Effects on mineral status and milking performance of feeding difructose anhydride to transition

The objective of this study was to assess the potential effect of difructose anhydride III (DFAIII) on calcemia, magnesemia, and milking performance of dairy cows. Sixty-six multiparous Holstein cows in late pregnancy (gestation days = 253.8 ± 2.13 d) were blocked according to their expected calving date and randomly assigned to either receiving no supplementation (Control) or receiving 40 g/d of DFAIII (DFA) between −14 and +7 d relative to calving following a complete randomized block design. Cows on Control received 640 g/d of a pellet containing no DFAIII, whereas DFA cows received the same pellet but containing 6.25%DFAIII. Pellets of each treatment were top-dressed on a daily basis while cows were dry, and were fed via an automatic feeding system twice daily (320 g each feeding) during milking. Dry cows were fed once a day, whereas lactating cows were fed twice daily. Dry matter intake was individually monitored on a daily basis. Cows were milked twice daily and milk production and milk protein and fat contents recorded at every milking. Cows were kept on the study until they reached 21 d postpartum. Cows were weighed at dry-off (about 45 d before study enrolment) and twice daily after calving at the exit of the milking parlor. Three days before the expected calving date and 6, 12, 24, 48 h and 7 and 14 d after calving cows were blood sampled for subsequent determination of serum Ca and Mg concentrations. There were no differences in DMI before calving, but DFA cows consumed more feed than Control cows about 15 DIM. All cows lost BW after calving but DFA cows lost slightly less BW during the first 5 DIM than Control cows. Cows on DFAIII produced more milk around 10 DIM compared with Control cows, and DFAIII cows produced more milk protein than Control cows after 3 d post-calving. Serum Ca concentrations were not affected by DFAIII supplementation; however, serum Mg concentrations at 6, 12, and 24 h after calving were greater in DFA than in Control cows. In conclusion, DFAIII did not affect postpartum calcemia but improved magnesemia between 6 and 24 h post-calving. Milk production in DFA cows was improved around 10 d post-calving and milk protein yield after 3 d postpartum compared with Control cows. The mechanism leading to increased Mg availability is not clear and warrants further research.


INTRODUCTION
Some degree of hypocalcemia as indicated by reduced serum concentrations of both total and ionized calcium the day after calving is commonly reported in the literature (Leno et al., 2017;Rodríguez et al., 2017;Robles-Guirado et al., 2023).This transient hypocalcemia is caused by a sudden increase in Ca demand to sustain colostrum and milk production coupled with an impaired efficiency of Ca absorption (Horst et al., 1990) and poor bone Ca resorption (Kamiya et al., 2005), especially in multiparous cows.Intestinal absorption of Ca can occur via transcellular and via paracellular pathways.The transcellular absorption of Ca is a rate-limiting active transport process dependent on energy and on a Ca-binding protein which activity is modulated by the availability of 1,25-dihydroxyvitamin D (Horst, 1986).The paracellular pathway is a passive transport through tight junctions mainly dependent on Ca concentration gradient between the lumen and the basolateral side of the intestinal mucosa (Bronner, 1998).Several factors may modulate paracellular intestinal transport.For example, lysophosphatidic acid increases the permeability of tight junctions by increasing phosphorylation of occludin (Hirase et al., 2001).Difructose anhydride III (DFAIII) is a crystal disaccharide obtained from inulin of chicory root formed by 2 fructoses (Kikuchi et al., 2004) that has been previously reported to be able to loosen intestinal tight junctions by changing the distribution of actin Effects on mineral status and milking performance of feeding difructose anhydride to transition cows Miguel Escartín, 1 Núria Rialp, 1 and Alex Bach 2 * filaments and claudin-1, but not occludin, resulting in a reduction of transepithelial electrical resistance (Suzuki and Hara, 2006).As a result, DFAIII has been reported to foster an increase in Ca absorption via the paracellular pathway in the intestine of rats (Mineo et al., 2002;Mitamura and Hara, 2005) and humans (Shigematsu et al., 2004;Suzuki and Hara, 2004).Similarly, Teramura et al. (2015a) reported, in an ex-vivo experiment using everted duodenal sacs of cattle, that Ca absorption increased in the presence of DFAIII.Difructose anhydride III reaches the duodenum of cattle 1 h after being consumed (Nakai et al., 2007), and about 70% of the intake of DFAIII escapes rumen degradation (Teramura et al., 2015b); thus, it can directly act on the intestine of cattle when provided through the ration.Teramura et al. (2015a) reported that provision of 40 g/d of DFAIII during 2 wk before and 1 wk after parturition improved calcemia of dairy cattle during the first 2 d post-calving.Previous studies have shown that cows that experience postpartum clinical calcemia produce less milk during the first weeks of lactation (Rajala-Schultz et al., 1999;Venjakob et al., 2018).However, the potential consequences of moderate or subclinical hypocalcemia postpartum (which occurs much more frequently than severe hypocalcemia) on subsequent milk production are less clear.Some studies report no effects (Østergaard and Larsen, 2000;Martinez et al., 2012), others report improvements (Jawor et al., 2012), and others report negative effects of postpartum subclinical hypocalcemia on milk yield (Chapinal et al., 2012).Differences between studies may be due to the threshold used and the duration of the decreased calcemia episode.On the other hand, the potential effect of molecules such as DFAIII on magnesium absorption in ruminants is lacking.Contrary to Ca, Mg absorption in ruminants takes place mainly in the rumen.Most studies indicate that Mg absorption in the rumen occurs predominantly by an active transport and is influenced by the concentrations of other molecules in the rumen fluid such as K (Leonhard-Marek and Martens, 1996) or volatile fatty acids (Schweigel and Martens, 2000).However, there are tight junctions in the stratum granulosum of the rumen, but not in the stratum corneum or the stratum basale (Graham and Simmons, 2005), and, thus it may plausible to hypothesize that DFAIII could also improve Mg absorption in the rumen by altering the permeability of the tight-junction in the stratum granulosum and then Mg could be absorbed by active transport from the stratum basale.Therefore, the hypothesis of this study was that supplementation of DFAIII around calving would improve calcemia, and magnesemia, and in turn improve milk yield following parturition because increasing calcium supply postpartum has been positively correlated with milk produc-tion (Oetzel, 2013).The objectives were to determine the effect of DFAIII on calcemia, magnesemia, and milking performance of dairy cows.

MATERIALS AND METHODS
The experimental protocol for this study was evaluated by the ethical committee of the Institut de Recerca i Tecnologia Agroalimentàries (Barcelona, Spain) and approved by the Catalan government under the code 11117.Sixty-six multiparous dry Holstein cows (2.7 ± 0.88 parities) in late pregnancy (gestation days = 253.8± 2.13 d) were enrolled in this study 21d before their expected calving date following a complete randomized block design between June and September 2021.There was a total of 10 blocks.Cows were blocked according to expected calving date and randomly assigned to either receiving no supplementation (Control) or receiving 40 g/d of difructose anhydride III (DFA).Twenty-one days before the expected calving date, cows were moved to individual pens (3 × 10 m) bedded with straw and equipped with troughs for ad libitum access to water.Cows in adjacent pens had physical contact.Cows were fed once a day while dry and twice daily after calving.Both TMR (Control and DFA) were calculated to be isonutritive, and met nutrient requirements recommended for dry or milking dairy cows (NASEM, 2021).After calving, cows were milked twice daily ca.0600 h and 1730 h in a rotary parlor and individual milk yield and milk fat and protein content were measured electronically (AfiMilk, Afikim Ltd., Israel).Cows were weighed at dry-off (about 45 d before study enrolment) and twice daily after calving using an electronic scale placed at the exit of the milking parlor.Cows on DFA received 40 g/d of DFAIII 14 d before the expected calving date, and supplementation continued for the first 7 d postpartum.Cows were kept on the study until they reached 21 DIM.Treatment application was performed by top-dressing 640 g/d of pellets while dry, and 640 g/d of split in 2 portions (one for each milking) of 320 g fed using automatic dispensers in the milking parlor.The pellets for the Control cows contained wheat bran meal (20%), soybean meal (78.7%), and salt (1.3%); whereas the pellets for DFAIII cows contained wheat bran meal (18.8%), soybean meal (73.8%), salt (1.15%), and DFAIII (6.25%).Personnel feeding the animals was blinded to treatment.
Samples of TMR (both for dry and lactating cows) were collected daily and pooled fortnightly for subsequent nutrient analysis.Ration samples were analyzed for DM content by drying at 103°C for 24 h, ash following AOAC method 942.05 (AOAC, 2000) at 550°C for 3 h, and weighing after cooling in a desiccator; ether extract using the AOAC method 920.30(AOAC, 2000); CP  EN 15510:2017(BSI, 2017) by ICP-AES.Lastly, the content of DFAIII was validated using HPLC.Ingredient and nutrient composition of the diets is presented in Table 1.
Dry matter intake was individually monitored on a daily basis.Feed intake of dry cows was determined by manually weighing feed offered and refused to each cow.After calving, cows were group-housed in a single freestall pen and feed intake was monitored using electronic bins (Moo Systems, Cortes, Spain).Blood samples were obtained from every cow by venipuncture of the coccygeal vessels using evacuated tubes (Vacutainer, Becton Dickinson, Madrid, Spain) at 3 d before the expected calving date (at 0800 h), at 6, 12, 24, 48 h and 7 and 14 d after calving (the last 2 samples also taken at 0800 h) for subsequent determination of serum Ca and Mg concentrations.Blood samples were maintained at room temperature 30 min to induce coagulation and serum was obtained after centrifugation at 2,000 x g for 10 min at 4°C, and then kept at −20°C until analysis.Serum Ca and Mg concentrations were measured using an AU400 analyzer (Beckman Coulter, Hamburg, Germany) following the Arsenazo III method (OSR 60117) and the xylidyl blue method (OSR 6189), respectively.Milk lactose, urea, and SCC contents on milk samples obtained about 10 DIM were determined using infrared spectroscopy.Lastly, postpartum energy balance was calculated as the difference between net energy consumed (i.e., DMI multiplied by the dietary content of net energy) and energy required, with the latter being estimated using milk yield, milk composition, and body weight and equations from NASEM (2021).

Calculations and statistical analysis
Based on previous literature (Hanada et al., 2021), it was assumed that DFAIII would elicit at least a 15% difference in serum Ca concentrations and considering a standard deviation in calcemia values of 0.31 mM (Rodríguez et al., 2017), to reach significance at P < 0.05 with a power = 0.8 a sample size of 15 cows per treatment would be needed.Similarly, to detect differences in milk yield assuming a standard deviation of 4.43 kg/d during early lactation (Bach et al., 2019), 21 cows per treatment would be needed to detect 10% difference in milk yield at P < 0.05 and an power = 0.8.
All data were checked for normality prior statistical analysis, and milk SCC were ln-transformed to reach normality.Energy-corrected milk was calculated following Bernard (1997).Cow was the experimental unit for statistical purposes.All parameters were analyzed using JMP (version 17, SAS Institute Inc., Cary, NC, USA) with a mixed-effects model for repeated measures with treatment, time, and their interaction as fixed effects, and cow and block as random effects using an antedependent structure with equal variances for all dependent variables except for serum Ca and Mg concentrations where an autoregressive structure of order 1.Non-repeated data (i.e., milk Ca, Mg, lactose, and urea contents and milk SCC counts) were analyzed using a mixed-effects model including block as random effect and treatment as a fixed effect.

RESULTS AND DISCUSSION
Actual days on treatment before calving was 16.4 ± 5.23 d, and actual day for the blood sample targeted at 3 d before calving was 5.3 ± 2.62 d.Four cows gave birth to twins (2 cows in the Control and 2 cows in the DFA treatment).Forty-nine and 46% of the calves born to cows in the Control and in the DFA group were males, respectively.Five cows in the Control group and one in the DFA group were treated for ketosis for 2 d, 1 cow in the Control group and 2 in the DFA treatment had retained placenta, and 2 cows in the Control group and one in the DFA treatment had mild mastitis (lasted < 3 d in all cases).There were no differences in DMI before calving between treatments (Table 2), but after calving there was an interaction (P < 0.01) between treatment and DIM resulting in greater DMI early after calving and about 15 DIM in DFA compared with Control cows (Figure 1).These results are in line with those reported by Wynn et al. (2015), who also observed greater DMI after calving, but not before, in cows supplemented with DFAIII.All cows lost BW after calving (Table 2), but this decrease was influenced by an interaction between treatment and DIM (Table 2), with DFA cows loosing slightly less BW during the first 5 DIM than Control cows (data not shown).These observations are in line with the study from Hanada et al. (2021) who reported a tendency for a lower BW loss early in lactation of cows supplemented with DFAIII.Despite the slightly lower BW loss in DFA cows compared with Control cows, there were no differences (P = 0.70) in energy balance after calving between treatments.Energy balance (considering DMI, ECM, and BW changes) for Control cows was −11.93 Mcal/d whereas for DFA cows was −11.24 Mcal/d over the first 21 DIM.There was an interaction between treatment and DIM for milk yield (P < 0.01), which resulted in greater milk yield in DFA than in Control cows around 10 DIM (Figure 2).Wynn et al. (2015) also reported increased milk yield early in lactation in cows supplemented with DFAII.A more recent study (Hanada et al., 2021) observed an increase in milk yield between 1 and 5 DIM in cows supplemented with DFAIII.The increase in milk yield in DFA cows herein was, at least partially, due to the a greater DMI (Figure 1).However, the reason behind the increased DMI observed herein and previous studies (Wynn et al., 2015;Hanada et al., 2021) is difficult to explain.Hanada et al. (2021) suggested that improvements in calcemia allowed for more efficient rumen contractions and that led to improvements in DMI.However, herein, calcemia (as discussed below) did not differ between treatment groups.Another reason for the increase in milk yield could be found in the lower milk SCC contents recorded in DFA compared with Control cows at around 10 DIM (Table 2), as it is commonly accepted that impairments in udder health around parturition result in decreased milk yields (Gonçalves et al., 2018).Several studies have reported a negative relationship between serum Mg concentrations and milk SCC (Qayyum et al., 2016;Jassim and Abdul-Wadood et al., 2019); thus, the improvement in milk SCC counts herein, could be partly attributed to the improved magnesemia observed in DFA cows (as discussed below).
Milk fat content, milk fat yield, and milk protein content were not influenced by dietary treatments; however, milk protein yield was greater (P < 0.05) in DFA than in Control cows for most of the days after 3 DIM (Figure 3).As a result, ECM yield was also affected by an interaction between treatment and DIM (Table 2) and followed the same pattern as milk yield (data not shown).There were no differences between treatments on feed efficiency (Table 2), but an interaction between treatment and DIM resulted in greater feed efficiency in Control than in DFA cows during the first 5 DIM (data not shown).Although feed efficiency is, in general, a desirable outcome, a high feed efficiency, especially in early lactation may be consequence of an excessive mobilization of body reserves, which may hamper the health of the cow (Bach et al., 2020).At ~10 DIM, milk Ca, Mg, and urea did not differ between treatments, but lactose content was greater in DFA than in Control cows (Table 2).
Serum Ca concentrations were not affected by DFAIII supplementation (Table 3); however, magnesemia was affected by an interaction between treatment and DIM, with DFA cows having greater serum Mg concentrations at 6, 12, and 24 h after calving than Control cows (Table 3; Figure 4).Previous studies report contradictory results in relation to serum Ca concentrations as a response to DFAIII supplementation.Wynn et al. (2015) did not report changes in serum Ca, but contrary to the observations herein, Hanada et al. (2021) observed increased serum Ca concentrations when supplementing DFAIII to cows.However, Teramura et al. (2015a) also reported an increase in serum Mg concentrations as observed in the current study, although the increase was much later (about 3 DIM) than in the    Hansen et al., 2002;Robles-Guirado et al., 2023).This increase in serum Mg is likely to be linked to the decrease in serum Ca levels that leads to a release of parathyroid hormone (secreted to increase Ca availability) which in turn exerts an increase in renal reabsorption of Mg (Riond et al., 1995a).Also, a shift of Mg from intracellular compartments to extracellular compartments during hypocalcemia may be also partially responsible for the increase of serum Mg after calving (Riond et al., 1995b).The fact that DFA cows had greater serum Mg levels could indicate that absorption of this mineral from the diet was increased, because Mg mobilization from bone is very minor in cattle (Houillier, 2014).Absorption of Mg takes place predominantly in the rumen in an unregulated manner and it is influenced by other dietary components such as potassium (Schonewille et al., 2008), but as hypothesized herein DFAIII may have increased the permeability of tight-junctions to Mg. Furtermore, Mg can also be absorbed also in the small (Harrison et al., 1963) and large intestine (Meyer and Busse, 1975).Intestinal Mg absorption is likely to take place through transient receptor potential melastatin  6 channel in the distal small intestine (Groenestege et al., 2007), for which there is no evidence that DFAIII may exert any effect.However, Mineo et al. (2004) reported increased rates of Mg absorption in the jejunum, ileum, and cecum of rats when exposing the tissues to DFAIII, thus it could also be speculated that the increased postpartum magnesium could also be partly due to increased intestinal Mg absorption.

CONCLUSIONS
Escartín et al.: DIFRUCTOSE ANHYDRIDE DURING TRANSITION using the Kjeldahl method 984.13 from AOAC (2000) with CuSO 4 as catalyst and a 2520 Digestor, Kjeltec 8400 Analyzer unit and a 8460 sampler unit (FOSS Analytical A/S Hilleröd, Denmark); NDF following Van Soest (1991); Ca and Mg following the method BS Escartín et al.: DIFRUCTOSE ANHYDRIDE DURING TRANSITION

2T:
Effect of treatment; D: Effect of day relative to calving, TxD: effect of interaction between treatment and day relative to calving.3Calculated as ECM/DMI.*Milk samples collected at 9 ± 2.5 DIM.

Figure 1 .
Figure 1.Dry matter intake of Holstein cows as affected by dietary treatments.Control: unsupplemented; DFA: supplemented with 40 g/d between −14 and 7 d relative to calving.* Denotes differences (P < 0.05) between Control and DFA within day.
Figure 2. Milk yield of Holstein cows as affected by dietary treatments.Control: unsupplemented; DFA: supplemented with 40 g/d between −14 and 7 d relative to calving.* Denotes differences (P < 0.05) between Control and DFA within day.

Figure 3 .
Figure 3. milk yield of Holstein cows as affected by dietary treatments.Control: unsupplemented; DFA: supplemented with 40 g/d between −14 and 7 d relative to calving.* Denotes differences (P < 0.05) between Control and DFA within day.
When feeding rations similar to the ones in this study, supplementation of DFAIII increases milk production around 10 d post-calving, and increases milk protein yield after 3 d postpartum.Serum calcium concentrations are not affected by DFAIII supplementation, but serum magnesium concentrations are greater in cows supplemented with DFAIII between 6 and 24 h postcalving.The exact mechanism leading to increased Mg availability is not clear and warrants further research.

2T:
Effect of treatment; D: Effect of day relative to calving, TxD: effect of interaction between treatment and day relative to calving.

Figure 4 .
Figure 4. Serum magnesium (solid bars) and calcium (dashed bars) concentrations of Holstein cows as affected by dietary treatments.Control: unsupplemented (gray bars); DFA: supplemented with 40 g/d between −14 and 7 d relative to calving (blue bars).a,b Different letters within hour indicates differences (P < 0.05) between Control and DFA.

Table 1 .
Escartín et al.: DIFRUCTOSE ANHYDRIDE DURING TRANSITION Ingredient and nutrient composition of the rations for dry and lactating cows (includes concentrate top-dressed during the dry period or fed in the milking parlor during lactation)

Table 2 .
Escartín et al.: DIFRUCTOSE ANHYDRIDE DURING TRANSITION Animal performance as affected by dietary treatments

Table 3 .
Serum calcium and magnesium concentrations as affected by treatments