum level. Of the four cows which displayed symptoms including; paresis, head tremors and the characteristic S-shaped neck due to the skeletal muscle weakness (Fox et al., 2015), three of the cows had calcium levels significantly lower than the cut-off value for clinical hypocalcaemia (<1.5mmol/l). This suggests that the IDEXX analysis machine is reliable and the biochemical breakdown was minimized through refrigeration.

Another further limitation of the study is the use of secondary data through using the on-farm databases to access information on the cows’ lactation and DTFS. As the secondary data was not collected by the researcher this may decrease the reliability (Boslaugh, 2007), however regarding lactation, there are little alternatives to obtain such data. Moreover, using secondary data allows the researcher to obtain a large data set, which with a restrictive time limit is a significant advantage. Another piece of secondary data was the calcium serum levels acquired from The George Veterinary Group. Again if the researcher had performed the calcium serum analysis, this may have improved the reliability however, the appropriate equipment was not available and therefore this was not possible. The IDEXX analysis machine used at The George Veterinary Group practice is widely available (IDEXX Laboratories, 2016) and considered reliable by Veterinarians to assess dairy herd health.

 

4.4 Future Research Considerations

With the limitations in mind, it would be beneficial to study all of the third parturition and above cows from the herd for a full 365 days. This would improve the reliability of the results due to creating a larger sample size (Kececioglu, 2002), and therefore would provide a better representation as to whether there is a relationship between hypocalcaemia and metritis. Additionally, seasonal bias on oestrus behaviour would also be minimised by following the herd through a full breeding season. Mahnani Sadeghi-Sefidmazgi and Cabrera (2015) found, within their five-year study, that seasonal difference had a significant effect on metritis. Considering the herd in a cohort study for an extensive period of time would also allow a greater volume of data to be collected and certain long-term effects to be considered. A 305-day milk yield could be extracted from the farm’s National Milk Records to see if there is a relationship between an increased milk yield as a potential risk factor for both hypocalcaemia and metritis. Further analysis could be made regarding the subsequent effects on oestrus through measuring not only the DTFS, but the number of serves to conception and the days from calving to conception. This increased data set would aid in providing a suggestion as to whether hypocalcaemia and/or metritis affected any of these key indicators of fertility.

Another prospect for further research would be to consider performing the study in several farms across the UK with similar management systems to Home Farm to reduce bias. This would allow direct comparisons to be made between farms and the successive results would be more representative of the UK dairy industry, which may help to improve current management techniques of these disease states if significant results were found.

 

5.0 Conclusion and Applications

Previous research has failed to agree on identifying whether or not a relationship exists between hypocalcaemia and metritis. Whilst it has been well documented that metritis has an effect on the subsequent initiation of the oestrus cycle, it is yet to be ascertained if calcium categories also have an effect. In this study, blood calcium serum levels, metritis scores and DTFS were analysed. The results found that there was no relationship between hypocalcaemia and metritis, which contradicts previous literature stating metabolic disorders such as hypocalcaemia can predispose postpartum inflammatory diseases due to compromising the cow’s immune system. There was no significant difference found between calcium categories; normal, subclinical and clinical, and DTFS or between metritis scores and DTFS. The results regarding hypocalcaemia corresponded with previous research in that it does not influence DTFS, however, there is very limited literature surrounding this topic and therefore a definite conclusion cannot be made. Finding no significant relationship between metritis and DTFS contradicts previous literature which has consistently identified metritis to prolong the initiation of the next oestrus cycle. Although, this result may be due to the small study population and an even smaller group of clinical metritis cases leading to statistical inaccuracy.

There were several limitations within the research which included; a specific time constraint, a varying population throughout the study, weather and seasonal bias, the use of secondary data and the undocumented effects of storing blood samples for up to two weeks. To overcome some of these limitations, it would be beneficial in future research to recruit all of the third parturition and above cows in the herd in a 365-day study period to reduce seasonal bias and allow a larger data set to be obtained. Particular emphasis on further key indicators of reproductive performance such as the number of serves and days to conception would also be beneficial. In order for the data to be more representative of the UK dairy population, it would also be beneficial to recruit other farms with similar management systems to increase the sample population. Both, hypocalcaemia and metritis remain prevalent within UK dairy farms, therefore further research is essential to develop a better understanding of preventative protocols. This would not only improve reproductive health and on-farm welfare, but also increase economic profits to the farmer to enable further investment into the dairy and farming industry.

 

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