Pontifícia Universidade Católica do Rio Grande do Sul Faculdade de Biociências Mestrado em Biologia Celular e Molecular - PPGBCM TRATAMENTO DE ESTRIAS ALBAS COM GALVANOPUNTURA: BENÉFICO PARA A ESTÉTICA, ESTRESSE OXIDATIVO E PERFIL LIPÍDICO Shanna Bitencourt Prof. Dr. Jarbas Rodrigues de Oliveira Porto Alegre, RS Pontifícia Universidade Católica do Rio Grande do Sul
Persiadan.irJ. Dairy Sci. 92 :1520–1531
American Dairy Science Association, 2009 .
Effect of fat source differing in fatty acid profile on metabolic parameters,
fertilization, and embryo quality in high-producing dairy cows
R. L. A. Cerri ,*† S. O. Juchem ,* R. C. Chebel ,* H. M. Rutigliano ,* R. G. S. Bruno ,* K. N. Galvão ,*
W. W. Thatcher ,† and J. E. P. Santos *†1
* School of Veterinary Medicine, University of California-Davis, Tulare 93274
† Department of Animal Sciences, University of Florida, Gainesville 32611
cows fed PO. Feeding a more unsaturated source of FA improved fertilization and embryo development in The objectives were to evaluate the effects of source lactating dairy cows, despite similar indicators of meta- of fatty acids (FA) on embryo quality of dairy cows. bolic status. A total of 154 Holstein cows were assigned randomly Key words: dairy cow , embryo quality , fatty acid ,
to 1 of 2 sources of FA supplemented at 2% of the reproduction dietary dry matter as calcium salts of either palm oil (PO) or linoleic and trans-octadecenoic acids (LTFA) INTRODUCTION
from 25 d prepartum to 80 d in milk (DIM). Cows were presynchronized beginning at 30 ± 3 DIM and Certain fatty acids (FA) have specific effects on dif-
then subjected to the Ovsynch protocol beginning on d ferent tissues, with potential benefits to the fertility 39 ± 3 postpartum. Timed artificial insemination was of dairy cows. These benefits seem to be independent performed 12 h after the final GnRH of the Ovsynch of the provision of calories and changes in the energy protocol with semen from a single sire of proven fertil- status of the cow (Staples et al., 1998; Santos et al., ity. The uteri of cows were nonsurgically flushed at 5 2008b). Differential effects of specific FA on reproduc- d after artificial insemination for collection of embryos- tive tissues have been demonstrated in vitro and in vivo oocytes. Ovaries were examined by ultrasonography on steroidogenesis (Wathes et al., 2007), metabolism throughout the synchronization protocol. Blood was of endometrial cells (Mattos et al., 2003, 2004), and sampled and plasma was analyzed for concentrations embryo quality and development (Thangavelu et al., of metabolites and hormones. The body condition score 2007; Santos et al., 2008b). and yields of milk and milk components were measured Linoleic acid (C18:2n-6) is an essential FA required throughout the first 90 DIM. Treatment did not affect for normal reproductive function in mammals (Burr concentrations of nonesterified FA, β-hydroxybutyrate, and Burr, 1930). Recently, high-producing dairy cows glucose, and progesterone in plasma. Body condition fed a supplemental fat enriched with linoleic acid and a was similar between treatments. Milk production was blend of trans-octadecenoic FA (LTFA) had increased
similar between treatments, but concentrations of fat pregnancy at 27 and 41 d after AI (Juchem et al., 2008); in milk and yields of fat and 3.5% fat-corrected milk however, the causes for such improvement were not decreased in cows fed LTFA, whereas concentration of clearly determined. As the precursor for endogenous true protein increased. Source of dietary FA did not synthesis of arachidonic acid (C20:4n-6), linoleic acid influence ovulatory responses, diameter of the ovula- could increase the incorporation of arachidonic acid tory follicle, and diameter of the corpus luteum dur- in membrane phospholipids of endometrial cells and, ing synchronization. Embryo-oocyte recovery relative consequently, PGF2α synthesis (Mattos et al., 2003), to the number of corpora lutea did not differ between with the latter playing an important role postpartum treatments. Fertilization tended to increase in cows in uterine involution (Kindahl et al., 1992). Enhancing fed LTFA compared with cows fed PO. Feeding LTFA uterine health, even past the immediate postpartum pe- improved the proportion of excellent-, good-, and fair- riod, has the potential to improve the fertility of dairy quality embryos, and embryos from cows fed LTFA had cows, as evidenced by better fertilization (Cerri et al., a greater number of blastomeres than embryos from 2009a) and pregnancy (Rutigliano et al., 2008), in cows not diagnosed with subclinical endometritis after 30 DIM. Furthermore, evidence of a direct beneficial effect Received August 8, 2008.
of linoleic acid during early embryo development has Accepted October 23, 2008.
1 Corresponding author: [email protected] been observed in more developed human embryos that FAT SOURCE ALTERS EMBRYO QUALITY IN DAIRY COWS incorporated more linoleic acid compared with their Holstein dairy cows (105 multiparous and 49 primipa-
undeveloped counterparts (Haggarty et al., 2006). An rous) were randomly assigned to initiate 1 of 2 treat-
increased number of blastomeres was observed when ments beginning at 25 d prepartum.
superstimulated embryo donor cows were fed diets en-
riched with either linoleic or α-linolenic acid (C18:3n-3) Treatment Diets
compared with a saturated source of FA (Thangavelu et al., 2007). Therefore, it is plausible to speculate All cows were fed the same diet as a TMR once daily that increased intake of linoleic acid could affect the prepartum and twice daily postpartum, except for the FA composition of reproductive tissues and, in turn, supplemental fat. Diets met or exceeded the dietary re-improve fertilization rate and embryonic development. quirements (NRC, 2001) for a nonlactating cow of 670 This might explain the improved pregnancy per AI kg consuming 12 kg of DM during the last trimester observed in lactating dairy cows fed a calcium salt of of pregnancy and for a lactating cow weighing 630 kg LTFA (Juchem et al., 2008).
consuming 24 kg of DM and producing 45 kg of milk Monoenoic FA containing a single double bond in containing 3.5% fat and 3.1% true protein in the first the trans configuration, such as trans-octadecenoic FA, 70 d of lactation. Diets were formulated by using the have distinct biological effects and can inhibit synthesis CPM-Dairy cattle ration analyzer (Cornell-Penn-Miner of fat in the mammary gland (Piperova et al., 2004), version 3.0.8; Miner Institute, Chazy, NY). Amounts although their effects on lipogenesis seem to be less dra- offered and refused were measured daily.
matic than those of specific conjugated linoleic acids. A Diets differed between treatments only by the nature concept that has been evaluated is the potential for FA of the FA profile contained in the supplemental fat that suppress milk fat synthesis to alleviate the negative offered to the cows. Supplemental fat in the form of energy balance and improve reproduction (Castañeda- calcium salt was offered from d −25 relative to calving Gutiérrez et al., 2007; Juchem et al., 2008). However, until 70 DIM, which was expected to result in an intake
suppressing milk fat synthesis by feeding trans FA does of supplemental fat of approximately 200 and 400 g/d
not seem to improve metabolism and energy balance for the pre- and postpartum periods, respectively. Cows
to justify the benefits to reproduction (Juchem et al., were fed a calcium salt of palm oil (PO; 2% of dietary
2004, 2008; Castañeda-Gutiérrez et al., 2007). Never-
DM; EnerG-II, Virtus Nutrition LLC, Fairlawn, OH) theless, if trans-octadecenoic FA fed in early lactation containing mostly saturated (palmitic acid: C16:0) and affect plasma concentrations of NEFA and BHBA, it monounsaturated FA (oleic acid: C18:1 cis-9), or a cal-is possible that they could decrease the negative ef- cium salt containing mostly linoleic acid (C18:2 cis-9, fects associated with excessive concentrations of these cis-12) and a blend of trans-octadecenoic (C18:1 trans) metabolites on the developmental capacity of oocytes FA (LTFA; 2% of dietary DM; EnerG-I Transition (Leroy et al., 2005) and pregnancy rates (Walsh et al., Formula, Virtus Nutrition LLC). A total of 76 cows 2007).
in the PO group (25 primiparous and 51 multiparous) The objectives of the present study were to deter- and 78 cows in the LTFA group (24 primiparous and mine the effects of feeding a rumen-inert fat enriched 54 multiparous) were enrolled in the study. Cows were with LTFA during late gestation and early lactation housed in 2 pre- and 2 postpartum pens/treatment, and on indicators of metabolic status, fertilization rate, all diets were rotated through all pre- and postpartum and embryo quality in dairy cows. It was hypothesized pens. Prepartum pens housed 38 to 44 cows any given that supplementation with the more unsaturated LTFA day, and postpartum pens housed 85 to 90 cows any would benefit the metabolic status of the cow by de- creasing milk fat synthesis and reducing concentrations Pre- and postpartum diets were sampled weekly from of NEFA and BHBA postpartum. Furthermore, it was the manger in each pen and then composited for 2-mo hypothesized that providing a diet enriched with LTFA periods. Every batch of calcium salts was sampled, and would improve the fertilization and embryo quality of a composite sample was analyzed for FA profile by GC nonsuperstimulated dairy cows.
(DePeters et al., 2001). Chemical composition of the experimental diets and FA profile of the calcium salts are shown in Table 1. Amounts of diets offered and MATERIALS AND METHODS
refused were measured daily.
Animals and Housing
BCS, Milk Yield, and Milk Components
The University of California-Davis Institutional Ani- mal Care and Use Committee approved all procedures The BCS (Ferguson et al., 1994) of all cows was de- involving cows in this study. A total of 154 lactating termined by the same person on d −25, 1, 40, and 70 Journal of Dairy Science Vol. 92 No. 4, 2009 CERRI ET AL.
Table 1. Chemical composition of experimental diets1 (mean ± SD), and fatty acid profile of the supplemental fat sources
NEL,2 Mcal/kg of DM Fatty acid, g/100 g of fatty acids C18:1, total cis C18:1, total trans C18:2 trans-10, cis-12 1PO = calcium salt of palm oil; LTFA = calcium salt of linoleic and trans-octadecenoic acids.
2Calculated according to NRC (2001).
relative to calving. Milk yields were measured weekly tions of NEFA, BHBA, and glucose. Plasma NEFA during the first 10 wk of lactation by the DHIA labora- concentrations were determined by using a colorimetric tory in Hanford, California. Milk samples were collected kit (NEFA-C, Wako Chemicals GmbH, Neuss, Ger-once weekly for the morning and afternoon milkings many). Concentrations of BHBA were determined by and analyzed for concentrations of fat, true protein, a colorimetric kit (Randox Laboratories Ltd., Crumlin, and SCC (Foss 303 Milk-O-Scan; Foss Foods Inc., Eden Ireland) following a modified kinetic protocol for a mi-Prairie, MN).
croplate reader. Glucose concentrations in plasma were analyzed based on the glucose oxidase reaction by using Blood Sampling and Analysis of Progesterone,
a biochemical analyzer (YSI 2700-S BioChem, Yellow NEFA, BHBA, and Glucose
Springs Instrument Co. Inc., Ohio OH).
Approximately 7 mL of blood was sampled from the Ovulation Synchronization Protocol and AI
coccygeal vein or artery into evacuated tubes contain-ing 17.55 mg of K2 EDTA (Vacutainer, Becton Dick- Cows in both treatments were subjected to an identi- inson, Franklin Lakes, NJ). Plasma was obtained from cal reproductive protocol for timed AI (Figure 1). The blood after centrifugation at 3,000 × g for 15 min in a estrous cycles of cows were presynchronized beginning refrigerated centrifuge at 5°C. Plasma was then stored at 30 ± 3 DIM, and the protocol consisted of an injec-at −25°C until further analyses for concentrations of tion of 100 μg of GnRH (gonadorelin diacetate tetra-progesterone, NEFA, BHBA, and glucose.
hydrate; Cystorelin, Merial Ltd., Iselin, NJ) and the Blood samples for progesterone analysis were col- placement of a controlled internal drug-releasing insert lected at the moment of PGF2α injection of the pre- containing 1.38 g of progesterone (CIDR; EAZI-Breed, synchronization and at every injection of the Ovsynch Pfizer Animal Health, New York, NY). An injection protocol to evaluate the ovarian response to the hor- of 25 mg of PGF2α (dinoprost tromethamine; Lutalyse monal treatments. Additional samples were collected at Sterile Solution, Pfizer Animal Health) was given 7 d d 3 and 5 after AI to determine the changes in plasma later concomitantly with the removal of the controlled progesterone concentration until the day of embryo- internal drug-releasing insert. The Ovsynch protocol oocyte collection. Plasma progesterone was analyzed by (Pursley et al., 1995) began 2 d after the end of pre-a validated ELISA (Cerri et al., 2004). The intra- and synchronization and consisted of an injection of 100 μg interassay CV in the study assays were, respectively, of GnRH, followed by 25 mg of PGF2α 7 d later and 4.9 and 7.3%. Individual samples with CV >15% or mi- a final injection of GnRH given 48 h after the PGF2α. croplates with interassay CV >15% were reanalyzed.
Cows were artificially inseminated 12 h after the last Blood was sampled at −22, −14, −7, 1, 6, 13, and injection of GnRH of the Ovsynch protocol. The same 27 d relative to calving and analyzed for concentra- person artificially inseminated all cows in the study. Journal of Dairy Science Vol. 92 No. 4, 2009 FAT SOURCE ALTERS EMBRYO QUALITY IN DAIRY COWS Figure 1. Diagram of the reproductive protocol used to synchronize ovulation in all cows. CIDR = controlled internal drug-releasing insert
containing 1.38 g of progesterone.
Semen from a single proven sire previously tested for horn in 20 cycles of infusion/recovery of 15 mL of solu-fertility from field inseminations in lactating cows was tion. Recovered embryos-oocytes were then evaluated used in both treatments.
for fertilization and grade quality (1 = excellent and good, 2 = fair, 3 = poor, and 4 = degenerated) ac- Ultrasound Evaluation of the Ovarian Structures
cording to the guidelines of the International Embryo Transfer Society (1998). Embryos were stained with 5 Cows had their ovaries examined by ultrasound (7.5- μg/mL of propidium iodide (Sigma, St. Louis, MO) to MHz transrectal linear probe; Sonovet 2000, Alliance determine the number of nonviable blastomeres and Medical, Bedford Hills, NY) for determination of ovar- then with 5 μg/mL of Hoechst 33342 stain (Molecular ian structures and response to hormonal treatments. Probes Inc., Eugene, OR) to determine the number of Maps of the ovaries were drawn for each individual cow, accessory spermatozoa by epifluorescence microscopy and the size and position of follicles ≥5 mm in diameter (365 nm excitation, >400 nm emission). The zona pel- and corpora lutea (CL) were recorded. Ultrasonographic
lucida was then dissolved with a solution of 0.02 N images were taken immediately before the injections of HCl in 0.1% Tween-20 (Sigma). The embryo was again GnRH and PGF2α of the presynchronization to deter- stained with 5 μg/mL of Hoechst 33342 stain, and the mine cyclic status. Cows with a visible CL in at least blastomeres were spread on a glass slide and counted by 1 of the 2 ultrasound examinations were considered cy- clic, and cows without a CL in both examinations were considered anovular. Ovaries were then scanned at the time of each injection of the Ovsynch protocol and again Experimental Design and Statistical Analyses
48 h after each GnRH injection to determine ovulation. The experimental design was a randomized complete An additional ultrasound evaluation was performed at block design. Cows were blocked according to parity the moment of embryo-oocyte collection to determine (first vs. greater than first lactation) and BCS (Fergu- the diameter of the CL. Occurrence of ovulation within son et al., 1994) at enrollment and, within each block, 48 h after each GnRH injection was characterized by were randomly assigned to 1 of the 2 treatments.
the disappearance of a previously recorded follicle ≥10 Binomial data were evaluated by logistic regression mm in diameter.
using the LOGISTIC procedure of SAS/STAT (SAS Embryo-Oocyte Collection and Evaluation
Inst. Inc., Cary, NC). The model included the effects of treatment, parity, average BCS postpartum, and BCS Cows were flushed on d 5 after AI by a transcervical change between calving and 70 DIM. Count data, such procedure using a silicone Foley catheter (18 French, as number of accessory sperm and number of total blas-30 mL, 56 cm). The balloon of the Foley catheter was tomeres, were analyzed by the GENMOD procedure placed approximately 3 cm past the external intercor- using a Poisson distribution with correction for over- nual ligament of the uterine horn ipsilateral to the CL. dispersion of data (SAS/STAT, SAS Inst. Inc.). The Approximately 300 mL of a flushing solution (ViGro model included the effects of treatment, parity, average complete flush solution, Bioniche Life Sciences Inc., BCS postpartum, and BCS change between calving and Belleville, Ontario, Canada) was used in each uterine 70 DIM. Median and mean values were obtained.
Journal of Dairy Science Vol. 92 No. 4, 2009 CERRI ET AL.
Blood metabolites (NEFA, BHBA, and glucose) and progesterone concentrations, as well as milk production and milk components were analyzed by ANOVA for repeated measures using PROC MIXED of the SAS/STAT program (SAS Inst. Inc.). The covariance struc-ture with the smallest Akaike's information criterion was used for the measurements used in the PROC MIXED model. The model included the effects of treatment, day of measurement, interaction of treatment × day of measurement, parity, and average BCS postpartum, with cow nested within treatment as the random error. For progesterone, analyses were performed separately for samples collected before and after PGF2α injections during the synchronization treatments.
Simple and partial correlations were performed to Figure 2. Body condition score of cows fed calcium salts of palm
assess the associations between NEFA, BHBA, and oil (PO) or linoleic and trans-octadecenoic acids (LTFA). Effects of glucose concentrations with outcomes of the collected treatment (P = 0.22), day relative to calving (P < 0.001), and the embryos-oocytes. Correlation analysis using Pearson interaction between treatment and day relative to calving (P = 0.40).
correlation was performed with PROC CORR of the SAS/STAT program (SAS Inst. Inc.). A multivariate ANOVA was used to evaluate the partial correlations by PROC GLM (SAS/STAT, SAS Inst. Inc.). Treat- Plasma NEFA, BHBA, and Glucose
ment differences with P ≤ 0.05 were considered signifi-cant, and from 0.05 < P ≤ 0.10 were designated as a Concentrations of NEFA and BHBA did not differ for cows fed PO or LTFA throughout the study (Figure 3). Similarly, no effect of treatment was observed for mean glucose concentration, but an interaction (P = 0.002) between treatment and day postpartum was observed, As expected, the chemical compositions of the pre- with a greater glucose concentration at d 1 postpartum and postpartum diets were similar (Table 1), but the in cows fed PO than those fed LTFA (74.1 ± 1.35 vs. FA profile of the supplemental fat sources differed 68.4 ± 1.30 mg/dL). In both treatments, concentra-markedly. The PO contained 50% of its FA as palmitic tion of NEFA increased (P < 0.001) sharply in the and oleic acids, whereas the LTFA contained more than week before calving, but that of BHBA increased (P 75% of its FA as mono- and polyunsaturated FA.
< 0.001) only after calving. Plasma NEFA and BHBA Cows received the prepartum diets for a similar concentrations peaked at 6 and 13 DIM, respectively. period of time, 25.6 ± 0.6 and 25.5 ± 0.6 d for PO Concentrations of glucose remained elevated until calv-
and LTFA, respectively. The mean (±SD) and median ing, followed by a steep decline (P < 0.001) in the first
lactation number did not differ (P > 0.54) between week postpartum, reaching a nadir at 13 DIM.
treatments and were 2.36 ± 1.34 and 2.00, respectively.
Dry matter intake did not differ for PO and LTFA in Milk Yield and Components
the prepartum (12.2 ± 0.4 and 12.6 ± 0.4 kg/d; P =
0.68) and postpartum (23.2 ± 0.5 and 22.8 ± 0.6 kg/d;
Average milk production during the first 10 wk of P = 0.32) periods.
lactation (Table 2) was similar between PO and LTFA; however, 3.5% FCM was greater (P = 0.001) for cows fed PO than for cows fed LTFA because of the reduced (P = 0.001) fat content when cows were fed calcium The mean BCS collected throughout the study (Fig- salts containing trans FA and linoleic acid. The reduc- ure 2) did not differ between treatments. There was an tion in milk fat content with feeding LTFA resulted in effect (P < 0.001) of day as cows lost BCS from calving a reduced (P = 0.001) fat yield when compared with to 40 DIM, but no interaction was observed between cows fed PO. On the other hand, milk true protein treatment and day of BCS evaluation. During the study concentration was less (P = 0.001) in cows fed PO than period, multiparous cows had a smaller (P < 0.001) in cows fed LTFA, although milk true protein yield did mean BCS than did primiparous cows (3.23 ± 0.01 vs. not differ between treatments. The SCC did not differ 3.55 ± 0.02).
between PO and LTFA.
Journal of Dairy Science Vol. 92 No. 4, 2009 FAT SOURCE ALTERS EMBRYO QUALITY IN DAIRY COWS Figure 3. Plasma concentrations of NEFA (panel A), BHBA (panel B), and glucose (panel C) during the pre- and postpartum periods for
cows fed calcium salts of palm oil (PO; ──) or linoleic and trans-octadecenoic acids (LTFA; ----). Plasma concentrations did not differ between PO and LTFA for NEFA (P = 0.80), BHBA (P = 0.58), and glucose (P = 0.20). There was an effect (P < 0.001) of day of sampling on concentrations of NEFA, BHBA, and glucose in plasma. There were no interactions between treatment and day for NEFA (P = 0.20) and BHBA (P = 0.23), but concentration of glucose was greater (*P = 0.002) on d 1 postpartum for cows fed PO compared with cows fed LTFA.
Journal of Dairy Science Vol. 92 No. 4, 2009 CERRI ET AL.
Table 2. Effect of source of dietary fatty acids on production and
18.3 ± 0.4 mm. Diameter of the CL at 5 d after AI, on composition of milk in the first 10 wk of lactation the day of uterine flushing, also was similar between treatments and averaged 21.8 ± 0.5 mm.
Concentration of progesterone was greater (P = 0.02) for cows fed PO compared with cows fed LTFA at the time of PGF2α injection (3.6 ± 0.1 vs. 2.8 ± 0.1 ng/mL) before initiation of the Ovsynch protocol (Figure 4). The concentrations of progesterone at different times in the Ovsynch protocol, at AI, and in the 5 d after AI Milk true protein were did not differ between the PO and LTFA groups. After AI, progesterone increased (P < 0.001) in the first 5 d, but no interaction between treatment and day after AI was detected.
PO = calcium salt of palm oil; LTFA = calcium salt of linoleic and Ovarian Structures, Responses to the Ovsynch
The proportion of embryos-oocytes recovered on d Protocol, and Progesterone Concentration
5 after AI relative to the number of CL was similar between treatments and averaged 52.5% (Table 4). The effect of fat source on ovarian follicles and CL, Fertilization tended (P = 0.10) to be greater for cows and responses to hormonal treatments are depicted fed LTFA than for those fed PO. Relative to embryos-in Table 3. The prevalence of cyclic cows tended (P oocytes, cows fed LTFA were 3.2 times more likely = 0.06) to differ, and more cows fed PO were cyclic (P = 0.02) to have embryos graded as 1 and 2 than than cows fed LTFA. Ovulations to the first and sec- cows fed PO. The proportion of degenerated embryos ond GnRH injections in the Ovsynch protocol, as well was similar between treatments, but when data from as CL regression after administration of PGF2α were both unfertilized and degenerate embryos-oocytes were not different between treatments. Synchronization of evaluated together, cows fed LTFA tended (P = 0.10) ovulation, which included CL regression and ovulation to have a smaller proportion of nonviable structures within 48 h after the second GnRH of the Ovsynch than cows fed PO.
protocol, was similar between treatments and averaged Relative to embryos only, cows fed LTFA had a 83.1%. Proportion of cows with double ovulation after greater (P = 0.05) proportion of grades 1 and 2 em-the second GnRH of the Ovsynch protocol did not dif- bryos than cows fed PO, but the proportions of de- fer between the PO and LTFA groups when all cows or generated embryos did not differ between treatments only ovulatory cows were considered. Diameter of the (Table 4). The mean number of blastomeres differed preovulatory follicle at the final GnRH of the Ovsynch (P = 0.01), and cows fed PO had fewer cells than cows protocol was similar between treatments and averaged fed LTFA; however, the median number of blastomeres Table 3. Effect of source of dietary fatty acids on cyclic status and on ovarian responses to the Ovsynch protocol
Treatment,1 % (n/n) Ovulation to first GnRH4 Ovulation to second GnRH Double—ovulatory cows Double—all cows Dominant follicle at AI, mm Corpus luteum diameter 5 d after AI, mm 1PO = calcium salt of palm oil; LTFA = calcium salt of linoleic and trans-octadecenoic acids.
2AOR = adjusted odds ratio. The PO treatment was used as the referent group.
3CI = confidence interval.
4One LTFA was not evaluated for ovulation to the first GnRH because this cow missed the ultrasonographic examination of the ovaries on the day of the first GnRH of the Ovsynch protocol.
Journal of Dairy Science Vol. 92 No. 4, 2009 FAT SOURCE ALTERS EMBRYO QUALITY IN DAIRY COWS Figure 4. Concentrations of progesterone in plasma during the synchronization protocol in cows fed calcium salts of palm oil (PO) or linoleic
and trans-octadecenoic acids (LTFA). Cows fed PO had greater (*P = 0.02) concentration of progesterone at the PGF2α of the presynchroniza-tion. Progesterone concentrations were similar (P > 0.68) between treatments on the remaining days. After AI, concentrations of progesterone increased (P < 0.001), but no interaction (P = 0.28) between treatment and day after AI was detected.
and proportion of live blastomeres remained similar Although treatments influenced embryo quality, no between treatments. The median number of accessory relationships were observed between the proportion of spermatozoa and embryos-oocytes with at least 1 ac- embryos graded as 1 and 2 and mean concentrations cessory spermatozoon were similar between treatments, of NEFA and BHBA during the pre- and postpartum but the mean number was greater (P < 0.01) in the periods or between NEFA and BHBA peak concentra-LTFA group compared with the PO group.
tions in the postpartum period. Simple and partial Table 4. Effect of source of dietary fatty acids on recovery, fertilization, and quality responses of embryos-oocytes
Treatment,1 % (n/n) Degenerated, unfertilized Embryos Grades 1 and 2 Blastomeres Mean ± SEM Acessory spermatozoa, n Mean ± SEM Embryo-oocyte ≥1 1PO = calcium salt of palm oil; LTFA = calcium salt of linoleic and trans-octadecenoic acids.
2AOR = adjusted odds ratio. The PO treatment was used as the referent group.
3CI = confidence interval.
Journal of Dairy Science Vol. 92 No. 4, 2009 CERRI ET AL.
correlations between plasma metabolites and embryo- observed by others who fed lactating dairy cows trans- oocyte responses (fertilization, grade quality, number of octadecenoic FA (Juchem et al., 2004; Piperova et al., blastomeres, percentage of live blastomeres, and acces- 2004). The decrease in milk energy output caused by sory spermatozoa) had a correlation coefficient of ≤0.3 the suppressed mammary lipogenesis was probably not and they were all nonsignificant (P > 0.10). Average sufficient to promote a significant decrease in body fat concentrations of NEFA during the postpartum period mobilization, as observed by the similar concentrations did not differ (P = 0.68) between cows with or without of NEFA, BHBA, and glucose, and the BCS between embryos graded as 1 and 2, and they were, respectively, the 2 treatments.
0.73 ± 0.05 and 0.76 ± 0.06 mEq/L. Similarly, mean Previous studies reported deleterious effects of NEFA BHBA concentrations during the postpartum period in the follicular fluid on oocyte competence (Leroy et were, respectively, 1,378.9 ± 120.8 and 1,150.0 ± 144.1 al., 2005) and of high BHBA concentrations from 3 μmol/L for cows with or without embryos graded 1 wk prepartum to 9 wk postpartum on the interval to and 2 (P = 0.22). Even when peak concentrations of pregnancy (Walsh et al., 2007). However, the indicators NEFA or BHBA during the postpartum period were of metabolic status of dairy cows during the transition analyzed in place of mean concentrations, no significant period were not correlated with fertilization and em-differences were observed between cows with or without bryo quality in the present study. In the current study, embryos graded 1 and 2, and they were, respectively, NEFA, BHBA, and glucose were measured during the 1.04 ± 0.07 and 1.14 ± 0.09 mEq/L of NEFA (P = transition period, but not when embryos-oocytes were 0.38), and 2,354.2 ± 214.7 and 1,786.0 ± 256.1 μmol/L collected. Thus, metabolic changes during the transi-of BHBA (P = 0.12).
tion period may not be good predictors to assess the effects of nutrient needs for lactation on early embryo development. In addition, it is possible that the effects of lactation and energetic status of the cows might Improvements in fertilization and early embryonic influence fertility in stages after the period of early development were observed in embryos-oocytes col- lected from cows fed a diet enriched with LTFA. The difference in proportion of cyclic cows between Embryos-oocytes from cows fed LTFA had an increased treatments was not expected because measures of en-proportion of fertilized oocytes and mean number of ergy status were mostly similar. Juchem et al. (2008) accessory spermatozoa. Furthermore, the proportion observed no effect of fat sources differing in FA profile of embryos-oocytes or only embryos graded as 1 and on the interval to first postpartum ovulation or pro-2 were greater when cows were fed LTFA than when portion of cows cyclic at 6 wk postpartum. Similarly, cows were fed PO. Diets were offered throughout the Santos et al. (2008b) indicated that fat feeding, but transition period up to 70 DIM, which is probably the not type of FA, affected the resumption of postpartum most appropriate time to positively affect the overall ovulation in lactating dairy cows. The responses to hor-metabolic status and the reestablishment of reproduc- monal treatments during synchronization of ovulation, tive functions in the cow.
ovulatory follicle and CL diameters, and concentrations Concentrations of NEFA and BHBA in plasma in- of progesterone in plasma were all similar for cows fed creased, whereas that of glucose decreased abruptly im- PO and those fed LTFA. Previous studies, however, mediately after calving, in a pattern already expected showed positive effects of supplementation with linoleic for a cow during the transition period (Grummer, and α-linolenic acids on preovulatory follicle diameter 1995). In the present study, however, no differences and CL volume on d 7 after AI (Bilby et al., 2006) were found in concentrations of NEFA, BHBA, and glu- and on progesterone concentration from d 6 to 8 after cose between treatments during the transition period. ovulation (Thangavelu et al., 2007) in lactating cows. These results are not in accordance with our initial hy- Increased preovulatory follicle diameter is expected to pothesis, although Juchem et al. (2004) also observed result in a larger CL, which explains the findings by a lack of differences in metabolic responses in cows fed Bilby et al. (2006). Although fat feeding clearly affects diets containing LTFA and PO during the transition follicle diameter (Santos et al., 2008b), it is less clear period. Furthermore, no difference in BCS was detected whether the type of FA has a differential effect on this throughout the experimental period, which reinforces response. The effect of polyunsaturated FA on steroido-the absence of a major alteration in the pattern of genesis (Wathes et al., 2007), particularly affecting the body fat mobilization. Milk fat synthesis decreased in regulation of the steroid acute regulatory protein, could cows supplemented with LTFA because of a reduction partially explain the improvements in CL function in in milk fat content, which resulted in reduced yields previous studies. Differences in responses between the of milk fat and 3.5% FCM. These results have been current study and the findings by others may be attrib- Journal of Dairy Science Vol. 92 No. 4, 2009 FAT SOURCE ALTERS EMBRYO QUALITY IN DAIRY COWS uted to when progesterone was measured. It is possible ments in fertilization were the result of improved uter-that dietary effects on CL function are not observed ine health (Cerri et al., 2009a), which subsequently led during metestrus and early diestrus. In addition, the to increased pregnancy per AI (Juchem et al., 2008). amounts of polyunsaturated FA fed by others (Bilby et In addition, the effects of LTFA on the cellular and al., 2006; Thangavelu et al., 2007) were substantially molecular pathways involved in the fertilization process greater than those used in the current study, and one in the cow are largely unknown. This possibility cannot cannot reject the view that the response to FA is dose be rejected because these FA can function as signaling dependent. The optimal level for supplementation of molecules and affect cell membrane properties (Wathes specific FA such as LTFA in the diet of dairy cows et al., 2007).
to improve fertility is unknown (Juchem et al., 2008; The present study initially hypothesized that the Santos et al., 2008b), and a major impediment is the beneficial effect on pregnancy observed by Juchem et lack of an accurate prediction of specific FA flow to the al. (2008) could be caused by improved fertilization and small intestine available for absorption (Santos et al., embryo quality. Cows fed LTFA indeed had improved 2008b).
fertilization and yielded a greater proportion of grades Cows were flushed 5 d after AI, and recovery rate 1 and 2 embryos, and their embryos had more blastom- averaged 54.5% for both treatments. Other studies that eres. Improvements observed in pregnancy in lactating attempted to recover embryos-oocytes from nonsupero- dairy cows fed LTFA might, in part, be attributed to vulated cows at a similar interval from induction of better embryo quality. There are no detailed descrip-ovulation had a similar recovery rate (Villaseñor et al., tions of FA content in bovine embryos from fertilization 2008; Cerri et al., 2009a,b). The reason for the rela- to d 5 of development, but there is a clear tendency to- tively low recovery is still unclear. A possibility is that ward the uptake of unsaturated FA by embryos as they a residual embryo-oocyte is still in the oviduct on d 5 develop from d 7 to 14 after AI (Menezo et al., 1982). after the induction of ovulation. Embryos-oocytes from For instance, the content of linoleic acid increased from cows fed LTFA had a greater mean number of accessory 0.5 to 5.7% of the total FA content of the embryo from spermatozoa and increased fertilization compared with d 7 to 13, suggesting increased uptake of linoleic acid as cows fed PO. It is possible that the increased number the embryo grows. Haggarty et al. (2006) observed an of spermatozoa reaching the oviduct might improve the increase in the incorporation of linoleic acid in cultured fertilization of oocytes. Cerri et al. (2009b) observed human embryos that developed beyond the 4-cell stage that the number of accessory spermatozoa that resulted compared with embryos that did not reached the 4-cell in the best sensitivity and specificity for fertilization stage. Moreover, an increase in the number of blasto-was 4, and cows with embryos-oocytes containing ≥7 meres was detected in embryos collected on d 7 after accessory spermatozoa had 100% fertilization. Further- AI in superovulated cows treated with diets contain- more, feeding the same types of supplemental FA used ing more linoleic or α-linolenic acids compared with a in the current study reduced the incidence of more se- diet containing more saturated FA (Thangavelu et al., vere metritis in dairy cows in the first 2 wk postpartum 2007).
(Juchem et al., 2008). Clinical uterine diseases post- Oocyte competence also seems to be affected by di- partum are known to increase the risk of subclinical etary supplemental fat in dairy cows. Increasing the endometritis in lactating dairy cows (Rutigliano et al., amount of dietary fat increased the number of trophec-2008), and the latter suppresses fertility (Rutigliano et toderm cells from in vitro-produced blastocysts origi-al., 2008), partly because of reduced fertilization rate nating from oocytes of cows fed a diet with increased (Cerri et al., 2009a). Subclinical endometritis was not fat content from calcium salts of PO (Fouladi-Nashta evaluated in the current study, but it is possible that et al., 2007). In fact, fat feeding has generally improved feeding LTFA changed uterine health or increased the embryo quality in embryo donor cows (Santos et al., concentration of unsaturated FA in endometrial cell 2008a). Interestingly, oocytes and the surrounding phospholipids (Wathes et al., 2007), which might have cells had a 3- to 4-fold increase in the concentrations favored sperm transport to the site of fertilization.
of linoleic acid when derived from cows in the winter Juchem et al. (2008) observed that cows fed LTFA months compared with the summer months (Zeron et had a small increase in plasma concentrations of PGF2α al., 2001). Such changes in oocyte membrane composi-metabolite in the first 2 d postpartum and a reduced tion may contribute to the increased fertility of dairy incidence of acute puerperal metritis compared with cows in the winter months compared with the sum-cows fed PO, although the incidence of retained placen- mer months (Zeron et al., 2001). Linoleic acid also has ta and the overall incidence of metritis were unaltered. been implicated in the regulation of meiotic arrest in Nevertheless, pregnancy to first postpartum AI was bovine oocytes at the germinal vesicle stage, because increased by feeding LTFA. It is possible that improve- Homa and Brown (1992) showed a negative correlation Journal of Dairy Science Vol. 92 No. 4, 2009 CERRI ET AL.
between diameter of the follicle and content of linoleic acid in the follicular fluid. Despite indications that Bilby, T. R., J. Block, B. C. do Amaral, O. Sá Filho, F. T. Silvestre, unsaturated FA have differential effects on the oocyte P. J. Hansen, C. R. Staples, and W. W. Thatcher. 2006. Effects (Wathes et al., 2007; Santos et al., 2008a), models in of dietary unsaturated fatty acids on oocyte quality and follicular vitro have not supported these suggestions. Bilby et development in lactating dairy cows in summer. J. Dairy Sci. al. (2006) observed no effect on in vitro oocyte nuclear Burr, G. O., and M. M. Burr. 1930. On the nature and role of the fatty maturation and apoptosis when cows were fed supple- acids essential in nutrition. J. Biol. Chem. 86:587–621.
mental FA with different degrees of unsaturation or on Castañeda-Gutiérrez, E., B. C. Benefield, M. J. de Veth, N. R. Santos, R. O. Gilbert, W. R. Butler, and D. E. Bauman. 2007. Evaluation in vitro embryonic development. Differences between of the mechanism of action of conjugated linoleic acid isomers on the findings of the current study and those of Bilby et reproduction in dairy cows. J. Dairy Sci. 90:4253–4264.
al. (2006) suggest either that an in vitro system may Cerri, R. L. A., H. M. Rutigliano, R. C. Chebel, and J. E. P. Santos. 2009b. Period of dominance of the ovulatory follicle not reflect in vivo findings (Santos et al., 2008a), or influences embryo quality in lactating dairy cows. Reproduction that beneficial effects observed in the current study and in others (Juchem et al., 2008) may be related to fac- Cerri, R. L. A., H. M. Rutigliano, F. S. Lima, D. B. Araujo, and J. E. P. Santos. 2009a. Effect of source of supplemental selenium on tors other than oocyte competence. Potential biological uterine health and embryo quality in high-producing dairy cows. windows are the oviduct and the uterine environment.
Cerri, R. L. A., J. E. P. Santos, S. O. Juchem, K. N. Galvão, and R. C. Chebel. 2004. Timed artificial insemination with estradiol cypionate or insemination at estrus in high-producing dairy cows. J. Dairy Sci. 87:3704–3715.
DePeters, E. J., J. B. German, S. J. Taylor, S. T. Essex, and H. Perez- Results from the current study demonstrate a dif- Monti. 2001. Fatty acid and triglyceride composition of milk fat ferential effect of type of FA on fertilization and embryo from lactating Holstein cows in response to supplemental canola quality in nonsuperstimulated lactating dairy cows. oil. J. Dairy Sci. 84:929–936.
Ferguson, J. D., D. T. Galligan, and N. Thomsen. 1994. Principal Feeding calcium salts of LTFA from late gestation to 70 descriptors of body condition score in Holstein cows. J. Dairy Sci. d postpartum improved fertilization and embryo qual- ity. Improvements in fertilization and embryo quality Fouladi-Nashta, A. A., C. G. Gutierrez, J. G. Gong, P. C. Garnsworthy, and R. Webb. 2007. Impact of dietary fatty acids on oocyte quality were not associated with responses of energy status, and and development in lactating dairy cows. Biol. Reprod. 77:9–17.
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pathways that led to improvements in fertilization and Juchem, S. O., R. L. A. Cerri, M. Villaseñor, K. N. Galvão, R. G. S. Bruno, H. M. Rutigliano, E. J. DePeters, F. T. Silvestre, W. W. embryo quality remain to be elucidated.
Thatcher, and J. E. P. Santos. 2008. Supplementation with calcium salts of linoleic and trans-octadecenoic acids improves fertility of lactating dairy cows. Reprod. Domest. Anim. doi:10.1111/j.1439- Juchem, S. O., R. L. A. Cerri, M. Villasenor, K. N. Galvão, R. G. This research was supported by grants from the Na- S. Bruno, H. M. Rutigliano, A. C. Coscioni, E. J. DePeters, W. tional Research Initiative USDA Research Grant no. W. Thatcher, D. Luchini, and J. E. P. Santos. 2004. Effect of feeding calcium salts of palm oil (PO) or of a blend of linoleic and 2004-35203-14137 from the USDA (Washington, DC), monoenoic trans-fatty acids (LTFA) on lactation and health of USDA formula funds, and the Center for Food Animal lactation Holstein cows. J. Dairy Sci. 87(Suppl. 1):95. (Abstr.) Health of the University of California-Davis. The au- Kindahl, H., K. Odensvik, S. Aiumlamai, and G. Fredriksson. 1992. Utero-ovarian relationship during the bovine post-partum period. thors thank Virtus Nutrition (Fairlawn, OH) for partial Anim. Reprod. Sci. 28:363–369.
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