Effect of capsaicin supplementation on lactational and reproductive performance of Holstein cows during summer

Keywords: capsaicin, oestrus synchronisation, summer season, Holstein cows.

Low fertility and production consequences are usually observed in dairy cows over summer in south of China owing to high ambient temperature and humidity. The effects of hot summer weather on dairy cows are not limited to tropical areas but also extend to cattle herds reared in mild climates. Generally, the low productivity in cows during summer is directly linked to metabolic functions, which in turn reduces lactational and reproductive performance (Bernabucci et al. 2014; Schüller et al. 2017). Additionally, increased blood circulation to the body surface for heat dissipation under hot conditions leads to insufficient blood supply to the digestive, reproductive and lactation systems of exposed animals. This phenomenon affects productive and reproductive efficiency in dairy animals (Das et al. 2016).

Various regimens are employed under commercialised and conventional dairy systems to combat conditions of high ambient temperature and humidity in dairy cows. Numerous reports illustrate the use of different dietary supplements to augment the productivity in dairy cows during summer heat conditions (Cheng et al. 2016; Hall et al. 2016; Cheng 2018; Nasiri et al. 2018). Oriental folk medicines have been widely used in the Asian region for the treatment of various diseases, including reproductive disorders, over the centuries (Cheng 2018). These traditional medicines are usually based on essences and secondary metabolites such as polysaccharides, saponins and essential oil (Cheng 2018). Capsaicin (CPS) is an active compound in Capsicum spp. and has a phenolic structure. It has been tested as an antimicrobial agent for enhancing the rumen fermentation (Jeeatid et al. 2018), as a stimulant for digestive enzyme secretion (Oh et al. 2017) and as a modulator for glucose homeostasis via an insulin secretion mechanism (Oh et al. 2017). Increased metabolic rate in response to CPS ingestion can be a good indicator for regulation of reproductive hormone production and secretory pattern.

The use of oestrus synchronisation protocols has been considered beneficial for improving oestrus response and pregnancy rates in dairy cows in summer (De Rensis et al. 2015; El-Tarabany and El-Tarabany 2015; Garciaispierto et al. 2018). Synchronisation regimens have been modified for successful ovulation induction in order to promote the efficacy of fixed-time artificial insemination (AI) (Dirandeh 2014; Dirandeh et al. 2015; Voelz et al. 2016; Garciaispierto et al. 2018). However, ovulation synchronisation protocols have not yet been tested in Chinese Holstein cows under conditions of high ambient temperature and humidity. For the present study, we hypothesised that fertility, metabolic functions and milk production could be maintained by the application of a modified Ovsynch protocol (Abulaiti et al. 2019) in combination with CPS as a dietary supplement in dairy cows under conditions of high ambient temperature and humidity. Hence, the study was designed to determine the beneficial effects of CPS on production and fertility in Chinese Holstein dairy cows under summer conditions of high ambient temperature and humidity.

The study was approved by the Ethical Committee of the Hubei Research Center of Experimental Animals (Approval ID: SCXK (Hubei) 20080005). All experimental protocols were performed in accordance with the guidelines of the Committee of Animal Research Institute, Huazhong Agricultural University, China.

The experiment was conducted during summer 2018, from 10 July to 24 August, at Hubei Tong Shun Co., Hubei province, China (30°32′N, 111°51′E). Hubei province has mountainous topography and is in the humid subtropical zone of central China. Climatic conditions are cool to cold in winter (average temperature 1–5°C) and hot in summer (average temperature 27–35°C, reaching 40°C during July and August). Average rainfall in Hubei province is 800–1600 mm (300–700 mm in summer vs 30–190 mm in winter) and maximum rainfall is recorded during the monsoon season.

During the experiment, the daily ambient temperature (T°C) and relative humidity (RH) were noted, and temperature– humidity index (THI) was calculated (Bohmanova et al. 2007) using the following formula:

Daily variations in T °C, RH and THI are presented in Fig. 1, with respective averages 35.6°C (± 0.2°C), 78.1% (± 1.3%) and 91.4 (±0.3) throughout the trial. The observed THI indicated severe heat stress for cows during the period of observations.

Fig. 1.  Daily variations of temperature (°C), humidity (%) and temperature–humidity index (THI) during the study period.

In total, 109 multiparous, lactating (34 ± 12.5 days in milking) Chinese Holstein cows with optimum body condition score (2.5–3.0) and average bodyweight (557.1 ± 89.5 kg) were selected. The selected cows were clinically and physically healthy with a normal history of calving and reproductive soundness. All of the cows were fed with total mixed ration (TMR) according to NRC recommendation (Table 1). Ad libitum fresh and clean water was provided routinely. The cows were housed in an open shed with a cemented roof top and two sides fenced by galvanised wire mesh. Exhaust fans for ventilation, sprinklers for shower and grooming brush facilities were also available at the farm. A tail-to-tail stall feeding system was in practice. Each cow was allocated an area of ~3.7 m² along with a manger 0.6 by 0.9 m.

Table 1.  Ingredients and composition of total mixed ration and concentrate for experimental cows in summer
Formulated according to NRC recommendations

The experimental cows (n = 109) were randomly divided into four groups and supplied with 0 (control, n = 27), 20 (CPS-20, n = 26), 40 (CPS-40, n = 28) and 60 (CPS-60, n = 28) mg of CPS (Guangzhu Pumai Biotechnology, China) per kg of TMR daily for continuous 30 days initiated on 10 July and terminate on 9 August 2018.

Cows were milked three times a day at 03:00, 11:00 and 19:00 daily. Milk yield was estimated in litters by using calibrated jars, and milk yield averages were calculated over intervals of 5 days from the start of supplementation to Day 45 of the trial. Milk composition parameters including total solids, lactose, fat, protein, somatic cell count, and urea nitrogen were determined by Milk Composition Analyzer (type 78110; Foss, Denmark).

Following the CPS supplementation from 10 July to 9 August, the cows were initially intramuscularly injected with gonadotropin-releasing hormone (GnRH, 200 µg) on 10 August (Day 0), followed by an intramuscular injection of prostaglandin F_2α (PGF_2α, 0.5 mg; Ningbo Sansheng Pharmaceutical Ltd, China ) on Day 7 of treatment. The second injection of GnRH (200 µg) and the first intramuscular injection of mifepristone (0.4 mg/kg, Hubei Yun Cheng Sai Technology, China) were given simultaneously on Day 9 of treatment. The cows were subjected to insemination 16 h post the second injection of GnRH. An intramuscular injection of human chorionic gonadotropin (hCG, 2000 IU, Ningbo Sansheng Pharmaceutical Ltd, China) was administered for each animal 5 days post-AI.

Ultrasonography (Desktop B-type veterinary ultrasound scanner, WED-9618-v, equipped with LV2-3/6.5 MHz rectal probe; Shenzhen Well D Medical Electronics, Guangdong, China) was used to monitor follicle development. The cows were scanned twice a day starting from Day 6 (the day before PGF_2α treatment) until Day12 (at 72 h after the second injection of GnRH) of treatment. Ovulation was considered by the sudden disappearance of dominant follicles at an ultrasonographic examination compared with the previous scan (Liu et al. 2016). The cows were observed for signs of oestrus visually twice a day (06:00 and 18:00). Pregnancy diagnosis was performed by ultrasonography 30 days post-AI.

Blood samples were collected from each cow at the start of CPS supplementation, at the time of AI, and 50 days after AI. Metabolic activity of serum glucose, lipoprotein esterase (LPL) and aspartate aminotransferase (AST) were assessed with ELISA kits (Shanghai Enzyme-linked Biotechnology Co.). Blood glucose values were assessed by use of the Ml076792 bovine blood glucose test kit. LPL estimation was performed by the Ml607622 bovine lipoprotein esterase test kit (sensitivity 3µg/mL, intra-assay variation <10%, inter-assay variation <15%); AST determination was done by the Ml063776 aspartate aminotransferase kit (sensitivity 0.5ng/mL, intra-assay variation <8%, inter-assay variation <10%).

Data were analysed using statistical software (SPSS version 17.0.1, Chicago, IL, USA). One-way analysis of variance (ANOVA) was applied to compare the diameter of the ovulatory follicle and growth rate of dominant follicles among groups. Two-way ANOVA was applied for the analysis of milk production, milk composition and metabolic activity at different intervals among the groups. The chi-square test was applied for comparison of pregnancy, oestrus and ovulation across the groups using Prism-6 software package (GraphPad Software). Values of P < 0.05 were considered statistically significant.

There was no effect of CPS on milk production among the groups during the first 2 weeks of supplementation. However, the CPS-40 group had significantly higher (P < 0.05) milk production than the control and CPS-60 groups on Days 20, 25, 30, 35, 40 and 45, and higher (P < 0.05) milk production than the CPS-20 group at most of these dates (Table 2). With regard to milk composition (Table 3), CPS-20 and CPS-40 groups had higher (P < 0.05) total solids than the control group on Day 20. Cows in CPS-60 group had higher (P < 0.05) lactose levels in milk than cows in group CPS-20 at both sampling times and higher (P < 0.05) lactose levels than cows in group CPS-40 on Day 20. There was no difference in milk protein level among treatment groups. Cows in groups CPS-20 and CPS-40 had higher (P < 0.05) milk fat than those in control group at both sampling times, and higher (P < 0.05) milk fat than those in group CPS-60 on Day 20. Milk urea nitrogen increased (P < 0.05) in groups CPS-20 and CPS-60 on Day 20 relative to the control. All CPS-supplemented groups had numerically higher somatic cell count than the control at both sampling times (Table 3).

Table 2.  Daily milk yield (kg) of cows supplemented with capsaicin (CPS) at different doses in summer
Within a row, means followed by the same letter are not significantly different (P > 0. 05)

Table 3.  Milk composition on Days 20 and 30 in cows supplemented with capsaicin (CPS) at different doses in summer
Within a row, means followed by the same letter are not significantly different (P > 0. 05)

The effect of CPS supplementation on reproductive variables in synchronised cows was presented in Table 4. The CPS-40 group showed greater (P < 0.05) oestrus response than other treatment groups. The CPS-40 group had significantly higher (P < 0.05) ovulation rate than other treatment groups, and higher (P < 0.05) ovulatory follicle diameter than the control group. However, duration of oestrus, follicle growth rate, and interval from second GnRH injection to oestrus and ovulation were similar (P > 0.05) across all groups. A higher (P < 0.05) pregnancy rate was observed in the CPS-40 group than control and CPS-20 groups.

Table 4.  Reproductive variables in cows treated with modified Ovsynch regimen after supplementation with capsaicin (CPS) in summer
Within a row, means followed by the same letter are not significantly different (P > 0. 05)

Cows in groups CPS-40 and CPS-60 had higher (P < 0.05) serum glucose levels than those in the control at the time of AI and at 50 days post-AI (Table 5). Cows in all CPS groups had significantly higher (P < 0.05) LPL levels than the control cows at both sampling times. However, only the CPS-40 treatment group had higher (P < 0.05) serum AST than the control at either sampling time.

Table 5.  Variation in serum glucose, lipoprotein esterase (LPL) and aspartate aminotransferase (AST) in cows on Day 0 of supplementation with capsaicin (CPS), the day before artificial insemination (AI), and 50 days post-AI
Within a column, means followed by the same letter are not significantly different (P > 0. 05)

Generally, the negative effects of heat stress are displayed by dairy cows in summer when THI values exceed 80 (Kadzere et al. 2002; Bohmanova et al. 2007). We recorded a higher THI value (91.0 ± 0.3), which indicates severe heat stress, and observed values that are higher than documented in earlier reports for dairy cows (Liu et al. 2020; Dehghan-Banadaky et al. 2013; Cheng et al. 2016). The THI values remained relatively constant during the experimental trial under local conditions. These extreme values of THI might be linked to location of study site, in the humid subtropical zone where the summer season coincides with higher humidity level.

Variation in milk production or milk composition is common in cows under heat stress condition (Ravagnolo and Misztal 2000; Joksimovic-Todorovic et al. 2011). There is an increasing trend with heat stress affecting not only the physiology, feed intake and milk production, but also reproduction efficiency of dairy cattle (Chen et al. 2018; Shenhe et al. 2018). Under heat stress, a sharp decrease in milk yield is observed in high producing dairy cows (Zimbelman et al. 2010; Dunn et al. 2014), and this reduction in milk yield is directly linked to low feed intake and reduced digestibility or absorption of nutrients (Boonkum and Duangjinda 2015). It is also reported that reduced flow of nutrients to the udder affects milk production and composition (Puggaard et al. 2014). Lower secretion of T₃ (triiodothyronine) or T₄ (thyroxine) affects protein metabolism by suppressing the appetite, and the release of prolactin is also influenced in heat-stressed cows (Cheng et al. 2016; Hall et al. 2016). Earlier use of GABA (gamma-aminobutyric acid) or betaine improved milk production and milk composition in cows during summer heat conditions (Cheng et al. 2016; Hall et al. 2016) because these compounds are involved in the metabolism of protein and enhancement of digestibility. CPS has also been used as a supplement for cows to test lipopolysaccharide (LPS) and glucose tolerance; however, no prominent effect on milk yield or milk composition was noted (Blanck et al. 2014; Oh et al. 2017). Unchanged milk production after CPS supplementation might increase the energy demand by the immune system following LPS challenge or glucose resistance test (Oh et al. 2017). In the present study, high milk production with improved milk fat, protein, lactose and urea nitrogen was achieved in CPS-supplemented cows. Ingestion of CPS in heat-stressed cows might improve the utilisation protein by enhancing rumen efficiency, thereby increasing milk protein and milk urea nitrogen level. The above findings suggest that CPS as a dietary supplement can effectively augment the gastrointestinal environment with an improvement in microbial protein synthesis in cows.

The application of exogenous hormones for the development of follicles with successful ovulation following fixed-time AI is a useful technique for reducing infertility rates in dairy cows during summer (Bilgen and Özenç 2009; Khodaeimotlagh et al. 2011; Dirandeh et al. 2015). In the present study, effects of the modified Ovsynch protocol in combination with mifepristone and hCG were evaluated on follicular dynamics, oestrus response and pregnancy rate in heat-stressed dairy cows. A better oestrus response with successful ovulation (60%) and pregnancy (40%) rates was achieved during the study. The present data on fertility reveal higher rates than in previous reports where ovulation synchronisation protocols were applied during the heat stress period (Dirandeh 2014; De Rensis et al. 2015; Dirandeh et al. 2015; El-Tarabany and El-Tarabany 2015). The higher pregnancy rates obtained in present study might be linked with the use of hCG injection post-AI. Accessory corpus luteum (CL) formation and increased CL life in response to hCG injection could be the pregnancy enhancing factor in the present study. The high progesterone secretion following hCG could reduce the incidence of early embryonic mortality during summer. Similar reports are documented where hCG was used for induction of ovulation in heat-stressed anoestrous cows (De Rensis et al. 2008; Honparkhe et al. 2009; Fischertenhagen et al. 2010; Garciaispierto et al. 2018). In addition, post-AI hCG in an Ovsynch protocol is reported as an appropriate strategy to improve fertility in cows during summer (de Rensis et al. 2008; Honparkhe et al. 2009).

On the other hand, achievement of optimal fertility responses following the Ovsynch protocol could be related to CPS supplementation of cows before initiation of the protocol in the present study. Earlier reports indicate that supplementation with dietary yeast significantly improved the pregnancy rates in heat-stressed cows (Dehghanbanadaky et al. 2013; Nasiri et al. 2018). Those authors speculated that improved metabolic status after yeast supplementation led to development of large ovulatory follicles with high circulation of steroid hormones in heat-stressed cows. CPS is also involved in the regulatory mechanism of the body, boosting the immune response with increased vascularity to internal tissues (Yoshioka et al. 2000; Sung Hyen et al. 2013). In this context, it has been speculated that the ingestion of CPS might increase the blood flow towards the internal organs (i.e. reproductive system), improving follicle development, meiotic maturation and embryonic development in heat-stressed cows. Incorporation of CPS in the diet of high producing cows could be an ameliorative strategy to improve the application of ovulation synchronisation protocols in terms of oestrus response, ovulation occurrence and pregnancy rate during stressful conditions of the summer season.

The present data on serum glucose, LPL and AST show that CPS has the capacity to improve their levels by enhancing the metabolic functions. The higher serum glucose level at breeding and post-breeding times in heat-stressed cows could be due to CPS supplementation improving the rumen fermentation efficiency. This higher level of glucose at or after AI is a good indicator for early embryo survival and maintenance of pregnancy. In addition, optimum serum glucose is the precondition for lactose synthesis, which maintains the osmotic pressure of milk. This indicates that better serum glucose levels with CPS supplementation enhanced lactose production and subsequent milk yield in lactating cows. The liver efficiency indictors AST and LPL also benefited by CPS in the diet , although, the variation in LPL or AST at the time of AI or post AI were observed which indicates that CPS supplementation might improve the metabolic activity of cows rather than the pathological level (Oh et al. 2017; Şekeroğlu et al. 2018). Hence, the provision of CPS efficiently regulated the different body functions without affecting liver functions.

In conclusion, CPS supplementation has the capacity to enhance milk production and maintain the metabolic function of cows during the heat exposure period. Moreover, supplementation with CPS is a useful strategy for improving follicular dynamics, oestrus, ovulation and pregnancy rate in synchronised cows during summer.

The authors declare no conflicts of interest.