Assessment of serum amyloid A concentrations and biochemical profiles in lactating jennies and newborn Ragusano donkey foals around parturition and one month after foaling in Sicily

Abstract A proper knowledge of biochemical parameters and inflammatory markers like serum amyloid A (SAA) is crucial in the monitoring of the first post‐partum period in equids. Since no information is available on SAA for donkeys at this stage, 50 animals including jennies (n.10) and newborn foals (n.10) within 48 hr from foaling, and jennies (n.10) and foals (n.20) after 30 days from parturition were enrolled in the study to assess routine biochemical profile including SAA. Jennies showed higher alkaline phosphatase and lower bilirubins and cholesterol at 30 days of lactation compared to post‐partum. Neonatal donkey foals showed significant higher concentrations of sodium, alkaline phosphatase, lactic dehydrogenase, blood urea nitrogen, creatinine and albumin within 48 hr of age, whilst higher values of phosphate and triglycerides were observed in older foals of 30 days of age. Significant higher SAA concentrations were recorded during the peripartum period in both jennies (25.95 ± 14.98 μg/ml) and newborn donkey foals (37.44 ± 19.75 μg/ml) compared to SAA values recorded in lactating jennies (2.38 ± 1.78 μg/ml) and in donkey foals (16.04 ± 18.14 μg/ml) at 30 days after parturition. The assessment of SAA in jennies and donkey foals around parturition and one month after foaling represents a valuable tool for the monitoring of health status during this stage when animals have to face with new challenges like the peak of lactation and extrauterine life adaptation respectively.


| INTRODUC TI ON
Pregnancy, parturition and lactation represent specific conditions that significantly influence animal metabolism. The effects of peripartum period on haematological and biochemical parameters have been studied in mares (Bazzano et al., 2014;Mariella et al., 2014) and more recently also in jennies (Bonelli et al., 2016).
Several biochemical parameters change in healthy pregnant subjects, thus confirming the need for proper reference ranges at this stage.
Similarly, when assessing both equine and donkey foals, clinicians need to refer to specific reference ranges according to animal age (Sgorbini et al., 2013;Veronesi et al., 2014).
In the last years, the assessment of specific acute phase proteins has been added to routine haematological and biochemical analysis to support clinicians in the early recognition and differentiation of acute infection and inflammation from other more benign clinical disease (Kay et al., 2019). Serum amyloid A (SAA) is an acute phase protein of the apolipoprotein family (APP), mainly produced by the liver, that rapidly increases in response to inflammation (Long & Nolen-Walston, 2020). Serum amyloid A is the only major positive APP in the horse as its concentrations are low or clinically undetectable in normal animals but rapidly increase from 10 up to 1,000 times following the onset of the acute phase response (Nolen-Walston, 2015).
Furthermore, SAA concentrations increase after 6 hr from the stimulus and decrease within 12 hr from the end of the disease because of its short half-life (30-120 min) (Long & Nolen-Walston, 2020).
Several studies have investigated SAA concentrations in horses affected by respiratory disease, colic, orthopaedic conditions or undergoing surgical procedures . Recently, a certain interest has been directed at SAA modifications in mares affected by reproductive diseases (Coutinho da Silva et al., 2013). Studies have also been performed in healthy horses during different type of exercise (Piccione et al., 2016;Witkowska-Piłaszewicz, Bąska, et al., 2019) or specific physiological state like peripartum in broodmares and neonatal foals (Chavatte et al., 1992;Krakowski et al., 2020;Stoneham et al., 2001). On the contrary, SAA concentrations have been scarcely investigated in domestic donkeys (Kay et al., 2019;McLean et al., 2016) and only one report exists on feral donkeys (Jerele et al., 2020).
Despite the increasing interest in donkey medicine and the need for specific reference range, no information is available approximately SAA values in healthy subjects. Considering the high potential of SAA for the early diagnosis of inflammatory diseases in equids, and the economic loss derived from a sick lactating jenny or a sick donkey foal, the early diagnosis of inflammatory disease in donkeys during this stage is of great importance.
Based on the current knowledge, the aim of this study was to investigate the main biochemical parameters and SAA levels in lactating Ragusana jennies and donkey foals reared for milk production during the first month after foaling.

| Animals
All animal housing, care and experimental procedures herein described were in accordance with the standards recommended by the EU Directive 2010/63/EU for experiments on animals. The research protocol was approved by Internal Animal Welfare Committee (approval number 5/2021).
A total of 50 Ragusana donkeys, reared in the same farm in Sicily, were enrolled in the study with the informed owner consent. All subjects underwent clinical examinations to confirm the health status, and only clinically healthy donkeys were included in the study.
Animals were divided into four groups according to the time of sampling: Group A consisted of n.10 jennies within 48 hr from delivery, Group B included n.10 jennies at 30 days of lactation, Group C consisted of n.10 (6 males, 4 females) newborn donkey foals (within 48 hr from birth), and Group D included n.20 (11 males, 9 females) donkey foals (within 1 month of age) ( Table 1). Jennies from Group A (mean age 7.5 ± 2 years, mean BCS (Evans & Crane, 2018) 2.8 ± 0.2) and Group B (mean age 7.7 ± 2.5 years, mean BCS 2.5 ± 0.2) were fed 6 kg/day dried grass hay and 0.5 kg/day concentrates. Donkey foals from both Group C and D were kept with their dams being allowed to nurse 24 hr/day during the experimental period. Animals were housed in individual straw-bedded boxes during the first week after foaling, and then they were moved to common paddocks shared with other dams and foals. Furthermore, animals were allowed to pasture 8 hr/day and water was provided ad libitum.
Blood samples were collected during the routine veterinary procedures performed in the donkey farm, in the morning (9-10:00 a.m.) before feeding, during the month of August 2019. A single blood sample was drawn from the jugular vein of each animal into 10 ml tubes containing clot activators (VACUETTE; Greiner Bio-One GmbH).

| Laboratory analysis
After collection, blood samples were placed on ice and delivered to the laboratory within 2 hr. At the laboratory, samples were centrifuged for 10 min at 1,000 g (Universal 32, Hettich Zentrifugen, Germany) and the obtained sera were divided into two 1.5 ml ali-  (days, d; years, y), body condition score (BCS, 5-point scale) (Evans & Crane, 2018)

| Statistical analysis
Data were analysed using statistical software Prism 8 (GraphPad Software Ltd).
Student's t tests were performed to highlight significant differences in studied blood parameters between Group A and B, and between Group C and D respectively. Values of p < .05 were considered statistically significant.
Statistical analysis was performed with one-way analysis of variance (ANOVA), followed by the Bonferroni test using Sigma-stat 3.1 software (SPSS, Chicago, IL, USA). Significantly different values (p < .05) were indicated in bold letters. Foals from group C showed higher serum concentration of Na, BUN, Crea, ALP, LDH, Alb and Ca/P ratio, and lower P and TG compared to Group D.

| RE SULTS
Mean values and standard deviations (SD) of biochemical parameters recorded for Group A and B are reported in Table 2 together with p values obtained from statistical analysis.
Mean values and SD of biochemical parameters together with p values recorded for group C and D are reported in Table 3.

| DISCUSS ION
Over the last years, several researchers investigated the haematological profile during the peripartum period in donkeys (Bonelli et al., 2016;Sgorbini et al., 2013;Veronesi et al., 2014); however, to the best authors knowledge, this is the first report including SAA assessment in the routine biochemical profile of lactating jennies and neonatal donkey foals.
Comparing our results with the existing literature, few parameters undergo significant modifications in jennies during lactation.
The decrease in serum cholesterol at 30 days of lactation compared to the post-partum observed in our study confirms previous findings in lactating jennies (Bonelli et al., 2016) and mares (Bazzano et al., 2014). According to Milonis and Polidori (Milonis & Polidori, 2021), despite donkey milk has lower fat percentage compared to other domestic species, the content of cholesterol in terms of g/100 g of fat ranges from 0.41 g/100 g to 0.97 g/100 g, that is even higher than cow and woman milk. Serum Alp values recorded in this study are higher compared to reference ranges for donkeys (Evans & Crane, 2018) and significantly increased at 30 days of lactation, in contrast with previous observations by Bonelli and colleagues (Bonelli et al., 2016). However, the tendency of serum Alp at increasing because of lactation has been demonstrated in other mammals like women and cows, by guessing that ALP originating from the mammary glands influenced serum Alp activity to some extent (Sato et al., 2005). Ragusana donkey is a breed mainly reared for milk production as jennies produce higher milk quantities compared to other breeds like the Amiata donkeys included in the study by we found higher mean concentrations compared to previous studies (Sgorbini et al., 2013;Veronesi et al., 2014). LDH tend to decrease significantly in Ragusano donkey foals whereas no significant modification was observed in Martina Franca ones (Veronesi et al., 2014).
Serum TG were found to increase in older Ragusano foals, but no significant difference was found by other authors (Sgorbini et al., 2013;Veronesi et al., 2014). Amongst serum electrolytes, we observed a slight decrease in Na at 30 days of life compared to neonatal period, and a significant increase of p as already found in Martina Franca donkey foals (Veronesi et al., 2014). Differently from other authors who found no significant change in albumin serum concentrations, we observed a slight decrease in Ragusano donkey foals at 30 days post-partum (Sgorbini et al., 2013;Veronesi et al., 2014).
Concerning SAA assessment in donkey species, we observed significant modifications both in jennies and donkey foals during the experimental period. Currently, the commonly accepted reference range for equids (0-20 ug/ml) has been assessed in healthy horses, with no sex-related differences, but an age-related effect has been reported with healthy neonatal foals (<1-week-old) show-  Despite the difference in methods can influence SAA assessment amongst studies, SAA concentrations tend to increase following parturition in mammals as this APP is involved in the process of parturition. According to a recent study by Gan and colleagues (Gan et al., 2020), SAA released by the placenta may participate in the onset of labour irrespective of infection by stimulating the expression of parturition-pertinent inflammatory factors with consequently increased production of proinflammatory cytokines and PGF2α in the placenta. According to our results, healthy foals showed slightly higher SAA concentrations compared to jennies within 48 hr from foaling that, in the authors' opinion, could be due to an early adaptation phase of new-borns to extrauterine life.
The present study presents some limitations: sample size was consistent for run statistical evaluations but not large enough to consider the obtained data suitable for reference range, and the animals included in the study belonged to a single donkey farm. Further studies with larger sample size are necessary in order to define a threshold for health and disease.

TA B L E 3
Mean values ± standard deviations (SD) and statistical significances of biochemical parameters recorded for Ragusano foals useful information approximately the monitoring of health status in these equids reared for milk production (Bordonaro et al., 2013).
Early detection of neonatal and post-partum diseases is to present a diagnostic challenge especially in donkey species. Because of their stoicism, clinical signs in these equids are often subtle and nonspecific and general conditions can worse rapidly. More robust reference ranges for SAA are needed to be used in clinical practice; despite this, the information herein provided could represent an initial help in early diagnosis and prompt therapeutical intervention that may have a major influence on jenny and foal survival, as well as on milk production, with significant economic consequences for donkey farmers.

ACK N OWLED G EM ENTS
The authors acknowledge the Azienda Agricola Arena farm and Mrs.
Mimma Arena for the collaboration during the study. Open Access funding provided by Universita degli Studi di Camerino within the CRUI-CARE Agreement.

CO N FLI C T O F I NTE R E S T
Authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Research data are not shared.