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Active immune system and dry matter intake during the transition period are associated with postpartum fertility in lactating Zebu cows
Livestock Science 228 (2019) 18–24
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Active immune system and dry matter intake during the transition period are associated with postpartum fertility in lactating Zebu cows
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Pratik Ramesh Wankhadea, Ayyasamy Manimaranb, , Arumugam Kumaresanb, Sakthivel Jeyakumarb, Veerasamy Sejianc, Duraisamy Rajendrand, Madiajagan Bagathd, Muniandy Sivarame, Kerekoppa P. Rameshae, Minu Rachel Varghesea a
Livestock Research Centre (LRC), Southern Regional Station (SRS), ICAR-National Dairy Research Institute (NDRI), Adugodi, Bengaluru 560 030, Karnataka, India LRC, SRS, ICAR-NDRI, Adugodi, Bengaluru, India c Division of Animal Physiology, ICAR-National Institute of Animal Nutrition and Physiology (NIANP), Adugodi, Bengaluru 560 030, Karnataka, India d Division of Animal Nutrition, ICAR-NIANP, Adugodi, Bengaluru, India e SRS, ICAR-NDRI, Adugodi, Bengaluru, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute phase proteins Inflammatory cytokines Energy indicators Transition period Reproductive performance Deoni cows
The aim of the present study was to assess if the metabolic adaptation and immune status during transition period influence the postpartum reproductive performance in dairy cattle. We assessed the levels of innate immune molecules (Haptoglobin: Hp, Serum Amyloid A: SAA, IL-6, TNF-α, IL-1β, and IL-8) and energy balance indicators [NEFA and Dry Matter Intake (DMI)] in transition Zebu (Deoni breed) cows that became pregnant within breeding period (n = 7) and remained non-pregnant (n = 10) even after the breeding period (i.e. from 45 to 180 days postpartum). Blood samples were collected at weekly intervals during transition period (−21 d before to 21 d after calving) and plasma variables were estimated using commercially available bovine specific ELISA kits. Plasma Hp concentration was significantly (P < 0.01) higher in cows that became pregnant during breeding period as compared to those that remained non-pregnant. While plasma TNF-α concentrations differed significantly (P = 0.05) between groups, such difference was not observed with SAA concentrations. Group x time interaction had a significant effect on plasma IL-6 (P < 0.01), IL-1β (P = 0.05), IL-8 (P = 0.05) concentrations and DMI (P = 0.001). Plasma NEFA concentrations differed significantly (P = 0.03) between groups, although all the experimental cows had NEFA concentrations within the physiological limits. Days in milk had a significant effect on milk yield (P = 0.001) and fat percentage (P = 0.03). It is concluded that active functioning of immune system, stable dry matter intake, and limited fat mobilization during transition period enabled the cows to conceive early during postpartum period.
1. Introduction The transition period in dairy cows is associated with an increased incidence of metabolic and infectious diseases due to metabolic, hormonal, immunological and behavioral changes (Van Saun, 2016). The metabolic changes start two weeks before calving and have effects on reproductive health up to nine weeks after calving, which in turn affects the postpartum reproductive performance (Leblanc, 2010). Similarly, alteration of immune function during the transition period is also a risk factor for postpartum diseases (Kerhli, 2015). Therefore, energy balance and immunity during transition period may influence postpartum reproductive performance of dairy animals. Among the various risk factors for poor reproductive performance, compromised energy status
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is believed to be the most important factor resulting in body fat mobilization. Since the liver's capacity is limited, the metabolites such as Non-Esterified Fatty Acids (NEFA) accumulate in the blood and consequently cause various adverse effects such as reduced feed intake, immune-suppression and decreased productive and reproductive performance (Adewuyi et al., 2005). Although exact reasons for decreased immune function during the transition period are not clearly known, it is reported that lactation and the metabolic changes occurring during transition period alter these functions (Nonnecke et al., 2003). Loor et al. (2005) reported that metabolic adaptations start well ahead of parturition and are mainly regulated through cytokines, acute phase proteins (APPs) and NEFA during peri‑parturient period. Negative Energy Balance (NEB), a hallmark of transition cow (Bell, 1995), is a
Corresponding author. E-mail address: [email protected] (A. Manimaran).
https://doi.org/10.1016/j.livsci.2019.07.020 Received 5 May 2018; Received in revised form 1 February 2019; Accepted 16 July 2019 Available online 17 July 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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followed up to 45 days after calving. All animals were apparently healthy and free from any diseases while enrollment. The experimental cows were maintained under individual tie barn housing system. The shed used for this experiment were open from all side, concrete paved with tiled roof. The housing space for cows was as per “Bureau of Indian Standards (BIS)”. All the cows were fed according to NRC (2001) recommendation using institute grown seasonal green fodders like maize, jowar, hybrid napier, paragrass, guinea grass and cowpea along with dry fodder (ragi straw) and commercially available concentrates (Nandini gold-cow feed containing 16–18% crude protein, 70–72% TDN, 2.5–3.5% fat, 5.5–6% crude fiber, 1–1.5% acid insoluble ash and 10–11% moisture, M/s Karnataka Milk Federation, Bengaluru). The cows were fed 15–20 kg green fodder, 2–3 kg ragi straw and 1.5–2.5 kg concentrates during prepartum transition period. After calving the animals were offered 18–22 kg green fodder, 2–3 kg ragi straw and 2.5–3 kg concentrate divided in equal proportion fed at the time of milking during morning and evening hours based on milk production of individual cow. The animals were provided with clean drinking water four times in a day.
metabolic adaptation process to adjust the imbalance between intake and demand, particularly in high yielding animals and, NEFA is traditionally used as indicators of NEB during transition period (Duffield et al., 2009). An elevation of NEFA during transition period is an indicator of adaptability of individual cows to NEB, but higher magnitude of elevation is often associated with poor productive and reproductive performance during later postpartum period (Ospina et al., 2010a, 2010b; Huzzey and Overton, 2013). APPs are non-specific innate immune molecules and their main functions are to restore the homeostasis of biological process. They are regarded as marker of inflammation (Eckersall and Bell, 2010) and the major bovine APPs, Haptoglobin: Hp and Serum Amyloid A: SAA, plays an important role in animal reproduction through endometrial reconstruction and intensification of the phagocytosis process against uterine pathogens. The role of APPs in prediction, diagnosis and treatment evaluation of postpartum uterine infections was reported by several researchers (Huzzey et al., 2011; Jeremejeva et al., 2012; Manimaran et al., 2016). Concentrations of Hp and SAA should be interpreted with caution as the physiological status of cows might influence their circulating concentrations; For instance, several researchers reported that parturition could have an influence on serum APPs concentrations (Alsemgeest et al., 1993; Regassa and Noakes, 1999; Humblet et al., 2006). Inflammatory cytokines are believed to be a central integrator of metabolism and immune function (Sordillo et al., 1995). Several researchers reported the relationship of inflammatory cytokines such as IL-1β, TNF-α and IL-6 with metabolic and infectious diseases during peri‑partum period (Boro et al., 2015; Zhang et al., 2016; Dervishi et al., 2016). In fact, the relationship between inflammation and metabolism during transition period was mostly explained through association studies under disease conditions (Ospina et al., 2010a, 2010b; Huzzy et al., 2011; Huzzey and Overton, 2013). The inflammatory response was also reported in apparently healthy transition cows (Bradford et al., 2015). Occurrences of overt inflammatory reaction without clear signs of infections or any pathological conditions were also reported during transition period (Bionaz et al., 2007). Though most of the transition cows undergo a brief period of inflammation, magnitude of the inflammatory condition is an ultimate determinant of its long-term outcome in terms of milk production and post-partum fertility. All the above studies were conducted in Holstein cows and such studies are very few in dual purpose breeds. Deoni is an important dual-purpose indigenous cattle breed of southern and western India (Kuralkar et al., 2014). Due to better disease resistance and heat tolerance of Deoni cattle, it is preferred by the small farmers in tropical drought-prone areas. This breed is well suited for low to medium input production system as compared to other breeds (Soumya et al., 2016). On the other hand, the reproductive efficiency of this breed is poor and hence, there is an ardent need for identification of factors associated with optimum fertility. With this backdrop, we hypothesized that the status of immune functions and metabolic adaptation during the transition period might influence the postpartum fertility in dual-purpose zebu cows. Thus, the aim of the study was to find out the alterations in the immune function and metabolic adaptation between the cows that did and did not conceive during early post-partum period.
2.2. Estimation of milk yield and fat per cent During the initial five days of postpartum colostrum period, the cows were allowed for suckling twice in a day by their calves. Thereafter, lactating cows were allowed for partial suckling for one minute by their calves for the letdown of milk, before actual milking by hand, and milk yield (Kg) was recorded with electronic weighing balance. The milk samples were collected in 50 ml sterilized centrifuge tubes from individual animals at 7 ± 1, 14 ± 1, 21 ± 1, 28 ± 1, 35 ± 1 and 42 ± 1 days of postpartum period for milk fat estimation using Gerber's method (IDF, 1991). 2.3. Reproduction management After a voluntary waiting period of 45 to 60 days, all the animals were routinely observed two times a day by trained personnel for estrus. Animals identified to be in estrus were confirmed for their proper stage to receive semen by an experienced Veterinarian. Animals detected in estrus in the early morning were artificially inseminated (AI) in morning of the same day with frozen semen and those cows detected in estrus in the late evening and night were inseminated in the morning of the next day. Two inseminations were done for each cow at 12 h interval. Pregnancy diagnosis was done 45 to 60 after AI through rectal palpation. The interval from calving to the first insemination, days open and services per conception were calculated for each animal. 2.4. Evaluation of cyclicity by trans-rectal ultrasonography The ultrasonography was performed for trans-rectal observation of reproductive organs of the animals after 42–47 days using M/s Aloka SSD 500, Aloka Co. Ltd. Tokya (Japan). The ovaries were examined for presence of follicle(s) and/or corpus luteum (CL). If an animal had normal sized ovaries with a CL in any one of the ovaries, then the animal was considered as cyclic.
2. Material and methods
2.5. Estimation of dry matter intake (DMI) and body condition score (BCS) of individual cows
2.1. Experiment animals and management The experiment was conducted at Livestock Research Centre (LRC), Southern Regional Station (SRS), ICAR-National Dairy Research Institute, Bengaluru, Karnataka (India), which is located in southern Plateau and Hill agro-climatic zone on 12° 97′N latitude and 77° 56′E longitude. The experimental procedures were duly approved by Institute Animal Ethical Committee (IAEC). A total 29 transition Deoni cows were enrolled a month before the expected date of calving and
The DMI of the individual cows was estimated on weekly basis before calving, on the day of calving, afterward on 3rd day and then on weekly basis up to 3rd week after calving. It was calculated by the amount of feed offered and amount of residue obtained on next day (i.e. 24 h) and then DMI was estimated per 100 kg body weight. All the cows were assessed for BCS (on a five-point scale) (Kellogg, 2010). 19
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Fig. 1. Plasma concentrations of Haptoglobin (1a) and Serum Amyloid A (1b) during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (**P < 0.01) between the group (G) at each sampling day (T) intervals.
interval between 1st and 2nd AI (N = 5) in pregnant and non-pregnant cows were 24.6 ± 10.1 and 45.2 ± 15.2 days, respectively.
2.6. Blood sampling and biochemical analysis Blood samples were collected from cows using vacutainer tubes with heparin as anti-coagulant on −21 ± 2, −14 ± 1, −7 ± 1 day before the expected date of calving, on the day of calving, and 3 ± 1, 7 ± 1, 14 ± 1 and 21 ± 2 days after calving. The blood samples were centrifuged within 30 min after collection, at 3000 rpm for 10 min and plasma was stored at −20 °C until further analysis. Blood plasma variables were analyzed using commercially available bovine specific ELISA kits and microplate reader (Multiskan GO, M/s Thermo scientific, USA). The ELISA kits used for Haptoglobin (Hp) was based on a solid phase ELISA and obtained from M/s Life Diagnostics, Inc., West Chester, PA (USA). The sandwich ELISA based kits for estimation of Serum Amyloid A (SAA), Interleukin 6 (IL-6), Interleukin 1β (IL-1β), Tumor Necrosis Factor α (TNF-α), Interleukin 8 (IL-8) were obtained from M/s Clound-Clone Corporation, Houston, TX (USA) and competitive ELISA based kit for NEFA estimation was obtained from M/s MyBiosource, Southern California, San Diego (USA). During study period, all the experimental cows were observed daily for any abnormalities. All primiparous, and animals suffered from any postpartum complication like abortion, dystocia, stillbirth, retained placenta, uterine infections (metritis, clinical endometritis) and clinical mastitis were excluded from study. Thus, seven pregnant, 10 nonpregnant cows were considered for biochemical assay and calculation of reproductive performance.
3.1. Ultrasonographic observation of ovarian structure during postpartum period in relation to pregnancy status in Deoni cows Upon ultrasonographic evaluation during 42–47 days postpartum, no clinical abnormality was detected in the genital tract of cows that eventually became pregnant within breeding period. Out of 7 cows that became pregnant within breeding period, three cows had CL while the remaining four cows had large size (>9 mm) follicles along with variable numbers of small (3–5 mm) and medium size (5–8 mm) follicles. Among 10 non-pregnant cows, seven cows had smooth ovaries with no large follicle or CL. 3.2. Association of bovine acute phase proteins and cytokines with pregnancy status of cows Plasma Hp concentration was significantly (P < 0.01) higher during immediate postpartum period (+7 d) in cows that became pregnant during breeding period as compared to those that remained non-pregnant (Fig. 1a). We found no significant influence of time, group and its interaction on SAA concentrations (Fig. 1b). Plasma IL-6 concentration was significantly influenced by the day of sampling (P = 0.001), group (P = 0.05) and their interaction (P = 0.01). Particularly, plasma IL-6 concentration was higher during pre-partum (−14th and −7th d) and postpartum period (3rd d) in cows that became pregnant within breeding period than that remained nonpregnant (Fig. 2a). Similarly, sampling day and group x time interactions had significant (P = 0.05) effect on IL-1β (Fig. 2c) and IL-8 concentrations (Fig. 2d). Plasma TNF-α concentration (Fig. 2b) was significantly influenced by group (P = 0.05).
2.7. Statistical analysis The animals were grouped into either pregnant or non-pregnant within the breeding period (i.e. from 45 to 180 days). To test the significant difference between pregnant and non-pregnant cows with respect to fertility parameters, a nonparametric test called Mann-Whitney U test was employed. Linear mixed model was used to test the significance of the differences between pregnant and non-pregnant cows for the parameters viz., APPs, cytokines, energy indicators, milk yield and fat percentage. The data were log-transformed before the analysis. The differences between groups were considered significant when P < 0.05. The statistical analysis was performed using SPSS version 16 software package (SPSS for windows, V16.0; M/s SPPS Inc., Chicago, IL, USA).
3.3. Association between energy indicators and pregnancy status in cows All the fixed factors such as group, sampling day and their interactions had significant (P = 0.001) influence on the DMI. The cows that became pregnant early during the breeding period consumed higher quantity of feed on dry matter basis (per 100 Kg body weight) during pre-partum period and maintained the stable DMI intake during postpartum period than the cows that remained open at the end of breeding period (Fig. 3a). Sampling day had significant (P = 0.001) effect on BCS during transition period where, it reduced from pre-partum period to the day of calving, and thereafter similar BCS was observed in both the groups (Fig. 3b). Plasma NEFA (P = 0.03) concentrations significantly differed between groups (Fig. 4). But, the observed levels were within physiological limits or lower than already reported threshold value for
3. Results Among pregnant cows, days to first artificial insemination (DTFAI), days open and number of services per conception were 68.4 ± 8.3, 95.4 ± 10.2 and 2.0 ± 0.4, respectively. Among non-pregnant cows, DTFAI and number of services per conception were 80.9 ± 9.2 days (12 days delayed than pregnant cows) and 2.0 ± 0.4, respectively. The 20
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Fig. 2. Plasma concentration of IL-6 (2a), TNF-α (2b), IL-1β (2c) and IL-8 (2d) during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (**P < 0.01; *P < 0.05) between the group (G) at each sampling day (T) intervals.
higher Hp concentration during first week of postpartum had more active innate immunity and suggested that acute phase response (APR) leads to prevention of uterine infections, clearance of pathogens and initiation of repair mechanism in the uterus. Humblet et al. (2006) also found significant increase in Hp concentrations in healthy cows during the first week of postpartum and suggested that parturition is associated with a physiological APR, as also observed in our study. We also found inter-individual variability in Hp levels as observed in previous studies (Humblet et al., 2006; Jacobsen et al., 2004). Among major APPs, SAA concentration was not found to be influenced by sampling day, group and interactions, indicating that Hp could be a more sensitive APP than SAA, particularly in apparently healthy cows. Several researchers also reported that Hp is a more appropriate and prominent proteins, while SAA is a low constitutive APP in dairy cattle. Longer half-life of Hp than SAA and stronger stimulation by IL-6, as observed in our study, could also be a reason for elevated Hp levels (Alsemgeest et al., 1994; MullerDoblies et al., 2004; Humblet et al., 2006). Hp is basically responsible
postpartum complications in dairy cattle. We found no effect of sampling day and group x time interactions on NEFA levels. 3.4. Association of milk yield and its fat percentage with pregnancy status There was no significant difference in average weekly milk yield and milk fat percentage between cows that became pregnant within breeding period as compared to those that remained non-pregnant (Fig. 5a and b). However, milk yield (P = 0.001) and its fat percentage (P = 0.03) were significantly influenced by time. 4. Discussion We observed a significantly higher concentration of bovine major APP during transition period in cows that later became pregnant within breeding period as compared to those cows remained non-pregnant during the period. Nightingale et al. (2015) reported that cows with
Fig. 3. Dry Matter Intake (DMI/100 Kg BW) (3a) and body condition score (BCS) (3b) during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (**P < 0.01; *P < 0.05) between the group (G) at each sampling day (T) intervals. 21
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We found that DMI was significantly higher in the cows that became pregnant within breeding period during prepartum period, which was maintained during postpartum period also. High DMI during the closeup period is better indicator of overall comfort of cows and thus prepartum DMI is more important than postpartum DMI (Drackley, 2003). NEFA is an energy indicator of fat mobilization in dairy cattle. Higher NEFA levels during transition period in cows that became pregnant within breeding period indicate that presence of mobilization of more adipose tissue to meet out the energy demand in these animals. Although cows which became pregnant within breeding period had higher concentrations of NEFA than the cows that remained open, the levels were within physiological limits or lower than already reported threshold value for postpartum complications. Earlier studies reported that the threshold level of NEFA for NEB was 0.3 mmol/l during early postpartum period (Adewuyi et al., 2005; Wittwer, 2012), which is well above the concentrations observed in the present study. Therefore, the observed levels of NEFA within physiological limit indicate controlled fat mobilization and it might have conferred a fertility advantage along with stable DMI in these cows. Holtenius et al. (2003) and Douglas et al. (2006) reported that cows fed with higher energy diet during close up period had a greater degree of insulin resistance, which allowed greater NEFA concentrations, while cows that fed limited quantity under requirements, had lower NEFA concentrations during periparturient period as was also observed in this study. Westwood et al. (2002) reported that shorter interval to ovulation as an important determinant of fertility even in cows with a higher NEFA level. In our study, ultrasonographic findings during 42–47 days postpartum confirmed the resumption of cyclicity in all cows that became pregnant than cows that remained non-pregnant (seven of 10 cows had smooth ovary). In fact, the cows that became pregnant were inseminated 12 days earlier than cows that remained non-pregnant. Reist et al. (2000) and Snijders et al. (2001) reported that reproductive performance was not associated with milk yield, milk fat and plasma metabolites level as was also observed in this study. The observed average milk yield in these dual-purpose cows (4–4.26 kg/day) agrees with those reported by Wankhade et al. (2018) and other researchers (Das et al., 2011; Kuralkar et al., 2014). Restricted or partial suckling by the calf is a common practice in dual purpose zebu (Bos indicus) cows as it is essential to initiate milk letdown and maintain the lactation in these cows (Tegegne et al., 1994). Indeed, partial suckling improved milk yield and udder health and reduced stress of calves and cows (Froberg, 2008; Hernandez et al., 2006). Although the role of inflammation-associated pathways in adaptation processes is not yet fully understood, Farney et al. (2013) suggested that inflammation at least in some cases, is an adaptive rather than pathological phenomenon, and some degree of inflammation might even be required for successful adaptation. Banos et al. (2013) reported that cellular immune traits associated with improved health or reproductive performances were not associated with reduced productivity
Fig. 4. Plasma concentration of NEFA during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (*P < 0.05) between the group (G) at each sampling day (T) intervals.
for anti-inflammatory, bacteriostatic and anti-oxidative activities. We observed that plasma concentrations of cytokines were significantly influenced by group, time and its interactions. IL-6 and TNFα concentrations were significantly (P < 0.05) higher in cows that later became pregnant within breeding period compared to those remained non-pregnant during that period. Ishikawa et al. (2004) also found that IL-6 level was higher during pre-partum than postpartum period as observed in the present study. Pro-inflammatory cytokines such as IL1β and IL-6 are known inducer of APR and consequent release of APPs from hepatocytes (Ametaj et al., 2005). In fact, in the present study, we found similar trends of higher concentrations of cytokines and APPs during early postpartum. Galvao et al. (2011) reported that healthy cows had a more robust pro-inflammatory response after calving, to eliminate the infections and returns to normal homeostasis more rapidly. Further, lower expression of pro-inflammatory cytokine genes (IL-1β, TNF-α, and IL-6) during peri‑parturient period caused the development of endometritis due to an intrinsic defect in endometrial cell functions (Nino-Soto et al., 2008). Aitken et al. (2011) also suggested that an efficient inflammatory response is critical for elimination of the invading pathogen, it restores immune homeostasis and returns tissues back to normal function and morphology. Therefore, the observed higher concentrations of cytokines and APPs in cows during transition period indicate more active functioning of immune system and the same could be attributed to early conception in these animals. Bradford and Yuan (2017) reported that low level of inflammation is an adaptive process and beneficial in very early lactation as activated inflammatory pathways can promote the resolution of problems even in absence of overt disease conditions.
Fig. 5. Weekly average of Milk yield (5a) and Milk Fat percent (5b) in pregnant (n = 7) and Non-pregnant (n = 10) Deoni cows. First week observation was considered after colostrum period of five days. 22
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such as poorer milk yield. In the present study, cows with higher levels of NEFA, but within physiological limit also had higher concentration of inflammatory markers such as Hp, IL-1β, IL-6, and TNF-α and therefore it is an adaptive rather than pathological mechanism in these cows. Carson (2008) reported that fat mobilization and partial oxidation to ketones are part of adaptive response to NEB. Loor et al. (2005) also reported that metabolic adaptation is mainly regulated by cytokines, APPs and serum NEFA as observed in this study. Kovac et al. (2009) suggested that higher concentration of NEFA in serum could be related to activation of the immune system. Recently we also reported that active functioning of the immune system, more dry matter intake and efficient metabolic adaptation in transition Deoni cows enabled to synthesis more milk during postpartum period (Wankhade et al., 2018). Since it will be difficult to ensure the randomness of sampling to relate the metabolic and inflammatory variables during transition period with pregnancy outcome, further studies are required in Zebu cows with optimum sampling size. An establishment of threshold value or likelihood ratio of these variables with pregnancy outcome is also required for more understanding. Taken together, it is concluded that more active functioning of immune system, stable dry matter intake, and limited fat mobilization during transition period enabled the cows to conceive early during postpartum period.
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Immunopathol. 164 (1), 16–23. Nino-Soto, M.I., Heriazon, A., Quinton, M., Miglior, F., Thompson, K., Mallard, B.A., 2008. Differential gene expression of high and low immune responder Canadian Holstein dairy cows. Animal Genomics for Animal Health 132. pp. 315–320. Nonnecke, B.J., Kimura, K., Goff, J.P., Kehrli, M.E., 2003. Effects of the mammary gland
Conflict of interest The authors declare that they have no competing interests. Acknowledgments The authors are thankful to Director, ICAR-NDRI, and Head, SRS of ICAR-NDRI for providing needful facilities. The fund for this study was provided by ICAR-Animal Science Divisionunder ICAR-extramural projects. References Adewuyi, A.A., Gruys, E., Van Eerdenburg, F.J.C.M., 2005. Non-esterified fatty acids (NEFA) in dairy cattle. A review. Vet. Q. 27 (3), 117–126. Aitken, S.L., Corl, C.M., Sordillo, L.M., 2011. Immunopathology of mastitis: insights into disease recognition and resolution. J. Mammary Gland Biol. Neoplasia 16 (4), 291–304. Alsemgeest, S.P.M., Kalsbeek, H.C., Wensing, T., Koeman, J.P., Van Ederen, A.M., Gruys, E., 1994. Concentrations of SAA (SAA) and haptoglobin (Hp) as parameters of inflammatory diseases in cattle. Vet. Q. 16, 21–23. Alsemgeest, S.P.M., Taverne, M.A.M., Boosman, R., Van Der Weyden, B.C., Gruys, E., 1993. Peripartum acute-phase protein serum amyloid a concentration in plasma of cows and fetuses. Am. J. Vet. Res. 54, 165–167. Ametaj, B.N., Bradford, B.J., Bobe, G., Nafikov, R.A., Lu, Y., Young, J.W., Beitz, D.C., 2005. Strong relationships between mediators of the acute phase response and fatty liver in dairy cows. Can. J. Anim. Sci. 85 (2), 165–175. Banos, G., Wall, E., Coffey, M.P., Bagnall, A., Gillespie, S., George, C., Russell, G.C., McNeilly, T.N., 2013. Identification of immune traits correlated with dairy cow Health, reproduction and productivity. PLoS One 8 (6), 65766. Bell, A.W., 1995. Regulation of organic nutrient metabolism during transition from late pregnancy to early lactation. J. Anim. Sci. 73 (9), 2804–2819. Bionaz, M., Trevisi, E., Calamari, L.U.I.G.I., Librandi, F., Ferrari, A., Bertoni, G., 2007. Plasma paraoxonase, health, inflammatory conditions, and liver function in transition dairy cows. J. Dairy Sci. 90 (4), 1740–1750. Boro, P., Kumaresan, A., Pathak, R., Patbandha, T.K., Kumari, S., Yadav, A., Manimaran, A., Baithalu, R.K., Attupuram, N.M., Mohanty, T.K., 2015. Alteration in peripheral blood concentration of certain pro-inflammatory cytokines in cows developing retention of fetal membranes. Anim. Reprod. Sci. 157, 11–16. Bradford, B.J., Yuan, K., 2017. Inflammation in the transition dairy cow: adaptation or pathology?Transitioncow.net/wpcontent/uploads/2014/05/Inflammation_ transition_dairy.pdfpp 1-9Accessed 5 September 2017. Bradford, B.J., Yuan, K., Farney, J.K., Mamedova, L.K., Carpenter, A.J., 2015. Invited review: inflammation during the transition to lactation: new adventures with an old flame. J. Dairy Sci. 98 (10), 6631–6650. Carson, M., 2008. The association of selected metabolites in peripartum dairy cattle with health and production. The University of Guelph. Das, D.N., Kataktalware, M.A., Ramesha, K.P., Reddy, A.O., 2011. Productive and reproductive performances of Deoni cattle under intensive management system. Indian J. Anim. Sci. 81, 1186–1188. Dervishi, E., Zhang, G., Hailemariam, D., Goldansaz, S.A., Deng, Q., Dunn, S.M., Ametaj, B.N., 2016. Alterations in innate immunity reactants and carbohydrate and lipid
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expression of ISG 15 mRNA in peripheral blood mononuclear cells of nulliparous and multiparous pregnant versus non-pregnant Bos indicus cattle. Reprod. Dom. Anim. 52 (1), 97–106. Tegegne, A., Geleto, A., Osuji, P.O., Kassa, T., Francesallin, R., 1994. Influence of dietary supplementation and partial suckling and body weight on lactation and reproductive performance of primiparous Boran (Bos indicus) cows in Ethiopia. J. Agric. Sci. 123 (2), 267–273. Van Saun, R.J., 2016. Indicators of dairy cow transition risks: metabolic profiling revisited. Tierarztl Prax. Ausg. G. Grosstiere Nutztiere 44 (2), 118–126. Wankhade, P.R., Manimaran, A., Kumaresan, A., Jeyakumar, S., Ramesha, K.P., Sejian, V., Rajendran, D., Bagath, M., Sivaram, M., 2018. Metabolism and immune status during transition period influences the lactation performance in Zebu (Bos indicus) cows. Indian J. Anim. Sci. 88 (9), 1064–1069. Westwood, C.T., Lean, I.J., Garvin, J.K., 2002. Factors influencing fertility of Holstein dairy cows: a multivariate description. J. Dairy Sci. 85, 3225–3237. Wittwer, F., 2012. Manual De Patologia Clinica Veterinaria, 2nd ed. Imprenta america, Valdivia, Chile. Zhang, G., Hailemariam, D., Dervishi, E., Goldansaz, S.A., Deng, Q., Dunn, S.M., Ametaj, B.N., 2016. Dairy cows affected by ketosis show alterations in innate immunity and lipid and carbohydrate metabolism during the dry off period and postpartum. Res. Vet. Sci. 107, 246–256.
on functional capacities of blood mononuclear leukocyte populations from periparturient cows. J. Dairy Sci. 86, 2359–2368. Ospina, P.A., Nydam, D.V., Stokol, T., Overton, T.R., 2010a. Evaluation of non-esterified fatty acids and β-hydroxybutyrate in transition dairy cattle in the northeastern United States: critical thresholds for prediction of clinical diseases. J. Dairy Sci. 93 (2), 546–554. Ospina, P.A., Nydam, D.V., Stokol, T., Overton, T.R., 2010b. Associations of elevated nonesterified fatty acids and β-hydroxybutyrate concentrations with early lactation reproductive performance and milk production in transition dairy cattle in the northeastern United States. J. Dairy Sci. 93 (4), 1596–1603. Regassa, F., Noakes, D., 1999. Release of PGFM in relation to uterine involution. Vet. Rec. 144, 502–506. Reist, M., Koller, A., Busato, A., Kupfer, U., Blum, J.W., 2000. First ovulation and ketone body status in the early postpartum period of dairy cows. Theriogenology 54 (5), 685–701. Snijders, S.E.M., Dillon, P.G., O'Farrell, K.J., Diskin, M., Wylie, A.R.G., O'Callaghan, D., Rath, M., Boland, M.P., 2001. Genetic merit for milk production and reproductive success in dairy cows. Anim. Reprod. Sci. 65, 17–31. Sordillo, L.M., Pighetti, G.M., Davis, M.R., 1995. Enhanced production of bovine tumor necrosis factor-α during the periparturient period. Vet. Immunol. Immunopathol. 49 (3), 263–270. Soumya, N.P., Das, D., Jeyakumar, S., Mondal, S., Mor, A., Mundhe, U., 2016. Differential
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Active immune system and dry matter intake during the transition period are associated with postpartum fertility in lactating Zebu cows
T
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Pratik Ramesh Wankhadea, Ayyasamy Manimaranb, , Arumugam Kumaresanb, Sakthivel Jeyakumarb, Veerasamy Sejianc, Duraisamy Rajendrand, Madiajagan Bagathd, Muniandy Sivarame, Kerekoppa P. Rameshae, Minu Rachel Varghesea a
Livestock Research Centre (LRC), Southern Regional Station (SRS), ICAR-National Dairy Research Institute (NDRI), Adugodi, Bengaluru 560 030, Karnataka, India LRC, SRS, ICAR-NDRI, Adugodi, Bengaluru, India c Division of Animal Physiology, ICAR-National Institute of Animal Nutrition and Physiology (NIANP), Adugodi, Bengaluru 560 030, Karnataka, India d Division of Animal Nutrition, ICAR-NIANP, Adugodi, Bengaluru, India e SRS, ICAR-NDRI, Adugodi, Bengaluru, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Acute phase proteins Inflammatory cytokines Energy indicators Transition period Reproductive performance Deoni cows
The aim of the present study was to assess if the metabolic adaptation and immune status during transition period influence the postpartum reproductive performance in dairy cattle. We assessed the levels of innate immune molecules (Haptoglobin: Hp, Serum Amyloid A: SAA, IL-6, TNF-α, IL-1β, and IL-8) and energy balance indicators [NEFA and Dry Matter Intake (DMI)] in transition Zebu (Deoni breed) cows that became pregnant within breeding period (n = 7) and remained non-pregnant (n = 10) even after the breeding period (i.e. from 45 to 180 days postpartum). Blood samples were collected at weekly intervals during transition period (−21 d before to 21 d after calving) and plasma variables were estimated using commercially available bovine specific ELISA kits. Plasma Hp concentration was significantly (P < 0.01) higher in cows that became pregnant during breeding period as compared to those that remained non-pregnant. While plasma TNF-α concentrations differed significantly (P = 0.05) between groups, such difference was not observed with SAA concentrations. Group x time interaction had a significant effect on plasma IL-6 (P < 0.01), IL-1β (P = 0.05), IL-8 (P = 0.05) concentrations and DMI (P = 0.001). Plasma NEFA concentrations differed significantly (P = 0.03) between groups, although all the experimental cows had NEFA concentrations within the physiological limits. Days in milk had a significant effect on milk yield (P = 0.001) and fat percentage (P = 0.03). It is concluded that active functioning of immune system, stable dry matter intake, and limited fat mobilization during transition period enabled the cows to conceive early during postpartum period.
1. Introduction The transition period in dairy cows is associated with an increased incidence of metabolic and infectious diseases due to metabolic, hormonal, immunological and behavioral changes (Van Saun, 2016). The metabolic changes start two weeks before calving and have effects on reproductive health up to nine weeks after calving, which in turn affects the postpartum reproductive performance (Leblanc, 2010). Similarly, alteration of immune function during the transition period is also a risk factor for postpartum diseases (Kerhli, 2015). Therefore, energy balance and immunity during transition period may influence postpartum reproductive performance of dairy animals. Among the various risk factors for poor reproductive performance, compromised energy status
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is believed to be the most important factor resulting in body fat mobilization. Since the liver's capacity is limited, the metabolites such as Non-Esterified Fatty Acids (NEFA) accumulate in the blood and consequently cause various adverse effects such as reduced feed intake, immune-suppression and decreased productive and reproductive performance (Adewuyi et al., 2005). Although exact reasons for decreased immune function during the transition period are not clearly known, it is reported that lactation and the metabolic changes occurring during transition period alter these functions (Nonnecke et al., 2003). Loor et al. (2005) reported that metabolic adaptations start well ahead of parturition and are mainly regulated through cytokines, acute phase proteins (APPs) and NEFA during peri‑parturient period. Negative Energy Balance (NEB), a hallmark of transition cow (Bell, 1995), is a
Corresponding author. E-mail address: [email protected] (A. Manimaran).
https://doi.org/10.1016/j.livsci.2019.07.020 Received 5 May 2018; Received in revised form 1 February 2019; Accepted 16 July 2019 Available online 17 July 2019 1871-1413/ © 2019 Elsevier B.V. All rights reserved.
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followed up to 45 days after calving. All animals were apparently healthy and free from any diseases while enrollment. The experimental cows were maintained under individual tie barn housing system. The shed used for this experiment were open from all side, concrete paved with tiled roof. The housing space for cows was as per “Bureau of Indian Standards (BIS)”. All the cows were fed according to NRC (2001) recommendation using institute grown seasonal green fodders like maize, jowar, hybrid napier, paragrass, guinea grass and cowpea along with dry fodder (ragi straw) and commercially available concentrates (Nandini gold-cow feed containing 16–18% crude protein, 70–72% TDN, 2.5–3.5% fat, 5.5–6% crude fiber, 1–1.5% acid insoluble ash and 10–11% moisture, M/s Karnataka Milk Federation, Bengaluru). The cows were fed 15–20 kg green fodder, 2–3 kg ragi straw and 1.5–2.5 kg concentrates during prepartum transition period. After calving the animals were offered 18–22 kg green fodder, 2–3 kg ragi straw and 2.5–3 kg concentrate divided in equal proportion fed at the time of milking during morning and evening hours based on milk production of individual cow. The animals were provided with clean drinking water four times in a day.
metabolic adaptation process to adjust the imbalance between intake and demand, particularly in high yielding animals and, NEFA is traditionally used as indicators of NEB during transition period (Duffield et al., 2009). An elevation of NEFA during transition period is an indicator of adaptability of individual cows to NEB, but higher magnitude of elevation is often associated with poor productive and reproductive performance during later postpartum period (Ospina et al., 2010a, 2010b; Huzzey and Overton, 2013). APPs are non-specific innate immune molecules and their main functions are to restore the homeostasis of biological process. They are regarded as marker of inflammation (Eckersall and Bell, 2010) and the major bovine APPs, Haptoglobin: Hp and Serum Amyloid A: SAA, plays an important role in animal reproduction through endometrial reconstruction and intensification of the phagocytosis process against uterine pathogens. The role of APPs in prediction, diagnosis and treatment evaluation of postpartum uterine infections was reported by several researchers (Huzzey et al., 2011; Jeremejeva et al., 2012; Manimaran et al., 2016). Concentrations of Hp and SAA should be interpreted with caution as the physiological status of cows might influence their circulating concentrations; For instance, several researchers reported that parturition could have an influence on serum APPs concentrations (Alsemgeest et al., 1993; Regassa and Noakes, 1999; Humblet et al., 2006). Inflammatory cytokines are believed to be a central integrator of metabolism and immune function (Sordillo et al., 1995). Several researchers reported the relationship of inflammatory cytokines such as IL-1β, TNF-α and IL-6 with metabolic and infectious diseases during peri‑partum period (Boro et al., 2015; Zhang et al., 2016; Dervishi et al., 2016). In fact, the relationship between inflammation and metabolism during transition period was mostly explained through association studies under disease conditions (Ospina et al., 2010a, 2010b; Huzzy et al., 2011; Huzzey and Overton, 2013). The inflammatory response was also reported in apparently healthy transition cows (Bradford et al., 2015). Occurrences of overt inflammatory reaction without clear signs of infections or any pathological conditions were also reported during transition period (Bionaz et al., 2007). Though most of the transition cows undergo a brief period of inflammation, magnitude of the inflammatory condition is an ultimate determinant of its long-term outcome in terms of milk production and post-partum fertility. All the above studies were conducted in Holstein cows and such studies are very few in dual purpose breeds. Deoni is an important dual-purpose indigenous cattle breed of southern and western India (Kuralkar et al., 2014). Due to better disease resistance and heat tolerance of Deoni cattle, it is preferred by the small farmers in tropical drought-prone areas. This breed is well suited for low to medium input production system as compared to other breeds (Soumya et al., 2016). On the other hand, the reproductive efficiency of this breed is poor and hence, there is an ardent need for identification of factors associated with optimum fertility. With this backdrop, we hypothesized that the status of immune functions and metabolic adaptation during the transition period might influence the postpartum fertility in dual-purpose zebu cows. Thus, the aim of the study was to find out the alterations in the immune function and metabolic adaptation between the cows that did and did not conceive during early post-partum period.
2.2. Estimation of milk yield and fat per cent During the initial five days of postpartum colostrum period, the cows were allowed for suckling twice in a day by their calves. Thereafter, lactating cows were allowed for partial suckling for one minute by their calves for the letdown of milk, before actual milking by hand, and milk yield (Kg) was recorded with electronic weighing balance. The milk samples were collected in 50 ml sterilized centrifuge tubes from individual animals at 7 ± 1, 14 ± 1, 21 ± 1, 28 ± 1, 35 ± 1 and 42 ± 1 days of postpartum period for milk fat estimation using Gerber's method (IDF, 1991). 2.3. Reproduction management After a voluntary waiting period of 45 to 60 days, all the animals were routinely observed two times a day by trained personnel for estrus. Animals identified to be in estrus were confirmed for their proper stage to receive semen by an experienced Veterinarian. Animals detected in estrus in the early morning were artificially inseminated (AI) in morning of the same day with frozen semen and those cows detected in estrus in the late evening and night were inseminated in the morning of the next day. Two inseminations were done for each cow at 12 h interval. Pregnancy diagnosis was done 45 to 60 after AI through rectal palpation. The interval from calving to the first insemination, days open and services per conception were calculated for each animal. 2.4. Evaluation of cyclicity by trans-rectal ultrasonography The ultrasonography was performed for trans-rectal observation of reproductive organs of the animals after 42–47 days using M/s Aloka SSD 500, Aloka Co. Ltd. Tokya (Japan). The ovaries were examined for presence of follicle(s) and/or corpus luteum (CL). If an animal had normal sized ovaries with a CL in any one of the ovaries, then the animal was considered as cyclic.
2. Material and methods
2.5. Estimation of dry matter intake (DMI) and body condition score (BCS) of individual cows
2.1. Experiment animals and management The experiment was conducted at Livestock Research Centre (LRC), Southern Regional Station (SRS), ICAR-National Dairy Research Institute, Bengaluru, Karnataka (India), which is located in southern Plateau and Hill agro-climatic zone on 12° 97′N latitude and 77° 56′E longitude. The experimental procedures were duly approved by Institute Animal Ethical Committee (IAEC). A total 29 transition Deoni cows were enrolled a month before the expected date of calving and
The DMI of the individual cows was estimated on weekly basis before calving, on the day of calving, afterward on 3rd day and then on weekly basis up to 3rd week after calving. It was calculated by the amount of feed offered and amount of residue obtained on next day (i.e. 24 h) and then DMI was estimated per 100 kg body weight. All the cows were assessed for BCS (on a five-point scale) (Kellogg, 2010). 19
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Fig. 1. Plasma concentrations of Haptoglobin (1a) and Serum Amyloid A (1b) during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (**P < 0.01) between the group (G) at each sampling day (T) intervals.
interval between 1st and 2nd AI (N = 5) in pregnant and non-pregnant cows were 24.6 ± 10.1 and 45.2 ± 15.2 days, respectively.
2.6. Blood sampling and biochemical analysis Blood samples were collected from cows using vacutainer tubes with heparin as anti-coagulant on −21 ± 2, −14 ± 1, −7 ± 1 day before the expected date of calving, on the day of calving, and 3 ± 1, 7 ± 1, 14 ± 1 and 21 ± 2 days after calving. The blood samples were centrifuged within 30 min after collection, at 3000 rpm for 10 min and plasma was stored at −20 °C until further analysis. Blood plasma variables were analyzed using commercially available bovine specific ELISA kits and microplate reader (Multiskan GO, M/s Thermo scientific, USA). The ELISA kits used for Haptoglobin (Hp) was based on a solid phase ELISA and obtained from M/s Life Diagnostics, Inc., West Chester, PA (USA). The sandwich ELISA based kits for estimation of Serum Amyloid A (SAA), Interleukin 6 (IL-6), Interleukin 1β (IL-1β), Tumor Necrosis Factor α (TNF-α), Interleukin 8 (IL-8) were obtained from M/s Clound-Clone Corporation, Houston, TX (USA) and competitive ELISA based kit for NEFA estimation was obtained from M/s MyBiosource, Southern California, San Diego (USA). During study period, all the experimental cows were observed daily for any abnormalities. All primiparous, and animals suffered from any postpartum complication like abortion, dystocia, stillbirth, retained placenta, uterine infections (metritis, clinical endometritis) and clinical mastitis were excluded from study. Thus, seven pregnant, 10 nonpregnant cows were considered for biochemical assay and calculation of reproductive performance.
3.1. Ultrasonographic observation of ovarian structure during postpartum period in relation to pregnancy status in Deoni cows Upon ultrasonographic evaluation during 42–47 days postpartum, no clinical abnormality was detected in the genital tract of cows that eventually became pregnant within breeding period. Out of 7 cows that became pregnant within breeding period, three cows had CL while the remaining four cows had large size (>9 mm) follicles along with variable numbers of small (3–5 mm) and medium size (5–8 mm) follicles. Among 10 non-pregnant cows, seven cows had smooth ovaries with no large follicle or CL. 3.2. Association of bovine acute phase proteins and cytokines with pregnancy status of cows Plasma Hp concentration was significantly (P < 0.01) higher during immediate postpartum period (+7 d) in cows that became pregnant during breeding period as compared to those that remained non-pregnant (Fig. 1a). We found no significant influence of time, group and its interaction on SAA concentrations (Fig. 1b). Plasma IL-6 concentration was significantly influenced by the day of sampling (P = 0.001), group (P = 0.05) and their interaction (P = 0.01). Particularly, plasma IL-6 concentration was higher during pre-partum (−14th and −7th d) and postpartum period (3rd d) in cows that became pregnant within breeding period than that remained nonpregnant (Fig. 2a). Similarly, sampling day and group x time interactions had significant (P = 0.05) effect on IL-1β (Fig. 2c) and IL-8 concentrations (Fig. 2d). Plasma TNF-α concentration (Fig. 2b) was significantly influenced by group (P = 0.05).
2.7. Statistical analysis The animals were grouped into either pregnant or non-pregnant within the breeding period (i.e. from 45 to 180 days). To test the significant difference between pregnant and non-pregnant cows with respect to fertility parameters, a nonparametric test called Mann-Whitney U test was employed. Linear mixed model was used to test the significance of the differences between pregnant and non-pregnant cows for the parameters viz., APPs, cytokines, energy indicators, milk yield and fat percentage. The data were log-transformed before the analysis. The differences between groups were considered significant when P < 0.05. The statistical analysis was performed using SPSS version 16 software package (SPSS for windows, V16.0; M/s SPPS Inc., Chicago, IL, USA).
3.3. Association between energy indicators and pregnancy status in cows All the fixed factors such as group, sampling day and their interactions had significant (P = 0.001) influence on the DMI. The cows that became pregnant early during the breeding period consumed higher quantity of feed on dry matter basis (per 100 Kg body weight) during pre-partum period and maintained the stable DMI intake during postpartum period than the cows that remained open at the end of breeding period (Fig. 3a). Sampling day had significant (P = 0.001) effect on BCS during transition period where, it reduced from pre-partum period to the day of calving, and thereafter similar BCS was observed in both the groups (Fig. 3b). Plasma NEFA (P = 0.03) concentrations significantly differed between groups (Fig. 4). But, the observed levels were within physiological limits or lower than already reported threshold value for
3. Results Among pregnant cows, days to first artificial insemination (DTFAI), days open and number of services per conception were 68.4 ± 8.3, 95.4 ± 10.2 and 2.0 ± 0.4, respectively. Among non-pregnant cows, DTFAI and number of services per conception were 80.9 ± 9.2 days (12 days delayed than pregnant cows) and 2.0 ± 0.4, respectively. The 20
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Fig. 2. Plasma concentration of IL-6 (2a), TNF-α (2b), IL-1β (2c) and IL-8 (2d) during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (**P < 0.01; *P < 0.05) between the group (G) at each sampling day (T) intervals.
higher Hp concentration during first week of postpartum had more active innate immunity and suggested that acute phase response (APR) leads to prevention of uterine infections, clearance of pathogens and initiation of repair mechanism in the uterus. Humblet et al. (2006) also found significant increase in Hp concentrations in healthy cows during the first week of postpartum and suggested that parturition is associated with a physiological APR, as also observed in our study. We also found inter-individual variability in Hp levels as observed in previous studies (Humblet et al., 2006; Jacobsen et al., 2004). Among major APPs, SAA concentration was not found to be influenced by sampling day, group and interactions, indicating that Hp could be a more sensitive APP than SAA, particularly in apparently healthy cows. Several researchers also reported that Hp is a more appropriate and prominent proteins, while SAA is a low constitutive APP in dairy cattle. Longer half-life of Hp than SAA and stronger stimulation by IL-6, as observed in our study, could also be a reason for elevated Hp levels (Alsemgeest et al., 1994; MullerDoblies et al., 2004; Humblet et al., 2006). Hp is basically responsible
postpartum complications in dairy cattle. We found no effect of sampling day and group x time interactions on NEFA levels. 3.4. Association of milk yield and its fat percentage with pregnancy status There was no significant difference in average weekly milk yield and milk fat percentage between cows that became pregnant within breeding period as compared to those that remained non-pregnant (Fig. 5a and b). However, milk yield (P = 0.001) and its fat percentage (P = 0.03) were significantly influenced by time. 4. Discussion We observed a significantly higher concentration of bovine major APP during transition period in cows that later became pregnant within breeding period as compared to those cows remained non-pregnant during the period. Nightingale et al. (2015) reported that cows with
Fig. 3. Dry Matter Intake (DMI/100 Kg BW) (3a) and body condition score (BCS) (3b) during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (**P < 0.01; *P < 0.05) between the group (G) at each sampling day (T) intervals. 21
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We found that DMI was significantly higher in the cows that became pregnant within breeding period during prepartum period, which was maintained during postpartum period also. High DMI during the closeup period is better indicator of overall comfort of cows and thus prepartum DMI is more important than postpartum DMI (Drackley, 2003). NEFA is an energy indicator of fat mobilization in dairy cattle. Higher NEFA levels during transition period in cows that became pregnant within breeding period indicate that presence of mobilization of more adipose tissue to meet out the energy demand in these animals. Although cows which became pregnant within breeding period had higher concentrations of NEFA than the cows that remained open, the levels were within physiological limits or lower than already reported threshold value for postpartum complications. Earlier studies reported that the threshold level of NEFA for NEB was 0.3 mmol/l during early postpartum period (Adewuyi et al., 2005; Wittwer, 2012), which is well above the concentrations observed in the present study. Therefore, the observed levels of NEFA within physiological limit indicate controlled fat mobilization and it might have conferred a fertility advantage along with stable DMI in these cows. Holtenius et al. (2003) and Douglas et al. (2006) reported that cows fed with higher energy diet during close up period had a greater degree of insulin resistance, which allowed greater NEFA concentrations, while cows that fed limited quantity under requirements, had lower NEFA concentrations during periparturient period as was also observed in this study. Westwood et al. (2002) reported that shorter interval to ovulation as an important determinant of fertility even in cows with a higher NEFA level. In our study, ultrasonographic findings during 42–47 days postpartum confirmed the resumption of cyclicity in all cows that became pregnant than cows that remained non-pregnant (seven of 10 cows had smooth ovary). In fact, the cows that became pregnant were inseminated 12 days earlier than cows that remained non-pregnant. Reist et al. (2000) and Snijders et al. (2001) reported that reproductive performance was not associated with milk yield, milk fat and plasma metabolites level as was also observed in this study. The observed average milk yield in these dual-purpose cows (4–4.26 kg/day) agrees with those reported by Wankhade et al. (2018) and other researchers (Das et al., 2011; Kuralkar et al., 2014). Restricted or partial suckling by the calf is a common practice in dual purpose zebu (Bos indicus) cows as it is essential to initiate milk letdown and maintain the lactation in these cows (Tegegne et al., 1994). Indeed, partial suckling improved milk yield and udder health and reduced stress of calves and cows (Froberg, 2008; Hernandez et al., 2006). Although the role of inflammation-associated pathways in adaptation processes is not yet fully understood, Farney et al. (2013) suggested that inflammation at least in some cases, is an adaptive rather than pathological phenomenon, and some degree of inflammation might even be required for successful adaptation. Banos et al. (2013) reported that cellular immune traits associated with improved health or reproductive performances were not associated with reduced productivity
Fig. 4. Plasma concentration of NEFA during transition period and its relationship with postpartum pregnancy status in Deoni cows (n: 7 Pregnant and 10 Non-pregnant). Lines (mean ± SE) bearing asterisk differ significantly (*P < 0.05) between the group (G) at each sampling day (T) intervals.
for anti-inflammatory, bacteriostatic and anti-oxidative activities. We observed that plasma concentrations of cytokines were significantly influenced by group, time and its interactions. IL-6 and TNFα concentrations were significantly (P < 0.05) higher in cows that later became pregnant within breeding period compared to those remained non-pregnant during that period. Ishikawa et al. (2004) also found that IL-6 level was higher during pre-partum than postpartum period as observed in the present study. Pro-inflammatory cytokines such as IL1β and IL-6 are known inducer of APR and consequent release of APPs from hepatocytes (Ametaj et al., 2005). In fact, in the present study, we found similar trends of higher concentrations of cytokines and APPs during early postpartum. Galvao et al. (2011) reported that healthy cows had a more robust pro-inflammatory response after calving, to eliminate the infections and returns to normal homeostasis more rapidly. Further, lower expression of pro-inflammatory cytokine genes (IL-1β, TNF-α, and IL-6) during peri‑parturient period caused the development of endometritis due to an intrinsic defect in endometrial cell functions (Nino-Soto et al., 2008). Aitken et al. (2011) also suggested that an efficient inflammatory response is critical for elimination of the invading pathogen, it restores immune homeostasis and returns tissues back to normal function and morphology. Therefore, the observed higher concentrations of cytokines and APPs in cows during transition period indicate more active functioning of immune system and the same could be attributed to early conception in these animals. Bradford and Yuan (2017) reported that low level of inflammation is an adaptive process and beneficial in very early lactation as activated inflammatory pathways can promote the resolution of problems even in absence of overt disease conditions.
Fig. 5. Weekly average of Milk yield (5a) and Milk Fat percent (5b) in pregnant (n = 7) and Non-pregnant (n = 10) Deoni cows. First week observation was considered after colostrum period of five days. 22
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such as poorer milk yield. In the present study, cows with higher levels of NEFA, but within physiological limit also had higher concentration of inflammatory markers such as Hp, IL-1β, IL-6, and TNF-α and therefore it is an adaptive rather than pathological mechanism in these cows. Carson (2008) reported that fat mobilization and partial oxidation to ketones are part of adaptive response to NEB. Loor et al. (2005) also reported that metabolic adaptation is mainly regulated by cytokines, APPs and serum NEFA as observed in this study. Kovac et al. (2009) suggested that higher concentration of NEFA in serum could be related to activation of the immune system. Recently we also reported that active functioning of the immune system, more dry matter intake and efficient metabolic adaptation in transition Deoni cows enabled to synthesis more milk during postpartum period (Wankhade et al., 2018). Since it will be difficult to ensure the randomness of sampling to relate the metabolic and inflammatory variables during transition period with pregnancy outcome, further studies are required in Zebu cows with optimum sampling size. An establishment of threshold value or likelihood ratio of these variables with pregnancy outcome is also required for more understanding. Taken together, it is concluded that more active functioning of immune system, stable dry matter intake, and limited fat mobilization during transition period enabled the cows to conceive early during postpartum period.
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