An assessment on the impact of weaning stress on the weaning age in Black Bengal goat kids

Bangladesh possesses a huge population of ruminant livestock, specifically, goats account for a remarkable total of 26.945 million [1]. Currently, goat farming is gaining popularity in Bangladesh to meet the increasing demand for animal protein, despite the hot and humid conditions of the country appearing to favor many diseases [2]. About 90% of goats belong to the Black Bengal breed [3, 4], which possess distinct characteristics that set them apart, including their remarkable ability to reproduce, their high fertility rates, their early onset of sexual maturity, their capacity to adapt to hot and humid environments, and the exceptional quality of their meat and skin [5, 6, 7]. Goat rearing is one of the major agricultural activities in the village areas of Bangladesh, which is conducted under a subsistence farming system. Commercial goat farming in intensive or semi-intensive settings is gaining popularity among medium and large farms. The standardization of various goat husbandry procedures is necessary for goat production under stall-fed or semi-intensive circumstances. 
Weaning, an unavoidable husbandry practice, typically results in the occurrence of weaning shock. The process of weaning is typically a challenging phase in a kid's life, sometimes accompanied by a reduction in weight gain, a cessation of growth, and, in certain instances, even weight loss [8, 9, 10], which is commonly known as weaning shock. There are several potential factors that could contribute to the observed phenomenon, including the weakening of the relationship between the dam and the kid, a transition from a diet mostly consisting of nutritious milk to solid feed, inadequate consumption of feed, gradual depletion of bodily reserves, compromised immune function, and the proliferation of opportunistic microorganisms [11, 12]. The outcomes of the post-weaning period are contingent upon various factors, including the feeding regimen implemented before weaning, the physical condition of the offspring, and their age and weight during the pre-weaning phase. These implications can extend for up to two months following the weaning process [12, 13]. The significance of weaning weight at the time of weaning surpasses that of age to mitigate the effects of weaning shock. Birth weight range lower than 1.5 kg had a higher mortality rate of 28.3% for Black Bengal goat (BBG) kids from birth to three months of age [14]. 
Animals have been observed to display behavioral and physiological reactions in response to acute stress circumstances [15, 16]. These behavioral features serve as the primary and visible parameters. In addition to evaluating behavioral traits, blood hormone levels are employed to evaluate animal well-being and stress levels [17, 18]. Stress leads to an elevation in cortisol levels, a suppression of thyroid hormone release, and an interruption of growth and development [19, 20]. It is widely acknowledged that thyroid hormones have the capacity to enhance oxygen consumption within the body and elevate body temperature. Thyroxine (T4) is the predominant hormone secreted by the thyroid gland. The conversion of T4 to triiodothyronine (T3) occurs in somatic cells, and as a result, the concentration of T4 serves as an indicator of the biochemical capacity for T3 transformation [21]. The assessment of stress levels in animals involves monitoring the hormones cortisol, T3, and T4 [22, 23]. The immune system has been observed to experience suppression in the presence of stress [24, 25, 26]. The level of stress experienced by individuals might differ depending on factors such as the age at which weaning occurs and the ability to consume feeds other than dam’s milk [27, 28]. The weaning period is a crucial stage in a kid's upbringing, characterized by a reduction or cessation of weight gain. The magnitude and manifestation of stress during the process of weaning are contingent upon various parameters, with particular emphasis on the age, body weight, and pre-weaning nutritional status of the offspring [29]. In the context of Bangladesh, it is commonly observed among goat farmers that there is a notable deceleration in development or complete retardation of growth, and in certain instances, even weight loss, following the weaning of kids. The mortality rate of kids experiences an upward trend throughout the transitional phase from pre-weaning to post-weaning, hence hindering farmers from attaining maximum profitability in goat farming. Despite the absence of comprehensive data, the issue of weaning shock and its consequential adverse impacts represents a significant challenge in the context of BBG production in Bangladesh. This study aimed to identify the most favorable age for weaning and evaluate the weaning stress on the following performance of BBG kids.

Study site and duration
The experiment was carried out at the Goat Research Farm in Bangladesh Livestock Research Institute (BLRI), Savar, Dhaka-1341, Bangladesh, from July 2018 to June 2020. 

Ethical statement
The Animal Experimentation Ethics Committee of Bangladesh Livestock Research Institute (BLRI) approved this research project (Reference no.: AEEC/ BLRI00104/2023). During sample collection, all the guidelines for animal care were carefully followed.

Animal selection and group allocation
A study was undertaken consisting of four distinct groups (A, B, C, and D) comprising eight kids in each group. Additionally, four lactating female goats were included in the study, where each group consisted of one lactating female goat and two kids (one male and one female). The birth weight of the kids in various groups had a comparable distribution. The kids belonging to various treatment groups were subjected to varied weaning ages following the birth of the kids. These ages were specifically set at 45, 60, 75, and 90 days, and were represented by the groups A, B, C, and D, respectively. The kids were then raised until they reached the age of six months. The control group in this study was designated as Group D. 

Housing and feeding of animals
The animals were housed in separate enclosures featuring slatted flooring, ensuring optimal hygiene and adhering to appropriate biosecurity measures. The feeding protocol was consistent across all groups, wherein kid individuals were permitted to remain in the presence of their mothers and engage in suckling behavior. After weaning, kids were fed ad libitum maize fodder and a concentrated mixture at the rate of 1.5% of their body weight and reared in an individual pen. The composition of concentrate feed was 30% of wheat bran, 27% of broken maize, 23% of soybean meal, 16% of khesari bran, 1.5% of protein concentrate, 0.5% of vitamin-mineral pre-mix, 1% of DCP, and 1% of salt.

Sample collection and data recording
Blood samples were obtained from the kids to analyze the levels of T3 (triiodothyronine), T4 (thyroxine), and cortisol hormones.  All blood samples were aseptically taken from the jugular vein using a vaccinator tube (Figure 1). The levels of T3 (triiodothyronine), T4 (thyroxine), and cortisol hormones were measured by using Biosystems-A15 Automated Biochemistry Analyser (Biosystems, Barcelona, Spain) according to manufacturer recommended commercial kit for veterinary use in blood samples collected from kids in different weaning schedules. The treatment groups were labeled as A, B, C, and D. The hormone analysis was conducted three days before the weaning schedule (-3) and three days after the weaning schedule (+3). The heart rate of the kid was measured using Polar-S810i Heart Rate Monitors (Polar, Kempele, Finland) (Figure 1) at three-time points: three days before weaning (-3), the day of weaning (0), and three days following weaning (+3) for various treatment groups. In addition to the mentioned factors, other parameters including birth weight, weaning weight, weight at 6 months, dry matter intake (DMI), daily growth rate at different developmental stages, incidence of diseases, and mortality rate among the kids were also recorded.

Statistics analysis
The data underwent analysis using the Generalized Linear Model (GLM) procedure in the Statistical Package for the Social Sciences (SPSS 2020). The analysis involved a factorial design with dimensions of 4x3 and 4x2, as well as an analysis of variance (ANOVA) conducted under a Completely Randomized Design (CRD). To ascertain the distinctions between means, Duncan's Least Significant Difference (LSD) test was employed.

Estimation of weaning stress
The T3, T4, and cortisol hormones were observed to assess the stress levels at different weaning ages in kids (Table 1). Although the concentration of T3 and T4 hormones didn’t differ significantly according to groups, the serum concentration of T3 hormones decreased significantly (p<0.05) at +3 days after weaning compared to -3 days before weaning. The results suggested that the kids under A, B, and C groups experienced more stress from weaning compared to those in group D.
The heart rate of kids also exhibited a significant rise (p<0.05) as a result of the stress experienced throughout the weaning process (Table 2 and Table 3). The maximum beats per minute (BPM_max) and average beats per minute (BPM_av) in groups C and D exhibited their highest values on the initial sampling day (day 0) and subsequently decreased by three units. However, in the case of groups A and B, a significant increase (p<0.01) was detected, starting from day 0 of the sample period, with the highest value recorded on day +3.

Weaning age and post-weaning performance of BBG kids 
Table 4 represents the post-weaning performances of different kid groups who were weaned at different ages. There was no significant difference (p>0.05) in the birth weight among the selected groups. However, there was a significant difference (p>0.05) in the average weaning weight of the kids across the various groups since they were weaned at different ages. 
Health issues for different groups of kids were monitored throughout the study period. Pneumonia, coccidiosis, dermatitis, and lameness were the most prevalent diseases or health problems observed during the experiment (Table 5). Two kids from the 45-day weaned group and one from the 60-day weaned group died between weaning to six months of age, respectively, from coccidiosis, pneumonia, and dermatitis with weakness. The kid mortality rate was 12.5% for the 60-day and 25% for the 45-day weaned groups, and 0% for the 90-day and 75-day weaned groups, respectively. 

The process of weaning is typically a challenging phase in the early life of a kid, generally accompanied by a reduction in the rate of weight increase, a complete cessation of growth, and occasionally, even a decline in body weight [8].
The study found a significant decrease (p<0.05) in thyroid hormone levels at +3 days following weaning. Thyroid hormone release decreases during stressful conditions [22, 30]. Cortisol concentration increased significantly (p<0.05) at +3 sampling days. This increment was 3 to 4-fold more from groups A and B. Heimbürge et al. [31], Gong et al. [32], and Bayazit et al. [33] reported that, in stressful conditions, cortisol concentration was also increased. However, in Saanen goats, significant changes were not observed in T3 and T4 hormones of the pre-and post-weaning periods [34]. Cortisol hormone levels may decrease with age [34, 35]. Hence, results suggested that kids in groups A and B were more stressed due to weaning than kids in the C and D groups. Right after birth, a goat kid's rumen is small and immature, and its digestive system is not yet entirely capable of processing complex plant materials. The maturation of the rumen is a gradual process influenced by the goat's diet [9]. As the kid consumes more solid food, especially fibrous plant material such as hay and grass, the rumen enlarges and develops a more extensive microbial population. These microbes break down cellulose and other complex carbohydrates in plants, aiding their digestion. The goat's rumen is generally well-developed and functioning efficiently around three months of age. At this stage, the goat primarily relies on its rumen for digestion and obtaining nutrients from plant-based diets [36].
Heart rate variability (HRV), which examines how the heart beats to show how the body reacts to stressors, is another method for detecting stress [37] and nowadays it is preferably used for stress assessment in animal husbandry. Measurement of HRV was done using a heart rate monitor Polar-S810i. [38], where heart rate, recorded as beats per minute (BPM), may regulate the workload during physical activity, exercise, or stress [39, 40]. As in humans, a linear relationship between BPM and maximum oxygen uptake has been shown among animals. So, if BPM increases, then animals are in stress conditions. Results showed that group C and group D had less BPM than groups A and B indicating that goat kids weaned at 75 to 90 days were under less stress than goat kids weaned at 45 to 60 days, and this result supports the findings of Hampson and McGowan [38], Sporkmann et al. [39] and Ogata et al. [40]. BPM has been used in the literature as a physiological measure to evaluate stress and welfare in dogs [41, 42]. There needs to be more studies about BPM measurement and its relation to stress in goats. In addition, current research suggests that animals with more BPM have a stress condition similar to the other animal study mentioned above. The findings indicated that kids belonging to groups A and B experienced the highest levels of stress as a result of early maternal separation. 
There was a significant difference in the average weaning weight of the kids across the various groups (p>0.05) as the kids in the various groups were weaned at different ages. Since a higher weaning weight helps a goat kid to attain the market and reproduction age faster, weaning weight is an important parameter for kids growing up in business enterprises. Typically, weaning weight depends on age, parity, litter size, milk production of the dam, and the sex of the kids, as well as the days the animals are weaned [10, 43]. This experiment found significant differences in weaning weights among the groups, with the highest value of 5.52kg for the 90-day weaned group and the lowest value of 3.15 kg for 45-day goat kids, which is comparable with Chakrabarti et al. [44], where authors showed an overall mean of 4.82±.20kg weaning weight of a BBG kid at three months of age is lower than what was found in this experiment. Solaiman et al. [45] found a 3-month weaning weight of 5.41kg and 4.97kg for male and female BBG kids, whereas Alam [46] found 5.70kg and 5.01 kg for male and female kids, which agreed with the finding of the present study. Higher weaning age, dam's age, and parity trigger the weaning weight to get high, while kids born from multiple births tend to attain lower weaning weight.  
Typically, BBG goat attains sexual maturity at six months of age. To attain sexual maturity at six months, BBG goat has to meet a certain body weight, at least half of the mature weight. Furthermore, a farmer might market his goats as early as six months if they weigh around 10-12 kg. Several factors such as weaning age, weight, sex of the animals, and system of rearing determine the value of six months body weight of BBG. In the experiment, kids' weight differed significantly among the groups, where the highest weight of 10.55kg was observed when kids were weaned at 90 days, and a lower value of 7.32kg was found when kids were weaned at 45 days and might affect the profitability of the farm. The findings are comparable with other authors. According to Solaiman et al. [45], BBG attained 9.18kg and 8.02kg of six months weight for male and female goats, respectively, whereas Alam [46] showed a six-month weight of 8.65kg and 7.40kg for male and female BBG. When a goat kid possesses a high weaning weight, then it may carry a higher six-month weight. Weaning age greatly influences six months of body weight of BBG. Typically, 90 days or more is needed for the complete development of reticule-rumen in goat kids [46]. As a result, a goat kid weaned below 90 days cannot utilize roughages, possesses an underdeveloped rumen, and may not attain optimum body weight at six months. 
Similar to six months’ body weight, it has been found huge variation among the groups for daily weight gain from weaning to six months of age, where 90 days weaned group showed the highest daily gain of 53.70g per day and the lowest value of 30.90g found for 45 days weaned groups. These results show consistency with Vickery et al. [8] and Ibrahim et al. [47], who stated that average daily gain was significantly higher for kids weaned at 8 and 10 weeks than those weaned at 4 and 6 weeks of age (143g, 144g, 125g, 126g). In an early weaning system retarded growth rate is experienced as milk consumed by kids is lower than delayed weaned kids. Also, the early weaned kids need help to utilize other feedstuffs properly due to incomplete development of the rumen reticulum. 
DMI of the animals mostly depends on the dam's age, body weight, rearing system, parity, and milk production. At an early age, a goat kid only relies on milk for survival, and then gradually develops the rumen reticulum [48]. In this experiment, the average DMI per day from weaning to six months of age tended to increase with increases in the weaning age of the kid, where 90 days weaned kids consumed a total of 170.27gm dry matter, and 45 days weaned kids consumed 130.03gm dry matter per day. However, the differences among the treatment groups were non-significant. Literature regarding average dry matter intake from weaning to six months of age is limited. An early-weaned goat kid not only consumes less milk than a late-weaned but also takes less other feedstuff due to incomplete rumen development [48, 49]. 
Early-weaned animals encountered more diseases compared to late-weaned animals [50, 51]. The result of the study also found in a similar vein that, the 45 days and 60 days-weaned groups encountered more health problems than the other two late-weaned groups of kid possibly due to their poor health and low immunity achieved from early weaning.

The growth and productivity of BBG kids could be negatively impacted if they are weaned too early specifically before 75 days of age. The study also suggested that the post-weaning performance of BBG kids is adversely affected by the age at which they were weaned. Kids who were weaned at 75 days to 90 days had fewer health and behavioral issues. In conclusion, the weaning age for BBG kids should not be less than 75 days. Further study is needed to minimize the weaning stress with proper management of BBG Kids.

The authors would like to thank all lab personnel of the Small Ruminants Research Laboratory, Goat Production Research Division, Bangladesh Livestock Research Institute (BLRI) for their valuable assistance with this research. The authors are also grateful to the BBG Conservation and Development Research Project (Grant No. 224289000) of the Ministry of Fisheries and Livestock, Bangladesh, for providing financial support for this research work.

SA: Conceptualization, designing the experiment, laboratory test, data analysis, and interpretation, critical review, and funding acquisition. MAH and MHR: Laboratory test, data analysis, drafted and revised the manuscript. MMHP and MHR: Farm experiment, revising the draft, and made it up to the final stage. NJ: Data analysis and result drafting. All authors have read, reviewed, and approved the final manuscript. 

There is no conflict of interest among the authors.