Effects of Different Feeding Methods on Behaviors，Immunities and Growth Performances of Suckling Calves

Bai Xiu-juan，Su Hang，Bao Jun，and Lv Jing*

College of Animal Sciences and Technology，Northeast Agricultural University，Harbin 150030，China

Abstract: Behaviors，immunities and growth performances of suckling calves to either fed milk through barrel feeding or artificial nipple feeding were investigated in this study. Seventy-two newborn female suckling Holstein calves were selected and divided into two treatments which fed regular milk through milk barrels or artificial nipples. The behaviors′ parameters were measured on 14，29 and 44 days. Body weight and body size of the calves were recorded on 15，30 and 45 days. Immune indicators were collected in quiet situation on 15，30 and 45 days. Compared with the barrel feeding method，calves fed by artificial nipples had less non-nutritive behaviors，stronger growth performances and higher immune levels. Artificial nipples gave calves sucking conditions and reduced the motivation of calves to suck each other，which might promote the development of complex stomachs，allow food to be better digested and more effective. It also reduced cross-infection indirectly which increased the calves' immunities.

Key words: growth performance，behavior，immunity，artificial nipple，barrel

Introduction

Calves are replacement groups of adult dairy cows on dairy farms. After birth，due to poor body resistance，incomplete development of the digestive system and other characteristics，calves are easily affected by the external environment，while the organs are in a state of development，suitable feeding conditions are very important for better growth，meanwhile suitable feeding conditions can lay a solid foundation for high yield (Xu，2011).

Because the 0-2 months old calves' digestive system are not well developed，the inappropriate feeding methods are likely to cause diseases (Xu，2011). For example，when feeding calves nowadays，maybe a small amount of milk into the rumen don't be digested and cause diarrhoea of calves (Bao，2019).In commerce，suckling calves have been replaced largely by artificial nipples or barrel feeding (Perssonet al.，2013) to improve calves' diets. Barrels were often used to feed calves in the early days，but later researches found that the incidence and the persistence of diarrhea are greater in calves fed from open pails than in those fed from artificial nipples (Wise and LaMaster，1968)，artificial nipples feeding calves show a numerically higher value of average day gain (ADG)than those fed with barrels (Donget al.，2017) and abnormal behaviors that are detrimental to the growth of the calves，due to the unsatisfied sucking motivation of the calve，such as sucking railings and excessive self-reformation. To solve these problems，people used some methods，such as slowing the flow rate of milk，introducing artificial nipples to feed and extending the retention time of the nipple，adjusting the size of the lap and the size of the space. From a behavioral perspective，feeding calves with artificial nipples can effectively reduce the duration of non-nutritive sucking behaviors at the edges of the bucket and sucking railing. Xu (2011) found that feeding with artificial nipples can effectively reduce the duration of nonnutritive sucking behaviors and the time of abnormal behaviors of sucking each other. From a nutritional point of view，teat-feeding has been presumed to be superior because of its favourable influence on digestive processes，e.g. secretion of saliva，salivary lipase and gastric fluid (Wiseet al.，1976; Roy，1980). Lidforset al. (1994) found if the calves eat for less than 1 min，they will hardly suck. If the time is too long，they will produce non-nutritive sucking，which will affect the growth and development. When calves are fed milk，feeding the calves with artificial nipples can reduce the time of mutual sucking (Jensen and Budde，2006)，indicating that different feeding methods can change the behaviors of the calves. The posture of calves using artificial nipples is similar to that of sucking cows' nipples under natural conditions，both raising and sucking，calves drink the milk and directly enter the abdomen with digestive enzymes(Wise and Anderson，1939; Friend and Dellmeier，1988). If calve bows its head，the emulsion will enter the rumen and cause diarrhea. Artificial nipple feeding will guide calve to drink milk to promote its growth and development. According to the above，the present experiment further predicted that the artificial nipple feeding would affect its immune level. In terms of growth performance，Applebyet al.(2001) found that calves fed freely gain more weights at 1 week of age than calves fed with milk barrels; however，some studies have shown that barrel feeding and bottle feeding have no significant effects on calves'weights (Veissieret al.，2002). The effects of different feeding methods on calve growth performances have not yet obtained exact results in the current study. It is hypothesised that it will have a positive effect and this aspect need further studies.

The understanding and mastery of livestock's behaviors，physiologists and immunities can directly or indirectly improve the productivity of the livestock industry. At present，there are reports on the effects of feeding methods on the behaviors and growth of calves，but there are few reports on behaviors，growth development and immune integration. How different feeding methods will affect calve behaviors，growth and development and the levels of immunities is a problem to be further confirmed. This experiment aimed to study the effects of different feeding methods(barrel feeding and artificial nipple feeding) on the behaviors，growth performances and immunities of Holstein calves to further guide productions.

Materials and Methods

Experimental design and animals

The experiments were conducted on a commercial dairy farm (calves were typically housed 6-10 per pen) from June to October，2016. Management of the experiment complied with the guidelines of the Institutional Animal Care and Use Committee of Northeast Agricultural University. Seventy-two calves were fed colostrum at postnatal 4 h and one day later，they were deprived from the cows，grouped in a herd of six calves received the same feeding regimes. Then，the calves were randomly divided into two treatments with six repetitions，fed regular milk through the milk barrel or artificial nipple，respectively. The pens(36 m2: 6.0 m (L)×6.0 m (W)×0.8 m (H)) consisted of an area with rice hull bedding (30 m2) and a feeding area (6 m2) with a solid concrete floor. Six stainless steel buckets were used to feed calves in each pen. The calves were fed at 7: 30 a.m. and 4: 30 p.m. each day.The amount of milk fed to the calves was adjusted as the followings: 6 L per day for weeks 0-2; 8 L per day for weeks 3-4; 10 L per day for weeks 5-7. From the 4th day of age，the calves received starter feed (CP of 20%，energy level of 1.66 mcal · kg-1，crude fiber of 12%，crude ash of 10% and lysine of 0.05%) and waterad libitum. During the experiment，the ambient temperature of calves was between 20℃ and 25℃.

Determination index and method

Growth performance measurement

Accurately recorded the daily feeding amount and surplus amount of calf opening materials. The calves were weighed at 12:00-2:30 p.m on 15，30 and 45 days of the experiment，and the weight gain of the calves were observed and recorded. The measurement time of the body size was the same as the weighing time，and the measurement of the body size included the body length，height and chest circumference.

Behavioral observation

Video surveillance system (model: JX-8702，Jixingweishi，Taiwan) was affixed on a post above each pen，allowing to record all the behavioral activities of the calves during the observation days.During the trial period，the behaviors sampling was conducted recorded for 12 h from 6: 00 a.m.-6: 00 p.m.on 14，29 and 44 days. Any occurrence of behaviors，such as self-grooming，exploring and locomotive play(Fukasawa amd Tsukada，2010)，head shaking (Sandemet al.，2006) and tongue-rolling (Redbo，1990)，were recorded by an experienced experimenter，and they were regarded as event behaviors represented as times(no.). Posture (lying and standing) with continuous observations represented as percentage. The behaviors of calves were observed by scanning sampling methods with 5 min interval.

Saliva and blood collection

At 12: 00-2: 30 p.m. on 15，30 and 45 days of the tests，the saliva of the calves was collected in a quiet state to make the calves chew the absorbent cotton ball wrapped by medical gauze until it was completely wetted for about 1 min. After sampling，the calves were placed in a centrifuge tube 2 000 r · min-1for 20 min to separate the saliva from the degreased cotton. The SIgA content was measured with a kit. Saw the instructions for the specific measurement steps.Ten mL blood samples were collected from the jugular vein，4.5 mL serum was collected by centrifuging the collected blood samples in 3 000 r · min-1for 15 min，and the contents of IgA，IgG，IL-2，IL-3，IL-6 and TNF-αin the serum were determined by the kits according to the kits′ instructions for the specific determination steps.

Data analysis

The experimental data were statistically analyzed by SPSS 17.0，the prevent sexual behaviors data were transformed by sqrt and Arcsin，and the normal distribution was tested by One-Sample Kolmogorov-Smirnov Test under Nonparametric Tests. The effects of bucket feeding/artificial nipple feeding on the behaviors，physiologists and immunities of experimental calves were tested by single factor analysis of variance (ANOVA). The main effects of various factors and the interaction of various factors were considered in data processing. The results were expressed as "mean±standard deviation" (p＜0.05). The analytical model was as the following:

Single factor:

Yi=μ+αi+e

Yi-individual observation value;μ-overall average;αi-positive/negative stimulus effect;e-random error.

Results

The results shown in Table 1 revealed that the barrel feeding calves showed significantly higher licking pen and higher crossing sucking than the artificial nipple feeding counterparts (p＜0.05)，whereas the barrel feeding calves had higher standing，self-grooming and locomotive playing behaviors than the nipple feeding calves on 14 and 29 days (p＜0.05). However，the results on the 44th day manifested that the above behaviors were not affected by different feeding methods (plying=0.098，pSelf-grooming=0.331 andpLocomotive play=0.458). The barrel feeding calves indicated significantly lower lying than the artificial nipple feeding counterparts on 14 and 29 days (p＜0.05)，but lying was not affected by different feeding methods on the 44th day (p=0.098).The barrel feeding calves revealed significantly lower exploring than the artificial nipple feeding counterparts on 14 and 44 days (p＜0.05)，but the barrel feeding calves showed significantly higher exploring than the artificial nipple feeding counterparts (p＜0.05).Nevertheless allo-grooming and social playing did not show to be affected by different feeding methods(pAllo-grooming=0.763 andpSocial play=0.485).

Table 1 Effects of feeding methods on behavior of calves (Mean±SD) (p＜0.05)

The results (Table 2) showed that the artificial nipple feeding calves revealed significantly higher feed intake than the barrel feeding counterparts (p＜0.05);body weight (BW) and average daily gain (ADG) did not show to be affected by different feeding methods on the 15th day (pBW=0.12 andpADG=0.07)，whereas the artificial nipple feeding calves revealed significantly higher BW and higher ADG than the barrel feeding calves on 30 and 45 days (p＜0.05). The barrel feeding calves indicated significantly lower body length and heart girth than the artificial nipple feeding counterparts on the 45th day (p＜0.05)，but on 15 and 30 days，body length did not show to be affected by different feeding methods (p15=0.26 andp30=0.91) and heart girth also showed that it did not affected by different feeding methods. Withers height did not affected by different feeding methods (p15=0.41，p30=0.32 andp45=0.06).

The results (Table 3) revealed that the serum IgG，IgA，saliva SIgA，IL-2，IL-3 and IL-6 level in the artificial nipple feeding group were significantly higher than those in the barrel feeding group (p＜0.05). The serum TNF-αlevel of the artificial nipple feeding group was significantly higher than that of the barrel feeding group on 30 and 45 days (p＜0.05)，whereas the level of serum TNF-αdid not show to be affected by different feeding methods (p=0.35).

Table 2 Effects of feeding methods on growth performance of calves (Mean±SD) (p＜0.05)

Table 3 Effects of different feeding methods on immune level of calves (Mean±SD) (p＜0.05)

Continued

Discussion

Behavior

The results showed that the artificial nipple feeding calves revealed significantly lower exploring，cross sucking and licking pen than the barrel feeding counterparts in the late experimental period，but other behaviors did not show to be affected by feeding methods in the late experimental period. Behaviors such as exploring，cross sucking or licking pen could be interpreted as a kind of high emotional arousal induced by negative stimuli (Westerathet al.，2014).Especially，the self-grooming behavior of animals seemed to be associated with unpleasant emotions(Sandemet al.，2006). Compared with the barrel feeding，the artificial nipple feeding could better satisfy the calves' sucking motivation and could make the calves have positive emotions. Calves fed milk with a teat performed less non-nutritive sucking of a dry nipple (Veissieret al.，2002) and less cross-sucking(Loberg and Lidfors，2001) than bucket-fed calves，Jensen and Budde (2006) found that irrespective of group size，nipple fed calves took longer to ingest the milk compared with barrel fed calves and they kept on sucking the empty teat after the milk was ingested，on the contrary，the bucket-fed calves left the bucket soon after it was empty and often turned to cross-suck on other calves. In a study by Lidfors (1993)，all the barrel feeding calves were observed to cross-suck after milk feeding，the results were consistent with what was observed in this experiment. In daily production，there was no guarantee that each calve could get sufficient sucking during barrel feeding，which caused negative emotion，so self-grooming，standing，exploring and locomotive playing behavior occurred more frequently than the barrel feeding group. Jensen and Budde (2006)also found that the barrel feeding calves were more often observed licking the fixtures of the pen and more barrel feeding calves were observed performing social grooming compared with the artificial nipple feeding calves. More oral activities directed toward fixtures of the barrel feeding compared with the artificial nipple feeding calves had been reported earlier (Veissieret al.，2002)，and the more social grooming might be a side effect of the cross-sucking. In summary，this study demonstrated that allo-grooming，cross sucking，licking pen and other behaviors were stereotyped behaviors that did not satisfy the calves' sucking motivation. Therefore，the above results might suggest that more expressions of these stereotypic behaviors indicated that an animal was exposed to a depressed situation. It was found that the artificial nipple could satisfy the calves' sucking motive，which would cause positive emotion and eventually lead to a reduction in the frequency of non-nutritive sucking and other stereotypical behaviors.

Growth performance

The results showed that the artificial nipple feeding calves revealed significantly higher feed intake than the barrel feeding counterparts. This might because calves sucked artificial nipples in the same way that calves suck cow nipples in their natural state.Some studies had pointed out that the esophageal sulcus groove could be fully closed，only when the animal voluntarily drunk liquid and was in a state of excitement unique to young animals. The esophageal sulcus reflex had nothing to do with the chemical composition of the liquid absorbed by the animal，and even water might cause the esophagus to close(Orskov and Ryle，1990). This might explain the increase in calves' feed intake due to sucking habits during weaning. Therefore，the increase in feed intake would affect BW and ADG of calves fed by different methods in the middle and late periods of the experiment. Similar results were also reported by Chigerweet al. (2009) that different feeding methods i.e. barrel feeding，artificial nipple feeding had effect on the BW of calf. The barrel feeding calves indicated significantly lower body length and heart girth than the artificial nipple feeding counterparts in the late experimental period and the similar findings were recorded by Chigerweet al. (2008)，Furmanet al.(1994) and Veissieret al(2013). The heart girth was not only depending upon the body weight gain，but also on the feeding and housing of calves (Quigleyet al.，2013; Chigerweet al.，2008; Furmanet al.，1994; Veissieret al.，2013). But the wither height and body length of calves fed by different methods showed non-significant difference among the groups during entire experimental period. Calves' height might not be as a useful parameter for measuring growth and development as weight. In summary，the growth performance of calves in the artificial nipple feeding group was generally better than that in the barrel feeding group. This might because the calves fed with the artificial nipples fed the same way in the natural state as sucking cows' nipples. When sucking milk，it was closed into a tube through reflex regulation，so the milk could go to the abomasum through the esophagus and omasum through the responsible gate. Therefore，the calves could show good growth performance，and should have strong esophageal groove reflex，especially when calves were used to this type of sucking. When it was fed with milk barrel or bottle，the calf still kept strong esophageal groove reflex，which could make the milk directly enter into the abomasum. It was consistent with the results of this study，the artificial nipple feeding could better promote the feeding of milk into the rumen with digestive enzymes，and had better growth performance compared with the barrel fed calves.

Immune level

The immune indicators (IgG，IgA，saliva SIgA，IL-2，IL-3，IL-6 and TNF-α) measured in this experiment were important indicators of immunities and important substances to improve the calves' auto-immune level.In this experiment，IgG，IgA，IL-2，3，6 and serum IgG mean±SD for different methods of colostrum feeding in calves (artificial nipple and barrel) were found significantly different among the groups. The serum TNF-αlevel of the artificial nipple feeding group was significantly higher than that of the barrel feeding group in the late experimental period. There were many factors that contributed to this result and from a nutrition perspective，inadequate nutrition could severely affect immune function and might increase the sensitivity of newborn calves to infectious diseases(Nonneckeet al.，2003). Nutrition was a critical determinant of immune responses，with the protein and energy supply influencing cell-mediated immunities，cytokine productions，phagocytic functions and secretory IgA antibody concentrations (Woodward，1998). In the first month after the calve was born，nutrient levels affected growth and development，and researchers found that increased nutrient intake was sufficient to optimize growth performance associated with elevated plasma IGF-I，insulin and glucose concentrations (Smithet al.，2002). Their data also showed that in well-nourished calves，the function of growth hormone was coordinated and sensitive to nutrient intake and growth hormone. Since the growth axis affected the developmental and functional aspects of the immune system (Clark，1997)，the artificial nipple feeding calves might be more beneficial to their immune systems by getting more milk. Calves fed milk replacerad libitumby an automatic feeder gained more BW and had an elevated IGF-I status (Schäffet al.，2016; Frietenet al.，2017; 2018)，which reflected improved body growth，but might also stimulate the immune system in calves (Clark，1997; Khanet al.，2011). Milk intake and immune levels might have a positive effect for each other. It could be said that the artificial nipple promoted the calf to suck milk，so that more nutrients reached the abdomen and promoted digestion，calves could absorb more nutrients，thereby improve the calves' immune levels，and the increase in immune levels increased the calves' feed intake.

Conclusions

The results of this trial with calves indicated that the artificial nipple feeding increased growth performance and the levels of IgG，IgA，saliva SIgA，IL-2，IL-3，IL-6 and TNF-α，while different feeding methods had no significant effects on calves' behaviors. The information obtained suggested that the calves'immune levels and growth performance might have a mutually reinforcing effect. The artificial nipple feeding was significantly better than the barrel feeding.From the present experiment，it was recommended to use the artificial nipple feeding on farms to improve the health status and performance of calves.