​Analysis of Uterine and Systemic Parameters in Fertile and Barren Mares

Mare fertility is paramount to the success of an equine breeding operation. Mares are typically classified into fertile and barren, depending on their pregnancy status after the breeding season. Establishment of a successful pregnancy is based on the interaction of local and systemic factors (Fox et al., 2016). Uterine status is directly related to fertility and, therefore, its evaluation is essential prior to breeding mares. Endometritis has been reported in about 25-60% of barren mares (Card, 2005) and, consequently, results in great losses to the equine breeding industry. The condition can be diagnosed on the basis of history coupled with external examination, transrectal palpation and ultrasonography, vaginal examination, endometrial cytology, bacterial culture and biopsy (McCue, 2008; LeBlanc and Causey, 2009; LeBlanc, 2010). Pregnancy failures resulting from endometritis have been attributed to conception failure (Bracher et al., 1996; Gilbert, 2011), direct negative effects on the embryo, increased prostaglandin production resulting in luteolysis, or a combination of these factors (Allen, 2001). Apart from the uterine factors, systemic status of the mares influences their fertility. Body condition score (BCS) appears to be important for successful reproduction and rearing of a healthy foal (Morley and Murray, 2014). The BCS is based on visual assessment and palpation of fat in six areas of the horse’s body, namely the lumbar spine, ribs, tail, area behind the shoulder, neck and withers (Henneke et al., 1983). Another factor that can influence the pregnancy outcome in mares is the presence of any systemic inflammatory or infectious condition, commonly characterized by the presence of fever and changes in the blood parameters such as CBC.
 
Considering that breeding success is influenced by multiple factors, the goal of this study was to evaluate the uterine and systemic status of fertile and barren mares. The specific objectives were to investigate the differences in uterine (ultrasonographic appearance of the uterus and endometrial cytology) and systemic parameters (BCS, rectal temperature and CBC) between fertile and barren mares. 

The present study was conducted during 2021-2022 at various stud farms in and around Pune and the Department of Animal Reproduction, Gynaecology and Obstetrics, Mumbai Veterinary College, Mumbai. Mares were classified into fertile (n=15) and barren (n=15) on the basis of their history and breeding records. All mares were reared under standard feeding and managemental conditions.
 
Evaluation and sample collection
 
Each mare was restrained in a travis and examined for body condition score and rectal temperature followed by transrectal ultrasonography and collection of uterine swabs for endometrial cytology. Blood samples were also collected in EDTA blood collection vials followed by CBC analysis using an automated CBC analyser.
 
Body condition score
The body condition of all mares were accessed on the basis of Henneke Body Condition Scoring System (Henneke et al.,1983; Hoopes et al., 2019).
 
Rectal temperature
 
Rectal temperature was measured using a calibrated digital thermometer manufactured by Omron Healthcare India Pvt. Ltd., Gurgaon, Haryana, India.
 
Ultrasonographic examination
 
A real-time B-mode ultrasonography machine (SonoScape A5) with a 5-7.5 MHz multi-frequency linear array trans-rectal transducer was used to scan the uterus (Fig 1).


 
Endometrial cytology
 
Samples were collected using double-guarded sterile swabs. Immediately after collection of the intra-uterine swabs, the swabs were rolled on a clean glass slide followed by fixing of smear with 10% buffered formalin and staining with Fields stain. The stained slides were observed under high power (40x) microscope (Fig 2). The number of neutrophils and epithelial cell in 10 microscopic fields on each slide were counted and recorded. The results were evaluated as per Ferris et al., 2015.

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Statistical analysis
 
The quantitative data were analysed by t-test to compare the mean values using SPSS 24 software (IBM SPSS for Windows, Version 24, IBM SPSS Inc.). Results were described as the mean±S.E. P-values less than 0.05 were considered significant.

Uterine parameters
 
On transrectal ultrasonographic examination of the uterus, intrauterine fluid was observed in 12 out of the 15 barren mares and none of the fertile mares (n=15) showed presence of intrauterine fluid. These findings are in agreement with previous studies that reported presence of intrauterine fluid in a vast majority of barren mares (Ibrahim et al., 2015; Barbary et al., 2016; Kouider et al., 2017; Derbala et al., 2018). The presence of intrauterine fluid in the barren mares suggests that they had endometritis because intrauterine fluid is considered as a reliable indicator of endometritis (Brinsko et al., 2003; Barbary et al., 2016). This assumption is further supported by the results of endometrial cytology. As compared to fertile mares, barren mares had significantly higher neutrophil number and neutrophil: epithelial cell ratio (Table 1). The greater abundance of neutrophils observed in the endometrial smears of barren mares is attributed to increased blood flow and diapedesis of the neutrophils associated with endometritis. All the barren mares in this study had severe inflammation of the uterus as indicated by an average of more than 5 neutrophils per high power field (Ferris et al., 2015). These mares also had a greater than 1% neutrophils: epithelial cells ratio (Table 1). This ratio is considered as a very sensitive cytological evaluation approach for diagnosing -endometritis in mares (Sikora et al., 2015). There is a variation in the cut-off percentages used for diagnosing endometritis in mares ranging between 1% and 5%. The barren mares in the present study had neutrophils: epithelial ratios that exceeded 5% and the average ratio for all 15 mares was in a close agreement with Rua et al., (2018) who reported an average of 10.1% in mares with endometritis.


 
Systemic parameters
 
As shown in Table 2, no differences in the rectal temperature, BCS and CBC parameters were observed between fertile and barren mares. Previous studies conducted by Henneke et al., 1983, Hoopes et al., 2019, Munoz et al., 2021 suggested that the BCS of the brood mares should lie between 4 to 7. In our study, the BCS of all the mares, regardless of their fertility status, was in the 4 to 7 range. Similarly, rectal temperature and CBC values were within the normal range. Hall et al., (2019) recorded that the normal rectal temperature in case of mares is 96.6 to 100.9°F. The blood parameters in equines suffering from endometritis were previously evaluated by Gavazza et al. (2002) who reported that all the blood parameters were within the normal range. Likewise, Gul et al., 2007 studied the haematological values in apparently healthy horses kept under field conditions. The CBC values observed in the present study were also in agreement with the reference values established by Pritchard et al., 2009. The results of the present study suggest that rectal temperature, BCS and CBC are not reliable for differentiating between barren and healthy mares.

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Based on the results of this study, it can be concluded that uterine health status is a major determinant of breeding success in mares. The absence of any difference in the systemic parameters between normal mares and mares with endometritis suggests that these parameters are not reliable indicators of endometritis or fertility in mares. Future studies involving larger sample sizes are required to further our understanding of the determinants of breeding success in the equine species.

The authors are thankful to the Mumbai Veterinary College for providing the research facilities and financial assistance to conduct the study.

 The authors declare that they have no conflict of interest.