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Animal Science Journal (2014) 85, 532–541
doi: 10.1111/asj.12181
ORIGINAL ARTICLE Plasma thiobarbituric acid reactive substances, vitamin A and vitamin E levels and resumption of postpartum ovarian activity in dairy cows Mari AOKI, Tomoko OHSHITA, Yasuhiro AOKI and Minoru SAKAGUCHI* Dairy Production Research Division, Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Sapporo, Japan
ABSTRACT Vitamins with antioxidative functions are commonly used as supplements to improve fertility in dairy cows. However, according to field test results uncertainty exists about the effect of these vitamins, especially in vitamin A and vitamin E, on ovarian functional activity. This study was performed to reveal the physiological characteristics of cows receiving enough feed and the ovaries of which were activated in the early postpartum period. Six of 12 primiparous cows showing the corpus luteum on 25 to 27 days after parturition were classified as early responders (PER); the remaining six were classified as late responders (PLR). Among 11 multiparous cows, nine were early responders (MER), and the remaining two were late responders (MLR). Plasma concentration of thiobarbituric acid reactive substances (TBARS) in the PER were lower than those in the PLR (P < 0.01). The ratio of plasma all-trans-retinol to intake α-tocopherol or β-carotene were increased in the following order: MER < PER < PLR (P < 0.01). The milk lactose (P < 0.025) and plasma glucose (P < 0.01) of the early responders tended to be lower than those of the late responders. These may have been associated with the availability of vitamins or energy balance. Thus, we suggest the possibility that the cows which were able to utilize antioxidants and energy from the feed efficiently may have earlier resumption of ovaries postpartum.
Key words: dairy cow, early responder, late responder, TBARS, vitamin.
INTRODUCTION Holstein dairy cows, not only in Japan but also in other countries, have recently experienced troublesome health issues caused by physiological stress (Nørgaard et al. 1999; Roche et al. 2000; Lucy 2001). These health problems have led to reproductive disturbances as the milk yield per cow has increased (Macmillan et al. 1996; Royal et al. 2000; Lucy 2001; Dochi 2010). Therefore, many researchers have attempted various approaches to improve reproductive performance while maintaining milk yield. Among them, many studies have proposed that oxidative stress (a type of physiological stress) is a cause of reproductive dysfunction in cattle (Miller & Brzezinska-Slebodzinska 1993; Bernabucci et al. 2005; Kankofer et al. 2010; Mantovani et al. 2010). To optimize performance, oxidative stress in highproducing cows must be controlled by supplying all known antioxidant nutrients (Miller & Brzezinska-Slebodzinska 1993). Vitamin A and E are well known antioxidants in terms of their function in reproductive improvement (Atwal et al. 1990; © 2014 Japanese Society of Animal Science
Campbell & Miller 1998; Ikeda et al. 2005; Sales et al. 2008) and are used as supplements. However, results of some feeding experiments supplying vitamin A (including β-carotene) and/or E did not produce improvements in cow reproduction (Greenberg et al. 1986; Tharnish & Larson 1992; Kaewlamun et al. 2011). There are divisions of opinion among researchers on the effects of supplying vitamin A and E for cows which do not have vitamin deficiency. Also, it has been debated whether early resumption of ovarian cycles improved cow fertility, but some researchers suggest that dairy cows which ovulate Correspondence: Mari Aoki, Dairy Production Research Division, Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Hitsujigaoka 1, Toyohira, Sapporo, Hokkaido 062-8555, Japan. (Email: [email protected]) *Present address: Laboratory of Theriogenology, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan. Received 28 January 2013; accepted for publication 19 November 2013.
PLASMA TBARS AND POSTPARTUM OVULATION
within 3 weeks postpartum were expected to increase fertility (Kawashima et al. 2006; Galvão et al. 2010). Because the first ovulation at early postpartum does not guarantee that the subsequent fertilization, implantation, embryo development, growth of fetus and so on, will work out, the early postpartum ovulation does not lead directly to the improvement of cow reproductive performance. However, there have been several reports that the first postpartum ovulation is considered to be one of the indicators for recovery of reproductive function in cows (Butler et al. 1981; Stevenson & Call 1982; Zurek et al. 1995; Kadokawa & Yamada 1999). Then, in this study, we divided the cows into good and poor fertility based on whether their first ovulation occurred within 3 weeks postpartum (early responder) or not (late responder), and we attempted to demonstrate the physiological differences between the early responders and the late responders.
MATERIAL AND METHODS Animals and feeding All procedures used in this experiment were approved by the Guide for the Care and Use of Experimental Animals (Animal Care Committee, Hokkaido Agricultural Research Center, Sapporo, Japan). This study was carried out at the Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (Japan). Twelve primiparous and 11 multiparous (mean parity, 3.4 ± 0.2) Holstein cows calved between 2006 and 2009 were randomly selected for screening of postpartum ovarian activity. The 305-day milk yield averaged about 10 000 kg. The cows were fed grass silage and/or corn silage (approx. 75–80% on fresh matter (FM) basis) with hay (approx. 5–10%), concentrate (approx. 12%) and mixture of beet pulp, lucerne, soybean flakes and corn (approx. < 5%); averages of nutrient components in the daily feed were approximately 33% dry matter (DM), 37.6% acid detergent fiber, 62% neutral detergent fiber and 11.5% crude protein. The cows were provided the feed twice daily (09.00 and 17.00 hours) until calving. After calving, they were given the feed twice daily after milking (09.00 and 19.00 hours). They were allowed free access to their own feed and water ad libitum in a tie-stall. The feed was of sufficient quantity to meet the cows’ requirements according to the Japanese Feeding Standard (Central Association of Livestock Industry 1999) in 2006 and 2007 and Nutrient Requirements of Dairy Cattle (National Research Council 2001) in 2008 and 2009. Daily feed intake was expressed as a percent of the amount of actual intake (the difference of amount of supplied feed and the residual) to the amount of supplied food. The mean daily intake per week (MDIW) was estimated using the daily intake of the last 7 days of the week (for example, the mean daily intake at 0 weeks after parturition was averaged for day −7 to day 0 before parturition).
Milk yield, milk composition, food intake, BCS and blood materials The milk composition (MilkoScan FT 120; Foss, Hillerød, Denmark) and somatic cell count (SCC) (Fossomatic 90; Animal Science Journal (2014) 85, 532–541
533
Foss) were determined by chemical analysis. The daily milk yield, milk composition, and SCC were obtained at 2 and 4 weeks after parturition. We recorded body condition score (BCS) (Gallo et al. 1996) each week and measured their MDIW from −4 to 4 weeks after parturition. We collected blood prior to milking and feeding in the morning (08.00 hour) each week from −4 to 4 weeks after parturition. Blood samples were taken from the jugular vein using evacuated tubes (Venoject II, Terumo, Tokyo, Japan) containing heparin for vitamin analysis or ethylenediaminetetraacetic acid (EDTA) for thiobarbituric acid reactive substances (TBARS), glucose, non-esterified fatty acids (NEFA), and 3-hydroxybutyrate (3-HB) analyses. Blood samples were centrifuged at 1600 × g for 20 min at 4°C and plasma was separated. The plasma samples were stored at −80°C until assayed.
Plasma and intake vitamins To estimate intake α-tocopherol (InVE) and the β-carotene (InBC) quantity from the feed, the samples of grass silage, corn silage and hay (main forage) were taken and pretreated as assay protocols recommended (Japan Grassland Agriculture & Forage Seed Association 2009). The α-tocopherol (VE) and β-carotene (BC) content of the main forage and plasma vitamin A as all-trans-retinol (PVA), plasma vitamin E as α-tocopherol (PVE) and plasma β-carotene (PBC) concentrations were analyzed by highperformance liquid chromatography (HPLC) (Landen 1981; Milne & Botnen 1986; Horwitz 2000). Each quantity of VE g or BC g in the main forage was calculated by the VE or BC content (g/kg) of main forage multiplied by the actual eating quantity of the main forage kg (supplied forage times a percent of the amount of actual intake), respectively. The quantities of vitamin E as InVE and InBC from the feed were determined as the sum of each actual intake VE g or BC g of the grass silage and/or the corn silage and the hay, respectively. The concentrate and others were present in very small amounts of VE and BC, and so we did not count them. The ratios of PVA, PVE and PBC concentrations to InVE and InBC (each expressed as PVA/InVE, PVE/InVE, PBC/InVE, PVA/ InBC, PVE/InBC and PBC/InBC) were estimated. The InVE and InBC per day were derived from the daily average of feed intake times the vitamin content of each feed (corn silage contained a mean 37 mg/FMkg of VE and mean 24 mg/ FMkg of BC, grass silage contained a mean 61 mg/FMkg of VE and mean 81 mg/FMkg of BC, and hay contained a mean 85.2 mg/DMkg of VE and mean 51.6 mg/DMkg of BC). The ratio of the concentration of plasma vitamins in the cows to intake of the vitamins was calculated.
Postpartum ovarian activity To evaluate the postpartum ovarian activity, the cows were classified as whether they ovulated within 3 weeks after parturition (early responders (ERs)) or not (late responders (LRs)) (Reist et al. 2000; Kawashima et al. 2006). The methods of determining the ERs or the LRs were referenced to previous reports (Leslie & Bosu 1983; Van de Wiel & Koops 1986; Caroll 1990; Taniguchi et al. 2007). To recognize the ERs, the presence of the corpus luteum on 25 to 27 days after parturition was determined by ultrasound examination (ECHOPAL EUB-405 7.5 MHz, Hitachi Medical Co., Tokyo, Japan). In addition, plasma progesterone concentrations were measured by enzyme immunoassay (Van de Wiel & © 2014 Japanese Society of Animal Science
534 M. AOKI et al.
Koops 1986; Taniguchi et al. 2007) and 1 ng/mL or higher concentrations were determined as the presence of luteal tissue.
Blood metabolite measurements The plasma concentrations of TBARS were measured fluorometrically using a TBARS assay kit (Oxitek TBARS Assay Kit; Zepto Metrix Co., New York, NY, USA) according to the manufacturer’s instructions. This represented an adaptation of the Yagi method (Yagi 1998). The concentrations of plasma glucose, NEFA and 3-HB were analyzed by an automatic analyzer (Nittobo Medical Co., Ltd, Tokyo, Japan) using commercially supplied reagent kits (N-assay GLU-UL; Nittobo Medical Co., Ltd.).
Statistical analysis First of all, we defined the experimental season when the cows calved as summer (from July to September) or winter (from November to March) and examined an effect of the season on the data. The effects of the experimental season (Season), experimental group (Group; primiparous ER (PER), primiparous LR (PLR) and multiparous ER (MER)), parity and experimental week (Week) were assessed by the analysis of variance (ANOVA) using SAS (ver. 9.2, SAS Institute, Cary, NC, USA). The data of the multiparous LR (MLR), only two cows, were not used for estimating statistical confidence measures and are shown as reference values. After that, the effects of Groups and experimental weeks (from −4 to 4 after parturition) on TBARS, PVE and MDIW or 2 weeks and 4 weeks after parturition on milk yield, lactose yield and solid non-fat (SNF) concentrations were analyzed by GLM with Tukey’s multiple comparison test when there was no interaction between Season and Group. For the items which had a significant effect of Group, it was also examined whether an effect of the parity (primiparous or multiparous) was significant using ANOVA. Means of the TBARS between the PERs and PLRs each week were compared using Student’s t-test. The correlation between variables was analyzed using the procedure CORR from SAS. The data were presented as means ± standard error of the mean (SEM).
RESULTS Seasonal differences and milk production Among 23 cows, six primiparous and nine multiparous cows (mean parity, 3.4 ± 0.2) were classified as ERs (n = 15), and six primiparous and two multiparous (parity = 3) were classified as LRs (n = 8). Thirteen of these cows calved in the winter (PERs = 4, PLRs = 6 and MERs = 3), and 10 cows calved in the summer (PERs = 2, MERs = 6 and MLRs = 2). The mean (at 2 and 4 weeks of lactation) milk yield, milk composition, SCC, concentration of plasma glucose and significance of the main effects of Season and Group are listed in Table 1. The main effect of Season was detected in some items. However, we could not analyze the interaction of the main effects (Season or Group, Week) between each other, because the data was too small to discuss them. The cows © 2014 Japanese Society of Animal Science
calved in the summer had higher 3-HB (P < 0.01), InVE (P < 0.01), InBC (P < 0.01), milk fat (P < 0.05), lactose (P < 0.05), total milk solid (TMS) (P < 0.01) and plasma glucose (P < 0.01) concentration than those in the winter, and PVA (P < 0.05), BCS (P < 0.01), PVA/InVE (P < 0.01), PBC/InVE (P < 0.01), PVA/InBC (P < 0.01), PVE/InBC (P < 0.05) and PBC/ InBC (P < 0.01) of the cows calved in the winter were higher than those in the summer. Main effect of Group on milk yield was significant. Although the milk yield of MERs was higher than that of the PERs and the PLRs (P < 0.01), a significant effect of the parity (P < 0.01) was observed. The lactose of the PLRs was higher than that of the PERs (P < 0.025) and MERs (P < 0.01), and the SNF of PLRs was higher than that of the MERs (P < 0.05). Although the plasma glucose was significantly affected by the parity (P < 0.01), the plasma glucose of the PLRs was higher than that of the PERs or the MERs, and that of the PERs was higher than the MERs. A positive correlation was obtained between the lactose and plasma glucose (r = 0.43, P < 0.01).
Body conditions and blood metabolites in early and late responders The BCS of the cows in all groups declined during the experimental period (Fig. 1). The means of the BCS in PERs, PLRs, MERs and MLRs were 3.4, 3.4, 3.2 and 3.3, respectively, and there was a difference due to the parity (P < 0.01). The BCS of the 12 primiparous cows (PERs and PLRs) was higher than that of the MERs (P < 0.01). However, there was no significant difference in BCS each week. The TBARS concentration of the PERs (mean ± SEM = 0.580 ± 0.027 nmol/mL) was lower than that of the PLRs (0.693 ± 0.027) (P < 0.01). The TBARS seemed to decrease during the preparturition period and to reach the lowest values around parturition, then that seemed to slightly return. In particular, although the TBARS concentration of the PERs had a tendency to be lower from 4 weeks preparturition to 1 week postparturition, there were no significant effects of Week and differences between the experimental groups in any given week. However, among primiparous cows, the TBARS of the PERs was lower than that of the PLRs (P < 0.01) at 0 week (P < 0.025; Student’s t-test) and 1 week (P < 0.05; Student’s t-test) postpartum (Fig. 2). There were weak positive correlations between the TBARS and BCS (r = 0.26, P < 0.01), PVA (r = 0.27, P < 0.01), PBC (r = 0.30, P < 0.01) and InVE (r = 0.15, P < 0.05). A main effect of Season was not significant on NEFA, but a main effect of Group (P < 0.01) was significant. The means ± SEM of the NEFA in the PERs, the PLRs, the MERs and the MLRs were 426 ± 41, 419 ± 22, 302 ± 26 and 280 ± 39 μEq/L, respectively. The NEFA of PERs and PLRs were higher than that of Animal Science Journal (2014) 85, 532–541
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Table 1 Milk yield, milk composition, and plasma glucose concentration of the primiparous early responders (PERs), primiparous late responders (PLRs) and multiparous early responders (MERs), and the significance of the effects of experimental season (differences between summer and winter; Season) and groups (differences between PERs, PLRs and MERs; Group)
Item
Daily milk yield (kg)
Milk fat (%)
Milk protein (%)
Lactose (%)
TMS (%)
SNF (%)
SCC (×103 cells/mL)§
Plasma glucose (mg/100 mL)
Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk
PER (n = 6)
PLR (n = 6)
MER (n = 9)
30.2 ± 1.4 30.4 ± 2.1 30.1 ± 2.1 4.6 ± 0.3 4.8 ± 0.5 4.3 ± 0.3 3.0 ± 0.1 3.1 ± 0.1 2.9 ± 0.1 4.5a ± 0.1 4.5 ± 0.1 4.5 ± 0.1 12.8 ± 0.3 13.1 ± 0.5 12.4 ± 0.4 8.2 ± 0.1 8.3 ± 0.1 8.1 ± 0.1 53.7 ± 0.5 53.3 ± 0.4 54.2 ± 0.7 62.4a ± 1.6 62.6 ± 2.0 62.3 ± 2.8
28.7 ± 0.7 28.8 ± 0.9 28.6 ± 1.0 4.3 ± 0.1 4.3 ± 0.2 4.2 ± 0.2 2.9 ± 0.1 3.0 ± 0.1 2.8 ± 0.1 4.7b ± 0.0 4.7 ± 0.1 4.8 ± 0.0 12.7 ± 0.1 12.8 ± 0.2 12.6 ± 0.2 8.3a ± 0.1 8.5 ± 0.1 8.2 ± 0.1 31.6 ± 0.3 33.6 ± 0.2 29.8 ± 0.3 70.1b ± 1.5 69.8 ± 1.7 70.4 ± 2.7
40.4 ± 0.0 39.9 ± 1.5 40.9 ± 1.9 4.2 ± 0.0 4.1 ± 0.2 4.3 ± 0.2 3.0 ± 0.0 3.2 ± 0.1 2.8 ± 0.1 4.4a ± 0.0 4.4 ± 0.1 4.5 ± 0.1 12.2 ± 0.0 12.4 ± 0.2 12.1 ± 0.2 8.1b ± 0.0 8.3 ± 0.1 7.9 ± 0.1 32.1 ± 0.5 35.3 ± 0.3 29.1 ± 0.7 55.9c ± 0.0 55.0 ± 1.2 56.8 ± 1.8
a
a
b
MLR‡ (n = 2) 42.0 ± 1.3 41.8 ± 2.0 42.3 ± 2.6 4.5 ± 0.4 4.8 ± 0.8 4.2 ± 0.5 2.8 ± 0.2 3.0 ± 0.3 2.7 ± 0.1 4.3 ± 0.1 4.2 ± 0.0 4.4 ± 0.1 12.3 ± 0.5 12.7 ± 1.1 11.9 ± 0.0 7.8 ± 0.2 7.9 ± 0.3 7.7 ± 0.5 19.7 ± 0.3 29.7 ± 0.0 13.0 ± 0.2 59.2 ± 4.2 56.5 ± 2.0 61.9 ± 9.3
P-values for main effect Season
Group
0.068
Animal Science Journal (2014) 85, 532–541
doi: 10.1111/asj.12181
ORIGINAL ARTICLE Plasma thiobarbituric acid reactive substances, vitamin A and vitamin E levels and resumption of postpartum ovarian activity in dairy cows Mari AOKI, Tomoko OHSHITA, Yasuhiro AOKI and Minoru SAKAGUCHI* Dairy Production Research Division, Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Sapporo, Japan
ABSTRACT Vitamins with antioxidative functions are commonly used as supplements to improve fertility in dairy cows. However, according to field test results uncertainty exists about the effect of these vitamins, especially in vitamin A and vitamin E, on ovarian functional activity. This study was performed to reveal the physiological characteristics of cows receiving enough feed and the ovaries of which were activated in the early postpartum period. Six of 12 primiparous cows showing the corpus luteum on 25 to 27 days after parturition were classified as early responders (PER); the remaining six were classified as late responders (PLR). Among 11 multiparous cows, nine were early responders (MER), and the remaining two were late responders (MLR). Plasma concentration of thiobarbituric acid reactive substances (TBARS) in the PER were lower than those in the PLR (P < 0.01). The ratio of plasma all-trans-retinol to intake α-tocopherol or β-carotene were increased in the following order: MER < PER < PLR (P < 0.01). The milk lactose (P < 0.025) and plasma glucose (P < 0.01) of the early responders tended to be lower than those of the late responders. These may have been associated with the availability of vitamins or energy balance. Thus, we suggest the possibility that the cows which were able to utilize antioxidants and energy from the feed efficiently may have earlier resumption of ovaries postpartum.
Key words: dairy cow, early responder, late responder, TBARS, vitamin.
INTRODUCTION Holstein dairy cows, not only in Japan but also in other countries, have recently experienced troublesome health issues caused by physiological stress (Nørgaard et al. 1999; Roche et al. 2000; Lucy 2001). These health problems have led to reproductive disturbances as the milk yield per cow has increased (Macmillan et al. 1996; Royal et al. 2000; Lucy 2001; Dochi 2010). Therefore, many researchers have attempted various approaches to improve reproductive performance while maintaining milk yield. Among them, many studies have proposed that oxidative stress (a type of physiological stress) is a cause of reproductive dysfunction in cattle (Miller & Brzezinska-Slebodzinska 1993; Bernabucci et al. 2005; Kankofer et al. 2010; Mantovani et al. 2010). To optimize performance, oxidative stress in highproducing cows must be controlled by supplying all known antioxidant nutrients (Miller & Brzezinska-Slebodzinska 1993). Vitamin A and E are well known antioxidants in terms of their function in reproductive improvement (Atwal et al. 1990; © 2014 Japanese Society of Animal Science
Campbell & Miller 1998; Ikeda et al. 2005; Sales et al. 2008) and are used as supplements. However, results of some feeding experiments supplying vitamin A (including β-carotene) and/or E did not produce improvements in cow reproduction (Greenberg et al. 1986; Tharnish & Larson 1992; Kaewlamun et al. 2011). There are divisions of opinion among researchers on the effects of supplying vitamin A and E for cows which do not have vitamin deficiency. Also, it has been debated whether early resumption of ovarian cycles improved cow fertility, but some researchers suggest that dairy cows which ovulate Correspondence: Mari Aoki, Dairy Production Research Division, Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (NARO), Hitsujigaoka 1, Toyohira, Sapporo, Hokkaido 062-8555, Japan. (Email: [email protected]) *Present address: Laboratory of Theriogenology, School of Veterinary Medicine, Kitasato University, Towada, Aomori, Japan. Received 28 January 2013; accepted for publication 19 November 2013.
PLASMA TBARS AND POSTPARTUM OVULATION
within 3 weeks postpartum were expected to increase fertility (Kawashima et al. 2006; Galvão et al. 2010). Because the first ovulation at early postpartum does not guarantee that the subsequent fertilization, implantation, embryo development, growth of fetus and so on, will work out, the early postpartum ovulation does not lead directly to the improvement of cow reproductive performance. However, there have been several reports that the first postpartum ovulation is considered to be one of the indicators for recovery of reproductive function in cows (Butler et al. 1981; Stevenson & Call 1982; Zurek et al. 1995; Kadokawa & Yamada 1999). Then, in this study, we divided the cows into good and poor fertility based on whether their first ovulation occurred within 3 weeks postpartum (early responder) or not (late responder), and we attempted to demonstrate the physiological differences between the early responders and the late responders.
MATERIAL AND METHODS Animals and feeding All procedures used in this experiment were approved by the Guide for the Care and Use of Experimental Animals (Animal Care Committee, Hokkaido Agricultural Research Center, Sapporo, Japan). This study was carried out at the Hokkaido Agricultural Research Center, National Agriculture and Food Research Organization (Japan). Twelve primiparous and 11 multiparous (mean parity, 3.4 ± 0.2) Holstein cows calved between 2006 and 2009 were randomly selected for screening of postpartum ovarian activity. The 305-day milk yield averaged about 10 000 kg. The cows were fed grass silage and/or corn silage (approx. 75–80% on fresh matter (FM) basis) with hay (approx. 5–10%), concentrate (approx. 12%) and mixture of beet pulp, lucerne, soybean flakes and corn (approx. < 5%); averages of nutrient components in the daily feed were approximately 33% dry matter (DM), 37.6% acid detergent fiber, 62% neutral detergent fiber and 11.5% crude protein. The cows were provided the feed twice daily (09.00 and 17.00 hours) until calving. After calving, they were given the feed twice daily after milking (09.00 and 19.00 hours). They were allowed free access to their own feed and water ad libitum in a tie-stall. The feed was of sufficient quantity to meet the cows’ requirements according to the Japanese Feeding Standard (Central Association of Livestock Industry 1999) in 2006 and 2007 and Nutrient Requirements of Dairy Cattle (National Research Council 2001) in 2008 and 2009. Daily feed intake was expressed as a percent of the amount of actual intake (the difference of amount of supplied feed and the residual) to the amount of supplied food. The mean daily intake per week (MDIW) was estimated using the daily intake of the last 7 days of the week (for example, the mean daily intake at 0 weeks after parturition was averaged for day −7 to day 0 before parturition).
Milk yield, milk composition, food intake, BCS and blood materials The milk composition (MilkoScan FT 120; Foss, Hillerød, Denmark) and somatic cell count (SCC) (Fossomatic 90; Animal Science Journal (2014) 85, 532–541
533
Foss) were determined by chemical analysis. The daily milk yield, milk composition, and SCC were obtained at 2 and 4 weeks after parturition. We recorded body condition score (BCS) (Gallo et al. 1996) each week and measured their MDIW from −4 to 4 weeks after parturition. We collected blood prior to milking and feeding in the morning (08.00 hour) each week from −4 to 4 weeks after parturition. Blood samples were taken from the jugular vein using evacuated tubes (Venoject II, Terumo, Tokyo, Japan) containing heparin for vitamin analysis or ethylenediaminetetraacetic acid (EDTA) for thiobarbituric acid reactive substances (TBARS), glucose, non-esterified fatty acids (NEFA), and 3-hydroxybutyrate (3-HB) analyses. Blood samples were centrifuged at 1600 × g for 20 min at 4°C and plasma was separated. The plasma samples were stored at −80°C until assayed.
Plasma and intake vitamins To estimate intake α-tocopherol (InVE) and the β-carotene (InBC) quantity from the feed, the samples of grass silage, corn silage and hay (main forage) were taken and pretreated as assay protocols recommended (Japan Grassland Agriculture & Forage Seed Association 2009). The α-tocopherol (VE) and β-carotene (BC) content of the main forage and plasma vitamin A as all-trans-retinol (PVA), plasma vitamin E as α-tocopherol (PVE) and plasma β-carotene (PBC) concentrations were analyzed by highperformance liquid chromatography (HPLC) (Landen 1981; Milne & Botnen 1986; Horwitz 2000). Each quantity of VE g or BC g in the main forage was calculated by the VE or BC content (g/kg) of main forage multiplied by the actual eating quantity of the main forage kg (supplied forage times a percent of the amount of actual intake), respectively. The quantities of vitamin E as InVE and InBC from the feed were determined as the sum of each actual intake VE g or BC g of the grass silage and/or the corn silage and the hay, respectively. The concentrate and others were present in very small amounts of VE and BC, and so we did not count them. The ratios of PVA, PVE and PBC concentrations to InVE and InBC (each expressed as PVA/InVE, PVE/InVE, PBC/InVE, PVA/ InBC, PVE/InBC and PBC/InBC) were estimated. The InVE and InBC per day were derived from the daily average of feed intake times the vitamin content of each feed (corn silage contained a mean 37 mg/FMkg of VE and mean 24 mg/ FMkg of BC, grass silage contained a mean 61 mg/FMkg of VE and mean 81 mg/FMkg of BC, and hay contained a mean 85.2 mg/DMkg of VE and mean 51.6 mg/DMkg of BC). The ratio of the concentration of plasma vitamins in the cows to intake of the vitamins was calculated.
Postpartum ovarian activity To evaluate the postpartum ovarian activity, the cows were classified as whether they ovulated within 3 weeks after parturition (early responders (ERs)) or not (late responders (LRs)) (Reist et al. 2000; Kawashima et al. 2006). The methods of determining the ERs or the LRs were referenced to previous reports (Leslie & Bosu 1983; Van de Wiel & Koops 1986; Caroll 1990; Taniguchi et al. 2007). To recognize the ERs, the presence of the corpus luteum on 25 to 27 days after parturition was determined by ultrasound examination (ECHOPAL EUB-405 7.5 MHz, Hitachi Medical Co., Tokyo, Japan). In addition, plasma progesterone concentrations were measured by enzyme immunoassay (Van de Wiel & © 2014 Japanese Society of Animal Science
534 M. AOKI et al.
Koops 1986; Taniguchi et al. 2007) and 1 ng/mL or higher concentrations were determined as the presence of luteal tissue.
Blood metabolite measurements The plasma concentrations of TBARS were measured fluorometrically using a TBARS assay kit (Oxitek TBARS Assay Kit; Zepto Metrix Co., New York, NY, USA) according to the manufacturer’s instructions. This represented an adaptation of the Yagi method (Yagi 1998). The concentrations of plasma glucose, NEFA and 3-HB were analyzed by an automatic analyzer (Nittobo Medical Co., Ltd, Tokyo, Japan) using commercially supplied reagent kits (N-assay GLU-UL; Nittobo Medical Co., Ltd.).
Statistical analysis First of all, we defined the experimental season when the cows calved as summer (from July to September) or winter (from November to March) and examined an effect of the season on the data. The effects of the experimental season (Season), experimental group (Group; primiparous ER (PER), primiparous LR (PLR) and multiparous ER (MER)), parity and experimental week (Week) were assessed by the analysis of variance (ANOVA) using SAS (ver. 9.2, SAS Institute, Cary, NC, USA). The data of the multiparous LR (MLR), only two cows, were not used for estimating statistical confidence measures and are shown as reference values. After that, the effects of Groups and experimental weeks (from −4 to 4 after parturition) on TBARS, PVE and MDIW or 2 weeks and 4 weeks after parturition on milk yield, lactose yield and solid non-fat (SNF) concentrations were analyzed by GLM with Tukey’s multiple comparison test when there was no interaction between Season and Group. For the items which had a significant effect of Group, it was also examined whether an effect of the parity (primiparous or multiparous) was significant using ANOVA. Means of the TBARS between the PERs and PLRs each week were compared using Student’s t-test. The correlation between variables was analyzed using the procedure CORR from SAS. The data were presented as means ± standard error of the mean (SEM).
RESULTS Seasonal differences and milk production Among 23 cows, six primiparous and nine multiparous cows (mean parity, 3.4 ± 0.2) were classified as ERs (n = 15), and six primiparous and two multiparous (parity = 3) were classified as LRs (n = 8). Thirteen of these cows calved in the winter (PERs = 4, PLRs = 6 and MERs = 3), and 10 cows calved in the summer (PERs = 2, MERs = 6 and MLRs = 2). The mean (at 2 and 4 weeks of lactation) milk yield, milk composition, SCC, concentration of plasma glucose and significance of the main effects of Season and Group are listed in Table 1. The main effect of Season was detected in some items. However, we could not analyze the interaction of the main effects (Season or Group, Week) between each other, because the data was too small to discuss them. The cows © 2014 Japanese Society of Animal Science
calved in the summer had higher 3-HB (P < 0.01), InVE (P < 0.01), InBC (P < 0.01), milk fat (P < 0.05), lactose (P < 0.05), total milk solid (TMS) (P < 0.01) and plasma glucose (P < 0.01) concentration than those in the winter, and PVA (P < 0.05), BCS (P < 0.01), PVA/InVE (P < 0.01), PBC/InVE (P < 0.01), PVA/InBC (P < 0.01), PVE/InBC (P < 0.05) and PBC/ InBC (P < 0.01) of the cows calved in the winter were higher than those in the summer. Main effect of Group on milk yield was significant. Although the milk yield of MERs was higher than that of the PERs and the PLRs (P < 0.01), a significant effect of the parity (P < 0.01) was observed. The lactose of the PLRs was higher than that of the PERs (P < 0.025) and MERs (P < 0.01), and the SNF of PLRs was higher than that of the MERs (P < 0.05). Although the plasma glucose was significantly affected by the parity (P < 0.01), the plasma glucose of the PLRs was higher than that of the PERs or the MERs, and that of the PERs was higher than the MERs. A positive correlation was obtained between the lactose and plasma glucose (r = 0.43, P < 0.01).
Body conditions and blood metabolites in early and late responders The BCS of the cows in all groups declined during the experimental period (Fig. 1). The means of the BCS in PERs, PLRs, MERs and MLRs were 3.4, 3.4, 3.2 and 3.3, respectively, and there was a difference due to the parity (P < 0.01). The BCS of the 12 primiparous cows (PERs and PLRs) was higher than that of the MERs (P < 0.01). However, there was no significant difference in BCS each week. The TBARS concentration of the PERs (mean ± SEM = 0.580 ± 0.027 nmol/mL) was lower than that of the PLRs (0.693 ± 0.027) (P < 0.01). The TBARS seemed to decrease during the preparturition period and to reach the lowest values around parturition, then that seemed to slightly return. In particular, although the TBARS concentration of the PERs had a tendency to be lower from 4 weeks preparturition to 1 week postparturition, there were no significant effects of Week and differences between the experimental groups in any given week. However, among primiparous cows, the TBARS of the PERs was lower than that of the PLRs (P < 0.01) at 0 week (P < 0.025; Student’s t-test) and 1 week (P < 0.05; Student’s t-test) postpartum (Fig. 2). There were weak positive correlations between the TBARS and BCS (r = 0.26, P < 0.01), PVA (r = 0.27, P < 0.01), PBC (r = 0.30, P < 0.01) and InVE (r = 0.15, P < 0.05). A main effect of Season was not significant on NEFA, but a main effect of Group (P < 0.01) was significant. The means ± SEM of the NEFA in the PERs, the PLRs, the MERs and the MLRs were 426 ± 41, 419 ± 22, 302 ± 26 and 280 ± 39 μEq/L, respectively. The NEFA of PERs and PLRs were higher than that of Animal Science Journal (2014) 85, 532–541
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Table 1 Milk yield, milk composition, and plasma glucose concentration of the primiparous early responders (PERs), primiparous late responders (PLRs) and multiparous early responders (MERs), and the significance of the effects of experimental season (differences between summer and winter; Season) and groups (differences between PERs, PLRs and MERs; Group)
Item
Daily milk yield (kg)
Milk fat (%)
Milk protein (%)
Lactose (%)
TMS (%)
SNF (%)
SCC (×103 cells/mL)§
Plasma glucose (mg/100 mL)
Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk Mean† At 2wk At 4wk
PER (n = 6)
PLR (n = 6)
MER (n = 9)
30.2 ± 1.4 30.4 ± 2.1 30.1 ± 2.1 4.6 ± 0.3 4.8 ± 0.5 4.3 ± 0.3 3.0 ± 0.1 3.1 ± 0.1 2.9 ± 0.1 4.5a ± 0.1 4.5 ± 0.1 4.5 ± 0.1 12.8 ± 0.3 13.1 ± 0.5 12.4 ± 0.4 8.2 ± 0.1 8.3 ± 0.1 8.1 ± 0.1 53.7 ± 0.5 53.3 ± 0.4 54.2 ± 0.7 62.4a ± 1.6 62.6 ± 2.0 62.3 ± 2.8
28.7 ± 0.7 28.8 ± 0.9 28.6 ± 1.0 4.3 ± 0.1 4.3 ± 0.2 4.2 ± 0.2 2.9 ± 0.1 3.0 ± 0.1 2.8 ± 0.1 4.7b ± 0.0 4.7 ± 0.1 4.8 ± 0.0 12.7 ± 0.1 12.8 ± 0.2 12.6 ± 0.2 8.3a ± 0.1 8.5 ± 0.1 8.2 ± 0.1 31.6 ± 0.3 33.6 ± 0.2 29.8 ± 0.3 70.1b ± 1.5 69.8 ± 1.7 70.4 ± 2.7
40.4 ± 0.0 39.9 ± 1.5 40.9 ± 1.9 4.2 ± 0.0 4.1 ± 0.2 4.3 ± 0.2 3.0 ± 0.0 3.2 ± 0.1 2.8 ± 0.1 4.4a ± 0.0 4.4 ± 0.1 4.5 ± 0.1 12.2 ± 0.0 12.4 ± 0.2 12.1 ± 0.2 8.1b ± 0.0 8.3 ± 0.1 7.9 ± 0.1 32.1 ± 0.5 35.3 ± 0.3 29.1 ± 0.7 55.9c ± 0.0 55.0 ± 1.2 56.8 ± 1.8
a
a
b
MLR‡ (n = 2) 42.0 ± 1.3 41.8 ± 2.0 42.3 ± 2.6 4.5 ± 0.4 4.8 ± 0.8 4.2 ± 0.5 2.8 ± 0.2 3.0 ± 0.3 2.7 ± 0.1 4.3 ± 0.1 4.2 ± 0.0 4.4 ± 0.1 12.3 ± 0.5 12.7 ± 1.1 11.9 ± 0.0 7.8 ± 0.2 7.9 ± 0.3 7.7 ± 0.5 19.7 ± 0.3 29.7 ± 0.0 13.0 ± 0.2 59.2 ± 4.2 56.5 ± 2.0 61.9 ± 9.3
P-values for main effect Season
Group
0.068
