J. Dairy Sci. 2007. 90:4253-4264. doi:10.3168/jds.2007-0117
© 2007 American Dairy Science Association ®
Evaluation of the Mechanism of Action of Conjugated Linoleic Acid Isomers on Reproduction in Dairy Cows1
E. Castañeda-Gutiérrez*,2,
B. C. Benefield*,
M. J. de Veth
,3,
N. R. Santos
,
R. O. Gilbert,
W. R. Butler* and
D. E. Bauman*,4
* Department of Animal Science, Cornell University, Ithaca, NY 14853-6401
BASF AG, Nutrition Research Station, Neumuehle 13, 76877, Offenbach/Queich, Germany
Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853-6401
4 Corresponding author: deb6{at}cornell.edu
 |
ABSTRACT
|
|---|
The objective of this study was to evaluate the mechanism of action through which conjugated linoleic acid (CLA) beneficially affects reproduction. Lactating Holstein cows (n = 45, 20 ± 1 DIM) were assigned to 1 of 3 treatments: 70 g/d of Ca salts of tallow (control); 63 g/d of lipid-encapsulated CLA providing 7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA (CLA 75:25); or 76 g/d of lipid-encapsulated CLA providing 7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA (CLA 50:50). Supplements were top-dressed for 37 d, milk production and DMI were recorded daily, and blood samples were taken 3 times per week. At 30 ± 3 DIM, ovulation was synchronized in all cows with a modified Ovsynch protocol, and on d 15 of the cycle cows received an oxytocin injection; blood samples were obtained frequently to measure 13,14 dihydro, 15-keto PGF2
. On d 16 of the cycle cows received a PGF2 injection and ovarian follicular aspiration was performed 54 h later. Follicular fluid was analyzed for fatty acids, progesterone, and estradiol. Endometrial biopsies were taken before and again near the end of the supplementation period for fatty acid analysis. The CLA resulted in decreased milk fat content of 14.1 and 6.1% at wk 5 of treatment of CLA 50:50 and CLA 75:25, respectively. There were no differences in energy balance or plasma nonesterified fatty acids; however, plasma IGF-I was greater in cows supplemented with CLA 50:50. The CLA isomers were not detectable in endometrial tissue, but cis-9, trans-11 CLA tended to be greater in follicular fluid of supplemented cows. Response to the oxytocin challenge was not different among treatments. Progesterone during the early luteal phase and the estradiol:progesterone ratio in follicular fluid tended to be greater in cows supplemented with CLA 50:50. Overall, these results indicate that short periods of CLA supplementation do not alter uterine secretion of PGF2 . The mechanism through which CLA affects reproduction may involve improved ovarian follicular steroidogenesis and increased circulating concentrations of IGF-I.
Key Words: conjugated linoleic acid • progesterone • insulin-like growth factor-I • prostaglandin F2
|
INTRODUCTION
|
|---|
Reproductive efficiency of dairy cattle has declined worldwide over the past 50 yr. Although several factors contribute to this problem, critical areas include metabolic effects of lactation on reproduction, mechanisms linking disease to reproduction, and early embryonic mortality (reviewed by Lucy, 2001). Dietary fat supplements in early lactation may benefit reproductive outcome by improving energy intake and reducing the extent of negative energy balance, as well as by increasing size of the ovulatory follicle and lifespan of the corpus luteum (reviewed by Mattos et al., 2000). Conjugated linoleic acid (CLA) is a fatty acid of 18 carbons with 2 conjugated double bonds. The most studied isomers are cis-9, trans-11 CLA and trans-10, cis-12 CLA, and their biological activity differs. The isomer trans-10, cis-12 CLA causes decreased milk fat yield (reviewed by Bauman and Lock, 2006), and previous studies in dairy cows have examined CLA-induced milk fat depression as an approach to decrease negative energy balance during early lactation. Supplementation with a mixture of CLA isomers providing less than 10 g/d of trans-10, cis-12 CLA did not affect energy balance (Bernal-Santos et al., 2003; Castañeda-Gutiérrez et al., 2005). Although the reproductive data in these experiments represented a limited number of cows, both studies observed a trend to decrease time to first ovulation postpartum and an increase in pregnancy rate.
Dietary polyunsaturated fatty acids (PUFA) may facilitate the suppression of uterine PGF2 synthesis induced by the early embryo, thereby having the potential to decrease embryo losses in early pregnancy (reviewed by Mattos et al., 2000). The PGF2 response to PUFA supplementation, however, has not been consistently observed across studies (Robinson et al., 2002; Petit et al., 2004), and these differences may be related to the effects of individual fatty acids. The CLA has been shown to inhibit prostaglandin synthesis in several systems (see review by Belury et al., 2002), but uterine PGF2 response has not been evaluated in vivo in dairy cows. Although there seems to be a positive effect of CLA on reproduction of dairy cows, the mechanism of action or whether this effect is associated with a particular CLA isomer has not been studied.
The objective of the present study was to evaluate the effect of dietary supplements of CLA on reproductive markers in dairy cows. Two mixtures that varied in proportion of cis-9, trans-11 CLA and trans-10, cis-12 CLA were used, and response variables included uterine PGF2 response to an oxytocin challenge, steroidogenesis in ovarian follicles and corpus luteum, and effects on circulating concentrations of reproductive and metabolic hormones.
|
MATERIALS AND METHODS
|
|---|
Animals and Design
All procedures involving animals were approved by the Cornell University Institutional Animal Care and Use Committee. Holstein cows (n = 45) from the Cornell University Dairy Teaching and Research facility were blocked by parity and 305-d mature-equivalent milk production, and assigned in a completely randomized block design to 1 of 3 treatments: 1) control (70 g/d of Ca salts of tallow), 2) CLA 75:25 (63 g/d of lipid-encapsulated CLA mixture that provided 7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA), and 3) CLA 50:50 (76 g/d of lipid-encapsulated CLA mixture that provided 7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA). The complete fatty acid profile of the supplements is provided in Table 1
. The supplements were topdressed on the feed once daily during the interval from 20 ± 1 DIM to 56 ± 1 DIM. The CLA mixtures were supplied by BASF AG (Ludwigshafen, Germany) and Ca salts of tallow by Virtus Nutrition LLC (Corcoran, CA). After supplementation, cows were kept in individual stalls for 4 wk (posttreatment period) and milk, milk composition, and DMI were recorded.
The basal diet was a TMR formulated using the Cornell Net Carbohydrate and Protein System (Fox et al., 2004) to meet or exceed nutrient requirements. The TMR was sampled weekly. The DM content was determined by drying at 54°C until constant weight, and then samples were ground and composited at 4-wk intervals. Feed composites were analyzed by wet chemistry methods for CP, ADF, NDF, and ether extract (Dairy One Cooperative Inc., Ithaca, NY). Ingredients and composition of the diet are reported in Table 2.
Cows were housed in individual stalls and fed ad libitum to allow 10% orts. Daily DMI was recorded during the supplementation and posttreatment periods. Water and mineral blocks were available throughout the study. Body weight and BCS (5-point system; Wildman et al., 1982) were recorded weekly. The BCS was recorded by 2 individuals, and the weekly average was used for statistical analysis. Cows were milked 3 times per day and milk production recorded electronically. One day per week, milk was sampled from each of the 3 milkings, and a composite was stored at 4°C with a preservative (bronopol tablet, D&F Control System, San Ramon, CA) until analyzed for fat, true protein, somatic cells, and lactose (Dairy One Cooperative Inc.) as described by Bernal-Santos et al. (2003). A second aliquot was stored without preservative at –20°C until analyzed for fatty acids.
Synchronization of Ovarian Cycles
At 30 ± 2 DIM, a controlled internal drug-releasing device (CIDR; Eazi-Breed, Pharmacia and Upjohn, Kalamazoo, MI) was inserted intravaginally and 100 mg of GnRH analogue (Cystorelin, Abbott Laboratories, North Chicago, IL) was injected (i.m.). Seven days later, the CIDR was removed and the cows received an i.m. injection (30 mg) of PGF2 (Lutalyse; Pharmacia and Upjohn). Cows received a second injection of GnRH analogue 2 d later (d 0 of the cycle), and ovaries were examined by linear array ultrasonography using a 7.5-MHz transrectal transducer (Aloka 210; Corometrics Medical Systems, Wallingford, CT). Ovarian activity was monitored every other day during the next 5 d until ovulation was confirmed by detection of a corpus luteum. Three cows in control and one cow in CLA 50:50 did not ovulate in response to synchronization. The procedure was therefore repeated 7 d later, and these cows continued to receive the treatment supplements for an additional 14 d; the second synchronization was successful in 3 of the cows. Data from the control cow that failed to respond to the second synchronization were excluded from all analysis of reproductive hormones.
Blood Sampling and Biopsy Procedures
Blood samples were taken during the treatment period 3 times per week via coccygeal venipuncture and collected into vacuum tubes containing sodium heparin (100 U/mL of blood). Plasma was harvested within 20 min after collection by centrifugation (2,800 x g for 15 min at 4°C) and stored at –20°C until analyzed for hormones and metabolites.
An endometrial biopsy was performed before starting treatment supplements (16 ± 2 DIM). For this procedure the external genitalia were cleaned and a biopsy tool covered by a plastic sheath was inserted into the vagina. The instrument was guided through the cervix by rectal palpation, and then introduced into the uterus after breaking the plastic sheath (to avoid bacterial contamination). From each cow 3 biopsies of uterine endome-trial tissue (
100 mg) were taken, and samples were immediately frozen in liquid nitrogen and stored at –80°C until being analyzed for fatty acid profile. The same biopsy procedure was repeated on d 15 of the synchronized cycle immediately after completion of the oxytocin challenge and blood sampling sequence.
Oxytocin Challenge
A jugular catheter was inserted on d 14 of the cycle, and on d 15 the cows received an i.v. injection of 3 mg of estradiol 17ß in a 50:50 ethanol:saline solution. Four hours later cows received an i.v. injection of 100 IU of oxytocin. Blood samples were collected 30 and 15 min before the oxytocin injection, every 15 min for 3 h after the injection, and every 30 min for an additional hour. Blood was centrifuged and plasma harvested and stored at –20°C until analyzed for 13,14 dihydro, 15-keto PGF2 (PGFM).
Follicular Aspiration
On d 16 of the cycle (the day after the oxytocin injection), cows received an injection of PGF2 (30 mg of Lutalyse; Pharmacia and Upjohn) to induce luteolysis and growth of ovarian follicles. About 54 h later trans-vaginal follicular aspirations were performed according to the procedure described by Manik et al. (2003). Briefly, cows received epidural anesthesia (5 cc 2% Lidocaine; The Butler Company, Dublin, OH), and a 7.5-MHz transvaginal convex transducer (Aloka 500V; Corometrics Medical Systems Inc., Wallingford, CT) fitted with an aspiration needle (17 g; Cook Pty Ltd, Australia), was inserted through the vagina. Ovaries were located and all follicles >9 mm were aspirated separately. Follicular fluids were centrifuged to precipitate cells and debris and stored at –20°C until analysis of steroid hormones and fatty acid profile.
Metabolite and Hormone Analyses
In all plasma samples taken during the supplementation period (3x per week), NEFA concentrations were quantified by enzymatic analysis (NEFA-C kit; Wako Chemicals, Richmond, VA) and IGF-I was quantified by RIA (Butler et al., 2006). Progesterone (P4) was determined by RIA (Elrod and Butler, 1993) in plasma samples taken during the luteal phase of the synchronized cycle to evaluate the functionality of the corpus luteum. In addition, P4 and estradiol (E2) concentration in each follicular fluid sample was determined by RIA (Elrod and Butler, 1993; Butler et al., 2006). Androstenedione in the follicular fluid was analyzed by a commercially available double antibody kit (DSL-4200; Diagnostic Systems Laboratories, Webster, TX) after diluting the sample 1:10 with 0 standard. A follicle was considered to be atretic when the E2:P4 ratio was less than 1 (Ireland and Roche, 1982). The PGFM in plasma collected during the oxytocin challenge was quantified by RIA as described by Meyer et al. (1995).
Fatty Acid Analysis
Fat content of the Ca salts of tallow and of the CLA supplements was determined (Dairy One Cooperative Inc.) by acid hydrolysis and ether extraction (Foss Tecator, Application Subnote AN 3414; Foss), respectively. The extraction of fatty acids from milk and uterine tissue was done according to the method of Hara and Radin (1978). For uterine samples the method was modified as follows: the uterine tissue was homogenized in 5.4 mL of hexane:isopropanol (3/2 vol/vol) and butylated hydroxytoluene (50 mg/L) using a tissue-tearer (985-370; Biospec Products Inc., Bartlesville, OK), followed by 0.24 mg of sodium sulfate solution. Fatty acids from follicular fluid were extracted according to Bligh and Dyer (1959) from 18 nonatretic follicles for which sufficient fluid was available (4 control, and 7 of each CLA 75:25 and CLA 50:50).
Methylation of fatty acids from the milk, the uterine tissue and the CLA supplements was performed by base-catalyzed transmethylation as described by Bernal-Santos et al. (2003). Methylation of fatty acids from calcium salts and from follicular fluid involved 1% methanolic sulfuric acid according to Christie (1989).
Fatty acid methyl esters were quantified by gas chromatograph (Hewlett Packard GCD system HP G1800 A, Avondale, PA) equipped with a CP-Sil 88 capillary column (100 m x 0.25 mm i.d. with 0.2-µm film thickness; Varian Instruments, Walnut Creek, CA). The oven temperature was set at 70°C for 4 min, then ramped to 170°C and maintained for 10 min, with a final increase to 225°C held for 15 min. Fatty acid peaks in chromatograms were identified using pure methyl ester standards (NuChek Prep, Elysian, MN). A butter oil reference standard (CRM 164; Commission of the European Community Bureau of References, Brussels, Belgium) was also analyzed periodically to control for column performance and to facilitate the calculation of recoveries and correction factors for individual fatty acids.
Statistical Analysis
Individual milk production and DMI values were reduced to weekly means before analysis. Yields of fat, protein, and lactose were calculated using the weekly mean for milk production and the milk composition (daily composite) obtained once per week. For all analyses, significance was declared at P 
0.05 and trends if P 0.10. Production variables, energy balance, plasma metabolites and hormones, and milk fatty acids were evaluated by ANOVA using the PROC MIXED procedure of SAS (2001) for repeated measures. The model included treatment, time, and treatment x time interaction. Cow within treatment was a random variable. The average milk production during the week before supplementation was used as a covariate in the analysis for milk production. Plasma concentrations of NEFA and IGF-I on d 0 of supplementation were used as covariates for these analyses.
Area under the curve was calculated for plasma progesterone during the luteal phase and for PGFM response after the oxytocin challenge using the trapezoidal method with PROC EXPAND of SAS, and ANOVA was performed with PROC GLM. Size of the ovulatory follicle, estradiol:progesterone ratio in the follicular fluid from nonatretic follicles, and uterine and follicular fatty acid composition were analyzed with PROC GLM. To determine main effects of treatment, values from the pretreatment biopsy were used as a covariate for the biopsy at the end of treatment. To test for differences in uterine fatty acid profile related to time after parturition, the 2 biopsies from the control cows were analyzed with a paired t-test using PROC TTEST.
|
RESULTS
|
|---|
Milk fat yield or content over the entire supplementation period were not significantly affected by supplementation with mixtures of CLA (Table 3). Milk fat content, however, decreased progressively over the treatment period and by wk 5 was significantly decreased by 14.1 and 6.1% for CLA 50:50 and CLA 75:25, compared with control, respectively (Figure 1; P = 0.005). After supplementation was terminated, fat content became similar to control. No difference was observed in milk protein or milk lactose content or yield (Table 3). Milk production was also unaffected by treatment, although there was a numerical increase in milk production in cows receiving the CLA supplements over the last few weeks of treatment (Figure 2).
View larger version (12K):
[in this window]
[in a new window]
|
Figure 1. Milk fat content during treatment and posttreatment periods. Commencing at 20 ± 1 DIM cows (n = 45) received a dietary fat supplement for 37 d. The supplement consisted of 76 g/d of lipid-encapsulated conjugated linoleic acid (CLA) for the CLA 50:50 treatment (7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA), 63 g/ d of lipid-encapsulated CLA for the CLA 75:25 treatment (7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA), or 70 g/d of Ca salts of tallow for the control treatment. The values shown are treatment by week least squares means and SEM for milk fat content averaged 0.09%. Over the entire treatment period, P-value for treatment effect was 0.17 and 0.64 for treatment by week interaction. The P-value for treatment effect for wk 5 of supplementation was 0.005.
|
|
View larger version (12K):
[in this window]
[in a new window]
|
Figure 2. Milk production during treatment and posttreatment periods. Commencing at 20 ± 1 DIM cows (n = 45) received a dietary fat supplement for 37 d. The supplement consisted of 76 g/d of lipid-encapsulated conjugated linoleic acid (CLA) for the CLA 50:50 treatment (7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA), 63 g/ d of lipid-encapsulated CLA for the CLA 75:25 treatment (7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA), or 70 g/d of Ca salts of tallow for the control treatment. The values shown are treatment x week least squares means and SEM for milk yield averaged 0.77 kg/d. During the supplementation period P-value for treatment effect was 0.20 and 0.52 for treatment by week interaction.
|
|
The DMI, BW, and BCS were not affected by treatment, and milk energy output and energy balance were similar among treatments (Table 3
). The similarity in energy balance is probably because of the small numerical increase in milk yield that followed the same time profile as the milk fat depression in the CLA treatments. Plasma NEFA concentrations were not affected by treatment consistent with the lack of treatment effect on energy balance (Table 3). During the dietary treatment period, CLA 50:50 supplementation resulted in increased mean plasma concentrations of IGF-I compared with other treatments (CLA 50:50 = 80 ng/mL, CLA 75:25 = 76 ng/mL, and control = 70 ng/mL; SEM ± 2, P = 0.02).
The fatty acid profile of milk fat is presented in Table 4. Trans-10, cis-12 CLA was increased in milk fat of the cows receiving CLA 50:50 and CLA 75:25, and this increment was dose-dependent. There were no other significant changes, however, in the profile of milk fatty acids.
Fatty acid composition of the endometrial tissue for the different treatments is presented in Table 5. Five fatty acids comprised about 70% of the total fatty acids, these being stearic (C18:0), oleic (C18:1 cis-9), linoleic (C18:2 cis-9, cis-12), arachidonic (C20:4), and palmitic acids (C16:0). Supplementation with CLA increased the proportion of C16:1 and tended to increase C18:1 trans 6 to 9. The C20:0 was marginally greater for CLA 50:50 than CLA 75:25. Cis-9, trans-11 CLA, however, was not increased in endometrial tissue from supplemented cows compared with the control, and trans-10, cis-12 was below the limits of detection (<0.01% of total fatty acids) for all treatments. Proportions of saturated, monounsaturated, or polyunsaturated fatty acids were not different among treatments. Fatty acid profile for control cows changed between the pretreatment and the end of treatment biopsy (Table 6), indicating changes in fatty acid composition of endometrial tissue during early lactation possibly related to differences in endocrine environment and physiological state of uterine involution.
Follicular fluid samples were obtained from 39 of the 45 cows, and the fatty acid profile in follicular fluid from 18 non-atretic follicles (4 control, and 7 of each CLA 75:25 and CLA 50:50) is presented in Table 7. Linoleic acid (C18:2 cis-9, cis-12) was the major fatty acid representing around 50% of total fatty acids, followed by stearic (C18:0) at 12%. Fatty acids of 20 carbons or longer represented about 7% of total fatty acids, and among the longer chain fatty acids, linolenic acid (C18:3) and C20:2 were significantly increased by the CLA 50:50 treatment. There was also a trend (P 0.09) for cis-9, trans-11 CLA and C22:2 to be increased in cows receiving CLA treatments. Overall, there were no treatment differences in the proportions of saturated, monounsaturated, or polyunsaturated fatty acids in the follicular fluid.
Diameter of the ovulating follicle in the synchronized cycle was not different among treatments (Table 8). The ratios of estradiol to progesterone and androstenedione to progesterone are also reported in Table 8. When only non-atretic follicles were analyzed cows supplemented with CLA 50:50 had numerically increased estradiol and androstenedione relative to progesterone, but this difference was not significant.
The plasma progesterone profile during the luteal phase is presented in Figure 3. There was no difference related to treatment in progesterone concentration over the total cycle (P = 0.12), but there was a trend for cows receiving the CLA 50:50 treatment to have greater concentrations of progesterone during the early luteal phase (d 6 to 8 of the cycle; P = 0.08). Injection of oxytocin resulted in an immediate increase in plasma PGFM followed by a gradual return to baseline by 240 min, and there was no significant time x treatment interaction (P = 0.91, data not shown). The PGFM area under the curve after oxytocin injection was not significantly affected by treatment (Table 8).
View larger version (11K):
[in this window]
[in a new window]
|
Figure 3. Plasma progesterone during the luteal phase of the estrus cycle. Commencing at 20 ± 1 DIM cows (n = 45) received a dietary fat supplement for 37 d. The supplement consisted of 76 g/d of lipid-encapsulated conjugated linoleic acid (CLA) for the CLA 50:50 treatment (7.1 g/d each of cis-9, trans-11 and trans-10, cis-12 CLA), 63 g/d of lipid-encapsulated CLA for the CLA 75:25 treatment (7.1 g/d of cis-9, trans-11 CLA and 2.4 g/d of trans-10, cis-12 CLA), or 70 g/d of Ca salts of tallow for the control treatment. Values shown are treatment x day least squares means and SEM for plasma progesterone averaged 0.4 ng/mL; the P-value was 0.12 for treatment effect and 0.44 for treatment by day interaction.
|
|
|
DISCUSSION
|
|---|
Previous studies have evaluated CLA-induced milk fat depression as an approach to reduce negative energy balance in early lactation. Although no significant changes in energy balance were observed, a trend for beneficial effects on reproduction was reported when the mixture of CLA isomers provided doses smaller than 10 g/d of each trans-10, cis-12 CLA and cis-9, trans-11 CLA (Bernal-Santos et al., 2003; Castañeda-Gutiérrez et al., 2005). Lipid encapsulation and calcium salts of fatty acids provide partial protection from rumen biohydrogenation (Perfield et al., 2004; de Veth et al., 2005), and the aim of this study was to evaluate physiological and hormonal responses to the same doses of CLA for which beneficial effects were observed previously. In addition, the use of 2 mixtures with different proportions of trans-10, cis-12 and cis-9, trans-11 CLA allowed for initial insight into isomer specific effects.
We calculated the amount of trans-10, cis-12 that bypassed the rumen based on the equations developed by de Veth et al. (2004) relating milk fat content of trans-10, cis-12 CLA to the abomasally infused dose of this CLA isomer. In the present study 1.32 and 0.44 g/ d bypassed the rumen for CLA 50:50 and CLA 75:25, respectively. The amount of trans-10, cis-12 CLA with the CLA 50:50 treatment is somewhat comparable to the 1.95 and 1.29 g/d trans-10, cis-12 in the studies of Bernal-Santos et al. (2003) and Castañeda-Gutiérrez et al. (2005), respectively. Based on summary equations (de Veth et al., 2004), the predicted reduction in milk fat yield would be 14 and 5% for CLA 50:50 and CLA 75:25, respectively, and this compares closely with the reduction in milk fat yield of 11 and 4% observed at wk 5 of treatment.
Previous studies have reported modest increases of about 3 to 10% in milk yield when cows were fed similar amounts of trans-10, cis-12 (Bernal-Santos et al., 2003; Mackle et al., 2003; de Veth et al., 2006). A numerical increase in milk yield of 4.5% from wk 2 to 5 of supplementation was observed in the present study. It has been suggested that energy spared from milk fat depression is repartitioned for milk production (Bernal-Santos et al., 2003; de Veth et al., 2006), and this is in accord with the present study in which milk energy output and energy balance were similar for CLA and control treatments and cows in all groups had similar circulating NEFA concentrations. Plasma IGF-I is positively correlated with energy balance and increases in plasma concentrations are associated with earlier resumption of ovulation (Butler, 2000). In the ovary, granulosa cells and corpus lutea have receptors for IGF-I, and intrafollicular and plasma concentrations are positively associated (see review by Zulu et al., 2002). The elevated plasma concentrations of IGF-I observed in the present study with CLA supplementation may be related to the trend reported in previous studies for earlier postpartum ovulation and increased pregnancy rates (Bernal-Santos et al., 2003; Castañeda-Gutiérrez et al., 2005), but the design of the present experiment did not allow for evaluation of these variables. Taylor et al. (2004) reported that cows with greater circulating IGF-I during the first 12 wk postpartum were more likely to conceive than those with lesser IGF-I. The mechanism through which CLA increased circulating IGF-I is unknown, but was associated with the mixture that provided larger amounts of trans-10, cis-12 CLA (CLA 50:50). During negative energy balance in early lactation, the liver is refractory to GH resulting in low concentrations of circulating IGF-I, but greater insulin availability restores coupling of the GH-IGF-I axis increasing circulating IGF-I concentrations (Butler et al., 2003). In other studies a 50:50 mixture of cis-9, trans-11 and trans-10, cis-12 CLA increased insulin sensitivity in muscle (Ryder et al., 2001) and increased genes related to insulin sensitivity in Zucker diabetic rats (Inoue et al., 2006), but these changes in insulin sensitivity were not observed with cis-9, trans-11 CLA alone. It is possible, therefore, that the effects of CLA for increasing plasma IGF-I in lactating cows may be mediated by subtle changes on hepatic sensitivity to insulin action that are specific to the trans-10, cis-12 CLA isomer.
The presence of dose-related amounts of trans-10, cis-12 CLA in milk fat confirmed absorption and utilization of the CLA supplements by the cows. Neither CLA isomer, however, was incorporated into endometrial tissue. In follicular fluid, there was a trend for enrichment of the cis-9 trans-11 isomer, but not trans-10, cis-12 CLA. The incorporation of CLA isomers into milk fat and tissues may be related to the plasma lipid fraction in which they are transported. Various studies have found preferential incorporation of specific isomers into specific lipid fractions and this seems to differ among tissues and species. Kramer et al. (1998) studied incorporation of CLA in lipid classes of liver and heart of CLA-supplemented pigs and reported that trans-10, cis-12 CLA was mainly incorporated in the triglyceride fraction in these tissues whereas cis-9, trans-11 was incorporated in phospholipids; also, Tischendorf et al. (2002) found that the trans-10, cis-12 CLA isomer was not incorporated in cell membranes. In contrast, Banni et al. (2001) found that cis-9, trans-11 CLA and its metabolites 18:2, 18:3, and 20:3 were preferentially incorporated into neutral lipids in the liver of rats. Incorporation of cis-9, trans-11 and trans-10, cis-12 CLA was reported in uterine tissue of pregnant rats fed CLA at 1.1% of the diet (Harris et al., 2001), but in the present study the cows received a much smaller dose (0.06% of DMI), and much of this was metabolized in the rumen. Thus, the smaller amount of CLA that was being absorbed at the small intestine of the cows in our study may explain the lack of incorporation in endometrium. Supplementation with CLA isomers influenced fatty acid profile in liver of rats; trans-10, cis-12 CLA induced a decrease in C16:0, C18:1, and C20:4 and increased C22 PUFA in the phospholipid fraction of liver (Sebedio et al., 2001). In the present experiment, significant changes occurred in the fatty acid profile of follicular fluid, including increases of C18:3 and C20:2 and a trend to increase C22:2 in cows supplemented with CLA 50:50.
The PGFM production in response to an oxytocin challenge did not differ among treatments. This differs from results in vitro in which a mixture of cis-9, trans-11 CLA and trans-10, cis-12 CLA, as well as either of the individual isomers decreased PGF2 synthesis in ewe cotyledonary endometrium (Cheng et al., 2003) and in BEND cells (Rodriguez-Sallaberry et al., 2006). It has also been reported that CLA decreased uterine prostaglandin production in pregnant rats (Harris et al., 2001), but not in gilts (Chartrand et al., 2003). In the present experiment, no change in fatty acid profile of the endometrial tissue was observed, and this may explain the similar response in PGFM to an oxytocin challenge among treatments.
Progesterone concentrations tended to be greater during the early luteal phase in cows supplemented with CLA 50:50. Higher progesterone concentration during early luteal phase (d 5 to 9 of the cycle) in cows resulted in a larger trophoblast and better embryo survival (Mann et al., 2006). The trend to have increased progesterone concentration in cows supplemented with CLA 50:50 may be related to greater circulating IGF-I. It has been shown that IGF-I plays a significant role in follicular and luteal development (Perks et al., 1999), promoting proliferation, progesterone production, and increased LH binding sites of bovine thecal cells (Stewart et al., 1995) as well as increasing estradiol production by bovine granulosa cells in vitro (Spicer et al., 2002). In addition, intrafollicular infusion of IGF-I increased the size of the dominant follicle and estradiol concentration in medium follicles in Holstein cows (Spicer et al., 2000). In the present study, the size of the ovulatory follicle did not differ amongst treatments, and there was no significant difference in concentrations of estradiol and androstenedione relative to progesterone in nonatretic follicles. The design and sampling in the present study did not allow evaluation of pregnancy outcome, but in previous studies, a trend to have fewer days to pregnancy was observed after supplementation was terminated (Bernal-Santos et al., 2003; Castañeda-Gutiérrez et al., 2005), thus raising the possibility that CLA exerted beneficial effects during development of the ovarian follicle and improved embryo viability.
|
CONCLUSIONS
|
|---|
Supplementation with trans-10, cis-12 CLA did not result in differences in estimated net energy balance or in plasma concentrations of NEFA. The CLA isomers were not detectable in endometrium, and there was no difference in the prostaglandin response to an oxytocin challenge. Plasma IGF-I concentrations were greater for cows supplemented with CLA 50:50. Cows supplemented with CLA 50:50 tended to have elevated plasma progesterone during the early luteal phase. The CLA may improve endocrine signals that can be beneficial to reproduction. Furthermore the benefits seem to be associated with the trans-10, cis-12 CLA isomer and are independent of energy balance.
|
ACKNOWLEDGEMENTS
|
|---|
The authors would like to acknowledge the contribution of Virtus Nutrition LLC for supplying the Ca salts of tallow and the following students and staff at Cornell University: Ray Axtell, Bruce Berggren-Thomas, Gladys Birdsall, Debra Dwyer, Walter Jones, Dana Muir, Susanne Pelton, Heather Roman, Cynthia Tyburczy, and Rebecca Waddle.
|
FOOTNOTES
|
|---|
1 Supported in part by Cornell Agricultural Experimental Station and BASF AG. Research was also supported by Smith Lever funds from the Cooperative State Research, Education, and Extension Service, US Department of Agriculture, under Agreement No. NYC-127437. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the US Department of Agriculture. 
2 Present address: Nestlé Research Centre, Lausanne, Switzerland.
3 Present address: Balchem Encapsulates, New Hampton, NY.
Received for publication February 15, 2007.
Accepted for publication May 11, 2007.
|
REFERENCES
|
|---|
Banni, S., G. Carta, E. Angioni, E. Murru, P. Scanu, M. P. Melis, D. E. Bauman, S. M. Fischer, and C. Ip. 2001. Distribution of conjugated linoleic acid and metabolites in different lipid fractions in the rat liver. J. Lipid Res. 42:1056–1061.[Abstract/Free Full Text]
Bauman, D. E., and A. L. Lock. 2006. Conjugated linoleic acid: Biosynthesis and nutritional significance. Pages 93–136 in Advanced Dairy Chemistry. Volume 2: Lipids. 3rd ed. P. F. Fox and P. L. H. McSweeney, ed. Springer, New York, NY.
Belury, M. A., S. Y. Moya-Camarena, M. Lu, L. L. Shi, L. M. Leesnitzer, and S. G. Blanchard. 2002. Conjugated linoleic acid is an activator and ligand for peroxisome proliferator-activated receptor-gamma (PPAR gamma). Nutr. Res. 22:817–824.[CrossRef]
Bernal-Santos, G., J. W. Perfield, D. M. Barbano, D. E. Bauman, and T. R. Overton. 2003. Production responses of dairy cows to dietary supplementation with conjugated linoleic acid (CLA) during the transition period and early lactation. J. Dairy Sci. 86:3218–3228.[Abstract/Free Full Text]
Bligh, E. G., and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911–917.[Medline]
Butler, W. R. 2000. Nutritional interactions with reproductive performance in dairy cattle. Anim. Reprod. Sci. 60–61:449–457.
Butler, S. T., A. L. Marr, S. H. Pelton, R. P. Radcliff, and M. C. Lucy. 2003. Insulin restores GH responsiveness during lactation-induced negative energy balance in dairy cattle: Effects on expression of IGF-I and GH receptor 1A. J. Endocrinol. 176:205–217.[Abstract]
Butler, S. T., S. H. Pelton, and W. R. Butler. 2006. Energy balance, metabolic status, and the first postpartum ovarian follicle wave in cows administered propylene glycol. J. Dairy Sci. 89:2938–2951.[Abstract/Free Full Text]
Castañeda-Gutiérrez, E., T. R. Overton, W. R. Butler, and D. E. Bauman. 2005. Dietary supplements of two doses of calcium salts of conjugated linoleic acid during the transition period and early lactation. J. Dairy Sci. 88:1078–1089.[Abstract/Free Full Text]
Chartrand, R., J. J. Matte, M. Lessard, P. Y. Chouinard, A. Giguere, and J. P. Laforest. 2003. Effect of dietary fat sources on systemic and intrauterine synthesis of prostaglandins during early pregnancy in gilts. J. Anim. Sci. 81:726–734.[Abstract/Free Full Text]
Cheng, Z., M. Elmes, D. R. Abayasekara, and D. C. Wathes. 2003. Effects of conjugated linoleic acid on prostaglandins produced by cells isolated from maternal intercotyledonary endometrium, fetal allantochorion and amnion in late pregnant ewes. Biochim. Biophys. Acta 1633:170–178.[Medline]
Christie, W. W. 1989. Gas Chromatography and Lipids: A Practical Guide. The Oily Press, Ayr, UK.
de Veth, M. J., E. Castañeda-Gutiérrez, D. A. Dwyer, A. M. Pfeiffer, D. E. Putnam, and D. E. Bauman. 2006. Response to conjugated linoleic acid in dairy cows differing in energy and protein status. J. Dairy Sci. 89:4620–4631.[Abstract/Free Full Text]
de Veth, M. J., J. M. Griinari, A. M. Pfeiffer, and D. E. Bauman. 2004. Effect of CLA on milk fat synthesis in dairy cows: Comparison of inhibition by methyl esters and free fatty acids, and relationships among studies. Lipids 39:365–372.[CrossRef][Medline]
de Veth, M. J., S. K. Gulati, N. D. Luchini, and D. E. Bauman. 2005. Comparison of calcium salts and formaldehyde-protected conjugated linoleic acid in inducing milk fat depression. J. Dairy Sci. 88:1685–1693.[Abstract/Free Full Text]
Elrod, C. C., and W. R. Butler. 1993. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J. Anim. Sci. 71:694–701.[Abstract]
Fox, D. G., L. O. Tedeschi, T. P. Tylutki, J. B. Russell, M. E. Van Amburgh, L. E. Chase, A. N. Pell, and T. R. Overton. 2004. The Cornell net carbohydrate and protein system model for evaluating herd nutrition and nutrient excretion. Anim. Feed Sci. Technol. 112:29–78.[CrossRef]
Hara, A., and N. S. Radin. 1978. Lipid extraction of tissues with a low-toxicity solvent. Anal. Biochem. 90:420–426.[CrossRef][Medline]
Harris, M. A., R. A. Hansen, P. Vidsudhiphan, J. L. Koslo, J. B. Thomas, B. A. Watkins, and K. G. D. Allen. 2001. Effects of conjugated linoleic acids and docosahexaenoic acid on rat liver and reproductive tissue fatty acids, prostaglandins and matrix metalloproteinase production. Prostaglandins Leukotr. Essent. Fatty Acids 65:23–29.[CrossRef]
Inoue, N., K. Nagao, Y. M. Wang, H. Noguchi, B. Shirouchi, and T. Yanagita. 2006. Dietary conjugated linoleic acid lowered tumor necrosis factor- content and altered expression of genes related to lipid metabolism and insulin sensitivity in the skeletal muscle of Zucker rats. J. Agric. Food Chem. 54:7935–7939.[CrossRef][Medline]
Ireland, J. J., and J. F. Roche. 1982. Development of antral follicles in cattle after prostaglandin-induced luteolysis—Changes in serum hormones, steroids in follicular-fluid, and gonadotropin receptors. Endocrinology 111:2077–2086.[Abstract]
Kramer, J. K. G., N. Sehat, M. E. R. Dugan, M. M. Mossoba, M. P. Yurawecz, J. A. G. Roach, K. Eulitz, J. L. Aalhus, A. L. Schaefer, and Y. Ku. 1998. Distributions of conjugated linoleic acid (CLA) isomers in tissue lipid classes of pigs fed a commercial CLA mixture determined by gas chromatography and silver ion high-performance liquid chromatography. Lipids 33:549–558.[Medline]
Lucy, M. C. 2001. ADSA Foundation Scholar Award—Reproductive loss in high-producing dairy cattle: Where will it end? J. Dairy Sci. 84:1277–1293.[Abstract]
Mackle, T. R., J. K. Kay, M. J. Auldist, A. K. McGibbon, B. A. Philpott, L. H. Baumgard, and D. E. Bauman. 2003. Effects of abomasal infusion of conjugated linoleic acid on milk fat concentration and yield from pasture-fed dairy cows. J. Dairy Sci. 86:644–652.[Abstract/Free Full Text]
Manik, R. S., S. K. Singla, and P. Palta. 2003. Collection of oocytes through transvaginal ultrasound-guided aspiration of follicles in an Indian breed of cattle. Anim. Reprod. Sci. 76:155–161.[CrossRef][Medline]
Mann, G. E., M. D. Fray, and G. E. Lamming. 2006. Effects of time of progesterone supplementation on embryo development and interferon-tau production in the cow. Vet. J. 171:500–503.[CrossRef][Medline]
Mattos, R., C. R. Staples, and W. W. Thatcher. 2000. Effects of dietary fatty acids on reproduction in ruminants. Rev. Reprod. 5:38–45.[Abstract]
Meyer, M. D., P. J. Hansen, W. W. Thatcher, M. Drost, L. Badinga, R. M. Roberts, J. Li, T. L. Ott, and F. W. Bazer. 1995. Extension of corpus luteum lifespan and reduction of uterine secretion of prostaglandin F2 of cows in response to a recombinant interferon-t. J. Dairy Sci. 78:1921–1931.[Abstract]
Perfield, J. W., A. L. Lock, A. M. Pfeiffer, and D. E. Bauman. 2004. Effects of amide-protected and lipid-encapsulated conjugated linoleic acid (CLA) supplements on milk fat synthesis. J. Dairy Sci. 87:3010–3016.[Abstract/Free Full Text]
Perks, C. M., A. R. Peters, and D. C. Wathes. 1999. Follicular and luteal expression of insulin-like growth factors I and II and the type 1 IGF receptor in the bovine ovary. J. Reprod. Fertil. 116:157–165.[Abstract]
Petit, H. V., C. Germiquet, and D. Lebel. 2004. Effect of feeding whole, unprocessed sunflower seeds and flaxseed on milk production, milk composition, and prostaglandin secretion in dairy cows. J. Dairy Sci. 87:3889–3898.[Abstract/Free Full Text]
Robinson, R. S., P. G. Pushpakumara, Z. Cheng, A. R. Peters, D. R. Abayasekara, and D. C. Wathes. 2002. Effects of dietary polyun-saturated fatty acids on ovarian and uterine function in lactating dairy cows. Reproduction 124:119–131.[Abstract]
Rodriguez-Sallaberry, C., C. Caldari-Torres, E. S. Greene, and L. Badinga. 2006. Conjugated linoleic acid reduced phorbol ester-induced prostaglandin F2 production by bovine endometrial cells. J. Dairy Sci. 89:3826–3832.[Abstract/Free Full Text]
Ryder, J. W., C. P. Portocarrero, X. M. Song, L. Cui, M. Yu, T. Combatsiaris, D. Galuska, D. E. Bauman, D. M. Barbano, M. J. Charron, J. R. Zierath, and K. L. Houseknecht. 2001. Isomer-specific antidiabetic properties of conjugated linoleic acid. Diabetes 50:1149–1157.[Abstract/Free Full Text]
SAS User’s Guide. Statistics, Version 8. 2001. SAS Inst. Inc., Cary, NC.
Sebedio, J. L., E. Angioni, J. M. Chardigny, S. Gregoire, P. Juaneda, and O. Berdeaux. 2001. The effect of conjugated linoleic acid isomers on fatty acid profiles of liver and adipose tissues and their conversion to isomers of 16:2 and 18:3 conjugated fatty acids in rats. Lipids 36:575–582.[Medline]
Spicer, L. J., P. Alvarez, T. M. Prado, G. L. Morgan, and T. D. Hamilton. 2000. Effects of intraovarian infusion of insulin-like growth factor-I on ovarian follicular function in cattle. Domest. Anim. Endocrinol. 18:265–278.[CrossRef][Medline]
Spicer, L. J., C. S. Chamberlain, and S. M. Maciel. 2002. Influence of gonadotropins on insulin- and insulin-like growth factor-I (IGF-I)-induced steroid production by bovine granulosa cells. Domest. Anim. Endocrinol. 22:237–254.[CrossRef][Medline]
Stewart, R. E., L. J. Spicer, T. D. Hamilton, and B. E. Keefer. 1995. Effects of insulin-like growth factor I and insulin on proliferation and on basal and luteinizing hormone-induced steroidogenesis of bovine thecal cells: Involvement of glucose and receptors for insulin-like growth factor I an luteinizing hormone. J. Anim. Sci. 73:3719–3731.[Abstract]
Taylor, V. J., Z. Cheng, P. G. Pushpakumara, D. E. Beever, and D. C. Wathes. 2004. Relationships between the plasma concentrations of insulin-like growth factor-I in dairy cows and their fertility and milk yield. Vet. Rec. 155:583–588.[Abstract/Free Full Text]
Tischendorf, F., P. Mockel, F. Schone, M. Plonne, and G. Jahreis. 2002. Effect of dietary conjugated linoleic acids on the distribution of fatty acids in serum lipoprotein fractions and different tissues of growing pigs. J. Anim. Physiol. Anim. Nutr. (Berl.) 86:313–325.[Medline]
Wildman, E. E., G. M. Jones, P. E. Wagner, R. L. Boman, H. F. Trout, and T. N. Lesch. 1982. A dairy cow body condition scoring system and its relationship to selected production variables in high producing Holstein dairy cattle. J. Dairy Sci. 65:495–501.[Abstract/Free Full Text]
Zulu, V. C., T. Nakao, and Y. Sawamukai. 2002. Insulin-like growth factor-I as a possible hormonal mediator of nutritional regulation of reproduction in cattle. J. Vet. Med. Sci. 64:657–665.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
A. L. Lock, M. Rovai, T. A. Gipson, M. J. de Veth, and D. E. Bauman
A Conjugated Linoleic Acid Supplement Containing Trans-10, Cis-12 Conjugated Linoleic Acid Reduces Milk Fat Synthesis in Lactating Goats
J Dairy Sci,
September 1, 2008;
91(9):
3291 - 3299.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Gervais and P. Y. Chouinard
Effects of Intravenous Infusion of Conjugated Diene 18:3 Isomers on Milk Fat Synthesis in Lactating Dairy Cows
J Dairy Sci,
September 1, 2008;
91(9):
3568 - 3578.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
