Effects of soaked hay on lung function and airway inflammation in horses with severe asthma

2.1 Horses

Ten horses with severe asthma belonging to the Equine Asthma Research Laboratory of the Université de Montréal were studied. All horses had history of severe asthma with documented clinical signs (breathing difficulty at rest and coughing), pulmonary neutrophilia, and difference in transpulmonary pressure (ΔP_L) >15 cmH₂0 when exposed to dry hay. Other than their respiratory condition, they were deemed healthy based on history, physical examination and complete blood count. The number of horses was selected based on an expected 50% to 65% decrease in lung resistance, based on previous studies. All experimental procedures were performed in accordance with the Canadian Council for Animal Care guidelines and were approved by the animal care committee of the Université de Montréal.

2.2 Study design

Exacerbation was induced over a 4-week period during which horses were housed indoors and fed dry hay. Throughout the study, they were housed on wood shavings, had access to a dry paddock for 2 to 6 hours a day, and received 500 mg of supplements with vitamins and minerals daily. Horses received either “dusty hay” (hay that had been bailed wet the previous summer), “normal hay” (subjectively less dusty), or a mix of both, based on their history of previous exacerbations over the previous 2 years (type of hay and turnouts duration typically inducing a clinical exacerbation over a 4-week period). The same hay given during the exacerbation period was used during the study period, that is, horses receiving dusty hay for exacerbation received soaked dusty hay if they were allocated to the soaked hay group. In compliance with the animal care protocol, clinical scores, attitude, and appetite were monitored at least 3 times a week, in addition to daily observations by barn personnel. Horses with severe and persistent clinical signs were required to have increased turnout time, to receive short-acting bronchodilators (salbutamol), or to be excluded from of the study and receive intravenous dexamethasone if high scores persisted despite initial interventions. After the exacerbation period, turnouts varied between horses but remained the same for an individual horse from weeks 0 to 6.

Once in exacerbation (week 0), horses were ranked according to the severity of their respiratory obstruction (resistance [R_L] value). The “odd” group consisted of horses 1 (highest resistance), 3 (3rd highest resistance), 5, 7, and 9, and the “even” group consisted of horses 2 (2nd highest resistance), 4, 6, 8, and 10. Group allocation was then done by flipping a coin: the odd group received alfalfa pellets and the even group received soaked hay. Horses received approximately 2% of their body weight in hay or pellets daily. The hay was completely immersed in cold water for 45 minutes in a large storage container. It was then coarsely drained before being placed at ground level in the stalls. The remaining hay that had dried was discarded before the next meal. Pellets were placed in a feeder. Respiratory clinical scores and weight were assessed weekly and lung function was measured on weeks 0, 2, 4, and 6. Pulmonary inflammation was evaluated on weeks 0 and 6 with bronchoalveolar lavage fluid (BALF) cytology and tracheal mucus scoring. To evaluate the persistence of residual bronchoconstriction, pulmonary function was measured at the end of the study, before, and 10 minutes after the administration of a bronchodilator (hyoscine butylbromide, 0.3 mg/kg, IV, Buscopan, Boehringer Ingelheim Vetmedica). Evaluators were blinded to group allocation for clinical scores and lung function, and to group and time points for BALF cytology and mucus scoring.

2.3 Pulmonary function

Respiratory clinical scores ranged from 2 to 8, based on nasal flare and abdominal effort during breathing.^{25, 26} The measurement of lung function was performed on horses at rest, without sedation. The difference in transpulmonary pressure (∆P_L) was estimated using a rigid esophageal balloon catheter, introduced to mid-thorax and connected to a differential pressure transducer. The flow of air was measured using a pneumotachograph connected to a rigid face mask that allows tidal volume measurement (Flexiware 7.6, SCIREQ, Montréal, Quebec, Canada). Lung resistance (R_L) and lung elastance (E_L) were determined using the change in transpulmonary pressure and flow via a multiple regression equation for the single compartment model of the lung (ΔP_L = E_LV + R_LV˙ + k; V is the volume, V˙ the airflow, and k the transpulmonary end-expiratory pressure).

2.4 Tracheal mucus scoring and bronchoalveolar lavages

The amount of mucus was assessed blindly by a veterinarian (RW) from the images taken during bronchoscopy according to a score ranging from 1 to 5 as previously published.²⁷ Bronchoalveolar lavage was performed under sedation with xylazine (0.4 mg/kg, IV) and butorphanol (10-20 μg/kg, IV), using a 1.6 m video-endoscope introduced through the nose and into the right main bronchus. A local anesthetic was infused in the distal trachea and bronchi (60 mL of 0.5% lidocaine hydrochloride). Two boluses of 250 mL of warm isotonic saline were instilled and re-aspirated. Cytocentrifugations from unfiltered BALF were prepared and stained with a modified Wright-Giemsa solution. Bronchoalveolar lavage cytology differential was evaluated on 400 cells, excluding epithelial cells, by a board-certified clinical pathologist (CB). The proportion of neutrophils was considered normal when below or equal to 5%.

2.5 Statistical analysis

Lung function, differential cell count and weight were analyzed with a linear mixed model with treatment (soaked hay vs pellets), time and the interaction between treatment and time as fixed effects, and with the treatment nested within the subject as random effect (this accounts for repeated measures for each subject over time with each treatment). A priori contrasts were performed to compare the means within the same treatment at different time points compared with week 0, and the means between treatments at each time point. The alpha level was adjusted for multiple comparisons using the Benjamini-Hochberg method. For BALF cytology, normality of the residual values was evaluated visually with quantile-quantile plots. Lung function data was log-10 transformed to normalize the distributions. Ordinal data, mucus and respiratory clinical scores were analyzed with the Cochran-Mantel-Haenszel test. Spearman correlation test was used to evaluate the association between pulmonary function parameters, clinical scores, and cell count differential in bronchoalveolar lavage. Pre- and post-bronchodilation data were compared with paired t-tests. Where appropriate, data are represented as the difference between group means with a confidence interval (CI) of 95%. P value <.05 was considered statistically significant. Analyses and figures were performed using Statistical Analysis Software (SAS vs. 9.3, Cary, North Carolina) and GraphPad Prism (9.0.1, GraphPad Software).

3.1 Horses

Six mares and 4 geldings were studied. They were 14.2 (mean) ± 2.4 (SD) years old, weighed 540 ± 63 kg, and were of various breeds (8 Quarter Horses or Paint Horses, 1 Canadian Horse, 1 Thoroughbred). During the exacerbation period, turnouts were adjusted as described above, but no medication was administered, and no horse was excluded.

3.2 Respiratory clinical scores

Clinical scores decreased gradually over time in both groups (P = .0002 and <.0001 for soaked hay and pellets, respectively), with no significant difference between groups. Contrasts comparing scores to baseline in each group were not significantly different, after adjustments with the Benjamini-Hochberg method (Figure 1).

3.3 Pulmonary function

The ∆P_L and E_L were significantly different from baseline at all time points, in both groups. The R_L was significantly different from baseline at all time points for the group fed soaked hay but was only different from baseline at week 4 for the group fed pellets (not significant after correction at week 6). There were no significant differences between the groups at each time point (Figure 2). At the end of the study, 4 out of 5 horses fed soaked hay had ∆P_L ≤ 10 cm H₂O, R_L ≤ 1.0 cm H₂O/L/s, and E_L ≤ 1.0 cm H₂O/L, while in the group fed pellets, only 1 horse was within normal ranges for all 3 parameters, and 2 others were in the normal range for elastance.

In the pellet-fed group, a significant decrease in ∆P_L and R_L were observed after hyoscine butylbromide administration, indicating the presence of residual bronchoconstriction on week 6 in this group. In the soaked hay group, there were no significant changes and the only horse who showed a marked improvement in lung function after administration of the bronchodilator was the one with lung function data outside the reference ranges (Figure 3).

3.4 BALF cytology

At baseline, all horses had BALF neutrophils >10% and they showed a decrease in pulmonary neutrophilia over the study period. The pulmonary neutrophilia was higher in the pellet group compared with soaked hay group at baseline (mean difference [95% CI]: 31.1 [7.3, 54.9], P = .01), and decreased only in the pellet group over time (P = .001). The decrease in the soaked hay group (P = .04) was not significant after adjustment. At week 6, 1 and 3 horses belonging to the pellet-fed group had BALF neutrophils below 5% and 10%, respectively, while in the soaked hay group, 2 and 5 horses had BALF neutrophils below 5% and 10%, respectively. There was no significant difference for mast cells, and eosinophils were absent from BALF (Figure 4).

For macrophages and lymphocytes, the mean was significantly higher at week 6 compared with baseline in the pellet group (P = .005 and P = .009, respectively). The soaked hay group showed the same result only for lymphocytes (P = .01).

3.5 Mucus scores

The mucus score decreased gradually over time in the soaked hay group (P = .03) but not in the pellet group (P = .75; Figure 5).

3.6 Body weight

In the soaked hay group, body weight decreased significantly between weeks 0 and 6 (P = .003), while there was no significant difference in the pellet group (Figure 6). The weight loss was 20.5 kg on average and ranged from 12 to 30 kg.

4.1 Summary

In this study, soaked hay improved respiratory clinical scores, lung function, and tracheal mucus scores, but not pulmonary neutrophilia, while pellets improved clinical scores, lung function, and pulmonary neutrophilia.

4.2 Soaked hay improved clinical signs and lung function

The improvement in respiratory clinical scores is consistent with the decrease in cough, nasal discharge, and overall clinical improvement reported by owners of horses with asthma after soaking hay.²⁰ However, perceived poor response in some horses is also reported,^{17, 19, 20} and this could contribute to a general impression of ineffectiveness, despite evidence of dust reduction with soaking.^21-24 Several elements could explain the relatively high efficacy of hay soaking in this study compared with the common perception. First, some owners will only briefly water down the hay, leaving much of a bale or flakes dry in the middle,¹¹ or will immerse the hay for a short period of time (<30 minutes).¹⁰ It is also unlikely that dried out hay would be consistently discarded by owners. In addition to this, the improvement in clinical scores was observed only after 2 weeks, which could be longer than owners might be willing to wait before concluding whether a treatment is effective or not. Finally, all the horses in the study were fed a low-dust diet of either soaked hay or pellets and were housed on wood shavings, which decreased the overall dust in the barn. Wood chip bedding decreases respirable dust in the breathing area⁵ and in the ambient air,^{7, 28} and the combination of wood shavings and hay pellets reduces respirable particles in the breathing zone by up to 50%.³ It was also shown that optimizing the management with soaked hay and wood shavings resulted not only in a significant reduction of respirable dust in the main stable, but also in an adjacent building with common airspace.⁵

Thus, since all horses were subjected to a low-dust diet and benefited from the low-dust environment, the improvement in function and inflammation observed in both groups could be greater than achievable in practice. In a large stable, it would be difficult to feed all horses with soaked hay or other low-dust diets just to accommodate a small number of asthmatic horses.

4.3 Lung inflammation decreased with pellets

Even if BALF neutrophilia decreased for all horses over the 6-week period, the comparison of the effects of soaked hay and pellets on pulmonary neutrophilia was complicated by the fact that the neutrophilia was unexpectedly higher in the pellet group at baseline. Pulmonary neutrophilia is positively correlated with respirable dust in the horse's breathing zone^{9, 29} and is therefore expected to decrease with environmental avoidance strategies. The persistence of mild pulmonary neutrophilia in some horses in the soaked hay group could be explained by the transient increase in respirable particles that become airborne when the hay dries out.³⁰ This might not have played a major role in this study because of hay removal, but it could have an impact in barns with less stringent practices. In the pellet group, the 2 horses that had close to 20% neutrophils in their BALF at the end of the study were also amongst the 3 horses with the highest percentage at baseline. Pulmonary neutrophil percentage is usually positively correlated with mucus score^{27, 31} but here, the mucus scores decreased only in the soaked hay group. This could be in part due to the head position during feeding as horses in the soaked hay group were fed on the ground while those in the pellet group ate from a feeder. Keeping the head up for longer periods of time could have impaired the mucociliary system, which could lead to a decreased clearance of particles and debris in the trachea.³²

It should also be considered that the study duration might not have been sufficient to observe a normalization of pulmonary neutrophilia. In a study on an oiled mixed hay feeding system, pulmonary neutrophilia was significantly improved only after 2 months of treatment with either oiled mixed hay or pellets,⁴ and it can take a few months of grazing to observe pulmonary neutrophils close to 5%.¹² Finally, the small number of horses combined with the relatively low BALF neutrophilia at baseline in the soaked hay group is likely to have affected our ability to detect a significant decrease in pulmonary neutrophilia in this group.

4.4 Limitations of hay soaking and practical considerations

Despite clinical improvement of horses in this study, it is important to remember that soaked hay increases the number of bacteria by 1.5 to 5 times for 10 minutes and 9 hours of soaking, respectively, suggesting rapid replication of bacteria from the time the hay is immersed in water and in the 2 hours after the hay soaking treatment.^{10, 22, 33} This suggests an increase in exposure of horses to endotoxins that will either be ingested or be resuspended when the hay starts drying. This could lead to persistent neutrophilia as exposure to endotoxins results in a dose-dependent increase in neutrophils in BALF.^{6, 11, 21} Since soaking hay causes a rapid increase in bacteria, this requires rapid distribution after soaking and removal of leftovers between meals. Leftovers removal increased labor but the volume discarded was difficult to quantify since leftovers were often scattered in the stall and had variable residual humidity. Disposal of post-soaking water effluent, which is an environmental pollutant^{34, 35} can also represent a challenge, especially when outdoor temperatures reach freezing point. All this requires personnel, adapted equipment (large and water-resistant container) and time. Handling is also made more difficult by the added weight, that can reach 4 times its original weight after 20 minutes of soaking.³⁶

4.5 Weight loss in the soaked hay group

Soaking hay provides hygienic qualities but also has undesirable consequences such as loss of water-soluble carbohydrates (WSC), minerals and crude proteins. The report decrease in WSC with soaking is highly variable: 2% to 4% after 12 hours,³⁴ 34% on average after 9 hours,¹⁰ and from 6% to 54% after 16 hours.³⁶ In this study the soaking time was relatively short but losses of minerals, crude proteins and WSC have been observed as early as 10 to 40 minutes following water immersion.^{10, 21, 23, 34, 37} The changes in nutrient composition of hay not only decreases the caloric intake but can also alter the palatability and consumption of hay by changing its taste.^{37, 38} Horses seem to prefer steamed and dry hay over soaked hay.³⁷ In this study the feeding behavior was not recorded, and leftovers were not weighed so we cannot conclude on the main factor driving the weight loss. The weight loss was not considered problematic since all horses had body condition scores of at least 5/9,³⁹ but in addition to usual recommendations of providing minerals and vitamin supplements to horses fed soaked hay, careful monitoring of their weight should be implemented to avoid unexpected weight loss.

In conclusion, soaked hay improves clinical scores, lung function and mucus scores of horses with severe asthma. The strict protocol for soaking and discarding dried-out hay in this study could however be considered too great of an inconvenience in some stables. In addition to this, feeding soaked hay or pellets could be less effective if only 1 or 2 horses are on a dust-reduction program, since there would still be dust from handling dry hay for the other horses. This treatment however consists in an alternative for horses with asthma that do not have access to grass pasture and when other medical conditions or budget constraints limit the use of other alternatives such as pellets, haylage, silage, or oiled mixed hay.