extfieldsofstudy: Medicine Biology

text:
Effects of Pair Versus Individual Housing on Performance, Health, and Behavior of Dairy Calves

Simple Summary In modern dairy farming systems, calves are often housed in individual pens or hutches, which results in less social interaction with their peers during the milk-feeding period. The aim of this study was to evaluate the effects of pair versus individual housing on performance, health, and behavior of dairy calves from the milk-feeding period to the first week after mixing. Results showed that pair versus individual housing had no effects on body weight, starter intake or average daily gain during the milk-feeding period, while pair housing increased the growth performance of calves during weaning and postweaning periods, and the beneficial effects of pair housing on growth faded after calves were mixed and moved to group housing. Paired calves showed higher diarrhea frequency only in week three. The behavior of calves was altered at different periods, including increased time spent in feeding, chewing and ruminating, and decreased self-grooming time, and a drop of non-nutritive manipulation for all calves after they were mixed and moved to group housing. We also found less social contact may lead to more non-nutritive manipulation. Abstract The aim of this study was to evaluate the effects of pair versus individual housing on performance, health, and behavior of dairy calves. Thirty female Holstein dairy calves were assigned to individual (n = 10) or pair housing (n = 10 pairs). The results showed that both treatments had a similar starter intake and average daily gain (ADG) during the preweaning period. During weaning and postweaning periods, paired calves had a higher starter intake, and the ADG of paired calves continued to increase but calves housed individually experienced a growth check. Paired calves showed higher diarrhea frequency only in week three. The results on behavior showed that feeding, chewing and ruminating time increased, and self-grooming time decreased with age during weaning and postweaning periods, and paired calves spent less time feeding, standing and self-grooming but more time lying during this time. After mixing, feeding, and chewing and ruminating time continued to rise, and self-grooming time continued to decline for both treatments. All calves spent less time standing and non-nutritive manipulation after mixing, and previously individually housed calves tended to increase non-nutritive manipulation. These results showed that pair housing improved growth during weaning and postweaning periods and that calves altered their behavior at different phases. Less social contact may lead to more non-nutritive manipulation.

Introduction
Under natural conditions, calves are nursed by the dam and tend to have social interactions with their peers or other animals [1,2]. In modern dairy farming systems, however, calves are often housed in individual pens or hutches. Hence, they are less likely to interact with their peers or other animals during the milk-feeding period.
Previous work has indicated that different housing systems (group versus individual housing) affect the performance and health of dairy calves. Some studies showed that compared with individual housing, group housing increased weight gains [3], starter intake [4] and hay intake of dairy calves [5]. Conversely, other studies showed no effects [6] or even negative effects on weight gain for group-housed calves [7]. Furthermore, respiratory diseases and diarrhea were reported to occur more frequently in group-housed veal calves [8]. On the contrary, Babu et al. [9] reported that rearing calves in a group resulted in a lower disease incidence. In other cases, health outcomes were similar between different housing systems [10]. The variability among studies may be related to differences in management (e.g., the number of animals per group, milk volume provided, duration of the feeding period, weaning method, and disease diagnosis). From a behavioral standpoint, weaning from a milk-based diet to a solid diet is one typical stressor faced by dairy calves, in which case, calves vocalize more (d 37 to 55) [11,12]. After weaning, calves are mixed with unfamiliar animals and moved to a novel environment, which may cause aggressive interactions (d 91 to 126) [13]. The stress resulted from weaning and mixing can negatively affect animal welfare [14]. Social housing during the milk-feeding period may have beneficial effects on behavior and cognition ability of calves even after they were weaned and mixed with unfamiliar animals in a group. Several studies have shown that social housing improved resilience to stress (d 51 to 53) [15], as well as increased competitive behavior (d 49 to 56) [16] and interactions (d 56 to 91) [17] after weaning. Furthermore, previous research has mainly clarified the effects of social housing on lying and feeding [17][18][19]. However, other behavioral responses, such as standing, chewing and ruminating, self-grooming, and non-nutritive manipulation have not been well characterized when calves were weaned and moved to group housing. The primary objective of this study was to compare growth, performance and health as well as evaluate the effects of paired versus individual housing on calves' behavior when they were weaned (d 42 to 56) and moved to group housing (d 63 to 70). We hypothesized that paired-housed calves would have better performance than individually housed calves.
When calves were weaned and moved to group housing, they experience changes in the way of feeding and management, especially when they are introduced to a different diet (from a milk-based diet to a solid diet or total mixed ration) and social environment. These changes may impact the behavior of calves. Overvest et al. [18] reported the day to day changes in lying and feeding during the weaning period (d 40 to 48), and Horvath and Miller-Cushon [14] described the day to day changes in standing time of calves mixed in a group (d 60 to 74 ± 5). However, how behavior would change from one period to another was still not clear. Therefore, the secondary objective focused on calf behavioral changes from the weaning period (d 42 to 56) to when they were mixed in a group (d 63 to 70). We hypothesized that calves would exhibit less socially affiliative behavior, such as self-grooming, which may be related to greater activity and exploratory behavior when calves were initially moved to group housing [14]. We also predicted that less non-nutritive manipulation would be observed after calves were moved to group housing, as non-nutritive manipulation often occurred among individually housed calves, especially during the milk-feeding period [20].  2016DR07). Thirty female Holstein dairy calves were collected from the end of March to the mid-April and were assigned to individual (n = 10) or pair housing (n = 10 pairs) based on birthdate and body weight (mean ± SEM; 43.5 ± 0.59 kg). The age difference between calves in the same pair was within 48 h. Only calves with successful passive transfer of immunity (mean ± SEM; 6.24 ± 0.09 g/dL), determined by clinical refractometer 24 h after birth, were included in the study (serum total protein ≥5.5 g/dL).

Preweaning, Weaning, and Postweaning
All calves were born in a calving pen and separated from their dams within 1 h after birth and weighed. After that, calves were moved to a separate and clean straw-bedded nursery room adjacent to the calving facility. If the younger calf of a pair was born within 24 h of the older calf of the pair, then the two calves were moved directly to the experimental calf barn less than 200 m from the nursery room. If the younger calf of a pair was not born within 24 h of the birth of the older calf, then the older calf was kept in the nursery room until the younger calf of the pair was born, accepting an age difference within a pair of maximum 48 h. Calves were transferred to the calf pens by a cart; paired-housed calves were transferred together within 3 h to 5 h after the birth of the younger calf, whereas individually housed calves were transferred alone.
Individually reared calves were kept in individual pens (1.5 m × 2.0 m), while paired-housed calves were provided twice the area (3.0 m × 2.0 m). Calf pens were located under a 3-sided (solid, 1.1 m in height), roofed shelter with a metal gate at the front. Calves could hear calves and see calves in neighboring pens through the openings in the gate. Openings provided access to buckets (10 L for each one) placed 35 cm apart in the center of the pen for water and starter. Calves housed individually had two buckets (one for water, one for starter), while pair housing calves were provided twice the feeding facilities. All calves had free access to water and pelleted starter feed. All the feeding facilities were cleaned daily. The interior of each pen was bedded with sand and bedding was replaced weekly.
Colostrum was heated to 39 • C in a water bath. After that, colostrum was transferred to 4-L esophageal tubing bottles and fed to the calf through a tube within 2 h after the calf was born on d 1. From d 2 to 56, pasteurized waste milk (nonsaleable milk) was provided 3 times daily at 08:00, 15:00 and 20:00 and the volume of milk for each time was equal. During the preweaning period, calves were fed 6 L/d from d 2 to 7, 7.5 L/d from d 8 to 42. Weaning was carried out by reducing milk volume on d 43 and calves were fed 6 L/d from d 43 to 49 and then 3 L/d until d 56. At each milk feeding, the buckets for water were removed temporarily and milk buckets (5 L for each one) were placed in the same position of the pen. For each pair, two milk buckets were used at each milk feeding, whereas calves housed in individual pens had a single milk bucket. Milk buckets were cleaned after each feeding. After weaning, calves remained in their pens during the postweaning period (d 57 to 63 ± 1). No forage was offered before mixing.

Mixing Period
On d 64 ± 1, individual calves were mixed with the paired-housed calves according to the age and moved to the calf barn. There were 5 groups and each group consisted of 6 calves: 2 previously housed in individual pens and 2 pairs previously housed in pairs. The age difference between calves in the same group was within 48 h. The back wall of the group pen (5.0 m × 4.0 m) was solid with two sides made from horizontal tubular metal bars (bar diameter: 5.0 cm; distance between bars: 12 cm) and a neck rail at the front. The length of the neck rail allowed all 6 calves to eat simultaneously (83 cm per calf). Total mixed ration (TMR) was delivered twice daily at 10:00 and16:00. Each group pen was equipped with one automatic water trough (length: 120 cm, width: 40 cm, height: 70 cm, depth: 20 cm) and calves had free access to water. Sand was used as bedding material and was replenished when the group was moved on d 70. Three Digital Thermometers (Deli Electronic Commerce Co., Ltd., Ningbo, China) were spaced evenly and mounted above (1.0 m) the sand bedding in the calf barn to record daily temperature and humidity (maximum and minimum).

Feed Sampling
Feed samples were collected weekly and immediately frozen at −20 • C until they were further analyzed. The nutritional composition (Table 1) of the dry matter, crude protein, neutral detergent fiber, acid detergent fiber, ether extract, crude ash, calcium and phosphorus were analyzed following the methods of AOAC International [21]. Throughout the study, starter intake was recorded daily based on the amount offered and refused by each calf from d 5 to 63 ± 1. TMR intake during the mixing period was not measured because of group housing. . Body length (shoulders to pins), withers height, hip height, and heart girth were also recorded at the same time points. Blood samples were collected via jugular venipuncture using vacutainer serum collection tubes containing no anticoagulant 24 h after birth. The blood samples were then centrifuged at 3500× g, 4 • C for 15 min. Serum total protein (TP) was determined by an optical refractometer (Honneur Nutritional Technology Co. Ltd., Beijing, China).

Health Check and Treatment
The health check consisted of three parts: (1) fecal scoring, (2) clinical examination of the respiratory system, (3) rectal temperature. Fecal scores were recorded daily at 10:00 each day until d 63 based on a 1 to 4 system according to the guidelines outlined by Larson et al. [22]. Scores were, 1 = firm, well-formed (not hard); 2 = soft, pudding-like; 3 = runny, pancake batter; and 4 = liquid, splatters, pulpy orange juice. Fecal score data were collected by one independent trained observer. All fecal scores were recorded by observing fecal matter on the ground of the pen or the tail and hindquarters of the calf. Fecal scoring was not conducted during the mixing period, because the fecal scores of an individual could not be accurately identified due to group housing. A diarrheic day was defined when the fecal score was >2. Weekly diarrhea frequency was calculated with the following equation: Diarrhea frequency = [(number of diarrhea calves × days of diarrhea) / (total number of calves × days of trial)] × 100%. Any calf with a fecal score >2 was treated according to the protocols established by the farm veterinarian (e.g., by administering antibiotic drugs and electrolytic solutions). Respiratory health was checked before each morning feeding through visually inspecting nasal discharge and Animals 2020, 10, 50 5 of 14 listening to breathing difficulties with auscultation by the farm veterinarian and a member of the research team before morning milk feeding. If calves had signs of respiratory disease such as nasal discharge, cough and breathing difficulties, and a rectal temperature ≥39.5 • C, they were treated using an Andrographis paniculata injection (10 mL; Dazheng Tec-Phar. Co. Ltd., Changchun, China) for a maximum of 48 d; if respiratory disease or pyrexia was not alleviated, the calf received antibiotics treatment for a maximum of 48 d. Electrolytes were also administered intravenously to calves that had a severe respiratory disease until fully recovered. Throughout the study, one calf from individual housing was treated for 3 d during the mixing period because of nasal discharge and breathing difficulties, and no other calves had respiratory disease.

Behavioral Observations
A digital color camera (DS-7800, HIKVISION, Hangzhou, China) was placed above each selected pen (placed 2.5 m in front of the pens and 3.5 m from the pen floor), monitoring the behavior of the calves. During nighttime hours (from 17:30 to 07:30), the infrared monitoring function of the camera would turn on automatically. The recorded behaviors ( Table 2) included feeding, chewing and ruminating, lying, standing, self-grooming, non-nutritive manipulation, and social contact. Six individually housed calves and 6 pairs of paired-housed calves were selected randomly for behavioral observations from d 43 to 70. Based on previous results [18,23,24], the sample sizes of behavior variables were estimated to obtain a power of 0.8 under a significance level of 0.05. During weaning and post-weaning periods, the behavioral data were recorded for 48 h on d 43, 50, and 57. For the mixing period, the behavior data were recorded for 48 h on the second day of mixing (d 65 ± 1) to avoid the effects of transition stress on calves. In order to clearly identify each selected calf from the groups during the mixing period, all selected calves were photographed from the front, back, left, right, and above. The observer could record behavior based on each calf's unique photos. For every 24 h duration (144 h in total for each calf), instantaneous scan-sampling with 5 min intervals was used to collect the lying, feeding, standing and chewing and ruminating data and continuous recording was used to collect the self-grooming, non-nutritive manipulation, and social contact data [25]. All behavioral data were recorded by one observer.
2.6. Statistical Analysis 2.6.1. Starter Intake, Growth, and Health Data Throughout the study, data were analyzed at the pen level (based on a single calf per pen for the individual treatment and the mean of the 2 calves per pen in the pair treatment). Starter intake data were averaged by the week, except for the first week data, which were averaged across the last three days (d 5 to 7). Continuous variables with repeated measurements, including starter intake, average daily gain (ADG), BW, and structural growth, were tested for normality using the UNIVARIATE procedure of SAS (version 9.2, SAS Institute Inc., Cary, NC, USA). These data were then analyzed from week 1 to week 10 (as a whole) using the MIXED procedure of SAS. The model included the fixed effects of time, treatment, and time × treatment interaction and the random effect of pen To account for the repeated measures within-subject, the covariance structures were chosen for each repeated variable on the basis of best fit which was determined from the Bayesian information criterion. The heterogeneous first-order autoregressive structure was selected for starter intake, BW, and structural growth data, and for ADG data, the first-order autoregressive structure was selected. Data for fecal scores were summarized by the week and analyzed using the Chi-squared test.

Behavioral Data
Behavioral data obtained for individual calves from video were also averaged by pen (a single calf per pen for the individual treatment and the mean of the 2 calves per pen in the pair treatment) across the 48 h in each observation week (week 7 and week 8 during weaning, and week 9 during postweaning and 10 during mixing). For each 48 h behavioral observation period, the average duration of each kind of behavior per 24 h was calculated. Behavioral data were analyzed separately by two stages: (1) weaning and postweaning, and (2) mixing. The comparison of social contact between two treatments was only analyzed during the mixing period, as individually housed calves had no social interaction before mixing. For stage 1, the effect of housing on behavior was tested using the MIXED procedure of SAS. The model included the fixed effects of treatment, week, and week × treatment interaction, and the random effect of pen. To account for the repeated measures within-subject, the first-order autoregressive structure was chosen for each behavior on the basis of best fit, which was determined from the Bayesian information criterion. For stage 2, the effect of housing on behavior was tested using one-way ANOVA. Lying, standing, non-nutritive manipulation and social contact data were normally distributed. Behavioral data of feeding, and chewing and ruminating were analyzed after logarithm transformation, and self-grooming data were analyzed after square root transformation to meet the normality assumption. The transformed data were back-transformed to report.
All data were reported as least squares mean. Differences of p < 0.05 were considered significant and 0.05 ≤ p < 0.10 was considered a tendency.

Starter Intake and Growth
As shown in Figure 1, starter intake showed an upward trend over time (p < 0.001) for both individually and pair housed calves with no difference in starter intake between treatments during the preweaning period (p > 0.05). During weaning and postweaning periods, starter intake tended to be higher for paired-housed calves during week seven (860.0 vs. 658.1 ± 80.1 g/d, p = 0.09), and than for individually housed calves during week eight (1461.4 vs. 1123.1 ± 97.0 g/d, p = 0.02) and week nine (2237.4 vs. 1899.5 ± 113.5 g/d, p = 0.04).
ADG increased over time (p < 0.001) for both treatments and no differences were found between treatments during the preweaning period (p > 0.05, Figure 2). During weaning and postweaning periods, the weight gain of paired-housed calves continued to increase, but individually housed calves experienced a growth check. The ADG for paired-housed calves tended to be higher during week seven (0.94 vs. 0.71 ± 0.07 kg/d, p = 0.08). Individually housed calves had higher ADG than calves housed in pairs during the mixing period (1.20 vs. 0.85 ± 0.09 kg/d, p = 0.01).

Health
Throughout the study, one calf from individual housing during the mixing period suffered from respiratory disease, and no other calves had respiratory disease. Diarrhea frequency is shown in Figure 3. Pair housing increased diarrhea frequency in comparison with individual housing of calves during week three (18.0% vs. 6.0%, p = 0.03), yet no differences were found between treatments in other weeks (p > 0.05). Figure 3. Effects of the housing system on diarrhea frequency before mixing for calves housed individually (n = 10 calves) or in pairs (n = 10 pairs). PH = calves housed in pairs; IH = calves housed individually; wk = week. Fecal scoring was not conducted during the mixing period (week 10), because the fecal scores of an individual could not be accurately identified due to group housing. * p < 0.05.

Behavior
As shown in Figure 4, during weaning (week 7-8) and postweaning (week 9) periods, feeding time increased (p < 0.001) for both treatments. Overall, individually housed calves spent more time feeding (83.0 vs. 53.1 ± 1.15 min/d, p = 0.04) compared with paired-housed calves during this period. After mixing, feeding time decreased for individually-housed calves but increased for paired-housed calves, and the previous housing system had no effects on feeding time after mixing (p = 0.82). Ruminating time increased over weaning and postweaning periods (p < 0.001) for both treatments

Health
Throughout the study, one calf from individual housing during the mixing period suffered from respiratory disease, and no other calves had respiratory disease. Diarrhea frequency is shown in Figure 3. Pair housing increased diarrhea frequency in comparison with individual housing of calves during week three (18.0% vs. 6.0%, p = 0.03), yet no differences were found between treatments in other weeks (p > 0.05).

Health
Throughout the study, one calf from individual housing during the mixing period suffered from respiratory disease, and no other calves had respiratory disease. Diarrhea frequency is shown in Figure 3. Pair housing increased diarrhea frequency in comparison with individual housing of calves during week three (18.0% vs. 6.0%, p = 0.03), yet no differences were found between treatments in other weeks (p > 0.05). Figure 3. Effects of the housing system on diarrhea frequency before mixing for calves housed individually (n = 10 calves) or in pairs (n = 10 pairs). PH = calves housed in pairs; IH = calves housed individually; wk = week. Fecal scoring was not conducted during the mixing period (week 10), because the fecal scores of an individual could not be accurately identified due to group housing. * p < 0.05.

Behavior
As shown in Figure 4, during weaning (week 7-8) and postweaning (week 9) periods, feeding time increased (p < 0.001) for both treatments. Overall, individually housed calves spent more time feeding (83.0 vs. 53.1 ± 1.15 min/d, p = 0.04) compared with paired-housed calves during this period. After mixing, feeding time decreased for individually-housed calves but increased for paired-housed calves, and the previous housing system had no effects on feeding time after mixing (p = 0.82). Ruminating time increased over weaning and postweaning periods (p < 0.001) for both treatments Figure 3. Effects of the housing system on diarrhea frequency before mixing for calves housed individually (n = 10 calves) or in pairs (n = 10 pairs). PH = calves housed in pairs; IH = calves housed individually; wk = week. Fecal scoring was not conducted during the mixing period (week 10), because the fecal scores of an individual could not be accurately identified due to group housing. * p < 0.05.

Behavior
As shown in Figure 4, during weaning (week 7-8) and postweaning (week 9) periods, feeding time increased (p < 0.001) for both treatments. Overall, individually housed calves spent more time feeding (83.0 vs. 53.1 ± 1.15 min/d, p = 0.04) compared with paired-housed calves during this period. After mixing, feeding time decreased for individually-housed calves but increased for paired-housed calves, and the previous housing system had no effects on feeding time after mixing (p = 0.82). Ruminating time increased over weaning and postweaning periods (p < 0.001) for both treatments and individually-housed calves tended to have greater ruminating time than paired-housed calves during week seven (2.56 vs. 1.79 ± 0.26 h/d, p = 0.09). After mixing, ruminating time continued to increase with age for all calves with no differences found between treatments (p = 0.61).
Lying time increased during the weaning period and decreased during the postweaning period for all calves. Standing time increased during the postweaning period for all calves. Calves housed in pairs spent more time lying (17.3 vs. 16.4 ± 0.27 h/d, p = 0.03) and less time standing (6.33 vs. 7.11 ± 0.18 h/d, p = 0.01) compared with calves housed individually during weaning and postweaning periods. After mixing, lying time remained stable and standing time decreased for all calves, and the previous housing system had no effect on lying (p = 0.56) and standing time (p = 0.84). There was a decrease in self-grooming time for both treatments over weaning and postweaning periods (p = 0.01), and calves housed individually exhibited more self-grooming than calves housed in pairs (40.7 vs. 20.6 ± 4.10 min/d, p = 0.02). After mixing, self-grooming time continued to decrease for all calves, with no differences between treatments (p = 0.65). In addition, non-nutritive manipulation time did not change with calf age during weaning and postweaning periods (p = 0.62), and treatment had no effects Animals 2020, 10, 50 9 of 14 on non-nutritive manipulation time during this period (p = 0.10). After mixing, all calves decreased non-nutritive manipulation time, and the non-nutritive manipulation time tended to be longer for calves that were previously individually-housed (33.0 vs. 16.9 ± 5.62 min/d, p = 0.07). During the mixing period, the previous housing system had no effect on social contact ( Figure 5; p = 0.53).   change with calf age during weaning and postweaning periods (p = 0.62), and treatment had no effects on non-nutritive manipulation time during this period (p = 0.10). After mixing, all calves decreased non-nutritive manipulation time, and the non-nutritive manipulation time tended to be longer for calves that were previously individually-housed (33.0 vs. 16.9 ± 5.62 min/d, p = 0.07). During the mixing period, the previous housing system had no effect on social contact ( Figure 5; p = 0.53).

Starter Intake and Growth
During the preweaning period, we did not observe any differences in starter intake, ADG or BW between treatments, yet during weaning and postweaning periods, pair housing improved growth performance. Our results were consistent with previous research that reported increased starter intake [6,26] and ADG [23] during the weaning period for paired-housed calves. Such improvements are likely due to social facilitation [12] and social learning [27], which allow calves housed in pairs to learn faster and eat more. Local enhancement is another factor affecting the feeding of calves, through which the behavior of one calf draws the attention of another in the same pair toward a particular food source [28,29]. In addition, paired calves might experience a lower level of stress during the weaning period because of social buffering [12]. The social buffering benefits of early pair housing have been discussed recently by Overvest et al. [18], who demonstrated that social housing might improve the ability to cope with the weaning stress via the positive effects on feed acceptance and behavioral flexibility. During the postweaning period, we observed greater starter intake in pair housing. Similar results were reported by Pempek et al. [20], who also attributed it to social facilitation. Besides, the competitive feeding environment among paired calves may also have resulted in more starter intake during the weaning and postweaning periods, as calves may increase the rate of feed intake in the competitive feeding environment [17]. Our results contribute to a body of evidence indicating that pair housing is particularly beneficial to solid-feed intake, growth, and supporting a smooth transition at weaning [3].
After mixing, calves were grouped together. Warnick et al. [30] and De Paula Vieira et al. [12] reported that calves previously housed in groups or pairs gained more than those previously housed in individual pens when they were mixed and placed together. Some studies attributed these results

Starter Intake and Growth
During the preweaning period, we did not observe any differences in starter intake, ADG or BW between treatments, yet during weaning and postweaning periods, pair housing improved growth performance. Our results were consistent with previous research that reported increased starter intake [6,26] and ADG [23] during the weaning period for paired-housed calves. Such improvements are likely due to social facilitation [12] and social learning [27], which allow calves housed in pairs to learn faster and eat more. Local enhancement is another factor affecting the feeding of calves, through which the behavior of one calf draws the attention of another in the same pair toward a particular food source [28,29]. In addition, paired calves might experience a lower level of stress during the weaning period because of social buffering [12]. The social buffering benefits of early pair housing have been discussed recently by Overvest et al. [18], who demonstrated that social housing might improve the ability to cope with the weaning stress via the positive effects on feed acceptance and behavioral flexibility. During the postweaning period, we observed greater starter intake in pair housing. Similar results were reported by Pempek et al. [20], who also attributed it to social facilitation. Besides, the competitive feeding environment among paired calves may also have resulted in more starter intake during the weaning and postweaning periods, as calves may increase the rate of feed intake in the competitive feeding environment [17]. Our results contribute to a body of evidence indicating that pair housing is particularly beneficial to solid-feed intake, growth, and supporting a smooth transition at weaning [3].
After mixing, calves were grouped together. Warnick et al. [30] and De Paula Vieira et al. [12] reported that calves previously housed in groups or pairs gained more than those previously housed in individual pens when they were mixed and placed together. Some studies attributed these results to the beneficial effects of social housing, such as reduced neophobia to new ration [31,32] and greater competitive success [16] when mixed with unfamiliar animals. On the contrary, we found that previously paired-housed calves had less ADG after mixing compared with calves housed individually, and the final BW was similar between treatments. Somewhat interestingly, Miller-Cushon and DeVries [4] reported that though paired calves had greater performance during the weaning period (d 39 to 49), previous housing (paired vs. individual) had no effect on DMI, ADG or final BW once previously individually housed calves were paired with unfamiliar calves after weaning (d 50 to 84). Similar results were reported by Overvest et al. [18], who also demonstrated that once calves previously housed individually were paired after weaning (d 49 to 56), they exhibited more feeding time and thus increased their solid feed DMI to a greater extent over time than paired-housed calves, and eventually resulted in similar DMI between treatments. These results suggested that previously individually housed calves could get the same performance (e.g., DMI, BW, and ADG) through modifying feeding behavior after they were exposed to social housing with unfamiliar calves. In this study, all calves experienced a sudden feed transition to TMR, in which case, the beneficial effects of social housing on food neophobia may be weakened by transition stress. Furthermore, we observed similar feeding time between treatments during the mixing period. Thus, we speculated that the higher ADG in calves previously housed individually may result from higher feeding rate, allowing them to consume sufficient TMR to meet or exceed their nutritional requirements and finally compensating for a previously lower starter intake. Further work to address this possibility is encouraged.

Health
In the current experiment, diarrhea frequency for calves housed in pairs was higher than that for calves housed individually in week three, yet no differences were found in other weeks. Some studies reported that housing calves in groups exhibited more health problems owing to higher levels of infectious agents and calf-calf transmission [33,34]. On the contrary, others reported a lower incidence of diarrhea for calves housed socially [9], and some found no differences in incidences of diarrhea and respiratory problems [35] between paired-housed calves and individually housed calves. The various results indicated that health problems were not consistently associated with social housing. The incidence of disease relies on many factors including calf immunity, environment management, disease diagnosis, and the ability of a calf to cope with stress [36]. These factors rather than the housing system may play a critical role in inducing health problems. Greater health problems in a group housing system may also stem from the difficulty of detecting disease in groups [23]. There is not enough evidence to support a diarrhea-increasing effect of pair housing, thus the higher incidence of diarrhea in pair housing in week three was probably due to low immunity to infection of calves aged from two to four weeks [37] and individual differences.

Behavior
Limited research has described how behavior would change at different periods from weaning to mixing, or the effects of paired or individual housing on behavior during these periods. Our results suggested that all calves experienced behavioral changes from weaning to mixing including increased feeding and ruminating time, and decreased self-grooming time.
The increase in feeding time and chewing and ruminating time over the weaning and postweaning periods aligned with the increase in feed intake. Besides, paired calves spent less time feeding but still had higher starter intake during weaning and postweaning periods, likely due to the competitive feeding environment as we discussed on starter intake and growth. Miller-Cushon et al. [17] found that calves housed in a competitive feeding environment had less time of feeding but an increased rate of feed intake compared with those housed in a noncompetitive feeding environment. Hence, paired-housed calves might increase their feeding rate rather than feeding time to consume more starter.
In the present study, lying time declined while standing time rose during the postweaning period, which could be attributed to the increase in feeding time with increasing age during this period. The previous study [18] suggested that calves may change their lying behavior to accustom themselves to feeding behavior. In addition, calves housed individually exhibited more lying time than paired calves during weaning and postweaning periods, which is contrary to previous studies [23,38]. Previous research [20] also mentioned no effects of individual vs. paired housing on lying. The variant space allowance for calves among studies may be responsible for the discrepancy in results, as space allowance was a vital factor for the expression of normal behavior [23,39]. Further research is encouraged to study the relationship between space allowance and lying. After mixing, standing time decreased for all calves. Previous studies reported that calves were more active and moved more followed by a reduction in activity after the first day of introduction to a group [14,29], and calves had diminished behavioral reactions after the first 24-h period following regrouping [40]. Thus, we speculated that calves might not be as active as the first day of introduction to a group as the behavior data were recorded for 48 h on the second day of mixing to avoid the effects of transition stress on calves in this study.
Self-grooming is expressed by calves as caring for their own body, and this behavior may be a means of satisfying socialization [27]. More self-grooming activities were observed in individually-housed calves in the present study, which was consistent with previous research [27], as the socialization was absent in these calves. In addition, self-grooming can also be an expression of stress. Taking rodent as a research model, previous studies [41][42][43] has reported that the relationship between stress and self-grooming can be described as an inverted U-shaped: Self-grooming typically occurs spontaneously at low stress and becomes longer during moderate stress and can be inhibited by high-stress states. Thus, the higher self-grooming of individually housed calves may respond to the higher stress (moderate stress) they faced compared with paired calves during weaning and postweaning periods.
Non-nutritive manipulation commonly occurs within artificial rearing systems [44], which can be strengthened by social deprivation [45]. In the current study, a drop in non-nutritive manipulation time for all calves after mixing was observed, which was likely due to more social interactions among calves after mixing. Bokkers and Koene [46] also indicated that less social interaction was an important factor causing dairy calves to lick objects (a nonnutritive manipulation behavior). In this study, no effects were found on non-nutritive manipulation during the weaning or postweaning period, whereas individually-housed calves tended to spend more time on non-nutritive manipulation compared with paired calves during the mixing period, which was similar to the previous study [47]. Although the effect of the previous housing system on social contact was not significant during the mixing period, calves housed in pairs previously still exhibited more social contact numerically, which may result in less non-nutritive manipulation.

Conclusions
Paired versus individual housing had no effects on body weight, starter intake or ADG during the preweaning period, while pair housing increased the growth performance of calves during weaning and postweaning periods, and the beneficial effects of pair housing on growth was weakened after mixing. Paired calves showed higher diarrhea frequency only in week three. Calves altered their behaviors at different periods from weaning to mixing, including increased feeding time and chewing and ruminating time, and decreased self-grooming time, and a drop of non-nutritive manipulation for all calves after mixing. Furthermore, less social contact may result in more non-nutritive manipulation.


extfieldsofstudy: Medicine Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2019.jsonl.gz.gz

text:
Utilization of Pectinase Cocktail and High Hydrostatic Pressure for the Production of Aged Black Garlic Juice with Improved Nutritional Value

In comparison with raw garlic, aged black garlic has been shown to display multiple pharmacological activities. We recently reported that pretreatment of pectinase cocktail with high hydrostatic pressure (HHP) before the process of aging garlic juice improves its antidiabetic activity and increases S-allylcysteine (SAC) content. Thus, this study was designed to investigate the influence of pectinase cocktail with HHP on the quality of aged black garlic juice formation and to identify the optimal manufacturing conditions. In the pretreatment step, garlic juice is heated at 55°C for 24 h. The contents of SAC and total polyphenols were increased with treatment of pectinase cocktail; this increase was greater under HHP processing. In contrast, the total flavonoid content was decreased in all pretreatment conditions. Garlic juice pretreated with pectinase cocktail and HHP had a significantly higher content of SAC in the early phase of aging than raw garlic juice, and the SAC was increased over time in both treatment groups. The total polyphenol content of garlic juice was significantly higher in the pretreatment group during the aging period, and the antioxidant activity of garlic juice showed a positive correlation with polyphenol content. Interestingly, HHP increased the enzymatic activity of the pectinase cocktail.

INTRODUCTION
Aged black garlic (ABG) is a type of processed garlic that is generally prepared by aging raw garlic at high temperature for 1∼3 months. During the aging process, non-enzymatic browning reactions, and enzymatic hydrolysis induce a variety of physicochemical changes to the raw garlic, including of color, texture, taste, flavor, and nutrient content (Ryu and Kang, 2017;Yuan et al., 2016). It has been reported that unstable and unpleasant compounds in raw garlic are converted into stable and tasteless compounds by heat treatment (Zhang et al., 2016). In addition, ABG extracts have been shown to exhibit multiple biological activities both in vitro and in vivo; specifically the extracts have more potent antioxidant and immunomodulatory properties than raw garlic extracts (Purev et al., 2012;Ryu and Kang, 2017). The improved biological activities of ABG may be associated with increased for-mation of antioxidants, including polyphenols and flavonoids (Choi et al., 2014;Lee et al., 2009). More importantly, S-allylcysteine (SAC), a sulfur-containing amino acid produced by enzymatic hydrolysis during raw garlic aging, has been considered a key compound responsible for the pharmacological activities of ABG (Colín-González et al., 2012). Along with the strong evidence for SAC bioactivity, clinical pharmacokinetic studies have shown that SAC is rapidly absorbed and very stable in the blood after consumption of ABG extracts (Kodera et al., 2002).
Owing to these beneficial changes in compounds during the raw garlic aging process, studies have been conducted to investigate the characteristics of ABG quality under different aging conditions (Bae et al., 2014;Choi et al., 2014;Toledano-Medina et al., 2016;Zhang et al., 2016). These reports have consistently shown that browning intensity, total polyphenol content, and antioxidant activity of garlic are increased with increased aging time and/or temperature, whereas moisture content and pH are decreased. Among these studies, only one reported changes in the SAC content of garlic in relation to aging time and temperature: SAC content gradually increased throughout the aging process, but was significantly higher when the garlic was exposed to lower temperatures (Bae et al., 2014). Taken together, these findings suggest that the conventional aging process, which occur at high temperatures for up to 3 months, do not produce the best quality ABG, and more efficient manufacturing process are needed to produce ABG with improved nutritional value.
Recently, we reported that pretreatment of pectinase cocktail with high hydrostatic pressure (HHP) increased the SAC content of ABG juice (ABGJ) with enhanced antidiabetic effects (Kim et al., 2017). The HHP process has been shown to contribute to stabilization of pectinase cocktails and influence enzymatic and nonenzymatic browning reactions during food processing and storage (Heberle et al., 2003;Ioannou and Ghoul, 2013;Tomlin et al., 2013). Thus, to develop optimal manufacturing conditions for ABGJ with improved nutritional value, we investigated changes in quality formation including SAC, polyphenols, and antioxidant capacity during the aging process after pectinase treatment with or without HHP. In addition, we observed the influence of HHP on the enzymatic activity of the pectinase cocktail used in this study.

Sample preparation
ABGJ was manufactured as previously described (Kim et al., 2017) with some modifications. Fresh peeled garlic (Uiseong, Korea) was ground using a laboratory blender (Waring Commercial, New Hartford, CT, USA), and 3% (v/v) commercial pectinase cocktail (Multifect Pectinase FE, Genencor, Palo Alto, CA, USA), containing pectinase, cellulase, and hemicellulose, was added into the mashed garlic juice. During pretreatment, the pectinase-added mashed garlic juice was incubated with or without HHP (50 or 100 MPa) at 55 o C for 24 h using a high-pressure liquefying extractor (DFS-2L, Toyo Koatsu Co., Ltd., Hiroshima, Japan). For aging, raw garlic juice and pectinase +HHP-treated garlic juice were incubated in a thermohydrostat chamber (TH-G-800, Jeio-Tech, Seoul, Korea) at 30 o C with 70% relative humidity for 35 days. The aging temperature used in this study was established based on the previous report that showed higher SAC contents are derived at lower temperatures (Bae et al., 2014). During the 35 days, all garlic samples were collected weekly, filtered, freeze-dried, and then stored at −20 o C until future use.

Analysis of SAC
SAC content of garlic juice was determined according to the method described previously (Kubec and Dadáková, 2008) with some modifications. The freeze-dried garlic powder was diluted with distilled water, and 0.1 mL of sample was mixed with 0.25 mL of 10 mM dansyl chloride in acetonitrile and 0.65 mL of 20 mM borate buffer (pH 9.2). The mixture was incubated at room temperature for 15 min, filtered, and subjected to an high-performance liquid chromatography system equipped with a diode-array detector (Nanospace SI-2, Shiseido, Tokyo, Japan) for SAC analysis. The analyte was separated using a Capcell Pak C18 UG120 column (250 mm×4.6 mm ID, 5 m, Shiseido). The solvent system consisted of two eluents: eluent A was 50 mM sodium acetate in distilled water (pH 5.0 adjusted by formic acid) and eluent B was methanol. A gradient elution was employed at different ratios of A : B (v/v); 7:3 (0 min), 6:4 (35 min), 2.5:7.5 (70 min), and 7:3 (95 min).

Determination of total polyphenols, total flavonoids, and antioxidant capacity
The total polyphenol content was determined using Folin-Ciocalteu's colorimetric method with some modifications as described in a previous report (Kim et al., 2010) and was expressed as mg of gallic acid equivalents (GAE). Total flavonoid content was measured using aluminum chloride colorimetric assays as previously reported (Kamtekar et al., 2014) and expressed as mg of rutin equivalents (RE).
The free radical scavenging activity of garlic juice was measured using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1,1-diphenyl-2-picrylhydrazil (DPPH) assays. For ABTS assays, 2,2'-azobis dihydrochloride (1 mM) and ABTS (2.5 mM) were dissolved in phosphate-buffered saline (100 mM), heated at 70 o C for 30 min, and then cooled to room temperature. The diluted ABTS solution (0.98 mL) was then mixed with sample solution (0.02 mL), and the absorbance was measured at 734 nm after 20 min using a spectrophotometer (UV 2100, Shimadzu Co., Kyoto, Japan). For DPPH assays, 1 mM of DPPH was dissolved in ethanol, and 1.95 mL of DPPH solution was mixed with 50 L of sample solution. The mixture was incubated at room temperature for 30 min, and then the absorbance was measured at 517 nm. The ABTS and DPPH radical scavenging activities were expressed as vitamin C equivalents.

Measurement of enzyme activity
The enzymatic activity of the pectinase cocktail was determined by the rate of release of reducing ends from polygalacturonate, according to methods described previously (Li et al., 2015;Reid and Ricard, 2000) with some modifications Briefly, two test tubes containing 2 mL of diluted enzyme stock were equilibrated to 55 o C and 10 mL of substrate stock solution was added to each tube. Tubes with or without HHP treatment (50 MPa) were incubated in a water bath at 55 o C for 0.5, 1, 2, 3, or 4 h. Solution A was prepared by sequentially dissolving Na 2 CO 3 , glycine, and CuSO 4 in distilled water and was used as a time 0 control. Neocuproine HCl was then added to each tube, and the tubes were mixed and incubated at 100 o C for 12 min. Finally, the tubes were cooled and diluted, and the absorbance was recorded at 450 nm. Enzyme activity (U/mL) was calculated as follows: Activity= g galacturonic acid released 0.2×194.1×t where 0.2 is the enzyme broth volume used in the assay, 194.1 is the molecular weight of galacturonic acid, and t is the reaction time in min. One unit (U) is equivalent to 1 mol product released per min.

Statistical analysis
Data were analyzed by one-way ANOVA using the SAS software for Windows release 9.2 (SAS Institute Inc., Cary, NC, USA). One-way ANOVA with repeated measures was performed to assess mean differences between groups. The least squares means option using a Tukey-Kramer adjustment was used for multiple comparisons between the experimental groups. Data are shown as mean±standard error (SE). P-values <0.05 are reported as statistically significant.

Effects of pectinase pretreatment with HHP on quality formation of ABGJ
To investigate the influence of pectinase pretreatment with HHP on quality formation of ABGJ, we examined changes in the contents of SAC, polyphenols, and flavonoids, and antioxidant capacity before and after the aging process. In the pretreatment step, incubation at 55 o C for 24 h with or without the pectinase cocktail significantly increased the SAC content of garlic juice (Fig. 1A).
After incubation with pectinase, the total polyphenol content was increased but this increase was not significant (Fig. 1B). Importantly, the contents of SAC and polyphenols were highest when garlic juice was subjected to cotreatment of pectinase with HHP at 50 or 100 MPa. In contrast, the total flavonoid content of garlic juice was decreased during the heating process; this change was greatest when heating with pectinase (Fig. 1C). The increase in phenolics and decrease in flavonoids heating are consistent with a previous study using onion, which was attributed to the hydrolysis of C-glycoside bonds of the flavonoids (Sharma et al., 2015).
Since the SAC content of garlic juice was increased by HHP at 50 MPa and slightly decreased at 100 MPa (Fig.  1A), we used garlic samples prepared at 50 MPa for further comparisons with raw garlic juice. As expected, the SAC content of garlic juice increased progressively over time throughout the aging process at 30 o C, although that of raw garlic juice was slightly decreased after 28 days ( Fig. 2A). In a previous study using different aging temperatures, garlic bulbs heated at low temperatures had higher amounts of SAC than those heated at higher temperatures: the SAC content increased up to 124.67 g/g when heated at 40 o C for 45 days (Bae et al., 2014). Of note, our current results show that the SAC content of raw garlic juice increased to 177.70 g/g when heated at 30 o C for 28 days. Importantly, garlic juice pretreated with pectinase under HHP showed 121.74 g/g of SAC before aging, and the SAC content increased to 181.42 g/g after aging at 30 o C for 35 days. These results suggest that cotreatment of pectinase with HHP before heating at 30 o C was more efficient for producing ABGJ with enhanced SAC content than the conventional aging process at higher temperatures.
A significant increase in total polyphenols in garlic juice was observed during aging when following pretreatment with pectinase and HHP (Fig. 2B). In addition, results from ABTS and DPPH radical scavenging activity assays showed that garlic juice had significantly higher antioxidant activity when pretreated with pectinase and HHP at different times during the aging process ( Fig. 2C and D). Regardless of pretreatment, the polyphenol content and antioxidant activity showed a similar pattern of change, whereby they increased until day 21 of aging and then decreased. This positive correlation between polyphenol content and antioxidant activity was also observed in a previous study using a whole bulb of garlic; both increased to day 24, and then decreased during the aging process at 72 o C (Toledano-Medina et al., 2016). Although SAC has been reported as a potent antioxidant compound (Dvořáková et al., 2016;Pérez-Severiano et al., 2004), our current data, combined with previous findings, indicate that polyphenols could be the major determinant of the radical scavenging potential of ABG.
The use of HHP as a nonthermal process for food preservation has been shown to inactivate microorganisms as well as enzymes responsible for shortening the shelf-life of food products (Garriga et al., 2004;San Martín et al., 2002). HHP inactivates microbes lethally and/or sub-lethally whilst minimizing degradation of flavor, color, and nutritional components (Yamamoto, 2017). These findings reveal the advantages of HHP technology as a promising nonthermal process for food processing. Therefore, further studies to evaluate the influences of HHP on microbial safety and the sensory properties of ABG may provide more meaningful data.

Influence of HHP on pectinase enzyme activity
Since pretreatment of pectinase with HHP resulted in significant increases in the contents of SAC and total polyphenols, and antioxidant activity of garlic juice before and after aging, we next determined whether HHP may enhance pectinase enzymatic activity. The content of released galacturonic acid and the corresponding enzymatic activity was higher in the HHP-treated group at 50 MPa than in the control group, with significant differences observed at 4 h (Fig. 3). Similarly, HHP treatment at 200∼ 400 MPa delayed the rate of pectinase cocktail inactivation when treated at 77 o C (Tomlin et al., 2013). These findings suggest that HHP could be effectively used to stabilize the commercial pectinase cocktail at a high temperature. Since pectinase cocktails are widely used in juice processing to improve extractability, clarity, and functional properties (Puri et al., 2012), our current findings provide useful information that could be applied to the juice industry, including to garlic juice processing. However, the question of how HHP stabilizes and increases pectinase activity remains unanswered, and further studies to investigate changes to enzyme structures with HHP are needed.


text:
Optimal method selection for biocompatible extraction of rosmarinic acid from mycorrhizal hairy roots of Ocimum basilicum

Highlights • Mycorrhizal hairy roots as a dual resource of biological and biochemical products.• Thirty degree is optimum temperature for biocompatible extraction.• Low percentage of methanol and DMSO as most suitable biocompatible solvent.

Introduction
Mycorrhizal co-cultures developed between hairy roots of medicinal plants and arbuscular mycorrhizal fungi in an in vitro culture system are potential resource of quality arbuscular mycorrhizal propagules and secondary metabolites that have agricultural and pharmaceutical applications respectively. Recently, we have shown that mycorrhizal hairy roots of Ocimum basilicum [1] are reserves of mycorrhizal propagules (Rhizophagus irregularis), antioxidants and rosmarinic acid (RA). In detail, arbuscular mycorrhizae (AM) as a biofertilizer promote plant growth, improve drought and stress tolerance and crop yield and also act as soil aggregator which prevents soil erosion and facilitates phytoremediation [2]. Biologically, RA possesses antioxidant, antiproliferative, anti-inflammatory, neuroprotective, hypoglycemic like properties and is also an active ingredient in a number of commercial products such as Neurex, Persen and Aquarox [3]. For the utilization of mycorrhizal hairy roots of O. basilicum as a continuous dual resource of agri-(mycorrhizae) and pharma-(rosmarinic acid) specific products, an essential requirement is development of a separation method that retains both mycorrhizal and host (hairy root) viability after extraction of secondary metabolites.
To date no study has provided a method for the simultaneous preservation of host viability (such as roots) and its symbiotic partner coupled with metabolite extraction however, a number of earlier studies had advocated development of biocompatible extraction methods to retain plant cell viability after metabolite extraction. For example, Weathers et al. [4] reported in hairy roots of Beta vulgaris (beetroot) use of three basic steps for extraction of a number of bioactive compounds such as betacyanin, betanin, betaxanthins and betalamic acid along with maintenance of plant cell viability. Similarly, Shotipruk et al. [5] reported the use of ultrasonication for menthol extraction from Mentha piperata (mint). Furthermore, Wang et al. [6] also reported use of a low percentage of methanol and increased temperature as being effective for extraction of isoflavonoids from Glycine max (soybean) while maintaining seed viability. Similar to plant cells, algae such as Dunaliella salina has also been tested for suitability of continuous extraction of β-carotene with its viability intact through utilization of biocompatible solvents [7,8].
Organic solvents such as ethanol and methanol are widely used for the extraction of RA [9][10][11]. Alternatives to organic solvents include ionic liquids [12] and Dimethyl Sulfoxide; DMSO [13] as potential green solvents for extraction of RA from Rosmarinus officinalis (rosemary) and suspension cultures of Coleus blumei (coleus) respectively. Ionic liquids are a class of organic salts that have an organic cation (such as dialkyl-imidazolium) and inorganic anion (such as Cl À ) and exist as liquids at a relatively low temperature (<100 C). They have gained attention as an attractive alternative to organic solvents due to their non-flammable, chemically and thermally stable nature [14]. In addition to solvents, heat treatment (within a range that maintains viability of the plant cell) has also been used to facilitate release of metabolites from hairy root systems [4]. Thus, for development of a biocompatible extraction technique with mycorrhizal hairy roots, determination of optimal temperature is one of the essential requirements.
In the present study we aim to develop a biocompatible extraction method that exhibits maximum metabolite solubility and extraction ability along with minimum loss of root biomass and low toxicity while maintaining root colonization potential of mycorrhizal fungi. The present study reports development of a biocompatible extraction method with mycorrhizal hairy roots of O. basilicum [1] by screening temperature-and sonication-assisted extraction methodologies through four sequential stages: 1) material preparation and selection, 2) assessment of the pretreatment regime, 3) extraction by utilization of two different agitation techniques: a) temperature-and b) sonication-assisted extraction with different solvents and 4) post treatment determination of extracted metabolite concentration and viability of both arbuscular mycorrhizae and host root.

Materials and methods
2.1. Establishment of mycorrhizal hairy roots for material selection, pretreatment and treatment analyses O. basilicum hairy root line 4 (HR 4) colonized with Rhizophagus irregularis [1] was used for the screening studies. To raise cocultures for experimental set up, a 6 week old co-culture of HR 4 raised in a culture jar (300 cm 3 , containing 100 mL minimal [M] semisolid medium) was divided into six equal sections and each section was inoculated to a freshly prepared culture jar containing 100 mL of M medium. Similar process was repeated to initiate cocultures for all experimental treatments. Inoculated jars were then incubated in the dark at 26 C (Incubator model: ET-650-8, Lovibond, Dortmund, Germany) for 120 days (d). The jars were observed weekly for any contamination and to monitor growth of the symbionts. After 120 d, to each mycorrhizal co-culture jar of HR 4; 100 mL of 10 mM sodium citrate buffer [15] was added and kept on a shaker (Kuhner Shaker, Basel, Switzerland) at 25 AE 2 C for 90 min at 140 rpm for deionization. The root material after deionizaton from all culture jars was passed through sterile 60 mM (BSS standard) sieve. Subsequently, the collected root material was washed with sterile distilled water twice. After washing, collected mycorrhizal roots were blot dried on a sterile filter paper (Whatman # 1) disc thrice to remove excess water. Blot dried roots were mixed and then divided into 1 AE 0.5 cm long segments for screening studies. To study the loss of rosmarinic acid (RA) into the deionization buffer, its concentration was quantified using HPLC as described in the post treatment analyses section.

Pretreatment analysis
To examine the effect of soaking on extraction of RA, harvested mycorrhizal root material (100 mg) was soaked in 1 mL of water for three time periods (6, 12 and 24 h) at room temperature on a rocker (Rockymax, Tarsons, New Delhi, India). Five replicates were taken for each treatment. The concentration of RA released in water was quantified by HPLC (High performance liquid chromatography) as described in the post treatment analyses Section (2.4).

Treatment analyses: temperature-and sonication-assisted extraction
All screening studies were carried out under aseptic conditions with fresh mycorrhizal roots (100 mg) of HR 4 in 10 mL of solvent in a 70 mL test tube (Borosil, New Delhi, India). Required percentages of all solvents used in the study were prepared in a total volume of 10 mL of autoclaved distilled water. Each extraction experiment was performed with five replicates. After every treatment, roots were separated from the extractant by forceps and the final volume of extractant was made to 10 mL in a volumetric flask with respective solvent. After separation, extractant, mycorrhizal propagules (spores and hyphal biomass) and roots were processed as described in the post treatment methodology (2.4). Four temperatures (25 C, 30 C, 40 C and 50 C) were studied. Test tubes with 10 mL water were maintained to required temperature in a water bath (Heto HMT 200, Heto Lab Equipment, Allerod, Denmark) before addition of fresh mycorrhizal roots segments. After addition of mycorrhizal roots (100 mg), the test tubes were incubated for 6 and 12 h in a shaker (Kuhner Shaker, Basel, Switzerland) at the required temperature and rotation (100 rpm). The treated roots were then processed as described in the post treatment methodology. Similar steps were followed for screening studies with 10, 20 and 30% (v/v) of methanol and ethanol in water at four different temperatures.

Sonication assisted extraction (SAE): solvents and surfactants
Sonication assisted extraction was performed using a Waterbath assisted sonicator (B3510E-DTH, Branson Ultrasonics, Danbury, USA) at 30 C for 15 min. The solvents, methanol (from 10 to 100% v/v in water), ethanol (10-100% v/v in water) (Merck, Mumbai, India), DMSO (1, 0.5, 0.1%; v/v in water), biocompatible solvents (nonane and dedecane; 100%) and surfactants (Triton X-100 and Tween-20 1, 2 and 3% v/v in water) (all from Sigma Aldrich, Bangalore, India) were screened. Sterile distilled water was used as a control for each of the above treatments. Each extraction experiment was performed with five replicates. After every treatment, the extractant (with final volume made to 10 mL in a volumetric flask), mycorrhizal propagules and roots were processed as described in the post treatment Section (2.4).

Sonication assisted extraction (SAE): ionic liquid as a green extractant
The potential of the ionic liquid 3 methyl-imidazolium bromide (1 M, Sigma-Aldrich, Bangalore, India) for extraction of RA from mycorrhizal roots of O. basilicum was examined in comparison with water and methanol (100%) using lyophilized mycorrhizal roots (lyophilzation was performed as described by Srivastava et al. [1]). In detail, 100 mg of the dried mycorrhizal roots were first soaked in 1 mL of water, methanol and ionic liquid respectively for 2 h on a rocker. After soaking, roots were submersed in 9 mL of the solvent in a test tube and sonicated (B3510E-DTH, Branson Ultrasonics, Danbury, USA) for 15 min at 25 AE 2 C. Following sonication, roots were separated using sterile forcep and 1 mL of the solution used for soaking was added to the 9 mL of sonicated solution to make total volume to 10 mL. The pooled extractant, mycorrhizal propagules and roots were processed as described in the post treatment methodology (2.4).

Comparison between low molarity ionic liquid
Three low molar (0.25, 0.50 and 0.75 M) solutions of 3 methylimidazolium bromide were prepared in ultrapure water. To 10 mL of each solution, 100 mg of fresh mycorrhizal roots was added and sonicated at 25 AE 2 C for 15 min. After sonication, final volume was made to 10 mL. The ionic liquid, mycorrhizal propagules and roots were processed as described in the post treatment methodology (2.4).

Post treatment analyses: RA concentration, mycorrhizal spore and host root viability assessment
After treatment three major analyses were performed to examine the effect of individual treatments on the RA concentration (HPLC analysis), mycorrhizal propagules viability (hyphal growth and spore germination on M medium) and colonized root viability (root growth).

Rosmarinic acid concentration: HPLC analysis
The concentration of RA was determined using HPLC. Briefly, an HPLC system (Shimadzu, Kyoto, Japan), equipped with a quaternary pump (LC -20AT), solvent degasser system (DGU -20 A5), autosampler (SIL -20A) and diode array detector (SPDM -20A) was used. Inbuilt software (LC solution, Shimadzu) was used to control the HPLC pump and acquire data from the diode array detector. Separations were performed on a C 18 Phenomenex column (Gemini-NX 250 mm Â 4.6 mm Â5 mm). A gradient program with HPLC grade water + 0.1% (v/v) ortho Phosphoric Acid (OPA) in water (Mobile phase A) and Methanol (HPLC grade, Merck, Mumbai, India) + 0.1% OPA (v/v) (Mobile phase B) in water as mobile phase was used for quantification of RA. A flow rate of 1.0 mL/ min, injection volume of 20 mL and detection wavelength of 280 nm was used. Unknown samples were identified and quantified by comparison of the retention times with those of a commercial standard of RA (Sigma, Bangalore, India) prepared in the range of 20-100 mg/L [1,3].

Mycorrhizal spore viability assay
Viability stain MTT [3-(4, 5 Dimethylthiazol-2-yl) -2, 5diphenyltetrazolium bromide], Sigma, Bangalore, India) was prepared as a 0.01% (w/v) solution in water and the treated mycorrhizal roots were then soaked in 1 mL of MTT solution for 40 h at 28 C. After incubation, the roots were observed under stereomicroscope (SZ16, Olympus, Japan) and viability percentage (viable spores/ total number of spores Â 100) was calculated. Two hundred spores per sample were observed. Red, pink or purple colored spores were considered viable while colorless, green or black colored spores were considered nonviable. C and e and f) 50 C. Data is represented as mean AE SEM of five replicates (n = 5). Bars topped by different letters indicate significant difference between 10, 20 and 30% of solvents for 6 (a, b) and12 (x, y, z) hours at p 0.05 by Tukey's HSD. "*" and "**" Indicates pair-wise comparison made between 6 and 12 h samples at p 0.01 and p 0.001 by t-test based on Holm's Sidak method.

In vitro mycorrhizal root viability studies
After treatment mycorrhizal roots were washed in sterile distil water thrice and then placed on M media plates and root tip positions were marked as a reference for root growth measurements. The plates were observed weekly for mycorrhizal (hyphal growth and spore germination) and root growth up to twelve weeks of age.

Statistical analysis
All statistical tests were performed using a commercial statistics software package (Prism 6.02, GraphPad Software Inc, La Jolla, USA). Prior to any statistical analyses, all data sets were tested for normality using the Shapiro-Wilk's test and homogeneity of variance by Levene's test. Mycorrhiza spore viability percentage data was arcsine square root transformed. All data are expressed in terms of mean AE SEM.
Pair-wise comparisons were made by the Holm's Sidak method to identify the difference (p 0.05, 0.01) between i) 6 and 12 h samples of ethanol and methanol in TAE, ii) ethanol and methanol at different percentage in TAE and iii) soaking and soaking with sonication assisted extraction in low molar ionic liquids. One way analysis of variance using Tukey's HSD (Honestly Significant Difference) at p 0.05 was used to determine significant differences in RA concentration between the different percentages used of ethanol, methanol, DMSO, ionic liquids Triton X-100 and Tween-20 after TAE and SAE.
Data obtained from the experiments on the effect of different treatments on mycorrhizal spore viability (viability percentage) were analyzed using Tukey's HSD test of significance at p 0.05.

Selection of starting material
No peak of RA (retention time, 15.38 min) was detected in the HPLC chromatogram of sodium citrate buffer obtained after deionization of solidified medium utilized for propagation of mycorrhizal hairy roots showing that deionization has no effect on RA extraction. However, other polyphenolics were detected at 280 nm (Fig. S1).

Pretreatment analysis: soaking in water
Soaking had no effect on the release of RA into water after 6, 12 and 24 h of incubation at room temperature. HPLC chromatograms obtained in pretreatment experiment at three different time periods were similar to that of sodium citrate buffer obtained after deionization of solidified medium used for propagation of mycorrhizal roots (Fig. S1).

RA concentration in the extractant
No RA was detected in any percentage of ethanol or methanol at 25 C after 6 and 12 h of incubation (results not shown). At 30 C, 30% methanol and 12 h was identified as the best solvent percentage and time combination for RA extraction (Fig. 1a, b). A significant difference in the concentration of RA extracted was found between 30% methanol and 30% ethanol after 6 and 12 h of incubation at 30 C. At higher temperatures of 40 and 50 C significant differences were found between 6 and 12 h samples at all percentages for both ethanol and methanol (Fig.1c-f). Avery low amount of RA (0.006 AE 0.001 mg/ L) was detected in water after 12 h of incubation at 50 C. On comparison between extraction with ethanol and methanol at 40 and 50 C, higher concentration of RA was found in all ethanol treatments. Among the four temperature studies, highest yield of RA was obtained in 30% ethanol at 50 C (0.44 mg/L). In conclusion, use of a higher percentage of solvent (methanol or ethanol) at a higher temperature (50 C > 40 C > 30 C) for a prolonged time period (12 h > 6 h) enhanced RA yield.

Mycorrhizal spore viability
Red colored spores (intra-radical and extra-radical) were considered viable (Fig. 2a, b). Highest viability percentage was found in control for all the treatments. Temperature clearly affects mycorrhizal viability as a decrease in viability percentage was observed with increased temperature. Minimal or no viability was found at 50 C. Decrease in mycorrhizal viability with increased percentage of solvent in the extraction medium and exposure time at all temperatures was also observed. 6 h treatment can be considered better than 12 h in terms of maintaining higher mycorrhizal viability percentage. Thus, solvent (10% methanol or ethanol), temperature (30 and 40 C) and time (6 h) were identified as optimal conditions for maintaining mycorrhizal viability (Table 1).

Root and mycorrhiza growth on M media
Root growth was observed for samples treated at 25 and 30 C, while no root growth or retarded growth was obtained for samples treated at 40 and 50 C after 6 and 12 h. Both root and hyphal growth was obtained only in 10% methanol at 25 and 30 C after 6 and 12 h of treatment (Fig. 2c-f). 10% methanol at 30 C was therefore found to be the optimal solvent and temperature combination for maintaining mycorrhizal and root growth.

RA concentration in the extractant
A low concentration (0.022 AE 0.001 mg/L) of RA was detected in water after sonication of mycorrhizal roots for 15 min. Significant differences were found between different percentages of all organic solvents used for SAE (Fig. 3a, b). At low percentages (10 and 20%) similar RA levels were found in methanol and ethanol while at 40 and 50%, a higher RA concentration was found in methanol than in ethanol.

Mycorrhizal spore viability
A lower spore viability percentage was found in ethanol than in methanol at all percentages (Table 2a). Higher mycorrhizal spore viability percentage was found in 10 and 20% methanol. Methanol was selected as the solvent system that retains the highest viability of mycorrhizal spores.

Root and mycorrhiza growth on M media
Similar to the mycorrhizal viability results, no root and hyphal growth was obtained in samples sonicated in ethanol. Growth of both roots and hyphae similar to that observed in TAE was found for samples sonicated in 10% methanol (Fig. 2c-f).

Treatment analyses: sonication assisted extraction (SAE) in ionic liquid
3.5.1. Comparison between water, ionic liquid and methanol for extraction of RA and mycorrhizal spore viability 3 methyl-imidazolium bromide (1 M) was compared with water and methanol as an extractant of RA from freeze-dried Table 1 Viability percentage of mycorrhizal spores after temperature-assisted extraction in three different percentages of ethanol and methanol at four different temperatures and two time periods. z z Data is represented as a mean AE SEM of each of five replicates (n = 5) in percentage. Same letters indicate no significant differences (p 0.05) between different percentages of solvent at one temperature according to Tukey's HSD. Highlighted figures show percentage and temperature which can be selected for further investigation studies. mycorrhizal roots (Fig. 3c). 0.97 AE 0.01 mg/g DW of RA was found in ionic liquid extracts. The RA concentration (mg/g DW) found in the ionic liquid was 4.5 fold higher than water but lower than methanol (two-fold). Viability studies showed no viable spores (Table 2b) in 1 M ionic liquid (similar to methanol), hence it was concluded that low molar ionic liquid can be used for screening studies as an alternative for biocompatible extraction method development.

Low molar ionic liquid extraction
3.5.2.1. RA concentration. An increase in RA concentration was found with increasing molarity of ionic liquid with both types of processes used (sonication and soaking + sonication) (Fig. 3d). On comparison between soaking plus sonication and sonication at three different molarities, a higher RA concentration was found in the soaking plus sonication treatment. At 0.75 M, almost double the amount (0.37 mg/L) of RA was found in soaking plus sonication in comparison to sonication alone (0.17 mg/L). The lowest RA was found at 0.25 M in sonication-assisted extraction (0.02 mg/L).

3.5.2.2.
Mycorrhizal spore viability. Viability of mycorrhizal spores was found to decrease with increase in molarity of the ionic liquid (Table 2c,   Nonane and Dodecane were not found suitable for biocompatible extraction method development in our study as no RA was detected in the extracted solution. Low levels of RA were found in 0.5% and 1.0% of DMSO (0.016 mg/L and 0.034 mg/L respectively). Further, no RA was detected in all perecentages of Triton X-100 and Tween-20.
Samples sonicated in biocompatible solvents and in three different percentages of DMSO showed 100% viable spores while no viability was observed with Triton X-100 and Tween 20 (Table 2d-f). Similar to viability results, root and hyphal growth was observed for samples treated in DMSO, showing its potential for selection as a solvent of choice.

Discussion
A range of options for the extraction of secondary metabolites by biocompatible extraction methods have been presented in the literature [7,8] but none have dealt with mycorrhizal roots and there are only four reports [4][5][6]13] with protocols that simultaneously maintain viability of the plant samples after extraction. For the development of an efficient extraction protocol, selection of starting material, pretreatment, solvent type and its percentage, extraction methodology and determination technique play a key role [16,17]. In our study different organic and biocompatible solvents and surfactants were screened with two agitation techniques that used variation in temperature and sonication to assist extraction of rosmarinic acid from mycorrhizal hairy roots.
Selection of starting material and its pretreatment play a critical role in development of the extraction methodologies [16]. Conventionally, dried and powdered plant materials are used for the extraction of polyphenolics. Being free from excess water and of homogenous particle size makes these materials highly suitable for secondary metabolite extraction [9]. However, in our study where maintenance of mycorrhizal and root viability for continuous propagation was the primary requirement, we utilized fresh and intact mycorrhizal roots (non-homogenized). Soaking as a pretreatment at room temperature was not found suitable for the release of RA from mycorrhizal roots of O. basilicum. Pretreatment analysis clearly suggested that processes other than soaking in water at room temperature which have ability to degrade or disrupt plant cell walls and membranes in a controlled manner such as chemical destabilizers (ammonium sulfate; EDTA), surfactants (Tween-80), pH or cellulolytic (cellulases; hemicellulases) or pectinolytic (pectinases) like enzyme factors [4,[18][19][20] can be explored in future as pretreatment alternatives with mycorrhizal roots for biocompatible extraction of RA.
We screened different percentages of aqueous ethanol and methanol with temperature (extended contact and slow release of secondary metabolites) and sonication (fast release of secondary metabolites) effect for the extraction of RA in a biocompatible way. Shaking ensured homogenous and continuous contact between the solvent and mycorrhized hairy roots for metabolite extraction. Ethanol was used because of commercial applicability, acceptability and GRAS (generally recognized as a safe) status in the food industry [10,21]. Polarity of a solvent affects extraction efficiency [17] hence; methanol was explored as an alternative to ethanol in our study. Increase in the concentration of RAwith increased temperature, percentage of solvent and exposure time was recorded, however, negative effect of the same was found on mycorrhiza and root viability. Extraction efficiency improves due to increase in the diffusion rate of the solvent and also due to decrease in its viscosityand surface tension [22,23]. Water alone as an extractant was not found to be effective for RA extraction with TAE. 10% methanol at 30 C and 6 h emerged as the most suitable treatment for root and mycorrhizal viability (Table 3). Through TAE analysis, 30 C was identified as the optimum temperature for sonication assisted extraction.
Many reports exist on the use of sonication assisted extraction for enhanced extraction of polyphenolics [10,20]. In contrast to TAE, RA was detected in water and sonication was found as a suitable technique for extraction of RA in lower percentages of organic solvents. Sonication was found to increase RA concentration with Table 2 Viability percentage of mycorrhizal spores after sonication-assisted extraction in organic solvents, ionic liquid, dimethyl sulfoxide, biocompatible solvents and surfactants. z z Data is represented as mean AE SEM of each of five replicates (n = 5) in percentage. In section (a) same letters indicate no significant differences (p 0.05) between different percentages of solvent according to Tukey's HSD. Highlighted figures show percentage of solvent which can be selected for further investigation. In section (c), different letters indicate significant differences (p 0.05) between different molarity of the ionic liquid for one process used. Highlighted figure shows highest viability obtained with ionic liquid and sonication assisted extraction. In section (d) and (e) similar letters indicate no significant differences (p 0.05) between different percentages of DMSO, biocompatible solvents. "Nil" indicates that no red (viable) spores were observed in the samples.
increased percentage of the organic solvent used. Although high concentration of RA was obtained in SAE treatment, it was found to have negative impact on viability of mycorrhizal roots. Ten percent methanol with fifteen minutes sonication was found as the best combination for biocompatible extraction in our study. A constant sonication frequency (45 KHz) was used in our study which indicates that increase in sonication frequency with continuous or periodic exposure can be an alternative for prospection in future. While we believe that we have analyzed the major aspects of a successful extraction regimes, clearly there are other possibilities such as higher sonication frequencies and refinement of the solid: liquid ratio (S/L), for further exploration that will provide further optimization of extraction conditions.
Ionic liquids with sonication are the most recently used green extraction solvents for polyphenolic extraction [12,22]. Ionic liquid was found to be effective for RA extraction from mycorrhizal roots however, an inverse relationship was found between molarity of ionic liquids and mycorrhizal viability. Low molar alkyl substituted ionic liquids such as 1-ethyl-3-methylimidazolium, 1-butyl-3methylimidazolium, 1-hexyl-3-methylimidazolium, 1-octyl-3methylimidazolium may provide alternative extraction opportunities for the maintenance of mycorrhizal viability along with metabolite extraction. No RA was found following extraction with the biocompatible solvents (nonane, dodecane) and this is likely due to their non-polar nature. Among all the solvents used DMSO showed highest mycorrhizal viability and root growth but very low amount of RA suggesting further exploration. Surfactants (Triton X-100 and Tween-20) at 1-3% were not found suitable for mycorrhizal viability hinting lowering of their percentage in future studies.

Conclusions
In conclusion, we explored several extraction alternatives that can both preserve and maintain the viability of the fungus and plant root with RA extraction and have opened up avenues for use of mycorrhizal roots as continuous resource of biological (mycorrhizal propagules) and biochemical (secondary metabolite) products. 10% methanol in water, higher percentages of DMSO and low molar ionic liquids with sonication are the leads for future exploration studies (Table 3).
While the extraction of RA used a suite of specifically optimised parameters, these are likely to vary with host plant and mycorrhiza species. The work presented here has been developed as a platform for the exploration of other protocols for biocompatible extraction of medicinally important compounds and also provides insights into the use of axenically developed mycorrhizal co-cultures for continuous mycorrhizae and metabolite production.

Funding source
Deakin University, Australia provided a post graduate scholarship to SS.

Conflict of interest
Authors declare no conflict of interest.